Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 963

______

Studies on the Transmembrane Signaling of β1

BY

ANNIKA ARMULIK

ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2000 Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Medical Biochemistry presented at Uppsala University in 2000

ABSTRACT

Armulik, A. 2000. Studies on the transmembrane signaling of β1 integrins. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 963. 92 pp. Uppsala. ISBN 91-544-4832-1.

Integrins are heterodimeric cell surface receptors, composed of an α and a β subunit, mainly for binding . subunit β1 can combine with at least 12 α subunits and thus form the biggest subfamily within the integrin family. In this thesis, functional properties of the splice variant β1B, and the effects of several mutations in the cytoplasmic tail of integrin subunit β1A were studied. In addition, the border between the transmembrane and cytoplasmic domains of several integrin subunits was determined. The β1B splice variant has been reported to have a dominant negative effect on functions of β1A integrins. In this study, it was studied if the expression of β1B had similar negative effects on the αvβ3 integrin functions since the β3 subunit is structurally similar to β1A. The β1B subunit was expressed in an integrin β1-deficient cell line and it was found that the presence of β1B does not interfere with adhesion or signaling of endogenous αvβ3. The border between the cytoplasmic domain and the C-terminal end of the transmembrane domain of integrin α and β subunits has been unclear. This question was experimentally addressed for integrin subunits β1, β2, α2 and α5. It was found that integrin subunits contain a positively charged lysine, which is embedded in the membrane in the absence of interacting proteins. The functional importance of the lysine in integrin transmembrane domains was investigated by mutating this to leucine in β1A. The mutation affected cell spreading and of the adapter CAS. The activation of kinase and tyrosine phosphorylation of was not affected. Furthermore, the mutation of two to phenylalanines in the β1A cytoplasmic tail was found to reduce the capability of β1A integrins to mediate cell spreading and migration. Activation of focal adhesion kinase in response to the later β1A mutant was shown to be impaired as well as tyrosine phosphorylation of adapter proteins paxillin and tensin, whereas overall tyrosine phosphorylation of CAS was unaffected. These data suggest the presence of focal adhesion kinase-dependent and -independent pathways for tyrosine phosphorylation of CAS after integrin β1A-mediated adhesion.

Key words: Integrin β1, integrin signaling, transmembrane domain, CAS, focal adhesion kinase.

Annika Armulik, Department of Medical Biochemistry and Microbiology, Biomedical Center, Box 582, Uppsala, SE-751 23, Sweden

Annika Armulik 2000 ISSN 0282-7476 ISBN 91-544-4832-1 Printed in Sweden by University Printers, Uppsala 2000 TToo tthhee mmeemmoorryy ooff mmyy GGrraannddmmootthheerr

KKaalllliillee vvaannaaeemmaallee This thesis is based on the following papers, referred to in the text by their roman numerals:

I Armulik, A., Svineng, G., Wennerberg, K., Fässler, R., and Johansson, S. (2000). Expression of integrin subunit β1B in integrin β1-deficient GD25 cells does not interfere with αvβ3 functions. Exp. Cell Res.254, 55-63

II Wennerberg, K., Armulik, A., Sakai, T., Karlsson, M., Fässler, R., Schaefer, E. M., Mosher, D. F., and Johansson, S. (2000). The cytoplasmic tyrosines of integrin subunit β1 are involved in FAK activation. Mol. Cell. Biol. 20, 5758-5765

III Armulik, A., Nilsson, I., von Heijne, G., and Johansson S.(1999). Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits. J. Biol. Chem. 274, 37030-37034

IV Armulik, A. and Johansson, S. (2000). Lysine 756 in the transmembrane domain of integrin subunit β1 is necessary for β1 integrin signaling. Manuscript

Published manuscripts have been reprinted with permission from the respective journals.

4 TABLE OF CONTENTS

ABBREVIATIONS……………………………………………………………………… 7 INTODUCTION………………………………………………………………………... 8 INTEGRINS…………………………………………………………………….. 8 Integrin structure………………………………………………………..…. 10 Extracellular domain…………………………………………………... 10 Transmembrane domain………………………………………………. 11 Cytoplasmic domain…………………………………………………… 13 SPLICE-VARIANTS OF β1…………………………………………………… 15 BIOSYNTHESIS OF INTEGRINS…………………………….………………. 17 INTEGRIN LIGAND BINDING……………………………………………… 19 MODULATION OF INTEGRIN LIGAND BINDING……………………… 21 Phosphorylation of integrin cytoplasmic tails…………………………….. 22 Lateral associations………………………………………………………... 23 Affinity/avidity modulation by cytoplasmic proteins…………………….. 24 Talin…………………………………………………………………… 24 Calreticulin…………………………………………………………….. 27 CIB…………………………………………………………………….. 27 ICAP-1α……………………………………………………………….. 28 Cytohesin-1…………………………………………………………….. 28 β3-endonexin…………………………………………………………... 28 TAP20………………………………………………………………….. 29 Ras family …………………………………………………… 29 INTGERINS AND RHO FAMILY GTPases…………………………………. 30 Regulation of Rho family proteins………………………………………… 30 Functions of RhoA………………………………………………………… 31 Functions of Cdc42 and Rac1……………………………………………... 33 INTEGRINS AND CELLULAR SIGNALING PATHWAYS………………... 35 Focal adhesion kinase……………………………………………………… 36 Structure……………………………………………………………… 36 FAK related proteins……………………………………………….…. 36 The FAF/Src complex……………………………………………….… 37 FAK activation………………………………………………………... 38 Cell survival………………………………………………………….. 38 Migration……………………………………………………………... 39 Activity regulation………………………………………………….…. 39 Crk and Crk-associated substrate (CAS)………………………………….. 41 CAS interacting proteins……………………………………………… 42 Regulation of CAS…………………………………………………….. 43 The CAS/Crk complex………………………………………………… 44 Paxillin and zyxin………………………………………………….………. 45 Tyrosine phosphorylation of paxillin………………………………….. 46 The LIM domains of paxillin………………………………………….. 46 The LD motifs of paxillin……………………………………………… 47 Cellular localization of paxillin……………………………………….. 47

5 Zyxin…………………………………………………………….…….. 48 Other non-receptor tyrosine kinases…………………………………… … 49 Mitogen-activated protein kinases………………………………………… 50 Activation of MAPK by integrins……………………………………… 50 Cell migration………………………………………………………… 51 ……………………………………………………………… 52 Protein kinase C…………………………………………………………… 53 Integrin linked kinase……………………………………………………… 55 Phosphatidylinositol kinases……………………………………………… 56 INTEGRINS AND CAVEOLAE……………………………………………... 58 PRESENT INVESTIGATION…………………………………………………………. 59 AIMS OF THE THESIS………………………………………………………. 59 RESULT AND DISCUSSION………………………………………………... 59 Paper I………………………………………………………………….……. 59 Paper II………………………………………………………………………. 60 Paper III……………………………………………………………………... 60 Paper IV……………………………………………………………………... 61 FUTURE PERSPECTIVES…………………………………………………… 61 ACKNOWLEDGEMENTS…………………………………………………………….. 63 REFERENCES………………………………………………………………………….. 64

6 ABBREVIATIONS a.a. amino acid ACK activated Cdc42-associated tyrosine kinase Arf ADP ribosylation factor CaMKII calcium- and calmodulin-dependent kinase II CAS Crk-associated substrate Cat Cool-associated, tyrosine-phosphorylated Cbp Csk binding protein Csk C-terminal Src kinase DAG diacylglycerol ECM extracellular matrix ER endoplasmatic reticulum ERK extracellular-regulated kinase FAK focal adhesion kinase FRNK FAK-related non-kinase CIB calcium- and integrin binding protein GAP GTPase-activating proteins GEF guanine-nucleotide exchange factor GDI GDP dissociation inhibitor ICAP integrin cytoplasmic domain-associated protein ILK integrin linked kinase JNK c-Jun N-terminal kinase MAPK mitogen-activated protein kinase MIDAS metal ion-dependent adhesion site MLC chain PAK p21-activated kinase PIP phosphatidyl inositol phosphate PI3K phosphatidyl inositol 3 kinase PKL paxillin kinase linker PKC protein kinase C PLC phospholipase C PTEN phosphatase and tensin homologue deleted on ten PTP protein tyrosine phosphatase Pyk proline rich tyrosine kinase RACK receptor for activated C-kinase SH Src homology STICK substrates that interact with C-kinase T M transmembrane

7 INTRODUCTION

Recently, while thinking about my son, I bought a book titled “ How do things work”. From this book one can read about working principles of many inventions that are part of our everyday life. Today’s medical science, even though it often only describes and observes biological processes, tries to answer questions why and how. This curiosity carries a noble philanthropic goal, to fight against diseases. But if the “Master of Life” would publish a book that gave us all the answers, tells us everything about everything, would we read it? Or would we continue to seek for answers ourselves even though such a book based on our search would most likely be entitled “How things might work”.

INTEGRINS

Most cells in multicellular organisms are in contact with each other and/or with a protein network called extracellular matrix (ECM). Extracellular matrices are highly ordered structures of proteins (e.g. collagens, , fibronectin) and glycosaminoglycans which are secreted and assembled by cells and have a remarkable control over cells (212). Cell-matrix and cell-cell contacts are critical for tissue integrity and homeostasis. Most of the cell surface receptors that mediate these interactions can be grouped into four protein families: , immunoglobilin superfamiliy members, and integrins. The term “integrin” was introduced by Richard Hynes’ group in 1986 and designates cell surface receptors that mainly bind extracellular matrix proteins. The fact that integrins are found in species from corals and sponges (the most primitive representatives of metazoan) to mammals indicates their importance in evolution of multicellular organisms (69). Since the publication of the full-length cDNAs of the fibronectin receptor (current name α5β1) in 1986 -1987 (16, 557), 18 α and 8 β subunits are now described. The known combinations of different α and β subunits give rise to 24 different integrin heterodimers (Fig. 1). Roughly, integrins fall into three subfamilies dependent on their subunit composition and ligand specificity. (i) The β1 subunit can combine with 12 α subunits and forms the biggest subfamily of the integrin family. The β1 integrins are widely expressed and mediate mainly to ECM proteins. (ii) The β2 and β7 integrins are expressed exclusively on blood cells and mediate interactions to ICAM-s, E- and fibrinogen. (iii) Integrins containing the αv subunit are expressed by blood cells, endothelial cells, epithelial cells and osteoclasts. This group of receptors recognizes a broad range of ligands containing an RGD motif. Two integrins with highly specialized functions fall outside these groups. The αIIbβ3 integrin, expressed by , has an important role in blood coagulation. The α6β4 integrin is an essential component of hemidesmosomes in keratinocytes. Integrins not only connect cells mechanically to the surrounding environment but also mediate signals from it that regulate growth, death, differentiation, and movement of cells. The short cytoplasmic tails of integrins are required for integrin signaling. Since integrins do not possess any known intrinsic kinase activity they transduce signals by spatially compartmentalizing docking and adapter proteins that link integrins to cytoplasmic kinases. Ligand bound integrins are clustered into aggregates that are connected to filaments, or intermediate filaments in the case of the α6β4 integrin. So, integrins not only act as molecular bridges that link intracellular filament systems with ECM but are also important for signal

8 α IIb αE

α3 β7 α2 α4 β α 3 α1 L α5 αM β5 αV β1 β2 α6 α β6 X α11 β8 α7 αD α10 β4 α8 α9

Figure 1. The integrin family. All presently known α and β subunits are shown as circles. Lines between α and β subunits show known associations. transduction from ECM by assembling cytoplasmic signaling molecules (outside-in signaling). Importantly, integrin-mediated cell adhesion is dynamically regulated by cells themselves (inside-out signaling). On the cell surface integrins acquire several conformations, corresponding to low or high affinity states for ligand binding. Modulation of integrin affinity by intracellular factors results in extensive conformational changes in the receptor that also affect the ligand-binding interface. A common theme in integrin signaling is “clustering”. What is the function of receptor clustering in integrin signaling? Why are integrins clustered at focal contacts? Many integrin mediated cell-matrix adhesion sites over the entire area would give a better “grip” for cells. Some likely reasons are: integrin clustering increases the avidity of the interactions with immoblized ligands (such as extracellular matrix). The tension emanating from strong and distinct connection sites with extracellular matrix has an important role in assembly of extracellular matrix. Integrin clustering promotes interaction between signaling molecules and thereby increases the efficacy of integrin signals. This may lead to a synergistic response and/or to a unique outcome of ramifying signaling pathways from clustered structures. What happens when integrins do not work properly? Integrin malfunction is linked to several genetic diseases. Leukocyte adhesion deficiency (LAD)-1 syndrome is an autosomal recessive disorder caused by mutations in the β2 that leads to absent /aberrant biosynthesis of the β2 subunit or to an integrin unable to bind ligands (9, 229, 230). These patients typically suffer from frequent infections due to the inability of to extravasate and kill microorganisms. Glanzmann thrombasthenia is an inherited bleeding disorder that is caused by decreased expression of functional integrin αIIbβ3 (37, 139, 552). Mutations causing this disease lie in the encoding either the α or the β3 subunit, leading to impaired aggregation with fibrinogen. Some forms of epidermolysis bullosa

9 (blistering diseases) are caused by mutations in the β4 gene (182, 587). In these patients, hemidesmosomes are unstable and keratinocytes easily detach from the underlying basement membrane.

Integrin structure

Integrins are heterodimers, composed of non-covalently associated α and β subunits. Each subunit consists of a large extracellular domain, a single transmembrane domain and a short cytoplasmic domain of usually less than 50 a.a. (Fig. 2). The only exception to this well conserved structure is the β4 subunit, which has a large cytoplasmic domain (more than 1000 a.a.). Even though the association between an α and a β subunit is mainly mediated by the N- terminal regions - expression of the extracellular domains of integrins results in formation of dimers that retain ligand binding activity (121, 358, 361) - the transmembrane and cytoplasmic domains also contribute to the receptor dimerization and/or stabilization (68, 425, 535).

b-propeller fold SS + + + + a N C subunit

I domain

b putative N I domain C subunit Disulphide-bonded region

2+ a chain N-terminal repeat EGF-like repeats Transmembrane domain + Putative Me binding site

Figure 2. Schematic representation of a generic integrin α and β subunit. The positions of the conserved domains in integrin subunits are shown (not to scale).

Extracellular domain

Extracellular domains of integrin α subunits contain seven homologous repeats at their N- terminus, each about 60 amino acid residues in length (Fig. 2). The four or three last repeats (depending on the integrin) contain potential divalent cation binding sites of the general structure DxDxDGxxD, where x represents any a.a. (EF-hand loops). The N-terminal repeats are predicted to fold into a seven-bladed β-propeller (424). The β-propeller is a cyclic

10 structure where each of the seven blades contains four β-strands that are tilted in a manner that brings the connecting loops either to the upper or the lower surface of the propeller. Nine α subunits (α1, α2, α10, α11, αD, αE, αL, αM, and αX) contain an additional domain that is inserted between the second and the third N-terminal repeats (Fig. 2). This domain is referred to as the I domain (from inserted) or the A domain (after the to the A domain of von Willebrand factor). Although the tertiary structure of integrins has not yet been determined, the crystal structure of I domains of αM, αL, α1, α2 subunits has been solved (153, 302, 403, 450). I domains form a so-called “Rossman fold” where five parallel and one-anti-parallel β strands are surrounded by seven α helices (302). Integrin I domains contain a metal ion-dependent adhesion site (MIDAS motif) that participates in integrin ligand binding (302, 574, 622). The insertion of the I domain does not interfere with the predicted folding of the seven N-terminal repeats; it will be located on the upper side of the propeller (338). Another group of α subunits (α3, α5, α6, α7, α8, αv) are proteolytically cleaved close to the transmembrane domain and the two chains are bridged by a disulfide bond. The functional significance of this cleavage is not known. Integrin β subunits contain a highly conserved sequence of about 200 a.a. in their N- terminal part which has been predicted to have a tertiary fold similar to that of the A domain in von Willebrand factor (Fig. 2). This domain contains a MIDAS motif which is part of the ligand binding pocket (324, 570). By reason of its structural similarity to the I domain of α subunit, this domain in β subunits has been denoted as a putative β I domain. Based on the data obtained by using a that recognizes an epitope comprising of regions from both α and β subunits, it was proposed that the putative β I domain associates with the side of the β-propeller domain at β-sheets 2 and 3 in α subunits (639). The C- terminal part of the extracellular domain of β subunits contains a cysteine rich region composed of four epidermal growth factor (EGF)-like repeats (stalk-like structure). Many growth factors and adhesion molecules contain EGF-like domains, which often participate in protein-protein interactions. The EGF-like repeats of integrin β subunits and the secreted protein TIED (ten β integrin epidermal growth factor-like repeat domains) share particular features not found in other EGF-like domains (49). This raises the interesting possibility that DNA encoding an ancestral TIED-like molecule might have integrated into the ancestor gene of β integrins and endowed integrins with novel features. For more then ten years it has been believed that integrins do not possess any enzymatic activity. A recent report by O’Neil et al. refutes this by showing that integrin subunit β3 possesses an endogenous thiol isomerase activity that is located in the cysteine repeats (408). Possibly, thiol bonding within the integrin stalk-like structure could be modified during integrin activation and thus contribute to the stabilization of an active conformation (408).

Transmembrane domain

Our present knowledge about the structure of the integrin transmembrane (TM) domains and their contribution to integrin signaling is very limited. In general, it has become clear that T M domains do not only passively anchor proteins to lipid bilayers but also serve important biological roles, e.g. in proteins such as the T-cell receptor and MH (major histocompatibility) complexes (113). For example, the T-cell receptor complex depends on charged amino acids in

11 the TM domains for assembly and cell surface expression (113). Alignment of the T M domains of integrin α and β subunits reveals some interesting conserved features. Firstly, they all are rather conserved in length. The transmembrane domain of the β2 and the β7 subunit seems to be 3-4 amino acids shorter than the TM domain of other β subunits (except for β4). Interestingly, these subunits are exclusively expressed in blood cells and acquire an inactive conformation until they are triggered. However, the exact length of the TM domain for integrin subunits is not known, primarily because the question of where the interface is between the extracellular and TM domain has not yet been experimentally addressed at all. Secondly, almost all transmembrane domains of α and β subunits contain small amino acids at three positions (Fig. 3). Notably, glycine is invariably found at the position no. 2 and no. 3 in α chains and in β chains, respectively (see Fig. 3). The functional importance of such an

α2 EVPTGVIIGSIIAGILLLLALVAILWKLGFFKRK α3 EIELWLVLVAVGAGLLLLGLIILLLWKCGFFKRA α4 YFTIVIISSSLLLGLIVLLLISYVMWKAGFFKRG α5 GVPLWIIILAILFGLLLLGLLIYILYKLGFFKRS α6 GVPWWIILVAILAGILMLALLVFILWKCGFFKRN αv PVPVWVIILAVLAGLLLLAVLVFVMYRMGFFKRV 12 3 β1 GPDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDR β2 GPNIAAIVGGTVAGIVLIGILLLVIWKALIHLS β3 GPDILVVLLSVMGAILLIGLAALLIWKLLITIHDR β5 TPNAMTILLAVVGSILLVGLALLAIWKLLVTIHDR β6 PPNIPMIMLGVSLATLLIGVVLLCIWKLLVSFHDR β7 DHTGQAIVLGCVGGIVAVGLGLVLAYRLSVEIV

extracellular transmembrane cytoplasmic

Figure 3. Amino acid alignment of the transmembrane domains of selected integrin subunits. Three boxes girdle the positions (marked as 1, 2, 3) of small amino acids in the T M domain. The conserved transmembrane lysine is indicated in bold. Three amino acids adjacent to the proposed TM domain are indicated in italics. arrangement has not been studied. The spacing between these small amino acids suggests that they are located at one side of the TM helix. Speculatively, they could form a flat surface on each integrin subunit, which could face the other subunit of the heterodimer or other interacting membrane proteins. Thirdly, the C-terminal segments of most integrin T M

12 domains exhibit a conserved pattern; one amino acid with a basic side chain is located in the continuous stretch of hydrophobic amino acids (Fig. 3). Generally, it has been suggested that the single lysine or arginine stops the TM domain whereas some investigators have proposed an alternative stop point 4-5 amino acids more distal from the lysine (Fig. 3). By applying the glycosylation mapping technique for determination of the border between the TM and cytoplasmic domains of different human α and β subunits, it was shown that the TM domains extend roughly to the second alternative startpoint leaving the conserved lysine in the membrane (paper III) (17). Interestingly, this kind of delineation places the highly conserved motif KXGFFKR in α subunits and the facing region in β subunits in the plasma membrane. These regions have been shown to be important for integrin dimerization and activation (127, 240, 339, 425, 542). They have also been reported to interact with different intracellular proteins such as skelemin, Rack1, WAIT-1, cytohesin-1 and calreticulin (Table 2). While most of the interactions characterized between integrins and other proteins occur via the extracellular or intracellular domain of an integrin, the association between the 16-kDa subunit of a protein called vacuolar H+-ATPase and integrin β1 subunit is mediated via TM domains of both proteins (531). The functional importance of this interaction is unclear but it might have a role in integrin internalization and receptor recycling (531).

Cytoplasmic domain

The cytoplasmic domains of integrins connect to the actin filament system and are required for integrin mediated signaling (outside-in signaling). In addition, through the interaction of specific intracellular proteins the cytoplasmic domains regulate the conformation of the extracellular domain (inside-out signaling). Cytoplasmic tails of β subunits (except β4) contain all the information necessary for integrin localization to focal contacts; a chimeric receptor, containing the extracellular and the transmembrane domain of the interleukin-2 receptor and the cytoplasmic tail of the β1 subunit, localizes to focal adhesions formed by endogenous integrins in fibroblasts (296). High levels of expression of the β subunit cytoplasmic tails inhibit functions mediated by endogenous intact integrins such as spreading, migration, and activation of FAK (99, 297, 342). In contrast to the β subunits, the cytoplasmic tail of integrin α subunits suppresses the localization to focal contacts of unoccupied integrins; deletion of the α tail results in ligand- independent recruitment to focal adhesions of the integrin (269, 296, 636). Cytoplasmic tails of integrin α and β subunits participate in intracellular signaling by recruiting structural and adapter proteins that link integrins to intracellular kinase cascades. Both the cytoplasmic tails of α and β subunits have in some cases been shown to activate pathways that lead to integrin specific cellular responses (255, 428, 603, 606, 621). The activation of integrins also involves the cytoplasmic domains of both α and β subunits (270, 341, 633). While deletion of the KXGFFKR motif in α TM domains results in constitutive high affinity conformation of the integrin (270, 409), deletion of the cytoplasmic tail of several α subunits results in defective ligand binding (265, 269, 270, 513). On the other hand, αIIb tail is not needed for ligand binding nor spreading (up to the KXGFFKR sequence) (309). Deletion of cytoplasmic domains of β2 and β3 abolishes the ligand binding of αLβ2 and αIIbβ3 integrins, respectively (218, 409). Cytoplasmic tails of α subunits differ in both length and amino acid sequence (Fig. 4). In marked contrast, six out of eight β subunits show a considerable degree of homology in length

13 cyto-1 cyto-2T/S cyto-3 β1 WKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK β2 WKALIHLSDLREYRRFEKEKLKSQWN-NDNPLFKSATTTVMNPKFAES β3 WKLLITIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFTNITYRGT β5 WKLLVTIHDRREFAKFQSERSRARYEMASNPLYRKPISTHTVDFTTNKFNKSYNGTTVDEGK β6 WKLLVSFHDRKEVAKFEAERSKAKWQTGTNPLYRGSTSTFKNVTYKHREKQKVDLSTDC β7 YRLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTINPRFQEADSPTL

α2 WKLGFFKRKYEKMTKNPDEIDETTELSS α5 YKLGFFKRSLPYGTAMEKAQLKPPATSDA α10 WKLGFFAHKKIPEEEKREEKLEQ

Figure 4. Amino acid alignment of the cytoplasmic domains of selected integrin subunits. The cyto-2, -3 and the T/S rich sequences in β subunits are boxed. The cyto-1 in β subunits and the KXGFFK motif in α subunits are indicated by arrows. The conserved transmembrane lysine is marked in bold. Vertical lines indicate the start sites for the cytoplasmic domains. and amino acid composition (Fig. 4). Great efforts have been made to identify residues in the cytoplasmic tail of β required for specific aspects of integrin function. In particular, the cytoplasmic domains of β1, β2, and β3 have been subjects for extensive studies regarding the effects of deletions and amino acid substitutions. Briefly, four conserved regions have been identified as having central roles in integrin β tail mediated functions (Fig. 4). The importance of these regions in integrin signaling is discussed under the section “Modulation of integrin ligand binding”. In addition, a sorting signal in β2 (YRRF) is required for recycling of spontaneously internalized receptors and for β2-integrin mediated cell migration (156). It is not known whether the related sequences in other β subunits have similar functions. In spite of somewhat clear knowledge of which regions in the cytoplasmic tails are essential for integrin functions, information on tertiary structures of integrin cytoplasmic domains is very limited. Recently, a first crystal structure of the two fibronectin type III domains from the cytoplasmic domain of integrin subunit β4 was resolved (129). The nuclear magnetic resonance structure for a myristoylated peptide corresponding to the cytoplasmic tail of αIIb has been resolved (589), and molecular models for cytoplasmic tails of the αIIbβ3 integrin have been presented (199).

14 SPLICE-VARIANTS OF β1

Identification of integrin splice-variants, both for extra- and intracellular domains has added an additional level of complexity in integrin signaling. Cytoplasmic splice-variants are described for β1, β3, β4, and for several α subunits (reviewed in 164). For human β1, five different splice-variants are characterized (Fig. 5) (11, 39, 299, 547, 582, 649). The cytoplasmic splice- variants do not change the ligand specificity for a given heterodimer, but they can modulate receptor affinity towards the ligand (paper I) (18, 43). All splice-variants of β1 share the common N-terminal part until the sequence WDT777 that corresponds to the 3’ end of exon 6 in the β1 gene (Fig. 5). The splice-variant A, mostly referred to as β1 only, is very conserved at the amino acid level amongst different species from sponge to human, particularly in the transmembrane and cytoplasmic domains (69). In mammals, all cells, except mature erythrocytes and myotubes express β1A. The expression of other splice-variants is more restricted.

752 777 β1A WKLLMIIHDRREFKAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK β1D WKLLMIIHDRREFKAKFEKEKMNAKWDTQENPIYKSPINNFKNPNYGRKAGL

β1B WKLLMIIHDRREFKAKFEKEKMNAKWDTVSYKTSKKQSGL β1C-1 β1C-2 WKLLMIIHDRREFKAKFEKEKMNAKWDTSLSVAQPGVQWCDISSLQPLTRSRFQQFSCLSLPSTWDYRVKILFIVRP

transmembrane cytoplasmic

Figure 5. The amino acid sequences of the cytoplasmic splice-variants of human integrin β1. The variant specific regions are shown in italics. The conserved transmembrane lysine is marked in bold. The NPXY motifs in β1A and β1D are underlined. A double lysine motif in β1B is in bold. The six amino acids absent in β1C-2 are underlined.

The β1B isoform was isolated from a human placenta library probed with a synthetic oligonucleotide corresponding to the cytoplasmic domain of β1A. The last 12 amino acids of β1B that are different from β1A are derived from the intronic sequence that follows immediately downstream of exon 6 (Fig. 5) (11). Analysis of the nucleotide sequence of the mouse β1 gene has revealed that mice have no homologue to human β1B. The mouse intronic sequence after exon 6 could potentially code for 15 amino acids (VSYETLLRAVGWFLK) that show no significant homology to human β1B, except for first three amino acids (39). The β1B specific transcript was detected at low levels in all human tissues and cell lines tested by

15 RT-PCR, but the protein was only detectable in skin (keratinocytes) and liver (hepatocytes) (11, 33). Expression of human β1B in CHO cells showed that β1B can dimerize with α subunits and bind to a fibronectin affinity matrix in an RGD-dependent manner in the presence of Mn2+. In contrast to β1A, the β1B integrins did not localize to focal contacts when cells were plated on fibronectin (33). Further analysis revealed that the human β1B isoform does not mediate cell spreading and activation of focal adhesion kinase in cells plated on anti-human β1 mAb (TS2/16) (34). In addition, expression of β1B in CHO cells reduced cell spreading on fibronectin and -1 but not on vitronectin. The cell attachment to fibronectin and laminin-1 was only affected in clones expressing high levels of β1B (50% of that of endogenous β1A). The migration on gelatin of CHO cells expressing the β1B was similar to that of CHO cells expressing β1A when vitronectin, but not fibronectin, was used as a chemoattractant. Again, higher β1B expression levels caused stronger inhibition. The so- called dominant negative effect of β1B on endogenous integrins was suggested to be caused by the competition of β1B with endogenous β1A for available α subunits (34) and subsequently a failure of β1B to activate intracellular signaling pathways. β1B expressed in β1-deficient cell line GD25, similar to β1A, dimerizes with α5, α3 and α6 subunits (464). GD25 cells do not adhere to laminin-1 but expression of β1A in GD25 cells restored the ability of these cells to attach to laminin-1 via α6β1A (613); however, the expression of β1B subunit did not promote cell adhesion unless Mn2+ was present in the medium (464). Analysis using antibodies recognizing epitopes exposed only in the ligand- competent/occupied integrins revealed that the extracellular domain of β1B integrins possesses an inactive conformation (paper I) (18, 464). The inactive ectodomain conformation could be changed to active by addition of Mn2+ or the GRGDS peptide (paper I) (18, 464). The spreading and organization of actin stress fibers of GD25-β1B cells on fibronectin was found to be impaired compared to GD25 cells (464). This indicated that β1B has a dominant negative effect not only on β1A integrins but also on αvβ3 integrin, since the attachment of GD25 to fibronectin is mediated via this latter integrin (613). However, the vitronectin substrate was not tested in the former report. In contrast to this finding (464) we have shown that β1B does not have a dominant-negative effect over the αv integrins (paper I) (18). The β1B integrins were found not only to be unable to mediate the assembly of fibronectin matrix but could also actually inhibit this process in CHO, GD25 and FRT cells (84, 464). Overexpression of constitutively active RhoA in FRT cells abrogated the negative effect of β1B on matrix assembly (83). Studies on human keratinocytes (one of the few cell types that reported to express the β1B variant at a detectable protein level) showed that overexpression of β1B in keratinocytes results in intracellular accumulation of the protein, which could be overcome by deleting the KK sequence (Fig. 5) (272). β1B has been suggested to have a regulatory role of adhesion-mediated signaling. However, the modulating effects of β1B over β1A have only been observed at expression levels many-fold higher than what occurs in vivo. Thus, the physiological relevance (if any) of human β1B remains to be established. Similar to β1B, the splice-variants β1C-1 and β1C-2 are only found in human (299, 547). The C-specific exon is part of an Alu element, and such DNA sequences are primate specific retrotransposable elements (547). The β1C-1 differs from β1C-2 by six amino acids (Fig. 5) that in β1C-2 are missing as a result of the utilization of a more distal 3’ splice acceptor site (547). Low levels of β1C-1 and β1C-2 transcripts have been identified by RT- PCR in many human cell lines and tissues (299, 353, 547). At the protein level β1C has been

16 detected in some cell lines (HEL, HUVEC) and in vivo in epithelial cells from tissues like breast, lung, gallbladder and prostate (168, 299). Immunohistological studies on prostate and breast cancer samples have shown that down-regulation of β1C expression correlates with invasive phenotype of these carcinomas (166, 353). Expression of β1C-1 in several cell lines has an inhibitory effect on but not on cell adhesion or organization of actin (168, 363). The cell-cycle inhibitor p27kip1 has been shown to be a potential downstream effector of β1C (167). The β1C-1 has an inhibitory effect on activation of ERK2 by fibronectin but not on activation of FAK or Akt. Moreover, ligation of β1C integrins leads to the activation of the Akt pathway (165). However, studies on β1C splice-variants expressed in β1-deficient GD25 cells showed that these subunits are retained in the cell and degraded rather than localized to the cell surface (548). The isoform β1D is the only splice-variant that shares significant homology with β1A (Fig. 5). The β1D specific part (the C-terminal 24 amino acids) is encoded by exon D, which is localized between exons 6 and 7 (582, 649). In vivo, the β1D isoform is only expressed in skeletal and cardiac muscles, and it completely displaces β1A in terminally differentiated muscle, where it associates with α7 (44). When expressed in non-muscle CHO and GD25 cells, β1D localizes at focal adhesions, and clustering of β1D triggers activation of FAK and MAPK pathways (43, 44). Cells expressing the β1D isoform showed reduced spreading and migration. It has been suggested that β1D integrins do not sense mechanical signals as efficiently as β1A integrins and therefore can not mediate cell migration as efficiently as β1A integrins (38). It is interesting that β1D integrins display an increased affinity for fibronectin and enhanced association with the actin cytoskeleton (43). In vitro binding studies have shown that the cytoplasmic domain of β1D binds the cytoskeletal proteins α-actinin, talin and with higher affinity than β1A (43, 438). It has been suggested that replacement of the β1A isoform in muscles with β1D might be necessary to strengthen the cytoskeletal-matrix link in muscle cells (43). However, the lack of β1D isoform in the mouse strain (due to the exon D knockout) did not affect muscle formation and did not cause muscular degeneration. In the converse situation, mice which express only the β1D variant (knock-in) were not viable and died in uteri because of a wide range of developmental defects (38). Embryonic β1D knock-in stem cells displayed reduced migratory activity. Expression levels of the β1D subunit were reduced when compared to β1A in wt ES cells; this could indicate that, when associated with other subunits than muscle-specific α7, the β1D protein is less stable (38).

BIOSYNTHESIS OF INTEGRINS

Expression of integrins is dynamically regulated during embryonic development (42). During cell differentiation a change in integrin repertoire takes place; for example, the splice-variant D of integrin subunit β1 replaces the variant β1A in mature muscle (44). However, only very limited information is presently available on the regulation of integrin genes. Promoter regions for several integrin subunits, including β1, β4, β2, α4, α5, are published (52, 92, 126, 481, 554). These promoter regions do not contain a TATA and CAAT-boxes and are very G+C rich, features that are common for housekeeping genes. Integrin β1 has two promoter regions and the distal promoter is highly active during development (92, 225).

17 The integrin αIIbβ3 is found exclusively in developing megakaryocytes and platelets. This is due to the cell-specific expression of the αIIb subunit. Studies on the promoter region of the αIIb subunit have revealed that the binding of the transcription factor Sp1 to a silencer element in the promoter inhibits the transcription of the αIIb gene (524). This inhibition can be overcome only in megakaryotic-like cell lines (524). Myeloid-specific expression of the αL gene is due to the activity of transcription factors Sp1 and Ap1 (406). Integrins are type-I transmembrane . During the protein synthesis in the ER individual integrin subunits are translocated through the ER membrane such that the C- terminus is located in the cytosol. Integrin α and β subunits dimerize in the ER and integrin dimers are transported from the ER via the Golgi compartment to the cell surface (213). Normally, a single integrin subunit can not leave the ER, but it has been shown that a single α or β subunit can occasionally escape from the ER, mostly localizing in the pre-Golgi compartment, when expressed in a cell type that does not express the right subunit for it to dimerize with (425). It seems that factors responsible for retention of unassembled β2 are displaced in α/β heterodimer (425). The retention signal of improperly paired α subunits has been shown to reside in the extracellular domain (68, 425). What determines that a given β subunit associates with a certain α subunit is not known. A chimeric β subunit possessing the extracellular part of β1, and the transmembrane and cytoplasmic parts of β3 did not associate with αIIb, the β3 specific α chain, indicating that α subunit selection is mostly determined by the extracellular domain (535). It is conceivable that other cellular proteins (chaperones or integrin-associated proteins) might have a regulatory role in this process. lines deficient in αL or β2 do not express β2 or αL subunit, respectively, on the cell surface. Northern blot analysis revealed an absence of the corresponding mRNA in each case (604). In contrast, both the αL and β2 subunits can be expressed on COS cells independently (143). Before the transport to cell surface takes place, integrin subunits, like all other synthesized proteins, must pass the “quality control” in the ER (reviewed in 152). Calnexin and calreticulin, together with glycosidases and glycosyltransferases, discern misfolded N-linked glycosylated proteins (reviewed in 214) in the ER as a part of the quality control. Indeed, calnexin interacts with immature integrin subunits before they are heterodimerized (308, 472). Immature β1 associates also with tetraspanin CD9 (482). Interestingly, recent data indicate that the cytoskeletal actin- binding protein talin is needed for the transfer of β1 integrins from ER to Golgi (8, 354). The authors suggest that the KXGFFKR motif in α chains may act as an export signal and that binding of talin to the β subunit unveils it (354). The finding that talin -/- embryonic stem cells show reduced levels of β1 integrin further demonstrates that talin is required at least for β1 integrin expression (447). In this case the amount of β1 subunit was reduced in whole cell lysates, possibly due to increased degradation. Some α chains (e.g. α3, α6) are endoproteolytically cleaved in extracellular domain close to the transmembrane domain. These subunits are therefore constituted by the transmembrane 25-30 kDa C-terminal light chain that is disulfide bonded to the N-terminal extracellular heavy chain. The endoproteolytic cleavage occurs after α/β subunit dimerization downstream of ER (305, 472). The protein convertase furin, mainly located in the trans-Golgi, processes proteins possessing the consensus sequence (RXR/KR), and this tetrabasic sequence is also found in α3 and α6 subunits. Endoproteolytic cleavage of α3, αv and α6 subunits was found not to occur in furin-deficient LoVo cells indicating that furin could be the convertase cleaving α subunits. The defective cleavage of these α subunits did not interfere with the integrin dimerization (305).

18 In the Golgi compartment terminal glycosylation of N-linked sugars takes place (reviewed in 427). The glycosylation has been reported to be unnecessary for integrin dimerization or endocytosis (304, 647). Studies on baby hamster kidney cells, which lack N- acetylglycosaminyl transferase I and hence are unable to complete the terminal processing of N-linked sugar chains, have shown that terminal sugars are not needed for α5β1 dimerization, surface expression or fibronectin binding (291). On the other hand, alterations in N-linked glycosylation have in some cases been shown to affect integrin ligand binding and cell spreading (304). The fact that cytoplasmic domains of integrin subunits are exposed to potentially interacting cytoplasmic proteins already when they reside in the ER raises the question of how these interactions are regulated. Are there specific escort proteins that mask integrin cytoplasmic tails and therefore prevent inappropriate binding of intracellular proteins to the cytoplasmic tails of integrin subunits until they have reached the plasma membrane? Taken all together, integrin biosynthesis is not well investigated yet, and further studies are needed to understand the regulation of the cell surface expression of integrins.

INTEGRIN LIGAND BINDING

Ligands recognized by integrins include a large number of ECM proteins (e.g. fibronectin, collagens, laminins, vitronectin, bone matrix proteins), cell-surface receptors belonging to the immunoglobulin superfamily (e.g. ICAMs, VCAMs), and the ADAM protein family (Table 1). Many pathogens use integrins to gain entry into the cell (275). The discovery that extracellular latent TGFβ (tumor growth factor) serves as a ligand for several integrins, and that binding of the avβ6 integrin to latent TGFβ induces its activity, augments the paradigm of integrin function (383, 384). In most cases an individual integrin can bind to multiple ligands and vice versa (Table 1). Interestingly, despite the variety of integrin ligands many of them possess similar short motifs as a recognition sequences for integrins. For example, the RGD sequence is present in many integrin ligands. However the flanking sequences and overall structure of the ligand, and also the individual features of the integrin ligand binding pockets, determine whether the interaction takes place or not. Our understanding of integrin tertiary structure as well as structures of their ligands is currently very limited. A combinational approach using different methods such as monoclonal antibody epitope mapping, analysis of integrin and ligand mutants has shed some light on these issues. Fibronectin is one of the most well studied integrin ligands. The major integrin binding sequence (RGD) is located in the tenth type III repeat in the fibronectin molecule. Although fibronectin can be recognized by at least eight integrins, formation of a stable interaction with fibronectin in some cases often requires other sites in the molecule, called synergy sites. For example, while integrin αvβ3 seems to bind exclusively to the RGD sequence in fibronectin, other integrins such as α5β1 and αIIbβ3 require also binding to the sequence PHSRN (the synergy site) for stable interactions (122). The crystal structure of a recombinant fibronectin fragment (type III repeats 7-10) revealed that the RGD and PHSRN sites are on the same side of the fibronectin molecule and could be reached by a single integrin (301). For the α5β1 integrin its has been proposed that the RGD sequence on fibronectin makes contacts with both subunits whereas the synergy site binds primarily to the α chain (380, 457).

19 Table 1. Integrins and their extracellular ligands

Integrin Matrix molecules Other ligands α1β1 Col I, IV, VI; Ln α2β1 Col I, II, III, IV, VII, XI; Ln α3β1 Ln 2/4, 5, 10/11 In α4β1 Fn, CSGAG VCAM-1, In, Im α5β1 Fn, Fg disintegrins, Im, In α6β1 Ln In, sperm fertilin β α7β1 Ln 1, 2/4 α8β1 Fn, Vn, Tn α9β1 Col I, Ln, Tn, OP VCAM-1 α10β1 Col II α11β1 Col I αvβ1 Fn, Vn TGFβ LAR αDβ2 ICAM-3 αLβ2 ICAM-1, 2, 3, 4, 5 αMβ2 Fg ICAM-1, iC3b, FX αXβ2 Fg iC3b αIIbβ3 Fg, Fn, Vn, TP, dCol vWF, Pl, disintegrins, - CAM αvβ3 Vn, Fg, Fn, bSp, Tn, TP, vWF, disintegrins, L1-CAM OP, MAGP-2, fibrillins, Del1 α6β4Ln αvβ5 Vn, bSp, αvβ6 Fn, Tn TGFβ LAR α4β7 Fn MadCAM-1, VCAM-1, disintegrins αEβ7 E-cadherin αvβ8 Col I, Fn, Ln

Abbreviations: bSp, bone sialoprotein; Del1, developmental endothelial -1; (d)Col, (denaturated) collagen; CSGAG, chondroitin sulfate glycosaminoglycan; Fg, fibrinogen; Fn, fibronectin; FX, Factor X; iC3b, inactivated fragment of complement factor C3; ICAM, intracellular adhesion molecule; Im, intimin; In, invasin; Ln, laminin; L1-CAM, neural L1; MAGP, microfibril-associated ; MadCAM, mucosal cell adhesion molecule; OP, osteopontin; Pl, plasminogen; TGFβ LAR, transforming growth factor β latency-associated peptide; Tn, tenascin-C; TP, trombospondin; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor

It should be emphasized that divalent cations are essential for integrin ligand binding; integrin function is completely inhibited in the presence of chelating agents (41, 378). Different divalent cations have distinct effects on ligand recognition. Under physiological conditions Mg2+ stimulates ligand binding, whereas Ca2+ generally has an inhibitory effect.

20 Mn2+ is a potent artificial promoter of ligand binding (40, 237, 378, 381, 393, 431, 533). Thus, divalent cations may induce a conformational change in the receptor that either favors or inhibits ligand binding. Divalent cations bind to two structurally different motifs within integrins. EF-hand motifs lie in the integrin α chain (Fig. 2). Although they lack a glutamic acid that is present at the twelfth position in other EF-hand motifs they do bind ions (35). Another ion binding region, the MIDAS motif, is located in integrin I domains (in α and β chains). Those integrins that contain the I domain in the α subunit bind ligands mainly via this domain (81). However, the importance of the I domain in integrin ligand binding relative to other integrin domains seems to be dependent on the ligand. A mutant integrin αLβ2 lacking the I domain does not recognize its ligands, whereas the I domain in αMβ2 is essential for binding to fibrinogen but not to other ligands, including Factor X and iC3b (307, 624). The binding of αMβ2 to Factor X and iC3b can be blocked by monoclonal antibodies to the β- propeller domain in α chain, indicating that multiple ligand binding sites are present in αMβ2 (624). The MIDAS motif and the exposed side chains in the surrounding surface in the I domain likely form major ligand contact sites (262). The first crystal structure of a complex ever between an integrin and a ligand, i.e. between the I domain of subunit α2 and a collagen peptide (GFOGER), further demonstrated a central role of the MIDAS motif in ligand binding (154). Comparison of the crystal structures of the unligated and collagen bound α2 I domain showed that changes in metal coordination are linked to conformational changes that result in formation of a collagen binding surface (154). The mechanism of ligand binding of those integrins that do not contain the I domain in their α chain is less clear. Nevertheless, the ligand-binding pocket seems to be formed by both subunits. The MIDAS motif on the top face of the putative I domain of β subunit, as well as putative loops on the upper side of the β-propeller of α chain, have been suggested to mediate ligand binding (244, 324, 379, 448). Although the ligand specificity is mostly determined by α chains (136, 219), β chains contribute also (553). A recent report by Mould et. al showed that ligand specificity for α5β1 is determined by the second and the third N-terminal repeat in the α chain (379).

MODULATION OF INTEGRIN LIGAND BINDING

Integrins are conformationally regulated proteins, existing in different conformational states (inactive, active and ligand occupied) (98, 626). The activation status of an integrin is largely dependent on cellular background. For example, T-cell β1 integrins bind their ligands only upon T-cell activation whereas in other cells ligand binding activity is constitutive (520). The activation state of different integrins is often regulated differently by the same stimuli. This is best exemplified by transendothelial migration of leukocytes into sites of inflammation, during which the affinity of β1 and β2 integrins is a dynamically regulated. For example, during the cytokine CC induced transendothelial chemotaxis of monocytes, the avidity of α4β1 for VCAM-1 was transiently up-regulated at the initial stage of migration whereas the avidity increase of α5β1 for fibronectin occurred at later stage and was prolonged (444, 602). Since these integrins share the same β subunit the α chains are most likely responsible for the different outcome. Activation of platelet integrin αIIbβ3 has also been subject to extensive investigation due to its critical role in blood clotting. The -IX (GPIb-IX)- mediated adhesion to von Willebrand factor at site of blood vessel injury activates the αIIbβ3

21 integrin via pathways where elevation of cytosolic Ca2+ levels is important. Studies on CHO cells expressing both receptors (GPIb-IX and αIIbβ3) have shown that the binding of a signaling molecule called 14-3-3, to GPIb-IX may have an important role in activation of integrin αIIbβ3 (194). The activation state of platelet integrin α2β1 was found to be dependent on the concentration of the stimuli. For example, low concentrations of ADP (an agonist to a G-protein coupled receptor) converted the non-active form of the α2β1 to an activated form 2+ able to bind soluble collagen, most likely as a result of a small increase of [Ca ]i. The collagen binding affinity could be further increased by high concentrations of ADP. This step involved the activation of several signaling pathways (PKC, PI3K, tyrosine kinase) (260). The mechanism for integrin activation is not known. Several models have been suggested (17, 439, 589, 615). The transition of conformational changes from the cytoplasmic part to the extracellular ligand-binding sites in the integrin molecule is believed to contribute the activation of integrins in response to cellular signals. The altered conformation of the cytoplasmic domain could be triggered by interaction with intracellular proteins or by enzymatic modification (e.g. phosphorylation). This topic will be discussed further below. The conserved sequences in the transmembrane/cytoplasmic domains of α and β subunits appear to have a central role in the control of integrin affinity-state. Deletion of the KXGFFKR motif in the α subunit or the KLLXXXHDR in the β subunit leads to constitutive activation of the receptor (241, 409, 410). Based on data obtained from the mutational analysis of the arginine in the KXGFFKR motif and aspartate in the KLLXXXHDR sequence it was suggested that the interactions mediated by these amino acids lock integrins in a low affinity conformation (240). Further support to this view comes from work by Haas and Plow who have shown that the cytoplasmic domains of αIIb and β3 form a ternary complex together with cations (199). Molecular modeling data indicated that the KLLVTI region in β3 (a.a. 716-721) is very labile. The KVGFFKR sequence in αIIb is positioned in parallel with this region and the interaction between these two sequences may be critical for maintaining a low affinity state (199). Regulation of the release of such a constraint can be part of integrin affinity modulation and/or outside-in signaling. Studies on the αIIb cytoplasmic domain using the NMR technique and monoclonal antibody mapping have provided experimental evidence that the spatial position of the cytoplasmic tails of αIIbβ3 changes upon ligand binding (306, 589).

Phosphorylation of integrin cytoplasmic tails

The cytoplasmic tails of integrin β subunits contain several potential phosphorylation sites, (Fig. 3) and phosphorylation of β subunits, as well as of some α subunits, has indeed been demonstrated (56, 79, 426). Calcylin A, a serine/threonine phosphatase inhibitor, decreases platelet adhesion and aggregation (310). Phosphoamino acid analysis of the cytoplasmic tail of β3 revealed that threonine 753 was phosphorylated after calcylin A treatment (310). A recent study has shown that kinases PDK1 and Akt (whose activity in platelets is stimulated by calcylin A) phosphorylated threonine 753 in the β3 tail and the presence of phosphorylated threonine inhibited the binding of Shc to tyrosyl phosphorylated β3 peptide (279). It is not clear if phosphorylation of threonine 753 affected the ligand binding ability of the receptor. A cluster of three threonines in β2 and two threonines in β1 are important for regulation of integrin adhesivness (217, 434, 612), which in the case of β1 was found to be due to effects on the extracellular conformation (612). In β2, phosphorylation of these threonines can be

22 detected in the presence of ocadaic acid (579). It is apparent that phosphorylation of integrin cytoplasmic domains occurs and that this can potentially regulate integrin function. Further studies are awaited to clarify the exact impact of phosphorylation/dephosphorylation of integrin cytoplasmic tails on integrin activation.

Lateral associations

In addition to the regulation of activation states by inside-out signaling, lateral associations with other cell surface molecules can also modulate ligand binding properties of a given integrin. Integrin-associated protein (IAP) or CD47 is a transmembrane glycoprotein consisting of an IgV-like amino terminal extracellular domain, a domain containing multiple membrane spanning segments and a short cytoplasmic tail. IAP is required for αvβ3 integrin binding to vitronectin (326). Furthermore, IAP is a receptor for thrombospondin (174,173) and binding of IAP to thrombospondin stimulates αvβ3 integrin-mediated cell spreading on vitronectin (173). IAP associates also with α2β1 integrin (596) and stimulates α2β1-mediated migration via Gi-mediated inhibition of ERK activity and suppression of cAMP levels (597). GPI-linked uPAR was shown to form a complex with β1-integrins and to alter its ligand binding ability. This association was promoted by caveolin (607), but it is also dependent on the direct binding of uPAR to the β-propeller domain of α-subunits (528). Integrin-mediated assembly of focal adhesions and stress fibers has, in some cell types, been shown to require syndecan-4 as co-receptor (146, 336, 491). However, cells derived from syndecan-4 -/- mice form focal adhesions and stress fibers (254). This unexpected finding may indicate that other syndecans can compensate for the lack of syndecan-4. A considerable body of data shows that cross talk between integrins and syndecans occurs (452). Tissue transglutaminase has been shown to form a complex with several β1 and β3 integrins and act as a co-receptor for fibronectin (5). Some integrins form complexes with several members of the transmembrane-4 superfamily proteins (TM4SF) or tetraspanins (46, 48, 510, 550). Integrin α3β1 has been shown to associate tightly with tetraspanin CD151 (632, 634) and this association is required for motility on fibronectin in response to fMLP (632) and contributes to the α3β1- dependent neurite outgrowth (47). CD151 was also shown to associate with integrin α6β4 and play a role in formation of hemidesmosomes (543). Co-operation between integrins and growth factor receptors is important for and migration and a firm connection between growth factor receptors and integrins has been demonstrated (197, 198, 372, 546). Some integrins have been shown to associate directly with growth factor receptors. Integrin αvβ3 can be coprecipitated with activated PDGFRβ, VEGFR-2, and the receptor and potentiate the biological activity of these receptors (60, 504, 534, 617). Several reports have demonstrated cross talk between different integrins (55, 57, 243, 444, 529). For example, it has been shown that engagement of αLβ2 decreases α4β1-mediated binding of T cells to fibronectin (444). The αvβ3 integrin has been shown to down-regulate α5β1-mediated migration of K562 cells by suppressing Ca2+- and calmodulin-dependent kinase (CaMKII) (57, 529).

23 Affinity modulation by cytoplasmic proteins

Since cytoplasmic domains of an integrin most likely regulate the ligand binding by specific protein interactions, an intensive search has been made to find such proteins. This has led to identification of a number of proteins that interact with integrins (Table 2). The implication of the majority of the listed proteins in integrin activity regulation is not clear. Nevertheless, some of these proteins (e.g. cytohesin-1) have clearly stated their role in regulation of integrin affinity whereas others seem to be required for integrin outside-in signaling. Many of these proteins are structural and bind to several integrin subunits. Binding of most cytoskeletal proteins to integrin cytoplasmic tails takes place after ligand binding and these interactions can contribute to integrin ligand binding by stabilizing an active conformation or by promoting increased avidity through the cooperative effect of clustered integrins.

Talin

The cytoskeletal protein talin can bind to cytoplasmic tails of several integrin β subunits (234, 288, 438, 489). Talin is a homodimer, with each subunit consisting of an N-terminal head domain and a C-terminal rod domain. Talin seems to have two integrin binding sites, one in the head domain and one in the C-terminal rod domain (82, 234). Overexpression of the talin head domain activates integrin αIIbβ3 in CHO cells (82). The mechanism of this activation is not clear but the isolated fragment may break interactions between the β3 tail and intact talin that possibly retain integrins in inactive state, or alternatively, the binding of talin head domain may alter integrin affinity or avidity (82). The membrane proximal region of the β3 cytoplasmic tail has been shown to interact with the N-terminal head domain of talin (430) but the binding of talin to the β tails also requires intact NPXY motifs (82, 261, 438). Talin binding to integrins is required but not sufficient to recruit integrins to focal adhesions (261, 588). Constitutive binding of integrin αLβ2 to actin filament system via talin in resting leukocytes was disrupted upon cell activation by proteolytic cleavage of talin, and reattachment of actin filaments to β2 was mediated by α-actinin (489). Ca2+-dependent activation of calpain, an intracellular protease that can cleave talin, increases the ligand binding avidity of the αLβ2 integrin by releasing it from cytoskeletal constraint that prevents receptor clustering (544). In agreement with this conclusion, deletion of cytoplasmic tails of integrin αLβ2 that obliterate association with cytoskeleton resulted in increased ligand binding avidity to ICAM-1. However, in this case, post-ligand binding events dependent on cytoskeletal interactions were also severely impaired (583). On the other hand, deletions of the cytoplasmic tail from other integrins generally result in loss of adhesion (217, 409). It is not yet clear if the above-described observations on β2-integrins in leukocytes are valid for other cells and/or integrins. It should be noted that reorganization of the actin cytoskeleton in lymphocytes is not only required for integrin activation but also for lymphocyte activation in general (reviewed in 432). Nevertheless, changes in the organization of actin cytoskeleton as well as the nature of the proteins that link integrin cytoplasmic tails to cytoskeleton can apparently modulate integrin affinity, and the physiological outcome of these interactions is dependent on the given integrin. Calpain has been suggested to cleave several focal adhesion proteins in addition to talin (e.g. paxillin and FAK) and even integrin cytoplasmic tails (87, 145, 362, 437, 506).

24 Table 2. Proteins interacting with the integrin cytoplasmic tails

Binding Cytoplas- Size Comments Ref. protein mic tail kDa bound β3- β3 13 Overexpression increases affinity of the αIIbβ3, 512 endonexin binds via NITY motif in β3 JAB1 β2 38 Transcriptional co-activator of AP-1, localizes to 51 nucleus and enhances AP-1 promoter transcription after integrin αLβ2 cross-linking α-actinin β1, β2, 100 Cytoskeletal actin binding protein, homodimer 421 β3 422 Calreticulin α tails 42 Could stabilize te high affinity state of an integrin 311 52/6 by binding to the KXGFFKR motif in ( tails 2 PI3K (p85) Tyr-P 85 Tyrosine phosphorylated β1 cytoplasmic peptide 258 β1 binds to the SH2 domain of the p85 in vitro CIB αIIb 22 Ca2+ binding protein, function in integrin signaling 388 is not known Cytohesin-1 β2 47 Overexpression induces activation of αLβ2 289 FAK β1, β2, 125 Protein tyrosine kinase localized at focal 500 β3 adhesions, implicated in cell migration and survival Filamin β1A, 280 Cytoskeletal protein, part of cortical actin 337 β1D, β7 network, homodimer 438 ICAP-1αβ1A20/2 Cell adhesion via β1 modulates phosphorylation 95 7/31 state of ICAP-1, binds via NPKY motif in β1 ILK β1, β2, 59 Overexpression of ILK induces anchorage 208 β3 independent growth IRS-1 αvβ3 185 Associates with the αvβ3 (and not with the αvβ5 593 or β1 integrins) in insulin stimulated cells Melusin β1 38 A cytosolic protein expressed in differentiated 66 myotubes MIBP β1A, β1D 19 A cytosolic protein expressed in striated muscle 321 tissue, downregulated during muscle differentiation Mss4 α3 14 Possible GEF for Rab3, binds to the KXGFFKR 616 motif in the α3 tail, function in integrin signaling is not known Paxillin α4 68 Interaction with paxillin results in decreased cell 335 spreading and increased migration via integrin 559 α4β1 Paxillin β1 68 Paxillin binds to a synthetic peptide derived from 500 the β1 subunit

25 Table 2. Continued

Rack1 β1, β2, 36 WD repeat protein; interaction with integrins in 323 β5 vivo requires phorbol ester stimulation of cells 643 α4, αv Shc Tyr-P 46/52 An adapter protein that links integrins to the 348 β4 /66 MAPK cascade Shc Tyr-P 46/52 An adapter protein that links integrins to the 116 β3 /66 MAPK cascade BP180 β4 180 A transmembrane component of 496 hemidesmosomes β4 >500 Stabilizes hemidesmosomes by bridging β4 to the 466 IF network Skelemin β1, β3 210 Myosin and IF-associated protein, binds to a 456 membrane proximal region of β3 Talin β1A 235 Cytoskeletal actin binding protein, localizes at 288 β1D, β2, focal adhesions 438 β3, β7 489 Nischarin α5 Overexpression of nischarin inhibits cell migration 7 (αv,α2) on FN; nischarin is not localized at focal adhesions PAK4 β5 68 Phosphorylates β5 in vitro. Overexpression of 640 PAK4 induces αvβ5-dependent cell migration on vitronectin PICK1 β6 45 PICK1 is a PKCα binding protein 538 TAP20 β5 20 Overexpression of TAP20 reduces cell adhesion 560 and increases cell migration via integrin αvβ5 WAIT-1 β7 (α4 49 Binds to a membrane proximal region in β7; a 471 and αE) mouse homologue EED is a transcriptional regulator of homeobox genes

Abbreviations: BP180, bullous pemphigioid 180 antigen; CIB, calcium and integrin binding protein; FAK- focal adhesion kinase; GEF, guanine nucleotide exchange factor; ICAP-1, integrin cytoplasmic domain-associated protein-1; IF, intermediate filament; ILK, integrin linked kinase; IRS-1, substrate-1; MIBP, muscle integrin binding protein; PAK, (p21- activated kinase; Rack-1, receptor for activated C-kinase; TAP20, theta-associated protein; Tyr-P, phospho-tyrosine; WAIT-1, WD protein associating with integrin cytoplasmic tails-1

There is an increasing body of evidence that the proteolytic activity of calpain is involved in integrin activation and post-ligand binding events. Calpain activity is important for platelet function and αIIbβ3 integrin-mediated platelet spreading (118, 638). In fibroblasts, calpain has been shown to be involved in regulation of focal contacts and organization of the actin cytoskeleton (248). A number of regulatory enzymes implicated in integrin signaling (kinases/phosphatases) are cleaved by calpain (109, 474). Cleavage of protein kinase C (PKC) by calpain generates a

26 constitutively active form which does not require diacyl glycerol for activity (442, 443). Calpain activity can also regulate PIP levels by inactivating phosphatidylinositol phosphatases and activating phosphatidylinositol kinases (405, 641).

Calreticulin

The intracellular calcium-binding protein calreticulin localizes in the ER lumen where it performs two major functions: chaperoning for glycoproteins and regulation of Ca2+ homeostasis (reviewed in 214, 365). In addition to these well-characterized functions, calreticulin is implicated in regulation of cell adhesion, although it has not been detected in the cytosol. Other observations suggest that calreticulin might regulate cell-substrate contacts indirectly via modulating the expression of vinculin (157). The first indication that calreticulin could be involved in cell adhesion came from affinity chromatography experiments where K calreticulin bound specifically to the synthetic peptide KXGFF( /R)R, corresponding to the conserved motif in α subunits, in a Ca2+-dependent manner (311). Further studies have shown that the association between integrins and calreticulin occurs in cells shortly after integrin ligand binding (110, 111). Furthermore, calreticulin-deficient embryonic stem cells were found to be defective in cell adhesion and also in adhesion-mediated Ca2+ influx, which could contribute to the impaired adhesion of these cells (112). Stimulation of calreticulin-deficient cells with lysophospatidic acid restored their ability to bind to fibronectin, possibly through a transient increase in cytosolic Ca2+ concentration (112). Studies on the E63 cells have shown that an initial intracellular Ca2+ release was a prerequisite for the integrin α7β1 induced Ca2+ influx, and that calreticulin mediated the coupling between these processes (295). Integrin α7β1, a cell surface form of calreticulin, and Ca2+ channel DHPR α1 were found to form a complex on the cell surface (295). Ectocalreticulin has also been reported to cooperate with the α6β1 integrin in triggering of cell spreading on laminin (652), possibly due its lectin-binding properties (614). All of these studies indicate a role of calreticulin in integrin mediated cell adhesion, but the mechanism by which calreticulin regulates integrin function remains abstruse.

CIB

Calcium- and integrin binding protein (CIB) was found to interact specifically with the cytoplasmic tail of αIIb (388). It is homologous to the Ca2+-binding proteins calcineurin B (a regulatory subunit of phospho-protein phosphatase 2B) and calmodulin, and has also Ca2+ binding capacities. Indeed, the interaction between CIB and the cytoplasmic tail of αIIb was found to be Ca2+-dependent. Furthermore, binding of CIB to integrin αIIbβ3 was not only dependent on integrin activation and ligand occupancy but also on platelet aggregation (521). When CIB was discovered it was suggested that CIB might have a role in inside-out signaling. However, the finding that association of CIB to the αIIbβ3 integrin does not activate the receptor indicates that it is not involved in inside-out signaling (578). The exact role of CIB in αIIbβ3-mediated processes is not known but it has been suggested that CIB might regulate signaling molecules associated with cytoskeleton (521). Interestingly, similar to CIB, localization of tyrosine phosphorylated β3 in the detergent-insoluble cytoskeletal fraction did not occur after ligand occupancy of αIIbβ3 unless platelet aggregation was allowed (257).

27 ICAP-1α

Interaction between integrin cytoplasmic domain-associated protein (ICAP-1α) and the cytoplasmic domain of β1 is highly specific and requires the NPXY motif in the C-terminus of the β1 subunit (95). ICAP-1 binds only to integrin β1A and not to β1B or β1D subunits (563). The major binding site in the β1A tail was actually mapped to the KSAVTTVV sequence between the NPXY motifs (563). ICAP-1α is a widely expressed serine-rich protein, and has no obvious protein domains implicated in (95, 643). ICAP-1α becomes phosphorylated after cell adhesion to fibronectin and may be required for β1-dependent cell migration (95, 643). It has been shown that CaMKII can phosphorylate threonine 38 in ICAP-1α that in turn results in decreased cell spreading (64). In this context, it is interesting that the activation of CaMKII was needed for α5β1-mediated migration and phagocytosis (57). Constitutively active CaMKII down-regulates adhesive properties of α5β1 (65). It is conceivable that phosphorylation of ICAP-1α by CaMKII contributes to the fluctuations in integrin affinity during cell migration. Interestingly, phosphorylation of ICAP-1α diminished in the presence of active RhoA (94).

Cytohesin-1

Cytohesin-1 is a cytosolic protein belonging to a family of guanine-nucleotide exchange factors (GEFs) for Arf (ADP ribosylation factor) GTPases. It consist of an N-terminal coiled- coil domain, a central Sec7 (sec-7 homology) and a C-terminal PH (pleckstrin-homology) domain. Cytohesin-1 binds specifically to the β2 cytoplasmic tail, and the binding appears to be dependent on the WKA (723-725) sequence in β2 (180, 289). Interestingly, this region has been shown to be buried in the plasma membrane in the absence of interacting proteins (paper III) (17). Overexpression of cytohesin-1 induces αLβ2 integrin dependent adhesion and has no effect on the affinity of β1 integrins (289). Ligand binding of αLβ2 is regulated directly by PI3K activity, through intermediate membrane recruitment of cytohesin-1 via its PH domain (226, 387). The ability of cytohesin-1 to activate β2 integrins is dependent on its GEF activity and direct binding to β2 subunit (180). The downstream ARF target of cytohesin-1 has not been identified yet. Although Arfs (reviewed in 377) have been mainly implicated in the regulation of intracellular vesicle transport they have also been shown to participate in actin remodeling. It is interesting that ARNO, related to cytohesin-1, induces cortical actin rearrangements by an as yet unidentified mechanism via its downstream effector Arf6 (170). Since the actin filament system modulates activity of β2 integrins, it is conceivable that cytohesin-1-induced integrin activation involves changes in the actin cytoskeleton implemented by a target Arf.

β3-endonexin

β3-endonexin, like many other proteins interacting with integrin cytoplasmic tails, was found by a yeast two-hybrid screening system. Its interaction with the integrin β3 cytoplasmic tail is dependent on an intact tertiary structure of β3 cytoplasmic tail (512) and on the cytoplasmic NITY motif (150). Overexpression of β3-endonexin activates the αIIbβ3 integrin in CHO cells, indicating that it is involved in affinity regulation of β3 integrins (264). β3-endonexin occurs as a short (111 a.a.) and a long (170 a.a.) form. Only the short isoform of

28 β3-endonexin binds to the β3 tail (512). β3-endonexin was shown to associate with cyclin A- Cdk2 via the N-terminal RXL sequence and to inhibit its activity (415). However, further studies are needed to understand the functional importance of this finding.

TAP20

TAP20 (theta-associated protein) shares a significant homology with human β3-endonexin at the amino acid level but TAP20 associates only with the cytoplasmic tail of integrin β5 subunit. Overexpression of TAP20 in endothelial cells was found to down-regulate the αvβ5- mediated cell adhesion whereas the migratory properties of the αvβ5 integrin were up- regulated (560). TAP-20 was found by the mRNA display method as a protein whose expression was dependent on the presence of active PKCθ (560). It has been shown previously that VEGF-induced angiogenesis dependent on αvβ5 could be mediated by PKCθ (561).

Ras family GTPases

Small GTPases of the are important regulators of cell growth and death (reviewed in 465, 519). Ras proteins have not been found to directly associate with integrins but they can modulate integrin affinity by mechanisms that are currently unknown. They function as molecular switches in intracellular signaling. In the GDP-bound form they are turned off, and in the GTP-bound form they are switched on. The switch between these two forms is controlled by the action of guanine-nucleotide exchange factors (GEFs) and GTPase- activating proteins (GAPs). The Ras family of the Ras superfamily consists of at least 14 members (reviewed in 465). Ras proteins can either suppress or promote integrin activation. Ha-Ras was shown to inhibit integrin αIIbβ3 activation in CHO cells and this suppression activity correlated with the activation of the MAPK pathway (242). Dominant negative mutants of Ha-Ras and the downstream effector Raf-1 enhanced hematopoetic cell adhesion via the α4β1 and α5β1 integrins to fibronectin (518). In an apparent contradiction to these results, expression of a constitutively active Ha-Ras has been shown to activate the α5β1 integrin in mast cells and the αLβ2 integrin in T-cells in a PI3K-dependent manner (266, 276). Overexpression of constitutively active R-Ras and reportedly activates integrins (266, 645). However, activated R-Ras stimulated cell adhesion on collagen but inhibited it on fibronectin, indicating that different integrins are subjects of different regulation (273). Stimulation of CD31 in T- cells propagates signals via Rap1 that induce cell adhesion through β1 integrins by stabilizing the active conformation of β1 integrins (458). Involvement of Rap1 in cell adhesion is further demonstrated by a study on SPA-1, a specific GAP for Rap1. Conditional overexpression of SPA-1 in adherent HeLa cells resulted in the inhibition of Rap1 activity and detachment from the matrix (571). In another study, the elevated Rap1 activity, in response to cell adhesion to fibronectin, was accompanied by decreased Ras activity (445). Findings that Rap1 can bind to Ras effectors but not activate them led to the conclusion that Rap1 antagonizes the Ras signaling by trapping Ras effectors (465). The molecular mechanism of R-Ras action is not known but it antagonizes the Raf-initiated integrin suppression pathway and seems to involve PEA-15, a small death effector domain containing protein (454, 511). Another study

29 demonstrated that R-Ras possesses a proline-rich motif that can bind the adapter protein Nck and this interaction was required for R-Ras-induced integrin activation (595). A tight bi-directional connection between the members of the Rho subfamily of Ras proteins and integrins has been established and this will be discussed in more detail below.

INTEGRINS AND RHO FAMILY GTPases

The Rho family GTPases are important regulators of the actin cytoskeleton (reviewed in 203). In addition to this they have been implicated in multiple cellular processes such as cell cycle control, transcriptional regulation and induction of apoptosis (347, 419, 505). At present more than 13 Rho GTPases are known in mammalian cells (reviewed in 24). The most studied members of this family, with respect to integrins, are RhoA, Rac and Cdc42. The Rho GTPases, like Ras GTPases, cycle between an active and an inactive state, the process being controlled by GEFs and GAPs. In addition, the cycling between the two nucleotide-bound states is accompanied by the cycling between the plasma membrane and cytosol (reviewed in 494). There are indications that the plasma membrane might contain receptors for GTP-bound Rho proteins (181). ERM (erzin, radixin, moesin) proteins have been shown to dissociate RhoGTPases from RhoGDI (GDP dissociation inhibitor) by direct binding to RhoGDI (reviewed in 350). The importance of the controlled regulation of Rho GTPases is illustrated by the fact that several Rho GTPase GEFs are oncogenes. Normal cells require anchorage to the extracellular matrix and signals from growth factors in order to proliferate. In tumor cells, signals from integrins and growth factors are constantly turned on even in the absence of growth factors and cell adhesion. Integrin-mediated adhesion leads to an activation of phosphatidyl-inositol 4-phosphate 5-kinase and accumulation of PI(4,5)P2. This effect is mediated by RhoA. Oncogenic variants of RhoA GEFs (Dbl and Lbc) were found to induce production of PI(4,5)P2 in suspended cells indicating that they transform cells by constitutive activation of an adhesion-dependent pathway (508). Loss of the tumor-suppressor protein hamartin is responsible for the development of benign tumors, namely hamartomas. Hamartin was shown to be involved in regulation of cell adhesion and stress fiber formation via RhoA in an ERM protein dependent manner (298).

Regulation of Rho family proteins

The mechanisms by which integrins and other plasma membrane receptors regulate activity of Rho GTPases are not fully elucidated. The obvious targets are GEFs and GAPs that regulate the activity of Rho GTPases. In order to become activated, Rho GTPases must be translocated to the plasma membrane. Thus, it is conceivable that integrins might also generate signals leading to a dissociation of Rho GTPases from GDI in the cytosol. Indeed, Rac translocates to the plasma membrane after integrin-mediated adhesion and this is needed for subsequent PAK activation (131). It is interesting that in vitro Rac bound only to cell membranes prepared from adherent cells and not to those from suspended cells (131). However, not all GDIs are cytosolic, GDIγ and GDI-3 are membrane bound (2, 639). Activation of GEFs requires tyrosine phosphorylation events (507). Indeed, in hematopoetic cells, Rac1 GEF Vav1 is phosphorylated and thereby activated by the tyrosine kinase Syk. Syk is activated by integrin clustering independently of actin cytoskeleton (175). GEFs

30 contain a pleckstrin homology domain (PH) and these domains are important for membrane localization by interacting with phosphatidyl inositol lipids. Integrins have been shown to induce PIK activity that leads to accumulation of PIPs (224, 360). By inhibiting or promoting GAP activity integrins could control the amount of GTP- bound Rho GTPases and therefore their signaling. The ligation of β1 (α6β1) in adherent melanoma cells stimulates tyrosine phosporylation of p190RhoGAP. Tyrosine phosphorylated p190RhoGAP localizes at membrane protrusions and is needed for matrix degradation (389). P190RhoGAP has been shown to be phosphorylated and thereby activated in a Src kinase- dependent manner after integrin engagement (22). Graf (GTPase regulator associated with FAK) stimulates GTPase activity of RhoA and Rac. It is interesting that Graf associates in an SH3 domain-dependent manner with the C-terminal domain of FAK (222). The functional importance of this association is not known. Loss of Graf function due to the deletion or mutations of the gene has been shown to be linked to the development of leukemias (61). Since the Rho activity is needed for cell cycle progression (419) elevated levels of Rho-GTP might contribute to tumor development.

Functions of RhoA

Integrin signaling and association with actin cytoskeleton are formation of stress fibers and focal adhesions by bifurcating pathways (398). The Rho-dependent stress fiber assembly can take place in the absence of integrin-matrix interactions and without formation of focal adhesions (162, 235, 398). On the other hand, formation of focal adhesions also requires integrin-mediated adhesion (36, 235). However, it should be pointed out that distribution of individual proteins to focal adhesions may be regulated in different ways. For example, constitutively active ARF1 induced paxillin recruitment to focal adhesions when Rho activity was suppressed (404). RhoA induces the formation of actin stress fibers and focal adhesions by stimulating actomyosin-based contractility via Rho kinase (103, 398) (Fig. 6). In addition, activation of the ubiquitously expressed Na-H exchanger NHE1 by Rho kinase has been shown to be important for focal adhesion assembly and integrin-mediated cell spreading. Interestingly, NHE1 activity is required for integrin-mediated tyrosine phosphorylation of FAK (568). The activity of the Na-H exchanger NHE3, which is expressed primarily in epithelial cells, is also regulated by RhoA and Rho kinase (549). However, the pertinence of this Na-H exchanger to integrin function has not been assessed. Contractility in smooth muscle (and non-muscle cells) is under the control of two independent mechanisms, one involves the RhoA activity and accumulation of phosphorylated myosin light chain (MLC), the other way involves caldesmon (Fig. 6). Binding of caldesmon to myosin blocks myosin ATPase activity necessary for actin-myosin based contraction (reviewed in 239). Overexpression of nonmuscle caldesmon in fibroblasts interferes in a Ca2+-sensitive manner with RhoA induced contractility. When the effect of caldesmon is blocked by a rising the Ca2+ level the stress fibers appear again, albeit with caldesmon is still associated (215). Besides the calcium-sensitivity, not much is known about the regulation of caldesmon. Caldesmon can be phosphorylated by kinases like PAK, MAPK (101, 169). Dominant-negative PAK increases stress fibers but decreases MLC phosphorylation (278).

31 LPA Bombesin ERM a a genistein b b GDI Rho-GDP + hamartin tyrosine phosphorylation

Rho-GDP GDI GEF GAP ERM P a b P Rho-GTP a PKC translocation kinase X ERM b + P PI(4,5)P 2 PI(4)5 kinase + Rho kinase + NHE1 P - P +

PI(4,5)P 2 - MPP P cofilin LIM-kinase + P P P

+ + MLC Contraction Assembly of stress fibers P 2+ + + P - Ca - MLCK calmodulin - 2+ Caldesmon [Ca ]i 2+ [Ca ]i

talin actin filaments vinculin homodimer actin monomers

Figure 6. RhoA mediated stressfiber and focal adhesion assembly. Activation of RhoA involves dissociation from the GDI in the and action of GEF. Activation of RhoA GEFs requires tyrosine kinase activity. RhoA-GTP binds and activates several serine/threonine kinases that in turn inactivate myosin phosphatase (MPP) by phosphorylation. Increased myosin light chain (MLC) phosphorylation promotes myosin filament assembly and actin-activated myosin ATPase activity. Binding of PI(4,5)P2 to focal adhesion proteins such as vinculin causes a conformational change in the molecule such that actin and talin binding sites are exposed. Activation of Rho results also in tyrosine phosphorylation of FAK, paxillin and p130CAS (not shown in the figure) in the absence of cell adhesion.

32 The assembly of fibronectin matrix is a multi-step process initiated by the binding of fibronectin to the integrins. α5β1 integrin is the main receptor for fibronectin matrix assembly, while integrin αvβ3 can support matrix assembly but not as efficiently as α5β1 (613). It has become clear that the increased cell contractility and tension emanating from RhoA activity contributes to the assembly of fibronectin matrix. Tension exerted by cells exposes cryptic self-assembly sites within the fibronectin molecule and therefore promotes matrix assembly (650).

Functions of Cdc42 and Rac1

Cytoskeletal changes induced by Rac (membrane ruffling, lamellipodia) and Cdc42 (filopodia) are associated with integrin-based adhesion complexes (398), but Rac-induced membrane ruffling, accompanied by rapid actin polymerization, can also occur in response to other stimuli independently of integrin-mediated adhesion (346). Integrins might be required for membrane ruffle stabilization and/or spatial regulation (346). Quite a number of downstream targets for Cdc42 and Rac1 are identified (Fig. 7) (reviewed in 23, 24, 53). WASP-family (Wiskott-Aldrich syndrome protein) proteins (WASP, N-WASP, WAVE) have shown to mediate morphological changes of cells caused by active Cdc42 and Rac (366, 367). WASP family proteins are regulators of the Arp2/3 complex (reviewed in 453) which in turn regulates actin polymerization by generating nucleation sites for rapid elongation and branching of actin filaments (reviewed in 220, 608). Binding of Rac and Cdc42 to another family of effector proteins, p21-activated kinase (PAK), is needed for Rac induced lamellipodia formation (Fig. 7). PAK also has to associate with PIX (PAK-interacting guanine nucleotide exchange factor) for this effect (411). GTP- bound Rac1 or Cdc42 activates PAK1, which subsequently phosphorylates and thereby activates LIM-kinase (147). LIM-kinase inactivates the F-actin depolymerizing activity of cofilin by phosphorylating an N-terminal serine residue on cofilin (628). Integrin-mediated directional cell motility promoted by constitutively active Rac and Cdc42 (274) is mediated via PAK1 (509). In directed cell movement membrane protrusions at the leading edge of lamellipodia have to get firmly attached via focal complexes in order to provide an anchorage point for contractile forces to pull off the rear end of the cell. PAK activity at the leading edge, which increases actin polymerization (via LIM kinase), could help to stabilize the leading edge (147). During the cell migration integrin activity has to fluctuate. Studies on living cells revealed that in stationary cell focal adhesions are highly motile whereas in the motile cells focal adhesions are stationary (532). This indicates that when cells start to migrate the integrin affinity to the matrix is regulated. Since increased integrin ligand-binding affinity decreases the migration rate (247), it is likely that focal contacts are dissolved at the rear end by down-regulating integrin activity. However, this has not yet been directly shown. It should be noted that in tissues cell migration/invasion is accompanied by matrix degradation, and several integrins have been shown to anchor protease activity at invapodia (382). The Cdc42 specific downstream effectors ACK-1 and ACK-2 (activated Cdc42- associated tyrosine kinase) have shown to be activated after cell adhesion as well as by EGF treatment, but the physiological significance of their activation is not well understood yet (151, 629, 630). ACK-2 has been shown to constitutively associate with β1 integrins and become activated after integrin-mediated adhesion in a Cdc42 dependent-manner (630). The activation of ACK-2 as well as Cdc42 occurs also in cells plated on poly-lysine indicating that

33 RTK a a ab b b Ras ERM 3 Sos GDI PI(3)K PI(3,4,5)P

Rac-GDP a b tyrosine phosphorylation Rac-GDP GDI a GEF GAP b ERM Rac-GTP

PI(4,5)P 2 PI(4,5)K PI(4,5)P 2

- cofilin LIM-kinase + PI(3)K PI(3,4,5)P 3 P PAK P P P

WASP - family P MLCK Raf JNK Contractility P Arp2/3 ERK Actin reorganization Migration

actin filaments vinculin talin homodimer actin monomers

Figure7. Rac/Cdc42 activated signaling pathways. The activity of Rac and Cdc42 is regulated by GDIs, GEFs and GAPs similarly to RhoA. Some of the effector proteins that bind to and become activated by GTP-bound Rac are shown. For formation of filopodia and lamellipodia the capped actin monomers have to be released. Elevated PI(4,5)P2 levels contribute to this by associating with actin-capping proteins (profilin, gelsolin), and thereby promoting their dissociation from actin, which in turn results in increased actin polymerization. The phosphorylation of LIM kinase by PAK1 increases its activity and subsequent LIM-kinase mediated cofilin phosphorylation. Rac binding to PI(3,4,5)P3 and PI(3,4)P3 increases its dissociation from GDP and might locate Rac to the plasma membrane.

34 integrin-mediated adhesion is not a prerequisite for this event. Interestingly, clustering of melanoma chondroitin sulfate proteoglycan in melanoma cells activates ACK-1 and promotes melanoma cell spreading via α4β1. Downregulation of ACK-1 by an antisense construct abolished cell spreading (151). Since proteoglycans are prime candidates for binding to poly- lysine, these observations may indicate that proteoglycans in combination with integrins activate ACKs. During the initial phase of cell spreading, which is accompanied by appearance of filopodia and extensive membrane ruffling, Cdc42 and Rac1 are activated (105), (120, 446) whereas RhoA activity remains low (461). Formation of stress fibers and focal adhesions takes place at later times corresponding to the period of high RhoA activity. Growth factor- induced RhoA activity in suspended cells is down-regulated by integrin ligation, indicating that during the initial phase of cell spreading on extracellular matrix the RhoA activity is kept low by specific signals (461). Down-regulation of RhoA activity after integrin engagement was shown to be Src kinase dependent (22). It is not known how, but Rac1 activity itself antagonizes RhoA effects, and dwindles stress fibers and focal adhesions (397, 490). In addition, integrins could activate the Rnd (“round”) GTPases (, , /RhoE) that also antagonize effects of RhoA (399). The Cat proteins (Cat 1, Cat 2) (Cool-associated, tyrosine-phosphorylated) regulate the association between Cool (cloned out of library) and PAK (30). Cool, PIX (PAK-interacting guanine nucleotide exchange factor) and p85SPR (85 kDa SH3 domain-containing proline-rich protein) all are same proteins found by different groups (31, 352, 413). (The present cell biology nomenclature is in desperate need for order. The field is waiting for a modern Carl von Linné who would organize the current, almost frantic, protein jargon). From here on I will use the name PIX because it at least tells something about the function of the protein even though it does not sound so “cool”. The Cat proteins are homologues to PKL (paxillin-kinase linker), a paxillin binding protein that mediates association between paxillin and the PAK/PIX complex (see further below) (576). Interestingly, the Cat proteins become tyrosine phosphorylated upon cell adhesion and also during the exit of cells from (30), which could possibly be a regulatory event in the assembly and/or activation of the PIX/PAK/PKL/paxillin complex. It is known that inhibition of polymerization inhibits cell spreading and membrane ruffling (50, 192). Microtubule depolymerization induces formation of stress fibers and focal adhesions via RhoA whereas microtubule growth activates Rac1 and promotes membrane ruffling (330, 600). Targeting of to focal adhesions correlates with instability of focal adhesions (267). Microtubules could therefore regulate cell motility and polarity by modulating interactions between actin fibers and cellular proteins (267). Interestingly, paxillin has been shown to colocalize with γ- at the cell microtubule organizing center in T-cells (216). Paxillin might bring the ends of microtubuli to cortical actin (216). However, how all these events are linked to integrin signaling remains elusive.

INTEGRINS AND CELLULAR SIGNALING PATHWAYS

Integrins activate common signaling pathways, which include activation of protein tyrosine kinases, serine/threonine kinases, elevation of levels of intracellular ions and phosphoinositides. These pathways are partially overlapping with pathways activated by

35 receptor tyrosine kinases such as growth factors receptors and G-protein coupled receptors. It has been known for some time that in order for a normal cell to proliferate it needs signals both from matrix and growth factors, whereas efficacy of these signals is synergistic. Cross-talk between integrins, growth factor receptors, G-protein coupled receptors, ephrin receptors, cadherins etc. leads to a response that is largely dependent on cell type (19, 133, 249, 364, 523). In the context of a whole cell and, even more importantly, in the context of an organism it is crucial that signals received and transmitted by cells are coordinately regulated. The section below deals mostly with signals sent by integrins.

Focal adhesion kinase

Focal adhesion kinase (FAK) plays a central role in integrin-mediated signaling linking integrins to a number of intercellular signaling pathways that lead to the reorganization of actin cytoskeleton, induction of survival signals and cell migration (Fig. 8). Studies on FAK -/- cells have shown that FAK activity is not needed for integrin activation and formation of focal contacts but is needed for focal adhesion turnover and cell migration (253).

Structure

FAK is a widely expressed 125 kDa cytosolic tyrosine kinase colocalizing with integrins at focal adhesions. FAK was originally identified as a protein with high phosphotyrosine content in v-Src transformed cells (196, 263, 497). In addition to integrin-mediated activation FAK can be activated by other receptor systems such as receptor tyrosine kinases and coupled receptors (475). FAK consists of a central catalytic domain and flanking regions. Unlike many other tyrosine kinases, FAK is devoid of SH2 or SH3 domains, but it contains phospho-tyrosines and proline-rich regions, which can bind to SH2 and SH3 domains. The major autophosphorylation site in FAK, tyrosine 397 (Y397), serves as a binding site for the SH2- domains of Src, Grb7, phospholipase C-γ-1 and PI3K (29, 97, 206, 499, 642). The adapter protein Grb2 binds to phosphorylated Y925 in FAK. Proline-rich regions in FAK mediate association with CAS, Src family kinases and Graf (GTPase regulator associated with FAK) (222, 441, 565). The N-terminal region of FAK has been shown to bind to integrin β subunits (β1, β3, β5) and the C-terminal region to paxillin and talin. The N-terminal domain also contains a so-called FAT sequence (focal adhesion targeting) required for FAK localization at focal contacts, and a domain with homology to band 4.1 (185). This region of FAK was shown to interact directly with EGF receptor and this association was required for EGF-stimulated cell motility (525). Thus, FAK seems to act as a unique scaffold molecule with kinase activity, which can recruit several signaling molecules into close proximity of integrins (Fig. 8). Although most integrins activate FAK in response to adhesion, activation of FAK does not seem to be critical for outside-in signaling of β2 integrins (210).

FAK related proteins

Several other kinases such as Pyk2 and FRNK have been described as sharing certain characteristics with FAK. FRNK (FAK-related non-kinase) consists only of the C-terminal domain of FAK thus lacks the catalytic domain (469). FRNK expression is directed by an

36 alternative intronic promoter residing between the exons encoding the catalytic domain and the C-terminal domain of FAK (401). Pyk2 (proline-rich t yrosine kinase), also named as CAKβ, RAFTK, CADTK, FAK2, is very homologous to FAK, sharing 60% amino acid sequence identity in the catalytic domain (28, 493). Pyk2, like FAK, contains proline-rich sequences capable of mediating interactions with SH3 domains and a putative focal adhesion-targeting domain. The major autophosphorylation site is also conserved between FAK and Pyk2. Pyk2 is activated by a wide variety of agents such as hormones, chemokines, growth factors and also by integrin ligation (25, 137, 148, 476, 562). In contrast to FAK, Pyk2 is a Ca2+- dependent kinase (reviewed in 27). It has previously been shown that G protein-coupled receptor (GPCR) signaling to MAP kinase is dependent on cell adhesion, intact focal contacts and the actin filament system, and PKC activity (133, 522, 523). Recent data indicate that Pyk2 is linking these two systems. In response to the GPCR agonist histamine, Pyk2 localizes to focal adhesions in a PKC-dependent manner. The translocation of Pyk2 to focal adhesions is required for histamine-evoked ERK activation (329). Pyk2 activates MAP kinase in a Src kinase-dependent manner via adapter proteins Shc and Grb2 (54). Pyk2 expression is mainly restricted to the central nervous system and blood cells (reviewed in 27). Despite the similarity to FAK, Pyk2 activities are regulated differently and it does not seem to be a critical mediator of matrix signals (reviewed in 27). Pyk2 can compensate for only some of the adhesion-induced signals mediated by FAK, e.g. activation of ERK2 via Shc/Grb2 complex, but this was not sufficient to overcome the FAK-/- cell migration defects (527).

The FAK/Src complex

Integrin clustering, either by ECM proteins or with antibodies, induces tyrosine phosphorylation of FAK (207, 497). The first step in FAK activation is autophosphorylation of the Y397 (149). The importance of the phosphorylation of Y397 for FAK function is confirmed by mutational studies where the mutation of tyrosine to phenylalanine in FAK disrupts activation of several integrin-mediated signaling pathways. Autophosphorylation of the Y397 in FAK creates a binding site for the SH2 domain of Src tyrosine kinases. Binding of Src kinases to FAK results in further tyrosine phosphorylation of FAK (tyrosines 407, 576, 577, 861, 925) (501). The phosphorylation of tyrosines 576 and 577 positively regulates FAK catalytic activity. In addition to the SH2 domain-mediated binding, Src family tyrosine kinases bind to FAK via their SH3 domain (565). Both the Src SH2- and SH3-binding sites of FAK are required for maximal induction of tyrosine phosphorylation of downstream targets by FAK/Src (565). Tyrosine 397 can be phosphorylated also by other unidentified kinases as shown by Sieg et al. (525). Kinase-inactive FAK expressed in FAK-deficient cells is trans- phosphorylated at Y397 after EGF treatment (525). Interestingly, although FAK is heavily tyrosine phosphorylated in v-Src transformed cells it was shown that v-Src does not induce phosphorylation of Y397 (359). The FAK/Src complex phosphorylates a number of FAK- associated proteins, including paxillin, CAS, tensin (Fig. 8). Studies on cells derived from Src-/- , FAK -/-, Csk-/-, and Src/Fyn/Yes-/- mice indicate that tyrosine phosphorylation of these proteins is largely dependent on Src kinase activity whereas FAK acts as a docking protein by activating and recruiting Src family kinases to their substrates (58, 253, 287, 498, 502, 591). The docking function for SH2 domain proteins is dependent on Src family kinases since

37 tyrosine phosphorylation of FAK in cells lacking Src, Fyn, and Yes is severely impaired (287). The impact of paxillin and CAS on integrin-mediated signaling will be discussed later.

FAK activation

The mechanism by which integrins activate FAK is presently not understood. The activation of FAK is dependent on cytoplasmic tails of integrin β subunits (6). Although synthetic peptides derived from the membrane proximal part of the cytoplasmic tails of integrin β1 subunit bind to FAK in vitro (500) it is unlikely that direct binding of FAK to integrin β tails is sufficient for FAK activation. Mutagenesis studies on the cytoplasmic tails of β subunits have shown that deletion of the last 13 a.a. from the β1 tail abrogates FAK activation and this site does not overlap with the FAK binding site mapped in the in vitro studies (317). Moreover, mutation of the membrane proximal region in the β3 tail (DRKE to ARKA and FAKF to AAEA) reduces formation of focal adhesions without affecting activation of FAK (343). In contrast to this, activation of FAK was impaired in a β1 mutant which lacked a part of the membrane proximal region (MIIHDR) (325). In some cell types constitutive association between FAK and β1 integrin subunit has been reported but activation of FAK takes place only when integrins are clustered on a solid support, e.g. when cells are plated on ECM or integrin antibodies (124). It has been suggested that FAK could be linked to integrins via talin. The talin binding sites in β tails partially overlap with the sites required for FAK activation (82, 261). It is known that activation of FAK is dependent on intact cytoskeleton; cytochalasin D treatment disrupts FAK activation, which could be due to impaired integrin clustering. FAK mutants that fail to target to focal adhesions are defective in inducing tyrosine phosphorylation of downstream substrates (515). The FAT sequence largely overlaps with the paxillin-binding site in FAK. It is likely that binding to paxillin contributes to FAK localization to focal adhesion, although probably not during the very early stages of focal adhesion formation (370, 371). Recent studies have shown that replacement of the two NPXY sequences with NPXF in the β1 tail abrogates FAK activation (paper II) (611). The NPXY motifs could serve as acceptor sites for PTB domains of an unknown FAK-regulating protein. The PTB domain can recognize sequences NPXpY but phosphorylation of the tyrosine is not always required (163). Integrin-mediated FAK activation has been shown to involve activity of Rho GTPases. The early phosphorylation of FAK after integrin ligation was shown to be Rho GTPase- independent whereas the later phase required Rho activity (412). Some studies have shown that inhibition of PKC activity inhibits also activation of FAK, thus placing PKC upstream of FAK (138, 201, 618).

Cell survival

Normal epithelial and endothelial cells require integrin-mediated adhesion for survival; detachment of cells from the matrix induces a form of apoptosis known as “anoikis” (171, 172). FAK activity is required for protecting cells from anoikis and expression of constitutively active FAK in epithelial cells prevents anoikis (172). Both Y397 and kinase activity of FAK were required to suppress anoikis (172). In addition to this, the necessity of FAK for cell survival has been demonstrated in a number of other systems using injection of

38 anti-FAK antibodies (245) and expression of dominant-negative FAK (252). During apoptosis, FAK has been shown to be proteolytically cleaved by caspases that results in disassembly of focal adhesions and detachment of cells (119, 312, 609). In renal epithelial cells, loss of focal adhesions and dephosphorylation of FAK preceded caspase-mediated cleavage of FAK during cell apoptosis (581). However, the mechanisms by which integrins and FAK prevent cells from apoptosis are not entirely clear. Several studies have shown that these pathways can differ between different integrins and different cell types (171, 252, 357, 537, 644). Adhesion-induced tyrosine phosphorylation of FAK promoted cell survival of mammary epithelial cells and human glioblastoma cell line by activating the PI3K-Akt survival pathway (184, 537), whereas in primary fibroblasts cell survival was dependent on activation of the JNK pathway (10). Integrin α5β1-mediated adhesion has been shown to up-regulate expression of Bcl-2, a protein that promotes cell survival, in CHO cells (644). In another study it was demonstrated that cell adhesion on fibronectin (via α5β1), but not on collagen (via α2β1), resulted in PI3K-dependent activation of Akt/PKB which protected cells from apoptosis evoked by different cytotoxic stimuli (303). Integrin-mediated cell survival of mammary epithelial cells is regulated via FAK by controlling the subcellular localization of an apoptosis promoter protein Bax (184). It should be noted that individual integrins have different capacities to protect cells from apoptosis (644). Some integrins (e.g. α6β4) can promote cell apoptosis (106).

Migration

The involvement of FAK in integrin-mediated migration is firmly established. FAK knockout cells display reduced migration (253). Re-expression of wt FAK in FAK -/- cells restores the ability of these cells to migrate on ECM (423, 526). FAK-induced cell migration is utterly dependent on the autophosphorylation site Y397 in FAK and its association with Src family kinases (88, 287, 423, 467, 526). It has been shown that CAS association with FAK is required for FAK-promoted cell migration (see below) (89). A FAK mutant selectively unable to bind PI3K cannot promote CHO cell migration on fibronectin showing that binding solely to Src and CAS is not sufficient for migration (460). Furthermore, overexpression of Grb7, which also binds phosphorylated Y397 in FAK promotes NIH 3T3 cell migration (206). The biochemical pathway downstream of Grb7 is not known yet. Even though a role for Grb2 in FAK-regulated migration has been ruled out (89) it is worth mentioning that Grb2 promotes actin assembly by enhancing the interaction between N-WASP and Arp2/3 complex (86). Recently, FAK was shown to be required also for growth factor-stimulated cell migration by acting as a molecular bridge between integrins and growth factor receptors (525).

Activity regulation

How is FAK activity regulated? In order to become active FAK must localize to focal contacts. How this is achieved is still not completely clear (see above). Observations that FAK requires intact actin stress-fibers for its activity and that FAK can be activated by constitutively active RhoA points strongly to the involvement of actin filament system in the regulation of FAK activity (78, 105, 470). Since FAK activity is critically dependent on the phosphorylated tyrosines in the molecule it is likely that FAK activity can be down-regulated by phosphatase activity. PTEN

39 Figure 8. Intracellular signaling pathways activated by a generic integrin.

(phosphatase and tensin homologue deleted on chromosome ten) functions as a dual specificity phosphatase acting both on proteins and lipids (reviewed in 569, 586). PTEN dephosphorylates PI(3,4,5)P3, a product of PI3K, at the 3 position in the inositol ring. Phosphoinositide phospholipids are ligands for PH domains. Such associations recruit PH domain-containing proteins, such as Akt, to the plasma membrane which is important for their activation (569, 586). It has been shown that PTEN, by negatively regulating FAK activity as well as dephosphorylating PIP3, down-regulates the matrix-dependent PI3K/Akt cell survival pathway (558). Loss of PTEN advances G1 cell-cycle progression by down-regulating p27KIP1 and promotes growth factor and adhesion-dependent cell survival by up-regulating PI3K/Akt pathway (545). In order for cells to move, the focal adhesions have to be dissolved and rebuilt again. Accumulating evidence suggests that the tyrosine phosphatase SHP-2 has a role in regulating cell adhesion and migration. Fibroblasts lacking a functional SHP-2 are impaired in their ability to spread and migrate on fibronectin (637). A report by Yu et al. demonstrated that SHP-2 mutant cells show elevated FAK phosphorylation in suspension. They suggested that impaired regulation of FAK phosphorylation causes these observed defects (637). On the

40 other hand, a report by Oh et al. showed that SHP-2 -/- cells are defective in induction of tyrosine phosphorylation of FAK and other focal adhesion proteins, e.g. CAS and paxillin (412). SHP-2 has been demonstrated to be involved in growth factor-induced cell migration (349, 449). IGF-1-induced FAK dephosphorylation is impaired in SHP-2 -/- cells and these cells show reduced migration on vitronectin in response to insulin (349). Cells that express mutant forms of PDGFR-β that are defective in SHP-2 binding cannot induce FAK activity and do not migrate towards PDGF (449). In another study it was demonstrated that cell adhesion to fibronectin enhances SHP-2 recruitment to wt PDGFR-β. This positively correlated with PDGF stimulated ERK activation because SHP-2 dephosphorylated tyrosine in PDGFR-β that is a binding site for RasGAP (134). These data demonstrate again a tight interconnection between growth factor receptor and integrin signaling. During mitosis cells round up from matrix in order to divide. This is accompanied with dissociation of the FAK/Src/CAS complex (625). Mitosis-specific serine/threonine phosphorylation of FAK was found to disrupt the binding between FAK and CAS. During the mitosis FAK, CAS and paxillin were not only highly serine/threonine phosphorylated but also tyrosine dephosphorylated (625). The identities of the kinases and phosphatases responsible for such alterations in the phosphorylation states of these proteins are currently not known. It was recently shown that Pyk2 kinase activity is negatively regulated by a protein called FIP200. Pyk2 activation, in response to several stimuli, correlated with the dissociation of Pyk2/FIP200 complex (577). Interestingly, FIP200 could also associate with FAK and inhibit its kinase activity in vitro (577). Whether FIP200 could regulate FAK activity in vivo and how is this connected to integrin-mediated signaling remains to be investigated. One way to regulate FAK activity is via FRNK. Overexpression of FRNK in chicken embryo cells interferes with endogenous FAK function, possibly by competing for a common binding partner(s) necessary for FAK signaling (468). The fact that FRNK expression is developmentally regulated in tissue specific manners indicates that FRNK might play a role regulating FAK activity during (401).

Crk-associated substrate and Crk

CAS (Crk-associated substrate) has an important role in integrin-mediated cell spreading and migration (89). CAS was identified as a highly tyrosine phosphorylated 130 kDa protein in v- Src- and v-Crk-transformed cells (484). The Cas family consists of at least three members: CAS, HEF1/CAS-L, and Efs/Sin (embryonal Fyn substrate/Src-interacting). These structurally very similar proteins have different expression patterns. CAS is widely expressed whereas CAS-L and Efs show more restricted tissue distribution. The fact that CAS-/- embryos die in utero possibly due to the defects in development of cardiovascular system shows that other CAS family members can not fully compensate for CAS (233). CAS proteins contain multiple interaction domains: an N-terminal SH3 domain, proline-rich regions, a substrate domain, a serine-rich region and a C-terminal domain that mediates homo- and heterodimerization (407, 484). CAS possesses several potential SH2 domain-binding sites, 15 in the substrate domain and one at the C-terminus.

41 CAS interacting proteins

CAS has been shown to bind many cellular proteins. Although CAS becomes heavily tyrosine phosphorylated upon various stimuli most of the interactions occur via the SH3 domain and the proline rich-regions in CAS. The SH3 domain of CAS can bind to FAK, PTP-1B, PTP- PEST, C3G, CMS (178, 211, 280, 281, 331, 248). The proline-rich regions of CAS have been shown to mediate interactions with Src-family kinases and PI3K (319, 390, 484). Proteins that have been shown to bind CAS via the phospho-tyrosines in vivo are Crk proteins, Grb2, and Src (77, 202, 390, 407, 591). In addition, SH2 domains of the p85 subunit of PI3 kinase, PLC γ, and Nck have shown to bind CAS from cell lysates prepared from adherent cells (591). CAS associates with the tyrosine phosphatase SHP2 in myoeloma cells after integrin ligation but the nature of this interaction is not known (351). It could be dependent on phosphotyrosines on CAS, similarly to the association between SHP2 and CAS-L (368). The serine-rich region of CAS interacts with the 14-3-3ζ protein in a phosphoserine-dependent manner (176). The interaction between CAS and 14-3-3ζ is regulated by cell adhesion. These two proteins colocalize at membrane ruffles in spreading cells and in the cytosol in stationary spread cells (176). During the last two years, several groups have identified new adapter proteins that associate with CAS and could potentially connect CAS to several cellular signaling pathways. One interesting class of adapter proteins (with no common name as yet) that bind to CAS is composed of an SH2 domain, a proline-rich region and a GEF domain. (80, 340, 486). The NSP (novel SH2-containing protein) proteins were found by searching EST databases for sequences that share homology with SH2-containing adapter proteins (340). Three adapter proteins (NSP1, 2, 3) from human fetal kidney library were cloned and further characterized. Insulin or EGF treatment and cell attachment to fibronectin induced tyrosine phosphorylation of NSP1, and NSP1 was found to associate with CAS in vivo. Overexpression of NSP1 in 293 cells was shown to result in an increased JNK kinase activity (340). NSP1 is also known as BCAR3, a protein that gives resistance to cytotoxic agent tamoxifen (580). Approximately at the same time a sequence of the mouse protein AND-34 was published (80) which turned out to be the orthologue to human NSP2. AND-34 associates with several tyrosine phosphorylated proteins, one of which is CAS, and cell attachment to fibronectin induces tyrosine phosphorylation of AND-34. Recently a mouse orthologue to human NSP3, Chat (CAS/HEF1-associated signal transducer) was shown to associate with CAS via the C- terminus of both proteins (486). Chat becomes serine/threonine phosphorylated upon growth factor treatment and this phosphorylation was abolished by a MEK inhibitor. Overexpression of Chat induced elevated JNK activity. Chat and CAS colocalized in the cytoplasm in COS-7 cells and upon growth factor treatment they became localized at membrane ruffles (486). A recent publication about AND-34 reveals some very interesting aspects of the functions of these new adapter proteins. AND-34 was shown to display GEF activity towards RalA and to a lesser extent towards Rap1A and R-Ras (190). Association between CAS and AND-34 occurs via the C-terminus of both proteins, similarly to that between Chat and CAS. Furthermore, overexpression of CAS blocks the ability of AND-34 to increase levels of Ral- GTP (190). The importance of the association between CAS proteins and the NSF/Chat family members in integrin-mediated signaling remains to be explored. The adapter molecule CMS, which consists of three SH3 domains, a proline-rich region and a coiled-coil domain (CC), associates with CAS via its proline-rich region and colocalizes

42 with CAS at membrane ruffles (281). Overexpression of CMS in COS-7 cells induces membrane ruffling and vesicle formation. The SH3 domains of CMS are most related to those in various , PIX and POSH, and interact in vitro with Src family kinases and PI3 kinase. CMS also contains several putative actin binding sites (281). Again, the functional role of CMS is yet not known but it could mediate assembly of signaling complexes that link CAS to actin cytoskeleton. The C-terminal tail of CAS was found to interact with the adapter protein nephrocystin (141). Nephrocystin is an SH3 domain-containing protein whose absence correlates with a cystic kidney disease (223).

Regulation of CAS

The regulation of CAS tyrosine phosphorylation is interesting. The generally accepted view is that integrin-induced tyrosine phosphorylation of CAS is predominantly evoked by FAK. Accordingly, FAK would phosphorylate the C-terminal tyrosine that would then be recognized by the SH2 domain of Src kinase (551). Binding of Src to CAS could then augment further CAS phosphorylation. However, CAS is tyrosine phosphorylated in FAK-/- cells, Fyn-/- and Abl-/- cells but not in Src-/- cells. Enhanced tyrosine phosphorylation of CAS occurs in Csk-/- cells (205, 591). These data indicate the importance of Src in CAS tyrosine phosphorylation. CAS is also differently tyrosine and serine phosphorylated in v-Crk and v- Src cells (63). CAS/14-3-3ζ complex can be observed only in lysates prepared from v-Crk transformed cells but not from v-Src transformed cells (176). v-Crk, when overexpressed in NIH 3T3 cells, coimmunoprecipitates with protein tyrosine kinase activity that phosphorylates CAS (394). FAK was not detected in these immunoprecipitates. Several lines of evidence suggests that CAS can be tyrosine phosphorylated in response to integrin- mediated cell adhesion in both FAK-dependent and -independent ways (paper II, paper IV) (611). A mutant of β1 integrin that is unable to activate FAK still induces tyrosine phosphorylation of CAS (paper II) (611). Interestingly, CAS from these cell lysates did not bind the SH2 domain of CrkII efficiently in vitro indicating that CrkII binding sites are phosphorylated by the FAK/Src complex (paper II) (611). Another integrin mutant that induces FAK tyrosine phosphorylation shows reduced tyrosine phosphorylation of CAS but not reduced association with CrkII (paper IV). In addition to these data several other reports indicate that CAS can be phosphorylated independently of FAK. Overexpression of the non- receptor tyrosine kinase Fes in the macrophage cell line BAC1.2F5 resulted in tyrosine phosphorylation of CAS (259). On the other hand, overexpression of Fer kinase, closely related to Fes kinase, in Rat-2 fibroblasts resulted in hypophosphorylation of CAS and subsequent cell detachment of cells. In this latter study the phosphorylation status of FAK was not affected (479). Fer kinase has been shown to mediate cross-talk between cadherins and β1 integrins, and accumulation of the Fer kinase in the integrin complex inhibits β1-integrin function (19). Clustering of a melanoma chondroitin sulfate proteoglycan (MCSP) was shown to induce melanoma cell spreading via α4β1, accompanied with activation of ACK-1 and tyrosine phosphorylation of CAS. Downregulation of ACK-1 by an antisense construct abolished tyrosine phosphorylation of CAS upon MCSP clustering, indicating that CAS may be a downstream target of ACK-1 (151). Adenovirus entry via αvβ5 integrins induces tyrosine phosphorylation of CAS in SW480 cells (319). Interestingly, adenovirus internalization induces CAS phosphorylation also in the FAK-/- fibroblasts. In platelets, tyrosine

43 phosphorylation of CAS occurs before FAK activation and seems to be dependent on phosphoinositide turnover (414). In this context it is notable that PI3K activity has been shown to be necessary for growth factor-induced tyrosine phosphorylation of CAS (90, 416), and that Ret kinase mediated tyrosine phosphorylation of CAS is sensitive to PI3K inhibitors (385). Whether integrin-induced CAS phosphorylation requires PI3K activity is not known. Several phosphatases have been shown to dephosphorylate CAS (75, 178, 331, 610). CAS has been demonstrated by many studies to be a target of PTP-PEST (114, 177-179). Overexpression of PTP-PEST in Rat1 cells reduced CAS phosphorylation in response to adhesion to fibronectin and reduced cell migration (179). PTP-PEST -/- fibroblasts also show reduced migratory properties but in this case CAS was hyperphosphorylated (14). These data show that CAS has to be continuously phosphorylated and dephosphorylated during cell movement and that PTP-PEST is involved in this process. In addition to PTP-PEST, several other phosphatases, such as PTPα, PTP1B, LAR have shown to dephosphorylate CAS (75, 331, 332, 610). An interesting possibility is that different PTPs may dephosphorylate different phosphotyrosines in CAS, which may regulate separate signaling complexes formed on CAS. It is also possible that some of these phosphatases do not dephosphorylate CAS directly but instead regulate the activity of kinases phosphorylating CAS. Association of PTP1B with CAS has been shown to be important for inhibiting transformation of rat 3Y1 fibroblasts by v-Crk and v-Src. Overexpression of PTP1B reduces formation of CAS/Crk complex (332). On the other hand, loss of PTP1B activity down-regulates Src activity (20). Tyrosine phosphorylated CAS localizes at focal contacts and at membrane ruffles (407, 440). In v-Crk transformed cells hyperphosphorylated CAS is predominantly localized at the cytoskeletal fraction (394). It should be noted that localization of CAS-L varies during the cell cycle. It gets cleaved and the N-terminal fragment migrates to the mitotic spindle (300). The SH3 domain of CAS is required for its localization to focal adhesions (211). It is therefore suggested that CAS is recruited to focal adhesions via FAK. It would be interesting to examine whether intracellular localization of CAS proteins is at least partially regulated via Arfs similarly to paxillin (look further below). CAS-/- cells show reduced cell spreading and migration on fibronectin (232). CAS-/- fibroblasts showed focal adhesions, indicating that CAS is not required for focal adhesion formation. CAS-/- primary fibroblasts show thin irregularly assembled actin filaments at the cell periphery whereas thick, long actin fibers traversing the cells are missing (233).

The CAS/Crk complex

Integrin-mediated cell adhesion or integrin clustering induces tyrosine phosphorylation of CAS (400, 594) and its subsequent interaction with the adapter protein CrkII (591). The CrkII/CAS complex has been implicated in integrin-mediated cell migration (286). In addition to this, the CAS/Crk complex has been shown to be partially required for protecting cells from apoptosis in a three-dimensional collagen matrix (102) (Fig. 8). The Crk family has three members (CrkI, CrkII, CrkL) each of which consisting entirely of SH2 and SH3 domains (reviewed in 160, 284). CrkI and CrkII are similar, generated by . The C-terminus of CrkII has an extra SH3 domain and a tyrosine (Tyr 221) located between two SH3 domains that is an important autoregulatory site. CrkL plays a more prominent role in hematopoetic cells than in other cell types. The phosphorylated tyrosine 221 is recognized by the SH2 domain of CrkII itself. This intramolecular interaction

44 inhibits CrkII binding to other proteins and therefore inhibits CrkII function. Regulated phosphorylation/dephosphorylation of CrkII has been shown to be important for integrin- mediated cell adhesion and migration. Mutation of tyrosine 221 in avian CrkII to phenylalanine inhibits PC12 cell adhesion to collagen IV and NGF-induced neurite outgrowth. CrkIIY221F was found to constitutively associate with paxillin at the perinuclear region (155). A phosphatase that dephosphorylates tyrosine 221 in CrkII has not been identified. The N- terminal SH3 domain of Crk can bind the GTPase regulators C3G, DOCK180, ASAP1 and tyrosine kinases of the Abl-family (73, 159, 283, 355). The SH2 domain of Crk binds paxillin and CAS (484). Thus, recruitment of Crk coupled to GEFs can link integrins to signaling pathways that regulate actin reorganization and cell migration, and lead to the activation of the JNK cascade. The CAS/Crk complex localizes to membrane ruffles in migratory cells. CAS/Crk induced COS cell migration on vitronectin was found to be blocked by a dominant negative Rac (286). In another report it was shown that Rac activation alone in the absence of the CAS/Crk complex is not sufficient to induce COS cell migration (102). This indicates that an unknown downstream effector(s) of the CAS/Crk complex is needed for cell migration in addition to Rac. The N-terminal SH3 domain of Crk is indispensable for CAS/Crk-induced cell migration (286). Overexpression of DOCK180 that binds to the SH3 domain of Crk up-regulates signals from CAS/Crk complex (283) and later it was shown that DOCK180 activates Rac1 (282) (Fig. 8). An understanding of the mechanism by which DOCK180 activates Rac1 is still lacking. Since DOCK180 does not possess the Dbl-homology domain and binds only to guanine nucleotide free Rac1, it has been suggested that DOCK180 may promote Rac activity by stabilizing the nucleotide free intermediate of Rac during the guanine exchange reaction catalyzed by GEFs (282). The CrkII/DOCK180/Rac1 pathway is evolutionarily conserved, existing in C. elegans and Drosophila and is required for morphogenetic processes during embryogenesis (402, 455). Surprisingly, expression of v-Crk in PC12 cells induces activation of Rho kinase and not PAK (12). The Crk/CAS complex seems also to mediate integrin-induced activation of the JNK pathway (140). One way to activate JNK from CAS/Crk is via Rac, and another way could be via C3G (Fig. 8). The substrate activity of the guanine nucleotide exchange factor C3G is directed against Rap1 and R-Ras. In NIH 3T3 cells activation of JNK pathway by the v- Crk/C3G complex was mediated via R-Ras and not via Rap (373). On the other hand, expression of v-Crk in chicken embryo fibroblasts did not induce JNK activation but instead induced activation of the PI3K/Akt pathway (3).

Paxillin and zyxin

Paxillin, like FAK and CAS was first identified as a protein with increased tyrosine phosphorylation in v-Src transformed chicken embryo fibroblasts (187). Paxillin is an important scaffold molecule that locates several kinase activities in close proximity to integrins at focal adhesions. Paxillin is a member of a protein family that includes Hic-5 and leupaxin (575). Human paxillin has been shown to exists in three isoforms (α, β, γ) resulting from differential splicing (356). The α isoform is the most common. Paxillin isoforms exhibit different binding properties toward cellular proteins and have different expression patterns

45 (356). Leupaxin is expressed in hematopoetic cells that do not express FAK and can associate with Pyk2 (328). Hic-5 is very similar to paxillin at the amino acid level and it also localizes to focal contacts (200, 567). Furthermore, Hic-5 associates with the proteins FAK, vinculin, Src family members, and Csk, as does paxillin (except for Crk) (567). It has been observed that expression levels of Hic-5 correlate with cell senescence (567).

Tyrosine phosphorylation of paxillin

Paxillin becomes tyrosine phosphorylated upon integrin-mediated adhesion to extracellular matrix and tyrosine phosphorylated paxillin preferentially locates to focal adhesions (91). However, in v-Crk transformed NIH 3T3 cells most of the phosphorylated paxillin did not localize in the cytoskeletal fraction (394). Tyrosine phosphorylation of paxillin occurs concomitantly with activation of FAK. FAK activation is not essential for paxillin tyrosine phosphorylation, but it does promote this process (205, 564). It has been suggested that paxillin binding to FAK could be a mechanism bringing paxillin in close vicinity to Src family tyrosine kinases that would implement paxillin tyrosine phosphorylation (467). This model is supported by the observations that expression of FRNK inhibits cell spreading and paxillin tyrosine phosphorylation, and these effects can be overcome by expression of Src or kinase- inactive FAK (467). In addition to FAK and Src, paxillin has been shown to be a substrate for the non-receptor tyrosine kinases Abl and Csk (488, 555). Integrin-mediated cell adhesion to fibronectin has been shown to induce transient localization to focal contacts and activation of Abl (314), and paxillin has been shown to co-precipitate with Abl in response to cell adhesion and to be phosphorylated by Abl in in vitro assay (316). It has been suggested that tyrosine phosphorylation of paxillin by Src is a critical step in mediating focal adhesion assembly (467). However, overexpression of Csk in astrocytes, a negative regulator of Src activity, abrogates cell attachment and spreading on ECM while tyrosine phosphorylation of paxillin is not affected (555). As discussed later, reduced Src activity might affect intracellular trafficking of paxillin and thereby inhibit focal adhesion formation even though paxillin is tyrosine phosphorylated by Csk. In addition to connecting integrins to kinase cascades that regulate organization of actin filament system, paxillin could potentially link integrins to the JNK cascade. FAK-mediated activation of JNK kinase pathway was shown to be dependent on paxillin localization to the plasma membrane (251). Furthermore, recruitment of paxillin alone to the plasma membrane was sufficient to induce JNK but not ERK activation (251).

The LIM domains of paxillin

Paxillin can be tentatively divided into two parts, the C-terminus and the N-terminus. The C- terminus consists of four LIM (Lin-11, Isl-1, Mec-3) domains. The LIM domains are cysteine/histidine-rich double zinc fingers that are found in transcription factors and in several actin filament-associated proteins such as zyxin, CRP-1 (cysteine-rich protein), LPP (LIM- containing lipoma-preferred partner). LIM domains 2 and 3 in paxillin determine its localization at focal contacts (72). Localization to focal contacts was shown to be partially dependent on serine and threonine phosphorylation of these LIM domains. Furthermore, this was also required for efficient cell adhesion to fibronectin (72). The serine/threonine kinase(s) that phosphorylate(s) these residues is not known, but there are indications that paxillin can

46 be phosphorylated by ERK2 (292). Paxillin becomes serine/threonine phosphorylated in response to cell adhesion (45, 128) and is heavily serine/threonine phosphorylated in mitotic cells (324). PTP-PEST was found to be in a complex with FAK and the formation of this complex was mediated by paxillin (516). Later studies have revealed that the association between paxillin and PTP-PEST is mediated via the LIM-domains 3 and 4 in paxillin and a proline-rich sequence in PTP-PEST (115, 516). Overexpression of PTP-PEST in Rat1 cells was reported not to cause reduction in paxillin phosphotyrosine content (179). On the other hand, a recent report by Chen et al. demonstrates that PTP-PEST dephosphorylates paxillin in vivo and this requires association between these two proteins (514). This result is supported by the fact that paxillin is constitutively hyperphosphorylated in PTP-PEST -/- fibroblasts, as are FAK and CAS (14).

The LD motifs of paxillin

Binding of paxillin to vinculin, talin, Csk, FAK, Pyk2, Src family kinases, PKL is mediated via the N-terminal domain of paxillin which contains five leucine-rich LD motifs (LDXLLXXL) and a proline-rich sequence (221, 322, 420, 487, 576). The association between paxillin and kinases FAK and Pyk2 seems to be constitutive and does not require protein phosphorylation (376, (420). Paxillin has two binding sites for FAK and Pyk2 (71) with LD domains 2 and 3 being the principal determinants for binding (564). The LD motif 4 of paxillin associates with the Arf GAP protein PKL (576). PKL has binding sites also for PIX, Nck and PAK. The binding of paxillin to the complex PKL/PIX/PAK could locate the PAK actin reorganization activity at focal adhesions (see above) (Fig. 8). Microinjection of GST-LD4 into NIH3T3 cells decreases cell migration into a wound in in vitro assays. The cytoplasmic tail of integrin β1 subunit has been shown in vitro to interact with paxillin but the binding site in paxillin has not been mapped (500). Unexpectedly, the cytoplasmic tail of integrin α4 subunit was shown to associate specifically with paxillin (334, 335). The α4β1 integrin has an important role in several physiological processes, e.g. in immune response (21, 231); it increases cell migration and reverses focal adhesion formation. The cytoplasmic tail of integrin α4 subunit is indispensable for these functions. The interaction between paxillin and α4 was shown to increase the rate of activation of FAK, which in turn leads to increased cell migration and thus explains some of these specific features of integrin α4 tail. The paxillin binding motif in α4 is conserved in all species tested so far (334).

Cellular localization of paxillin

Tyrosine phosphorylation of tyrosines 118 and 31 in paxillin creates binding sites for the adapter protein CrkII, and this association was shown to be required for migration of Nara Bladder Tumor II cells on collagen but not for adhesion or spreading (436). In contrast, in another study overexpression of paxillin inhibited cell hapotactic migration on collagen, vitronectin and fibronectin (631). Overexpression of paxillin in which four N-terminal phosphorylation sites, including 118 and 31, were mutated neither inhibited nor promoted cell migration (631). Paxillin phosphorylated on tyrosines 31 and 118 has been shown to

47 specifically localize at focal adhesions and at the cell periphery (391). This demonstrates that tyrosine phosphorylation of paxillin can regulate localization of paxillin in the cell. Work in recent years has revealed that the recruitment of paxillin to focal adhesions is dependent on the activity of ADP-ribosylation factors, Arfs. Arf proteins are small GTPases that regulate membrane traffic by controlling vesicle coat assembly and disassembly (reviewed in 142). The Arf family of proteins can be divided into three classes. Class I proteins (Arf1, 2, 3) regulate mostly vesicle trafficking in the ER-Golgi and endosomal compartments. The class III protein, Arf6, has been implicated in regulation of the endosomal-plasma membrane system and the actin filament system. Arfs, like Ras family proteins, are regulated by GEFs and GAPs. Initial studies showed that recruitment of paxillin from the Golgi compartment to focal adhesions in serum-starved Swiss 3T3 fibroblasts required the activity of Arf1 (404). Recently, a protein named PAG3 (paxillin-associated protein with Arf GTPase-activating protein activity, number 3) was shown to associate with paxillin (290). Expression of PAG3 was up-regulated concomitantly with appearance of the invasive phenotype during monocyte maturation. PAG3 overexpression inhibits paxillin recruitment to focal contacts and cell migration. PAG3 could mediate its effects via regulating the activity of Arf6. The fact that PAG3 colocalized with paxillin in the cytoplasm but not in the focal adhesions in COS-7 cells indicates that this association is regulated. The phosphotyrosine content of PAG3 was elevated during monocyte maturation and so was binding to paxillin. The question of whether phosphorylation regulates association between these two proteins was not addressed. Src has been shown to bind and phosphorylate ASAP1, a PI(4,5)P2-dependent Arf GAP (73). By phosphorylating GAPs, Src might down-regulate their activity and thereby increase membrane trafficking and promote migration. In addition to PAG3, the same group identified several other paxillin binding proteins sharing the conserved zinc finger motif. These were named PAG1, PAG2 and PAG4 (290). PAG1 was reported to be active primarily towards Arf1. The above mentioned-paxillin binding protein PKL is also a GAP for Arfs. In in vitro kinase assays PKL is phosphorylated both on tyrosine and serine residues (576). Whether it is phosphorylated upon cell attachment or during cell migration remains to be determined. PKL homologues, the Cat proteins (see above), have been shown to become tyrosine phosphorylated in response to cell adhesion to fibronectin (30). How phosphorylation regulates the activity of these proteins remains to be determined. It is conceivable that several Arfs and Arf GAPs (possibly also Arf GEFs) regulate the subcellular localization of paxillin. Some could regulate the cycling from Golgi to focal adhesions upon cell adhesion (like Arf1), whereas others could regulate the endocytotic cycle during cell migration (like Arf6).

Zyxin

Other LIM domain-containing proteins have several interesting features. Zyxin is a focal adhesion protein that is comprised of an N-terminal proline-rich region and C-terminal LIM domains and possesses a nuclear import signal (117). An interaction between zyxin and α- actinin seems to be required for its localization to focal contacts (459). α-Actinin is an actin cross-linking protein that has been shown in in vitro assays to directly bind to the cytoplasmic tail of integrin subunit β1 (421, 422). The N-terminal proline-rich region of zyxin interacts directly with Ena/Vasp family members. Proteins of the Ena/Vasp family serve as

48 ligands for profilin and can modulate actin reorganization (reviewed in 345). Inhibition of zyxin function, by interfering with its cellular localization, inhibits cell migration and spreading (144). Zyxin has also been reported to interact with Vav, a GEF for Rho GTPases (228). Interestingly, zyxin has been shown to shuttle between the cytoplasm and nucleus (396). LIM domains are often found in transcription factors and can mediate interactions with DNA (reviewed in 125). Another LIM domain protein, LPP, has a very similar cellular localization to zyxin (435). Transcriptional activation capacity contributed by both proline- rich region and LIM domains has been demonstrated for LPP (435). Furthermore, LIM domains of Hic-5 were found to recognize specific DNA fragments in vitro (395). Hic-5 accumulates in the nuclei in response to hydrogen peroxide and could stimulate of c-Fos and p21 (517). The N-terminal half of Hic-5 was permissive for nuclear localization. Nuclear localization of paxillin has not been observed.

Other non-receptor tyrosine kinases

In addition to FAK a number of other protein tyrosine kinases have been implicated in integrin-mediated signaling. These include Src family kinases, Abl (314, 316), ACK (see above), Syk (189, 492, 627). Below the role of Src kinases in integrin-mediated signaling is summarized. Src family protein kinases are activated not only by integrin engagement but also by many other classes of cellular receptors (reviewed in 1, 566). Three members of the family, Src, Fyn, Yes are ubiquitously expressed, whereas others are mainly expressed in hematopoetic cells. They consist of an SH4 domain that contains signals for lipid modification, an SH3 domain followed by an SH2 domain, a catalytic domain (SH1 homology domain), and a C-terminal negative regulatory sequence. A large number of studies have established that Src family kinase activity is required for most integrin-mediated processes, including adhesion, migration, survival, and proliferation. Src, when activated, forms a complex with FAK. However, as shown by several studies, Src family kinases can act without FAK after integrin-mediated adhesion (348, 503, 598). How integrins activate Src family kinases independently of FAK is not known. This is quite surprising considering the importance of these kinases in integrin-mediated signaling. In general, the activity of Src kinases is regulated by phosphorylation and dephosphorylation. In its inactive state the SH2 domain is interacting with phosphorylated tyrosine 527 (in chicken Src) and the SH3 interacts with proline-rich sequence in the linker region. Csk (C-terminal Src kinase) phosphorylates tyrosine 527 and therefore inactivates Src kinases. Csk is a cytosolic protein and needs to be translocated to the plasma membrane in order to phosphorylate Src. Csk is located to the membrane by binding via its SH2 domain to the transmembrane protein Cbp (Csk-binding protein), also called PAG (67, 268). Cbp is phosphorylated, possibly by Src kinases themselves. Phosphorylated Cbp then serves as an anchor for Csk, which then can down-regulate Src activity (268). Autophosphorylation of tyrosine 416 in Src is important for regulation of Src kinase activity. Src activation involves displacement of intramolecular SH2 and SH3 domain interactions and dephosphorylation of tyrosine 527. The membrane phosphatase PTPα and cytosolic phosphatase SHP-1 have been shown to dephosphorylate Src (209), (536, 648). The binding of the SH2 domain of Src to the C-terminal phosphotyrosine of PTPα displaces the intramolecular bridging in Src and allows dephosphorylation of tyrosine 527 in Src (648). PTP1B activity appears to be important for integrin-mediated cell adhesion and spreading.

49 Loss of PTP1B resulted in reduced activity of Src showing that Src could be a physiological PTP1B target (20). These data also indicate that PTP1B could be the phosphatase that mediates activation of Src induced by integrins. CAS is not only a target for Src, it seems to be an important regulator of Src function as well. CAS-/- fibroblasts are resistant to Src mediated cellular transformation (233). Recently it was reported that overexpression of CAS activates Src, and a mutation in the SH3 domain of Src that abolished binding to CAS inhibited also the ability of Src to phosphorylate its downstream targets (76). CAS could possibly regulate Src activity by displacing intramolecular interactions and by bringing phosphatases that could activate Src. Src activity has been shown to regulate the activity of small Rho proteins in response to integrin engagement. Integrin stimulation with the soluble GRGDS peptide is sufficient to suppress RhoA activity via a Src-dependent mechanism (22). Distribution of the tyrosine kinases Syk, Lyn and Fgr to the Triton-X100 insoluble fraction after integrin β2 clustering was independent of actin polymerization (627). These data indicate that integrin-mediated activation of Src kinases does not require integrin clustering nor de novo actin polymerization, which is different from FAK activation.

Mitogen-activated protein kinases

The mitogen-activated protein (MAP) kinases are important mediators in signal transduction pathways from the plasma membrane to the cell nucleus. They are involved in many cellular processes such as survival and proliferation, and they are activated by a range of diverse stimuli. Three major MAPK family subgroups are: ERKs (1 and 2), JNKs (more then 10 splice-variants), and four isoforms of p38 (α, β, γ, δ) (reviewed in 318). ERK1 and ERK2 are particularly linked to cell proliferation, while JNK and p38 kinases are often activated by cellular stresses and also referred to as stress-activated protein kinases. MAPK cascades consist of at least three protein kinases that work in sequence forming a “module”. ERK kinase activation requires phosphorylation of two residues within the activation loop by MEKK1/2. MEKK1/2 in turn is activated by Raf. The JNK and p38 kinases are activated in the same manner but by different kinases. In addition to kinases, scaffold proteins are required that bind all kinases in the module, plus additional regulatory factors that organize and compartmentalize MAP kinase activity in the cell. Activated MAP kinases phosphorylate a large number of downstream targets such as transcription factors (Elk-1, c-Jun) and kinases (Mnk1, 2).

Activation of MAP kinases by integrins

The mechanisms by which integrins initiate signals that lead to activation of the ERK signaling pathway are diverse (Fig. 8). Some studies have shown the involvement of FAK whereas other exclude it. Binding of the adapter protein Grb2 via its SH2 domain to phosphorylated tyrosine 925 in FAK could link integrins directly to Ras/ERK pathway. However, fibronectin- stimulated activation of ERK2 in human 293 cells was dependent on FAK Y397 and not on Y925, via a mechanism that required intact cytoskeleton and involved tyrosine phosphorylation of Shc and its association with Grb2 (502, 503). Integrin-mediated activation of MAP kinase can occur also in the absence of FAK activity; levels of FRNK expression in

50 NIH 3T3 cells sufficient to block adhesion-dependent phosphorylation of FAK have no effect on integrin-mediated activation of MAP kinase (503). The necessity of FAK in ERK activation seems to be either integrin and/or cell type specific. However, FAK has been shown to be required for growth factor activated ERK in stably adherent cells (462) indicating that under “normal conditions” FAK activity is required. Certain integrins (α4β6, α1β1, α5β1, αvβ3) are linked to the ERK pathway by the adapter protein Shc apparently in an α subunit-dependent manner (Fig. 8) (348, 598). Ligation of these integrins activates Fyn, which is associated with caveolin-1 in close proximity to integrins, leading to tyrosine phosphorylation of Shc and formation of Shc/Grb2 complex (599). It has also been shown that Src-mediated tyrosine phosphorylation of Shc promotes Grb2 binding and signaling to ERK after cell adhesion to fibronectin (503). The involvement of caveolin-1 in this study was not addressed. There are also indications that Shc can directly bind to integrin β subunit. Binding of Shc to the phosphorylated tyrosine in the NPXY motif in β4 directly links β4 to Ras signaling pathway (348). Phosphorylation of these tyrosines in β3 tail has been shown to generate docking sites for signaling proteins Grb2 and Shc (116). Tyrosine phosphorylation of the cytoplasmic domain of β1 has been detected only in v-Src transformed fibroblasts (258) and it is still unclear whether Shc can bind to β1. Integrin-mediated activation of ERK seems also to involve small GTPases from the Rho family. Dominant negative Rho blocks integrin-induced activation of ERK whereas Lbc enhances it (463). Cell attachment to fibronectin was shown to send a co-stimulatory signal needed for Raf1 activation. Phosphorylation of serine 338 on Raf1 occurs upon cell adhesion to fibronectin and is required for Raf-1 activation. Raf1 was found to be phosphorylated by PAK1 in a PI3K-dependent manner (96). The involvement of the Rho family GTPases in integrin-mediated Raf and ERK activation could explain why Raf and ERK could be activated by integrins even when Ras activity is blocked.

Cell migration

The integrin-activated ERK pathway seems to have a role in hapotactic cell migration on ECM (285). Elevated ERK activity increased MLCK activity leading to phosphorylation of MLC and thereby promoted COS-7 cell motility (285) (Fig. 8). Dominant negative Shc inhibits cell migration without affecting tyrosine phosphorylation of FAK and CAS (193). It was shown that Shc-induced cell migration was random whereas FAK/CAS-induced cell motility was directional (108, 193). Both CAS/Crk complex and ERK have been shown to be important for cell migration (100, 285, 286). In two reports it has been stated that blocking each of these pathways (ERK or CAS/Crk) independently blocks cell migration even when the activity of the other pathway is not affected (100, 102). These authors have said that in the presence of ERK inhibitors cell migration is prevented, although by looking at the data, it seems that MEK inhibitor reduces cell migration by 40-50% only. In their previous work, the CAS/Crk-induced cell migration was not blocked by a dominant negative Ras (286). Similarly, FAK-promoted hapotactic cell migration is not inhibited in the presence of MEK inhibitor PD98059 (89). Thus, these data could point to the possibility that ERK activity is not absolutely required for FAK/CAS/Crk complex promoted cell migration on ECM. All these reports are very confusing, and make it difficult to judge whether the ERK activity is crucial for cell migration or not.

51 Cell cycle

In normal cells, signals from extracellular matrix and growth factors cooperatively regulate cell cycle progression. Upon activation MAP kinases translocate to the nucleus and regulate the activity of several transcriptor factors that direct the expression of cyclin-dependent kinases and cyclins. Integrin-mediated cell adhesion appears to be important for certain events during the cell cycle, such as the induction of the expression of cyclin D and cyclin E, and down- regulation of the cell cycle inhibitors p21 and p27 (59),(158, 195). Furthermore, it has been shown that integrin-mediated adhesion (via integrins α2β1 and α5β1) is required for epidermal growth factor-induced ERK activation (15). Interestingly, Rap1 activity was up-regulated in an integrin-dependent manner in suspended cells via the CrkII/C3G complex and this correlated with downregulation of ERK activity in suspended cells (74). On the other hand, insulin treatment dissociates the CrkII/C3G complex and down-regulates Rap1 activity, and up-regulates ERK activity (417). It appears as if different integrins have different capacities to activate ERK. Inhibition of binding of integrin α5β1, but not α4β1, to fibronectin stimulated ERK activity and upregulation of cyclin A/Cdk2 and cyclin E/Cdk2 complexes, which was accompanied with increased cell proliferation. Interestingly the stimulation only occurred when α5β1 integrin was inhibited in the early (188). On the other hand, exogenous expression of the α5 subunit in myoblasts promotes cell proliferation (495). In epithelial cells integrin α5β1 mediates cell proliferation via activation of ERK by the EGFR (294). Integrin-induced activation of EGFR has also previously been shown to be required for activation of ERK and cell survival (376). Thus, integrin α5β1 might not be a potent ERK activator by itself and it could use growth factor receptors for signal amplification. Integrin αIIbβ3 has been shown to down-regulate ERK activity after fibrinogen binding (386). Integrin α7-deficient muscle cells show elevated levels of activated ERKs, which then correlated with degeneration of muscle fibers (483). This data shows that in normal muscles the presence of integrin α7β1D down- regulates ERK activity, which is required to maintain tissue integrity. Furthermore, the integrin splice variant α6A, and not α6B, α3A, α3B has been reported to activate ERK (74, 606). Regulation of ERK activity by different Ras family proteins, integrins and growth factors is very complex and far from fully understood. Although necessary for cell-cycle progression, integrin-mediated activation of ERK seems to be relatively weak compared the response induced by growth factors. It has been suggested that the JNK pathway is the main target for integrins. Adhesion-mediated activation of the JNK pathway during the G1 phase of the cell cycle was needed for HUVEC cells to enter the S phase. Activation of JNK was evoked by the FAK/Src complex and CAS/Crk coupling (Fig. 8) (418). The signaling pathway(s), downstream of the CAS/Crk complex leading to JNK activation, required for cell-cycle progression is/are not known. Fibronectin- mediated activation of the JNK and not the PI3K/Akt pathway was shown to be necessary for survival of primary rat synovial fibroblasts (10). In these cells activation of JNK involved the Ras/Rac/Pak1/MKK4 pathway. Both active ERK and JNK have been shown to colocalize with integrins at focal contacts (10, 161). Targeting of active ERK to focal contacts in response to integrin engagement or activation of v-Src is required MLCK activity. Since ERK activity promotes MLCK activity this indicates a positive feedback loop in ERK regulation.

52 Collagen receptor α2β1 has been shown to specifically activate p38α (255). The activation was dependent on the cytoplasmic tail of α2. The mechanism by which integrin α2β1 activates p38α is not clear but it appears to involve Cdc42, MKK3 and MKK4 (255).

Protein kinase C

PKC activity is required for several aspects of integrin function such as cell spreading, activation of ERK and activation of FAK (104, 130, 138, 315, 369, 473). PKCs can modulate integrin ligand binding to fibronectin (123). PKC activation upon integrin clustering is an early event of adhesion-mediated signaling (592). It is not clear what the mechanisms are by which PKC exerts its effect on cell spreading and integrin-activation. PKC was originally identified as a phospholipid-dependent and diacylglycerol (DAG)- stimulated protein kinase activity. Today we know that PKC is a family of kinases; the PKC family can be divided into three subfamilies based on structural differences and on the requirement for calcium and lipids for catalytic activity. Conventional PKCs (isoforms α, βI, βII, γ) bind the co-activating factors Ca2+ and DAG (the pharmacological counterpart to DAG is PMA - phorbol myristic acid). Novel PKCs (δ, ε, η, θ) do not require Ca2+ for activation and atypical isoforms (ι/λ, ζ, µ) require neither Ca2+ nor DAG (256, 374). In addition to Ca2+ and DAG, PKC activity and localization is regulated by binding to intracellular proteins. Specific inducers of cellular responses give rise to different localization of individual PKCs within in the same cell and this is then due to the binding of PKC to different proteins. Therefore, PKC binding proteins bring PKCs in close contact with substrate proteins. It is also conceivable that in addition to directing PKC to the right substrates, PKC binding proteins may prevent inappropriate phosphorylation of other proteins. This would appear to be appropriate since PKCs are very promiscuous in in vitro assays (256, 374). Thus, in order for integrins to use PKCs in downstream signaling it is important to regulate intracellular levels Ca2+ and DAG and to be “friends” with PKC binding proteins. Current knowledge about the biochemical mechanism that leads to integrin-mediated activation of PKC has many gaps. This is largely due to the general limited knowledge about PKC regulation. It has been shown that cell spreading on gelatin requires PKC activity, which is dependent on arachidonic acid release and DAG production (104). The clustering of β1 integrins leads to activation of phospholipase A2 (PLA2) by unknown mechanisms (Fig. 8) (26). PLA2 removes arachidonic acid from phospholipids. It has been suggested that arachidonic acid could regulate the activity of phospholipase C, which then hydrolyses

PI(4,5)P2 to DAG and inositol trisphosphate (26). It is also known that integrin adhesion causes a transient increase of intracellular [Ca2+] by influx of extracellular Ca2+ (295, 477, 530). It is not known how integrins and Ca2+ channels in the plasma membrane are coupled but there are indications that calreticulin might be involved (see above). A recent report by Zhang et al. shows that the SH2-domain of PLCγ1 can bind to phosphorylated tyrosine 397 in FAK, and that binding of PLCγ1 to FAK increases tyrosine phosphorylation of PLCγ1 and its enzymatic activity (642). PLCγ activity would lead to a more general release of Ca2+ from intracellular stores. The Ca2+-independent isoforms of PKC have been shown to be activated by PI(3,4,5)P3 (135) which places PI3 kinase activity upstream of PKC.

53 Studies on COS-7 cells have shown that in these cells three PKC isoforms (α, β, ε) locate to the plasma membrane in response to cell adhesion. In these cells, inhibition of PKC did not affect tyrosine phosphorylation of FAK upon cell adhesion, but instead was found to affect activation of ERK2 via a Shc pathway (369). Vascular smooth muscle cell adhesion to fibronectin induced integrin-dependent translocation of PKCα and PKCε to the focal adhesions (204). Downregulation of these isozymes inhibited cell spreading (204). Colocalization of talin and α-actinin with integrins αvβ3 and αvβ5 is inhibited by blocking PKC activity with Calphostin C, suggesting that PKC may regulate the interaction of these proteins with integrins (315). Studies on Jurkat cells showed that PMA-stimulated leukocyte adhesion to fibronectin via β1 integrins was dependent on protein geranylgeranylation (333). Ras family proteins are not geranylgeranylated (they require prenylation for function) and because the authors excluded the ERK pathway and PI3K they suggest that PKC stimulates geranylgeranylation of Rho subfamily proteins (333). PKCα has been shown to regulate β1 integrin endocytosis and thereby β1-mediated cell migration (392). Both the regulatory and catalytic domain of PKCα were required for promoting cell motility whereas only the regulatory domain was required to form a complex with β1 integrins (392). In addition, overexpression of PKCα in MCF-7 cancer cells up- regulates β3 levels and down-regulates β5 levels by post-transcriptional and transcriptional mechanisms, respectively, which correlated with increased metastatic capacity of the cells (85). Another study on MCF-7 cells demonstrated that TPA treatment up-regulates α2β1 expression and alters the avidity of α2β1 integrin by a Rho-dependent mechanism (480). It is known that PKC-phosphorylated substrates are distinctly localized in the cell (256). There are two types of proteins that mediate substrate targeting. The first group is called STICKs (substrates that interact with C-kinase); they are phospholipid-binding proteins that mostly are localized at membranes and are substrates that interact with PKC. The second class includes non-substrate proteins that position PKC in the proximity of substrates (256). Generally, phosphorylation of STICKs by PKC modulates their activity. STICKs are implicated in various cellular processes such as spreading, vesicle trafficking etc. It is conceivable that some of these proteins are required for integrin function. For example, two focal adhesion proteins (vinculin and talin) and MARKCS (myristoylated alanine-rich C kinase substrate) are STICKs for PKCα (250). However, how PKC affects the activities of vinculin and talin is not known. Activation of β2 integrins involves release of a cytoskeletal constraint (see above), and activation of PKC stimulates β2-diffusion in the membrane. Recently it was shown that MacMARCKS (macrophage-enriched MARCKS) is required for activation of β2 in a PKC-dependent manner (651). In cells deficient in MacMARCKS or expressing a mutant form that can not be phosphorylated by PKC, the diffusion β2 integrins in the plasma membrane does not occur in response to PMA. Adding cytochalasin D bypasses the effect of MacMARKCS deficiency suggesting that MacMARCKS is required for releasing the cytoskeletal constraint during the activation of β2 integrins. Since PKC/STICK interactions in in vitro experiments do not show isozyme selectivity it is likely that additional factors are required for specific responses in cells. Unlike, STICKs, RACKs (receptor for activated C-kinase) bind PKC isoforms specifically, and thus localize them at the place of action. Inhibition of the interactions between RACKs and PKCs have shown to abrogate the cellular response that required the activity of a given PKC (478, 635). It has been reported that Rack1 binds to the cytoplasmic

54 tail of integrin subunits β1, β2, β5 via its WD repeats (323). Association of Rack1 with β1 integrins in cells required treatment with phorbol esters (323). Since Rack1 is the RACK for PKCβ (375), the binding of Rack1 to integrins could locate the activity of PKCβ next to integrins. Studies on Rack1 localization in CHO cells have shown that treatment of cells with phorbol ester induced movement of PKCβII and Rack1 to the Golgi-apparatus. It was demonstrated that association between Rack1 and PKCβII occurs before the transport and PKC kinase activity was not needed for the transport (477). In order for integrins to associate with Rack1 a specific signal from integrins, in addition to activation of PKC, is probably needed to direct the Rack1/PKCβ complex to the integrins. What regulates the transport of Rack1 in the cell is not known. Interestingly, Rack1 has been shown to bind Src kinases and to inhibit their kinase activity and cell growth (93). A PDZ domain-containing protein PICK1 (protein interacting with C kinase 1) that binds PKCα (539, 540) interacts with the cytoplasmic domain of integrin subunit β6 (538). The cytoplasmic tail of β6 can be specifically phosphorylated by PKCα in in vitro assay, which potentially could modulate functions of β6 integrins (538). PICK1 can bind to GTP-bound forms of Arf1 and Arf3 (556). Syndecans are transmembrane heparan sulfate proteoglycans that are involved in cell adhesion and/or migration. Syndecan-4 colocalizes with α5β1 and αvβ3 integrins on fibronectin or vitronectin, respectively (146, 336, 619). The cytoplasmic tail of syndecan-4 can bind PKCα and thus locate PKC activity close to integrins. Binding of PKCα to syndecan-4 potentates its activation by phospholipid PI(4,5)P2. Thus, syndecan-4 not only can localize PKC at focal contacts but can also modulate the catalytic activity of PKC. It is clear that studies on PKCs in integrin function deserve more attention and require much more careful approaches than what we have seen so far. For example, the regulatory domain of PKCs have been shown to interact with PH and LIM domains (293). It is highly probable that several interactions between PKCs and LIM and/or PH domain-containing proteins at focal contacts will be identified during the coming years. Finally, is has become evident that phorbol esters do not only bind and activate classical and novel PKC isozymes but also other cellular proteins. For example, some GEFs and GAPs for Ras and Rac, and several Ras family proteins (e.g. Rap1) have been shown to bind and become activated by DAG and PMA (62, 271).

Integrin-linked kinase

Integrin-linked kinase (ILK) was identified in a yeast two-hybrid screen as a protein that interacted with the cytoplasmic domain of integrin β1 (208). ILK is a cytosolic serine/threonine kinase that comprises three domains. The N-terminal part of ILK consists of four ankyrin repeats, these repeats are followed by a PH-like domain and a C-terminal catalytic domain that contains a sequence that binds to integrins. Overexpression of ILK in epithelial cells results in a loss of cell-cell contacts and a decrease in cell-matrix adhesion, and promotes anchorage-independent growth (208, 451, 620). Interestingly, ILK overexpressing cells are tumorigenic despite the fact that assembly of FN matrix was increased (620). The anchorage-independent growth was induced by expression of cyclin D1 and cyclin A in suspended cells (451). ILK has been shown to phosphorylate in vitro a peptide derived from the cytoplasmic tail of integrin β1 (208). Whether ILK phosphorylates β1 integrin in vivo is not known. It is also not known which residues in the cytoplasmic tail of β1 interact with

55 ILK. ILK has been demonstrated to localize at membrane ruffles and at focal contacts together with α5β1 (320). Recently, a protein called PINCH was found to interact with ILK (572). PINCH is an adapter protein built up from five LIM domains. The most N-terminal LIM domain interacts with ILK, and LIM domain number 4 interacts with Nck-2 (572, 573). Further studies revealed that ILK is targeted to focal contacts via PINCH. However, the C- terminal integrin-binding site is also required (320). It has been demonstrated that ILK becomes transiently activated upon cell adhesion to fibronectin in a PI3K dependent manner (132). It is not clear whether the PH domain of ILK binds PI(3,4,5)P3 in vivo, but PI(3,4,5)P3 stimulates the kinase activity of recombinant ILK in vitro (132). ILK could be involved in integrin-mediated activation of Akt/PKB in a PI3K- dependent manner. Akt/PKB is an intracellular serine/threonine kinase that has an anti- apoptotic effect, which is accomplished by ways that involve quite a number of targets

(reviewed in 584). Full activation of Akt/PKB requires PI(3,4,5)P3 and phosphorylation of two residues, Thr 308 and Ser 473. Threonine 308 in Akt/PKB is phosphorylated by the cytosolic serine/threonine kinase PDK1. A kinase, which has been named PDK2, that could phosphorylate serine 473 is less well characterized. Some data show that ILK is capable of phosphorylating serine 473 in Akt/PKB (132). PTEN-mutant cells exhibit elevated kinase activity of ILK and constitutive Akt/PKB serine 473 phosphorylation (433). Overexpression of dominant-negative or kinase-deficient ILK in PTEN-mutant cells suppresses the constitutive phosphorylation of serine 473 but not threonine 308 in Akt/PKB (433). However, some investigators are questioning the view that ILK is a “real” kinase. Several groups have failed to detect any kinase activity in ILK immunoprecipitates (32, 344). The kinase domain of ILK lacks certain motifs present in other protein kinases such as the conserved DFG motif and a catalytic aspartate residue. Instead it has been suggested that ILK regulates phosphorylation of serine 473 in Akt/PKB by an indirect mechanism (344). ILK from human, Drosophila, and C.elegans was found to possess a conserved FSF motif that might regulate ILK activity. Mutation of the FSF motif to FAF abolished ILK induced serine phosphorylation on Akt/PKB whereas FDF (mimicking FSpF) stimulated it even in dominant negative ILK. The authors suggest that the sequence FS(p)F in ILK could be required for inhibiting a serine 473 phosphatase activity rather than activating a kinase activity (PDK2) (344).There are indications that ILK may have a role in integrin inside-out signaling. Genetic studies in C.elegans have shown that loss of Unc-97 (PINCH homologue) results in phenotypes that are very similar to those of integrin-null mutants (227). It has also been shown that expression of ILK and PINCH is lost in keratinocytes that undergo terminal differentiation (623). Interestingly, loss of integrin β1 ligand-binding ability occurs concomitantly with terminal differentiation of keratinocytes (236, 601). Regions in the β1 cytoplasmic tail that are required for keratinocyte differentiation and proliferation have recently been mapped (313). It would be interesting to test whether regions required for proliferation are involved in binding of ILK to β1 cytoplasmic tail.

Phosphatidylinositol kinases

Phosphoinositides (PIs) have important roles in cell signaling, being required for many cellular functions such as migration, survival, vesicle trafficking, and proliferation. The synthesis of phosphoinositides is very complex, involving a number of different phosphoinositol kinases

56 and phosphatases (reviewed in 585). Many cellular proteins require localization to the membrane for their activation. These proteins (e.g. kinases, GTPases) often possess a PH- domain that binds to PIs and can therefore be recruited to the membrane. Several proteins controlling reorganization of the actin filament system also contain motifs that bind PIs. Activation of PI kinases and phosphatases is required for integrin-mediated cell migration and cell survival as discussed above in some detail (186, 303, 646). The importance of these phospholipids in integrin-mediated signaling is clearly established. I will confine myself to describing only some examples where PIs are regulating proteins involved in integrin function. Vinculin is an actin binding protein present in focal adhesions. Vinculin is not required for formation of focal adhesions but might strengthen the contact between actin filaments and integrins (107, 590). Vinculin is composed of an N-terminal head domain that is followed by a proline-rich region and a C-terminal region. Vinculin can bind to talin, F-actin, α-actinin, and Vasp (vasodilator-stimulated phosphoprotein) but the binding sites in vinculin are blocked by the intramolecular association of the N-terminal and the C-terminal domains. Binding of acidic phospholipids to vinculin, in particular PI(4,5)P2, disrupts the intramolecular interaction and therefore exposes binding sites in vinculin for Vasp and talin (183, 246). For the interaction with F-actin the situation does not appear to be so simple and a recent report by Steimer et al. shows that actin binding activity of vinculin requires steps other than binding to PI(4,5)P2 (541). PI(4,5)P2 binding to vinculin also promotes vinculin oligimerization (246). Another important binding partner of vinculin is the SH3 domain-containing adapter protein vinexin (277). Vinexin associates with Sos and can regulate JNK activity in response to epidermal growth factor (4). Hence, the vinculin/vinexin/Sos complex could possibly be involved also in integrin-mediated JNK activation. Cortactin (EMS-1) is a cytosolic protein that colocalizes with cortical actin and becomes tyrosine phosphorylated in response to integrin-mediated adhesion (594). Overexpression of cortactin has been shown to lead to increased cell migration that appears to be dependent on tyrosine phosphorylation of cortactin (238, 429). Localization of cortactin to membrane ruffles and at cell periphery has been shown to be mediated by Rac and PAK (605). The F- actin cross-linking activity of cortactin is inhibited in the presence of PI(4,5)P2. Several tetraspanins that form complexes with integrins have shown to associate with type II PI4 kinase (47, 543). It was noticed that integrin/tetraspanin/PI4K complexes were localized at the cell periphery rather than at focal adhesions. It has been suggested that this complex may direct membrane protrusions during cell migration (47).Integrin-mediated cell migration requires PI3 kinase activity. Activation of PI3 kinases leads to increased levels of

PIs phosphorylated at position 3. PI(3,4,5)P3 has been shown to promote cell migration by activating PKC (135). PI(3,4,5)P3 was also found to dissociate α-actinin and vinculin (not talin or paxillin) from focal contacts (192). It was shown that PI(3,4,5)P3 disrupted the binding of α-actinin to the cytoplasmic tail of integrin β1 (192). The authors suggest that binding of

PI(3,4,5)P3 also reduces the affinity of α-actinin for the actin filaments. Localization of zyxin to focal adhesions is dependent on its association with α-actinin and is therefore indirectly regulated by PI3K (459).

57 INTEGRINS AND CAVEOLAE

Caveolae are plasma membrane structures (invaginations) that were first described in epithelial cells (13). They exist in a variety of shapes (flat, vesicular, tubular). Caveolae are highly enriched with glycosphingolipids, cholesterol, GPI-linked proteins, caveolins, and proteins that associate with the plasma membrane through lipid modifications. In addition to these molecules, caveolae contain many membrane receptors (e.g. receptor tyrosine kinases), membrane transporters, kinases, and structural proteins (13). The caveolae membrane system plays a specific role and is essential for normal cell behavior. The emerging understanding is that caveolae at the plasma membrane are like signaling stations, they compartmentalize enzymatic reactions and are places were signal integration takes place (13). There is still no consensus how these specific plasma membrane domains should be called; in addition to caveolae, names like detergent-insoluble glycolipid domains, glycosphingolipid-enriched membranes or “rafts” are used. Integrin signaling and caveolae appear to be linked. Caveolin-1 binds to some integrin α subunits and links certain integrins to ERK activation via Shc (599). As mentioned, caveolae are enriched with kinases, including the Src family, implicated in integrin signaling. The recently described protein Cbp that has a central role in Src regulation is particularly located in caveolae (268). Binding of Csk to Cbp brings Csk in close vicinity to Src family kinases and allows phosphorylation and inactivation of Src. Integrin-associated protein (IAP) forms a complex with αvβ3 integrins (70, 327). The formation of a functional signaling complex between IAP and integrin αvβ3 requires cholesterol (191). Cholesterol interacts with the multiple membrane spanning domain of IAP and is probably required for maintaining a conformation necessary for binding to integrins (191). Depletion of caveolin in 293 cells disrupts β1 integrin signaling but not ligand binding or clustering (607). It was shown that caveolin-deficient 293 cells had no tyrosine kinase activity associated with β1-integrins (607). Thus, the caveolae membrane system appears to be important for integrin signaling and adds another level of complexity. The coming years will certainly improve our understanding about the role of caveolae in integrin signaling.

58 PRESENT INVESTIGATION

AIMS OF THE THESIS

The present project deals with aspects of structure and function of the integrin β1 subunit. Integrins are a family of cell surface receptors for extracellular matrix proteins and cell surface molecules. Each integrin consists of one α and one β subunit. The β1 subunit dimerizes with 12 different α subunits and thereby form the biggest integrin subfamily with broad ligand specificity. The β1 integrins are essential for embryonic development and for many physiological processes in an adult organism.

In the present study the following issues were addressed: - The functional properties of a cytoplasmic splice-variant of the integrin β1, namely β1B - The role of the two cytoplasmic tyrosines in the cytoplasmic domain of integrin β1A in integrin signaling - The position of the border between the transmembrane and the cytoplasmic domains of integrin subunits - The importance of a single conserved lysine in the transmembrane domain of integrin subunits in integrin signaling

RESULTS AND DISCUSSION

Paper I

Armulik, A., Svineng, G., Wennerberg, K., Fässler, R., and Johansson, S. (2000). Expression of integrin subunit β1B in integrin β1-deficient GD25 cells does not interfere with αvβ3 functions. Exp. Cell Res.254, 55-63

In this paper, the functional properties of the integrin β1 splice-variant β1B were studied. Human β1B was first described by Altruda et. al (11). Analysis of the genomic sequence of the mouse β1 gene revealed that no homologue to the human β1B specific part can be translated from mouse gene (39). Previous reports about β1B function showed that expression of β1B results in a dominant negative effect on β1A functions, possibly as a result of competition for interacting proteins for the common region (34). In our study we investigated if expression of β1B had similar negative effects on αvβ3/5 integrin functions since the β3 and β5 subunits are structurally similar to the β1A subunit. The β1B subunit was expressed in GD25 cells that are deficient in β1 expression due to the knockout of the β1 gene. Endogenous expression of αvβ3 and αvβ5 integrins by GD25 cells allows them to attach to fibronectin and vitronectin. As shown by others, we confirmed that integrins containing the β1B subunit possess an inactive conformation of the extracellular domain for ligand binding, and do not activate the focal adhesion kinase (FAK) pathway after clustering evoked by monoclonal antibodies. GD25 cells expressing the integrin subunit β1B exhibited a similar level of activation of FAK to that of parental cell line on the fibronectin and vitronectin. Moreover, tyrosine phosphorylation of two focal adhesion proteins, paxillin and

59 CAS, was found to be undisturbed by β1B, nor could we detect any negative effect of β1B on the organization of actin cytoskeleton and on cell proliferation. Thus, the function of αv integrins was found to be unaffected by the presence of β1B, although the expression level of β1B was much greater than that detected in vivo.

Paper II

Wennerberg, K., Armulik, A., Sakai, T., Karlsson, M., Fässler, R., Schaefer, E. M., Mosher, D. F., and Johansson, S. (2000) The cytoplasmic tyrosines of integrin subunit β1 are involved in FAK activation. Mol. Cell. Biol. 20, 5758-5765

It has been shown that mutation of the two tyrosines in the cytoplasmic domain in integrin subunit β1A (Y783, Y795) to phenylalanines markedly reduces the capability of β1A integrins to mediate directed cell migration, but not the capability to support cell attachment, to form focal contacts, and polymerize FN via β1A (485). In this study we have identified that activation of focal adhesion kinase (FAK) is severely impaired in response to β1-dependent adhesion in GD25-β1AY783/795F cells as compared to wild type GD25-β1A or mutants where only a single tyrosine was altered (β1AY783F or β1AY795F). Phosphorylation site-specific antibodies selective for FAK phospho- tyrosine 397 indicated that the defect in FAK activation via β1AY783/795F lies at the level of the initial autophosphorylation step. Although the GD25-β1AY783/795F cells have a defect in β1 integrin-dependent FAK activation, FAK is still present in focal adhesions. β1A dependent tyrosine phosphorylation of FAK substrates tensin and paxillin was lost in the β1AY783/795F cells, consistent with the defect in FAK activation. In contrast, overall tyrosine phosphorylation of CAS was unaffected by the mutation (YY783,795FF) in β1. These data strongly suggest that tyrosines Y783 and Y795 in the cytoplasmic tail of integrin subunit β1A are critical mediators of FAK activation via β1A integrins.

Paper III

Armulik, A., Nilsson, I., von Heijne, G., and Johansson S. (1999) Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits. J. Biol. Chem. 274, 37030-37034

The border between the cytoplasmic domain and the C-terminal end of the transmembrane domain of integrin α and β subunits has been unclear. The 26 presently known integrin subunits (excluding two) exhibit a similar pattern at membrane/cytoplasm interface: a single conserved, positively charged amino acid (Lys or Arg), a short stretch of hydrophobic amino acids, and a highly polar sequence unlikely to be buried in the plasma membrane. In this paper we have used a glycosylation mapping technique to determine the position of the C-terminal end of the transmembrane helices (TMHs) of human integrin α (α2, α5) and β (β1, β2) subunits in microsomal membranes. The TMHs were found to extend roughly to Phe1129 in α2, to Phe1026 in α5, to Ile757 in β1, and to His728 in β2. Interestingly, the α-carbon of the conserved lysine near the C-terminal end of the TMH (corresponding to Lys752 in β1) is

60 buried in the plasma membrane, and the charged amino group most likely reaches into the polar head-group region of the lipid bilayer. Delineation of the margin between the transmembrane and cytoplasmic domains of integrin subunits presented in this paper is the first based on experimental data.

Paper IV

Armulik, A. and Johansson, S. (2000) Lysine 756 in the transmembrane domain of integrin subunit β1 is necessary for β1 integrin signaling. Manuscript

The studies on the transmembrane domain of human integrin subunits in paper III showed that a single, positively charged amino acid (e.g. Lys1022 in human α5 and Lys752 in human β1) is positioned in the plasma membrane in the absence of interacting proteins. To investigate the functional importance of the transmembrane lysine 756 in the mouse integrin β1 subunit (corresponding to human 752) this amino acid was replaced with leucine (K756L), and the mutated β1 subunit was stably expressed in β1-deficient GD25 cells. Metabolic labeling of GD25 expressing wt or mutated β1A subunit showed that the mutant β1 subunit was processed less efficiently to the mature form compared to wt β1. The extracellular domain of β1AK756L integrins possesses a ligand competent conformation as defined by the competence to mediate cell adhesion to laminin-1 and fibronectin and the presence of the mAb 9EG7 epitope. However, the spreading of GD25-β1AK756L cells on fibronectin and laminin-1 was impaired, and the rate of migration of GD25-β1AK756L cells was reduced compared to GD25-β1A cells. Tyrosine phosphorylation of focal adhesion kinase and paxillin in response to β1AK756L-mediated adhesion was similar to that induced by wt β1. Interestingly, tyrosine phosphorylation of CAS was reduced after β1-mediated adhesion in GD25-β1AK756L cells. Despite the reduced tyrosine phosphorylation of CAS, formation of the CrkII/CAS complex also occurred in mutant cells, indicating that the K756 in β1 subunit is necessary for FAK/Src independent tyrosine phosphorylation of CAS.

FUTURE PERSPECTIVES

This work has raised several questions, either directly or indirectly, that deserve further investigation. Very little is known about integrin structure. While crystal structures of a whole integrin in different conformational/activation states appears to be a distant goal, high-resolution structural information for the transmembrane and cytoplasmic domains may be within reach. The issue is complicated by the heterodimeric nature of integrins but it should be possible to overcome this problem by developing available protein expression techniques. During the course of my studies it has become clear that integrin biosynthesis is not well studied. There are many specific questions that are still not satisfactorily answered. What determines the selectivity of the association between α and β subunits? If the subunits are held together mainly via their extracellular domains (which is a common view) why then are mutations in the transmembrane and cytoplasmic domains often affecting the assembly of α-β

61 heterodimers? And which cytoplasmic and ER-located chaperons are involved in the folding, dimerization and transportation processes? What are the physiological functions of three conserved small amino acids in the transmembrane domains of integrin subunits? Do these amino acids contribute to the integrin heterodimer formation and/or propagation of conformational changes across the plasma membrane? The functional importance of the positive charge in the plasma membrane in integrin subunits deserves further investigation. Mutational analysis of this amino acid in the β1 subunit showed that this amino acid has a role in integrin outside-in signaling. This should be further explored and the corresponding (positively charged) amino acid in β subunits should be mutated and the consequences of this investigated. Is there really a repositioning of the transmembrane domain in the plasma membrane during integrin activation as we hypothesized in the paper III? This question is experimentally difficult to address, although some conventional/straight forward approaches are available. Introduction of an additional positive charge would keep transmembrane domain “shorter” - will this result in a constitutively active integrin? Will it affect integrin outside-in signaling? Our studies have also indicated that integrin-mediated tyrosine phosphorylation of several adapter protein could involve yet unidentified kinase pathways. What kinase(s) are involved in CAS tyrosine phosphorylation and what could be the functional importance of this? What is the identity of the 90 kDa tyrosine phosphorylated protein associated with the CAS/Crk complex? Mutational analysis of the NPXY motifs in the cytoplasmic tails of integrin β1 subunit in paper II clearly shows that tyrosine residues are important in integrin-mediated FAK activation. Is there a protein (containing PTP domains?) that binds to these tyrosine residues (either phosphorylated or not phosphorylated) and mediates integrin-induced activation of FAK?

62 ACKNOWLEDGEMENTS

This study was carried out at the Department of Medical Biochemistry and Microbiology. I would like to thank all the people at the department for a pleasant working atmosphere and their help. Particularly I would like to extend my gratitude to

Staffan Johansson for being an excellent supervisor

Present and past members of “our” group; Birgitta, Sophie, Stina, Paola, Teet, Gunbjørg, Peter, Dorota, Lasse, Marjam, and Krister

All my co-authors

Dr. Ivan Dikic and Dr. Pontus Aspenström from the Ludwig Institute for Cancer Research and Prof. Lena Claesson-Welsh and Helena Larsson from the Rudbeck Laboratory for stimulating discussions and help

I also would like to thank people from the Department of Veterinary Medical Chemistry, SLU

Special thanks to

Annica, Maria for your friendship and the help you have given to make Sweden become more like a second home to me

Staffan. The only “problem” we have had during these years is FIGURES and even this is not a real problem. Thank you for everything

All “my Estonians” in Sweden: Mart (extra thanks for making the Eurovision Song Contest into a long waited event), Taavo, Anu, Teet, Tanel, Sirje, Maido, Margit for help and company. All “my Estonians” in Estonia, particularly Piret, Martin, Kadri and Andres

My “officemates” Krister, Peter, Sophie and Paola. In Estonia we have a saying when translated into English ends up looking silly but still... “A small stable accommodates many good sheep”. Thanks

Lena. I wish that I were be so patient and friendly as you are, and have such a healthy view on life as you do

Fergal for your cheerfulness that blows away all “clouds of woe”

ESTLINE for safe trips between Stockholm and Tallinn

My family for never ending support and love

My son for giving the reason to live. You too deserve to get a doctoral hat, smaller though

63 REFERENCES

1. Abram, C. L., and S. A. Courtneidge. 2000. Src family tyrosine kinases and growth factor signaling. Exp. Cell Res. 254:1-13. 2. Adra, C. N., D. Manor, J. L. Ko, S. Zhu, T. Horiuchi, L. Van Aelst, R. A. Cerione, and B. Lim. 1997. RhoGDIgamma: a GDP-dissociation inhibitor for Rho proteins with preferential expression in brain and pancreas. Proc. Natl. Acad. Sci. U.S.A. 94:4279-4284. 3. Akagi, T., T. Shishido, K. Murata, and H. Hanafusa. 2000. v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation. Proc. Natl. Acad. Sci. U.S.A. 97:7290-7295. 4. Akamatsu, M., S. Aota, A. Suwa, K. Ueda, T. Amachi, K. M. Yamada, S. K. Akiyama, and N. Kioka. 1999. Vinexin forms a signaling complex with Sos and modulates epidermal growth factor- induced c-Jun N-terminal kinase/stress-activated protein kinase activities. J. Biol. Chem. 274:35933- 35937. 5. Akimov, S. S., D. Krylov, L. F. Fleischman, and A. M. Belkin. 2000. Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin. J. Cell Biol. 148:825-838. 6. Akiyama, S. K., S. S. Yamada, K. M. Yamada, and S. E. LaFlamme. 1994. Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras. J. Biol. Chem. 269:15961-15964. 7. Alahari, S., and R. Juliano. 2000. Nischarin, a novel protein that interacts with integrin alpha subunits and modulates cell migration, p. 67. Keystone Symposium, Joint Regulation of Signaling Pathways by Integrins and Growth Factors, Breckenridge, Colorado. 8. Albiges-Rizo, C., P. Frachet, and M. R. Block. 1995. Down regulation of talin alters cell adhesion and the processing of the α5β1 integrin. J. Cell Sci. 108:3317-3329. 9. Allende, L. M., M. Hernandez, A. Corell, M. A. Garcia-Perez, P. Varela, A. Moreno, I. Caragol, F. Garcia-Martin, J. Guillen-Perales, T. Olive, T. Espanol, and A. Arnaiz-Villena. 2000. A novel CD18 genomic deletion in a patient with severe leucocyte adhesion deficiency: a possible CD2/lymphocyte function-associated antigen-1 functional association in humans. 99:440- 450. 10. Almeida, E. A., D. Ilic, Q. Han, C. R. Hauck, F. Jin, H. Kawakatsu, D. D. Schlaepfer, and C. H. Damsky. 2000. Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase. J. Cell Biol. 149:741-754. 11. Altruda, F., P. Cervella, G. Tarone, C. Botta, F. Balzac, G. Stefanuto, and L. Silengo. 1990. A human integrin β1 subunit with a unique cytoplasmic domain generated by alternative mRNA processing. Gene 95:261-266. 12. Altun-Gultekin, Z. F., S. Chandriani, C. Bougeret, T. Ishizaki, S. Narumiya, P. de Graaf, P. Van Bergen en Henegouwen, H. Hanafusa, J. A. Wagner, and R. B. Birge. 1998. Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein. Mol. Cell. Biol. 18:3044-3058. 13. Anderson, R. G. 1998. The caveolae membrane system. Annu. Rev. Biochem. 67:199-225. 14. Angers-Loustau, A., J. F. Cote, A. Charest, D. Dowbenko, S. Spencer, L. A. Lasky, and M. L. Tremblay. 1999. Protein tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and cytokinesis in fibroblasts. J. Cell Biol. 144:1019-1031. 15. Aplin, A. E., S. M. Short, and R. L. Juliano. 1999. Anchorage-dependent regulation of the mitogen-activated protein kinase cascade by growth factors is supported by a variety of integrin alpha chains. J. Biol. Chem. 274:31223-31228. 16. Argraves, W. S., S. Suzuki, H. Arai, K. Thompson, M. D. Pierschbacher, and E. Ruoslahti. 1987. Amino acid sequence of the human fibronectin receptor. J. Cell Biol. 105:1183-1190. 17. Armulik, A., I. Nilsson, G. von Heijne, and S. Johansson. 1999. Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits. J. Biol. Chem. 274:37030-37034. 18. Armulik, A., G. Svineng, K. Wennerberg, R. Fassler, and S. Johansson. 2000. Expression of integrin subunit beta1B in integrin beta1-deficient GD25 cells does not interfere with alphaVbeta3 functions. Exp. Cell Res. 254:55-63. 19. Arregui, C., P. Pathre, J. Lilien, and J. Balsamo. 2000. The nonreceptor tyrosine kinase fer mediates cross-talk between N- cadherin and beta1-integrins. J. Cell Biol. 149:1263-1274. 20. Arregui, C. O., J. Balsamo, and J. Lilien. 1998. Impaired integrin-mediated adhesion and signaling in fibroblasts expressing a dominant-negative mutant PTP1B. J. Cell Biol. 143:861-873. 21. Arroyo, A. G., J. T. Yang, H. Rayburn, and R. O. Hynes. 1996. Differential requirements for alpha4 integrins during fetal and adult hematopoiesis. Cell 85:997-1008. 22. Arthur, W. T., L. A. Petch, and K. Burridge. 2000. Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism. Curr. Biol. 10:719-722. 23. Aspenstrom, P. 1999. Effectors for the Rho GTPases. Curr. Opin. Cell Biol. 11:95-102.

64 24. Aspenstrom, P. 1999. The Rho GTPases have multiple effects on the actin cytoskeleton. Exp. Cell Res. 246:20-25. 25. Astier, A., H. Avraham, S. N. Manie, J. Groopman, T. Canty, S. Avraham, and A. S. Freedman. 1997. The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after beta1- integrin stimulation in B cells and binds to p130cas. J. Biol. Chem. 272:228-232. 26. Auer, K. L., and B. S. Jacobson. 1995. Beta 1 integrins signal lipid second messengers required during cell adhesion. Mol. Biol. Cell 6:1305-1313. 27. Avraham, H., S. Y. Park, K. Schinkmann, and S. Avraham. 2000. RAFTK/Pyk2-mediated cellular signalling. Cell Signal 12:123-133. 28. Avraham, S., R. London, Y. Fu, S. Ota, D. Hiregowdara, J. Li, S. Jiang, L. M. Pasztor, R. A. White, J. E. Groopman, and et al. 1995. Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. J. Biol. Chem. 270:27742- 27751. 29. Bachelot, C., L. Rameh, T. Parsons, and L. C. Cantley. 1996. Association of phosphatidylinositol 3-kinase, via the SH2 domains of p85, with focal adhesion kinase in polyoma middle t-transformed fibroblasts. Biochim. Biophys. Acta 1311:45-52. 30. Bagrodia, S., D. Bailey, Z. Lenard, M. Hart, J. L. Guan, R. T. Premont, S. J. Taylor, and R. A. Cerione. 1999. A tyrosine-phosphorylated protein that binds to an important regulatory region on the cool family of p21-activated kinase-binding proteins. J. Biol. Chem. 274:22393-22400. 31. Bagrodia, S., S. J. Taylor, K. A. Jordon, L. Van Aelst, and R. A. Cerione. 1998. A novel regulator of p21-activated kinases. J. Biol. Chem. 273:23633-23636. 32. Balendran, A., A. Casamayor, M. Deak, A. Paterson, P. Gaffney, R. Currie, C. P. Downes, and D. R. Alessi. 1999. PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr. Biol. 9:393-404. 33. Balzac, F., A. M. Belkin, V. E. Koteliansky, Y. V. Balabanov, F. Altruda, L. Silengo, and G. Tarone. 1993. Expression and functional analysis of a cytoplasmic domain variant of the β1 integrin subunit. J. Cell Biol. 121:171-178. 34. Balzac, F., S. F. Retta, A. Albini, A. Melchiorri, V. E. Koteliansky, M. Geuna, L. Silengo, and G. Tarone. 1994. Expression of β1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility. J. Cell Biol. 127:557-565. 35. Baneres, J. L., F. Roquet, M. Green, H. LeCalvez, and J. Parello. 1998. The cation-binding domain from the alpha subunit of integrin alpha5 beta1 is a minimal domain for fibronectin recognition. J. Biol. Chem. 273:24744-24753. 36. Barry, S. T., H. M. Flinn, M. J. Humphries, D. R. Critchley, and A. J. Ridley. 1997. Requirement for Rho in integrin signalling. Cell Adhes. Commun. 4:387-398. 37. Basani, R. B., D. L. French, G. Vilaire, D. L. Brown, F. Chen, B. S. Coller, J. M. Derrick, T. K. Gartner, J. S. Bennett, and M. Poncz. 2000. A naturally occurring mutation near the amino terminus of alphaIIb defines a new region involved in ligand binding to alphaIIbbeta3. Blood 95:180- 188. 38. Baudoin, C., M. J. Goumans, C. Mummery, and A. Sonnenberg. 1998. Knockout and knockin of the β1 exon D define distinct roles for integrin splice variants in heart function and embryonic development. Genes Dev. 12:1202-1216. 39. Baudoin, C., A. Van der Flier, L. Borradori, and A. Sonnenberg. 1996. Genomic organization of the mouse β1 gene: conservation of the β1D but not of the β1B and β1C integrin splice variants. Cell Adhes. Commun. 4:1-11. 40. Bazzoni, G., L. Ma, M. L. Blue, and M. E. Hemler. 1998. Divalent cations and ligands induce conformational changes that are highly divergent among beta1 integrins. J. Biol. Chem. 273:6670- 6678. 41. Bazzoni, G., D. T. Shih, C. A. Buck, and M. E. Hemler. 1995. Monoclonal antibody 9EG7 defines a novel β1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium. J. Biol. Chem. 270:25570-25577. 42. Beauvais-Jouneau, A., and J. P. Thiery. 1997. Multiple roles for integrins during development. Biol Cell 89:5-11. 43. Belkin, A. M., S. F. Retta, O. Y. Pletjushkina, F. Balzac, L. Silengo, R. Fassler, V. E. Koteliansky, K. Burridge, and G. Tarone. 1997. Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative splicing. J. Cell Biol. 139:1583-1595. 44. Belkin, A. M., N. I. Zhidkova, F. Balzac, F. Altruda, D. Tomatis, A. Maier, G. Tarone, V. E. Koteliansky, and K. Burridge. 1996. Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells. J. Cell Biol. 132:211-226. 45. Bellis, S. L., J. A. Perrotta, M. S. Curtis, and C. E. Turner. 1997. Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin. Biochem. J. 325:375-381.

65 46. Berditchevski, F., and E. Odintsova. 1999. Characterization of integrin-tetraspanin adhesion complexes: role of tetraspanins in integrin signaling. J. Cell Biol. 146:477-492. 47. Berditchevski, F., K. F. Tolias, K. Wong, C. L. Carpenter, and M. E. Hemler. 1997. A novel link between integrins, transmembrane-4 superfamily proteins (CD63 and CD81), and phosphatidylinositol 4-kinase. J. Biol. Chem. 272:2595-2598. 48. Berditchevski, F., M. M. Zutter, and M. E. Hemler. 1996. Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins). Mol. Biol. Cell 7:193-207. 49. Berg, R. W., E. Leung, S. Gough, C. Morris, W. P. Yao, S. X. Wang, J. Ni, and G. W. Krissansen. 1999. Cloning and characterization of a novel beta integrin-related cDNA coding for the protein TIED ("ten beta integrin EGF-like repeat domains") that maps to chromosome band 13q33: A divergent stand-alone integrin stalk structure. Genomics 56:169-178. 50. Bershadsky, A. D., E. A. Vaisberg, and J. M. Vasiliev. 1991. Pseudopodial activity at the active edge of migrating fibroblast is decreased after drug-induced microtubule depolymerization. Cell Motil. Cytoskeleton 19:152-158. 51. Bianchi, E., S. Denti, A. Granata, G. Bossi, J. Geginat, A. Villa, L. Rogge, and R. Pardi. 2000. Integrin LFA-1 interacts with the transcriptional co-activator JAB1 to modulate AP-1 activity. Nature 404:617-621. 52. Birkenmeier, T. M., J. J. McQuillan, E. D. Boedeker, W. S. Argraves, E. Ruoslahti, and D. C. Dean. 1991. The alpha 5 beta 1 fibronectin receptor. Characterization of the alpha 5 gene promoter. J. Biol. Chem. 266:20544-20549. 53. Bishop, A. L., and A. Hall. 2000. Rho GTPases and their effector proteins. Biochem. J. 348 Pt 2:241-255. 54. Blaukat, A., I. Ivankovic-Dikic, E. Gronroos, F. Dolfi, G. Tokiwa, K. Vuori, and I. Dikic. 1999. Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades. J. Biol. Chem. 274:14893-14901. 55. Blystone, S. D., I. L. Graham, F. P. Lindberg, and E. J. Brown. 1994. beta 3 differentially regulates adhesive and phagocytic functions of the fibronectin receptor alpha 5 beta 1. J. Cell Biol. 127:1129-1137. 56. Blystone, S. D., F. P. Lindberg, M. P. Williams, K. P. McHugh, and E. J. Brown. 1996. Inducible tyrosine phosphorylation of the β3 integrin requires the αV integrin cytoplasmic tail. J. Biol. Chem. 271:31458-31462. 57. Blystone, S. D., S. E. Slater, M. P. Williams, M. T. Crow, and E. J. Brown. 1999. A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function. J. Cell Biol. 145:889-897. 58. Bockholt, S. M., and K. Burridge. 1993. Cell spreading on extracellular matrix proteins induces tyrosine phosphorylation of tensin. J. Biol. Chem. 268:14565-14567. 59. Bohmer, R. M., E. Scharf, and R. K. Assoian. 1996. Cytoskeletal integrity is required throughout the mitogen stimulation phase of the cell cycle and mediates the anchorage-dependent expression of cyclin D1. Mol. Biol. Cell 7:101-111. 60. Borges, E., Y. Jan, and E. Ruoslahti. 2000. PDGF-receptor-(beta) and EGF-receptor-2 bind to the beta3 integrin through its extracellular domain. J. Biol. Chem. . 61. Borkhardt, A., S. Bojesen, O. A. Haas, U. Fuchs, D. Bartelheimer, I. F. Loncarevic, R. M. Bohle, J. Harbott, R. Repp, U. Jaeger, S. Viehmann, T. Henn, P. Korth, D. Scharr, and F. Lampert. 2000. The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q. Proc. Natl. Acad. Sci. U.S.A. 97:9168-9173. 62. Bos, J. L. 1998. All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral. EMBO J. 17:6776-6782. 63. Bouton, A. H., and M. R. Burnham. 1997. Detection of distinct pools of the adapter protein p130CAS using a panel of monoclonal antibodies. Hybridoma 16:403-411. 64. Bouvard, D., and M. R. Block. 1998. Calcium/calmodulin-dependent protein kinase II controls integrin α5β1-mediated cell adhesion through the integrin cytoplasmic domain associated protein-1α. Biochem. Biophys. Res. Commun. 252:46-50. 65. Bouvard, D., A. Molla, and M. R. Block. 1998. Calcium/calmodulin-dependent protein kinase II controls α5β1 integrin-mediated inside-out signaling. J. Cell Sci. 111:657-665. 66. Brancaccio, M., S. Guazzone, N. Menini, E. Sibona, E. Hirsch, M. De Andrea, M. Rocchi, F. Altruda, G. Tarone, and L. Silengo. 1999. Melusin is a new muscle-specific interactor for beta(1) integrin cytoplasmic domain. J. Biol. Chem. 274:29282-29288. 67. Brdicka, T., D. Pavlistova, A. Leo, E. Bruyns, V. Korinek, P. Angelisova, J. Scherer, A. Shevchenko, I. Hilgert, J. Cerny, K. Drbal, Y. Kuramitsu, B. Kornacker, V. Horejsi, and B. Schraven. 2000. Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a

66 novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation. J. Exp. Med. 191:1591-1604. 68. Briesewitz, R., M. R. Epstein, and E. E. Marcantonio. 1993. Expression of native and truncated forms of the human subunit. J. Biol. Chem. 268:2989-2996. 69. Brower, D. L., S. M. Brower, D. C. Hayward, and E. E. Ball. 1997. Molecular evolution of integrins: genes encoding integrin beta subunits from a coral and a sponge. Proc. Natl. Acad. Sci. U.S.A. 94:9182-9187. 70. Brown, E., L. Hooper, T. Ho, and H. Gresham. 1990. Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins. J. Cell Biol. 111:2785-2794. 71. Brown, M. C., J. A. Perrotta, and C. E. Turner. 1996. Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding. J. Cell Biol. 135:1109-1123. 72. Brown, M. C., J. A. Perrotta, and C. E. Turner. 1998. Serine and threonine phosphorylation of the paxillin LIM domains regulates paxillin focal adhesion localization and cell adhesion to fibronectin. Mol. Biol. Cell 9:1803-1816. 73. Brown, M. T., J. Andrade, H. Radhakrishna, J. G. Donaldson, J. A. Cooper, and P. A. Randazzo. 1998. ASAP1, a phospholipid-dependent arf GTPase-activating protein that associates with and is phosphorylated by Src. Mol. Cell. Biol. 18:7038-7051. 74. Buensuceso, C. S., and T. E. O'Toole. 2000. The association of CRKII with C3G can be regulated by integrins and defines a novel means to regulate the mitogen-activated protein kinases. J. Biol. Chem. 275:13118-13125. 75. Buist, A., C. Blanchetot, L. G. Tertoolen, and J. den Hertog. 2000. Identification of p130cas as an in vivo substrate of receptor protein- tyrosine phosphatase alpha. J. Biol. Chem. 275:20754-20761. 76. Burnham, M. R., P. J. Bruce-Staskal, M. T. Harte, C. L. Weidow, A. Ma, S. A. Weed, and A. H. Bouton. 2000. Regulation of c-SRC activity and function by the adapter protein CAS. Mol. Cell. Biol. 20:5865-5878. 77. Burnham, M. R., M. T. Harte, A. Richardson, J. T. Parsons, and A. H. Bouton. 1996. The identification of p130cas-binding proteins and their role in cellular transformation. Oncogene 12:2467- 2472. 78. Burridge, K., C. E. Turner, and L. H. Romer. 1992. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J. Cell Biol. 119:893-903. 79. Buyon, J. P., S. G. Slade, J. Reibman, S. B. Abramson, M. R. Philips, G. Weissmann, and R. Winchester. 1990. Constitutive and induced phosphorylation of the α- and β-chains of the CD11/CD18 leukocyte integrin family. Relationship to adhesion- dependent functions. J. Immunol. 144:191-197. 80. Cai, D., L. K. Clayton, A. Smolyar, and A. Lerner. 1999. AND-34, a novel p130Cas-binding thymic stromal cell protein regulated by adhesion and inflammatory cytokines. J. Immunol. 163:2104- 2112. 81. Calderwood, D. A., D. S. Tuckwell, J. Eble, K. Kuhn, and M. J. Humphries. 1997. The integrin alpha1 A-domain is a ligand binding site for collagens and laminin. J. Biol. Chem. 272:12311-12317. 82. Calderwood, D. A., R. Zent, R. Grant, D. J. Rees, R. O. Hynes, and M. H. Ginsberg. 1999. The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation. J. Biol. Chem. 274:28071-28074. 83. Cali, G., C. Mazzarella, M. Chiacchio, R. Negri, S. F. Retta, M. Zannini, F. Gentile, G. Tarone, L. Nitsch, and C. Garbi. 1999. RhoA activity is required for fibronectin assembly and counteracts beta1B integrin inhibitory effect in FRT epithelial cells. J. Cell Sci. 112:957-965. 84. Cali, G., S. F. Retta, R. Negri, I. Damiano, R. Gentile, G. Tarone, L. Nitsch, and C. Garbi. 1998. Beta1B integrin interferes with matrix assembly but not with confluent monolayer polarity, and alters some morphogenetic properties of FRT epithelial cells. Eur. J. Cell Biol. 75:107-117. 85. Carey, I., and J. D. Noti. 1999. Isolation of protein kinase C-alpha-regulated cDNAs associated with breast tumor aggressiveness by differential mRNA display. Int J Oncol 14:951-956. 86. Carlier, M. F., P. Nioche, I. Broutin-L'Hermite, R. Boujemaa, C. Le Clainche, C. Egile, C. Garbay, A. Ducruix, P. Sansonetti, and D. Pantaloni. 2000. GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASp) with actin- related protein (ARP2/3) complex. J. Biol. Chem. 275:21946-21952. 87. Carragher, N. O., B. Levkau, R. Ross, and E. W. Raines. 1999. Degraded collagen fragments promote rapid disassembly of smooth muscle focal adhesions that correlates with cleavage of pp125(FAK), paxillin, and talin. J. Cell Biol. 147:619-630. 88. Cary, L. A., J. F. Chang, and J. L. Guan. 1996. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J. Cell Sci. 109:1787-1794. 89. Cary, L. A., D. C. Han, T. R. Polte, S. K. Hanks, and J. L. Guan. 1998. Identification of p130Cas as a mediator of focal adhesion kinase- promoted cell migration. J. Cell Biol. 140:211-221.

67 90. Casamassima, A., and E. Rozengurt. 1998. Insulin-like growth factor I stimulates tyrosine phosphorylation of p130(Cas), focal adhesion kinase, and paxillin. Role of phosphatidylinositol 3'- kinase and formation of a p130(Cas).Crk complex. J. Biol. Chem. 273:26149-26156. 91. Cattelino, A., S. Cairo, B. Malanchini, and I. de Curtis. 1997. Preferential localization of tyrosine- phosphorylated paxillin in focal adhesions. Cell Adhes. Commun. 4:457-467. 92. Cervella, P., L. Silengo, C. Pastore, and F. Altruda. 1993. Human beta 1-integrin gene expression is regulated by two promoter regions. J. Biol. Chem. 268:5148-5155. 93. Chang, B. Y., K. B. Conroy, E. M. Machleder, and C. A. Cartwright. 1998. RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells. Mol. Cell. Biol. 18:3245-3256. 94. Chang, D. D., C. Wong, H. Smith, and J. Liu. 1997. ICAP-1, a novel β1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of β1 integrin. J. Cell Biol. 138:1149-1157. 95. Chang, D. D., C. Wong, H. Smith, and J. Liu. 1997. ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin. J. Cell Biol. 138:1149-1157. 96. Chaudhary, A., W. G. King, M. D. Mattaliano, J. A. Frost, B. Diaz, D. K. Morrison, M. H. Cobb, M. S. Marshall, and J. S. Brugge. 2000. Phosphatidylinositol 3-kinase regulates raf1 through pak phosphorylation of serine 338. Curr. Biol. 10:551-554. 97. Chen, H. C., and J. L. Guan. 1994. Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. U.S.A. 91:10148-10152. 98. Chen, L. L., A. Whitty, R. R. Lobb, S. P. Adams, and R. B. Pepinsky. 1999. Multiple activation states of integrin alpha4beta1 detected through their different affinities for a small molecule ligand. J. Biol. Chem. 274:13167-13175. 99. Chen, Y. P., T. E. O'Toole, T. Shipley, J. Forsyth, S. E. LaFlamme, K. M. Yamada, S. J. Shattil, and i. M. G. 1994. "Inside-out" signal transduction inhibited by isolated integrin cytoplasmic domains. J. Biol. Chem. 269:18307-18310. 100. Cheresh, D. A., J. Leng, and R. L. Klemke. 1999. Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells. J. Cell Biol. 146:1107-1116. 101. Childs, T. J., M. H. Watson, J. S. Sanghera, D. L. Campbell, S. L. Pelech, and A. S. Mak. 1992. Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells. J. Biol. Chem. 267:22853-22859. 102. Cho, S. Y., and R. L. Klemke. 2000. Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell migration and suppress apoptosis during invasion of the extracellular matrix. J. Cell Biol. 149:223-236. 103. Chrzanowska-Wodnicka, M., and K. Burridge. 1996. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J. Cell Biol. 133:1403-1415. 104. Chun, J. S., and B. S. Jacobson. 1993. Requirement for diacylglycerol and protein kinase C in HeLa cell- substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading. Mol. Biol. Cell 4:271-281. 105. Clark, E. A., W. G. King, J. S. Brugge, M. Symons, and R. O. Hynes. 1998. Integrin-mediated signals regulated by members of the rho family of GTPases. J. Cell Biol. 142:573-586. 106. Clarke, A. S., M. M. Lotz, C. Chao, and A. M. Mercurio. 1995. Activation of the p21 pathway of growth arrest and apoptosis by the beta 4 integrin cytoplasmic domain. J. Biol. Chem. 270:22673- 22676. 107. Coll, J. L., A. Ben-Ze'ev, R. M. Ezzell, J. L. Rodriguez Fernandez, H. Baribault, R. G. Oshima, and E. D. Adamson. 1995. Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion. Proc. Natl. Acad. Sci. U.S.A. 92:9161- 9165. 108. Collins, L. R., W. A. Ricketts, L. Yeh, and D. Cheresh. 1999. Bifurcation of cell migratory and proliferative signaling by the adaptor protein Shc. J. Cell Biol. 147:1561-1568. 109. Cooray, P., Y. Yuan, S. M. Schoenwaelder, C. A. Mitchell, H. H. Salem, and S. P. Jackson. 1996. Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain. Biochem. J. 318:41-47. 110. Coppolino, M., C. Leung-Hagesteijn, S. Dedhar, and J. Wilkins. 1995. Inducible interaction of integrin α2β1 with calreticulin. Dependence on the activation state of the integrin. J. Biol. Chem. 270:23132-23138. 111. Coppolino, M. G., and S. Dedhar. 1999. Ligand-specific, transient interaction between integrins and calreticulin during cell adhesion to extracellular matrix proteins is dependent upon phosphorylation/dephosphorylation events. Biochem. J. 340:41-50. 112. Coppolino, M. G., M. J. Woodside, N. Demaurex, S. Grinstein, R. St-Arnaud, and S. Dedhar. 1997. Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion. Nature 386:843-847.

68 113. Cosson, P., S. P. Lankford, J. S. Bonifacino, and R. D. Klausner. 1991. Membrane protein association by potential intramembrane charge pairs. Nature 351:414-416. 114. Cote, J. F., A. Charest, J. Wagner, and M. L. Tremblay. 1998. Combination of gene targeting and substrate trapping to identify substrates of protein tyrosine phosphatases using PTP-PEST as a model. Biochemistry 37:13128-13137. 115. Cote, J. F., C. E. Turner, and M. L. Tremblay. 1999. Intact LIM 3 and LIM 4 domains of paxillin are required for the association to a novel polyproline region (Pro 2) of protein-tyrosine phosphatase- PEST. J. Biol. Chem. 274:20550-20560. 116. Cowan, K. J., D. A. Law, and D. R. Phillips. 2000. Identification of SHC as the primary protein binding to the tyrosine phosphorylated beta{sub3} subunit of alphalIbbeta{sub3} during outside- in integrin platelet signaling. J. Biol. Chem. in press. 117. Crawford, A. W., and M. C. Beckerle. 1991. Purification and characterization of zyxin, an 82,000- dalton component of adherens junctions. J. Biol. Chem. 266:5847-5853. 118. Croce, K., R. Flaumenhaft, M. Rivers, B. Furie, B. C. Furie, I. M. Herman, and D. A. Potter. 1999. Inhibition of calpain blocks platelet secretion, aggregation, and spreading. J. Biol. Chem. 274:36321-36327. 119. Crouch, D. H., V. J. Fincham, and M. C. Frame. 1996. Targeted proteolysis of the focal adhesion kinase pp125 FAK during c- MYC-induced apoptosis is suppressed by integrin signalling. Oncogene 12:2689-2696. 120. D'Souza-Schorey, C., B. Boettner, and L. Van Aelst. 1998. Rac regulates integrin-mediated spreading and increased adhesion of T lymphocytes. Mol. Cell. Biol. 18:3936-3946. 121. Dana, N., D. M. Fathallah, and M. A. Arnaout. 1991. Expression of a soluble and functional form of the human β2 integrin CD11b/CD18. Proc. Natl. Acad. Sci. U.S.A. 88:3106-3110. 122. Danen, E. H., S. Aota, A. A. van Kraats, K. M. Yamada, D. J. Ruiter, and G. N. van Muijen. 1995. Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of beta 1. J. Biol. Chem. 270:21612-21618. 123. Danilov, Y. N., and R. L. Juliano. 1989. Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event. J. Cell Biol. 108:1925-1933. 124. Danker, K., B. Gabriel, C. Heidrich, and W. Reutter. 1998. Focal adhesion kinase pp125FAK and the beta 1 integrin subunit are constitutively complexed in HaCaT cells. Exp. Cell Res. 239:326-331. 125. Dawid, I. B., R. Toyama, and M. Taira. 1995. LIM domain proteins. C R Acad Sci III 318:295- 306. 126. De Meirsman, C., E. Schollen, M. Jaspers, K. Ongena, G. Matthijs, P. Marynen, and J. J. Cassiman. 1994. Cloning and characterization of the promoter region of the murine alpha- 4 integrin subunit. DNA Cell Biol 13:743-754. 127. De Melker, A. A., D. Kramer, I. Kuikman, and A. Sonnenberg. 1997. The two phenylalanines in the GFFKR motif of the integrin alpha6A subunit are essential for heterodimerization. Biochem. J. 328:529-537. 128. De Nichilo, M. O., and K. M. Yamada. 1996. Integrin αvβ5-dependent serine phosphorylation of paxillin in cultured human macrophages adherent to vitronectin. J. Biol. Chem. 271:11016-11022. 129. de Pereda, J. M., G. Wiche, and R. C. Liddington. 1999. Crystal structure of a tandem pair of fibronectin type III domains from the cytoplasmic tail of integrin alpha6beta4. EMBO J. 18:4087-4095. 130. Defilippi, P., M. Venturino, D. Gulino, A. Duperray, P. Boquet, C. Fiorentini, G. Volpe, M. Palmieri, L. Silengo, and G. Tarone. 1997. Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation. J. Biol. Chem. 272:21726-21734. 131. del Pozo, M. A., L. S. Price, N. B. Alderson, X. D. Ren, and M. A. Schwartz. 2000. Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19:2008-2014. 132. Delcommenne, M., C. Tan, V. Gray, L. Rue, J. Woodgett, and S. Dedhar. 1998. Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and /AKT by the integrin-linked kinase. Proc. Natl. Acad. Sci. U.S.A. 95:11211-11216. 133. Della Rocca, G. J., S. Maudsley, Y. Daaka, R. J. Lefkowitz, and L. M. Luttrell. 1999. Pleiotropic coupling of G protein-coupled receptors to the mitogen- activated protein kinase cascade. Role of focal adhesions and receptor tyrosine kinases. J. Biol. Chem. 274:13978-13984. 134. DeMali, K. A., E. Balciunaite, and A. Kazlauskas. 1999. Integrins enhance platelet-derived growth factor (PDGF)-dependent responses by altering the signal relay enzymes that are recruited to the PDGF beta receptor. J. Biol. Chem. 274:19551-19558. 135. Derman, M. P., A. Toker, J. H. Hartwig, K. Spokes, J. R. Falck, C. S. Chen, L. C. Cantley, and L. G. Cantley. 1997. The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C. J. Biol. Chem. 272:6465-6470.

69 136. Dickeson, S. K., N. L. Mathis, M. Rahman, J. M. Bergelson, and S. A. Santoro. 1999. Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins. J. Biol. Chem. 274:32182-32191. 137. Dikic, I., and J. Schlessinger. 1998. Identification of a new Pyk2 isoform implicated in chemokine and antigen receptor signaling. J. Biol. Chem. 273:14301-14308. 138. Disatnik, M. H., and T. A. Rando. 1999. Integrin-mediated muscle cell spreading. The role of protein kinase c in outside-in and inside-out signaling and evidence of integrin cross- talk. J. Biol. Chem. 274:32486-32492. 139. Djaffar, I., and J. P. Rosa. 1993. A second case of variant of Glanzmann's thrombasthenia due to substitution of platelet GPIIIa () Arg214 by Trp. Hum Mol Genet 2:2179-2180. 140. Dolfi, F., M. Garcia-Guzman, M. Ojaniemi, H. Nakamura, M. Matsuda, and K. Vuori. 1998. The adaptor protein Crk connects multiple cellular stimuli to the JNK signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 95:15394-15399. 141. Donaldson, J. C., P. J. Dempsey, S. Reddy, A. H. Bouton, R. J. Coffey, and S. K. Hanks. 2000. Crk-associated substrate p130(Cas) interacts with nephrocystin and both proteins localize to cell- cell contacts of polarized epithelial cells. Exp. Cell Res. 256:168-178. 142. Donaldson, J. G., and C. L. Jackson. 2000. Regulators and effectors of the ARF GTPases. Curr. Opin. Cell Biol. 12:475-482. 143. Douglass, W. A., R. H. Hyland, C. D. Buckley, A. Al-Shamkhani, J. M. Shaw, S. L. Scarth, D. L. Simmons, and S. K. Law. 1998. The role of the cysteine-rich region of the beta2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, alphaLbeta2, CD11a/CD18) heterodimer formation and ligand binding. FEBS Lett. 440:414-418. 144. Drees, B. E., K. M. Andrews, and M. C. Beckerle. 1999. Molecular dissection of zyxin function reveals its involvement in cell motility. J. Cell Biol. 147:1549-1560. 145. Du, X., T. C. Saido, S. Tsubuki, F. E. Indig, M. J. Williams, and M. H. Ginsberg. 1995. Calpain cleavage of the cytoplasmic domain of the integrin beta 3 subunit. J. Biol. Chem. 270:26146- 26151. 146. Echtermeyer, F., P. C. Baciu, S. Saoncella, Y. Ge, and P. F. Goetinck. 1999. Syndecan-4 core protein is sufficient for the assembly of focal adhesions and actin stress fibers. J. Cell Sci. 112:3433- 3441. 147. Edwards, D. C., L. C. Sanders, G. M. Bokoch, and G. N. Gill. 1999. Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol 1:253- 259. 148. Eguchi, S., H. Iwasaki, T. Inagami, K. Numaguchi, T. Yamakawa, E. D. Motley, K. M. Owada, F. Marumo, and Y. Hirata. 1999. Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells. Hypertension 33:201-206. 149. Eide, B. L., C. W. Turck, and J. A. Escobedo. 1995. Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK. Mol. Cell. Biol. 15:2819-2827. 150. Eigenthaler, M., L. Hofferer, S. J. Shattil, and M. H. Ginsberg. 1997. A conserved sequence motif in the integrin β3 cytoplasmic domain is required for its specific interaction with β3-endonexin. J. Biol. Chem. 272:7693-7698. 151. Eisenmann, K. M., J. B. McCarthy, M. A. Simpson, P. J. Keely, J. L. Guan, K. Tachibana, L. Lim, E. Manser, L. T. Furcht, and J. Iida. 1999. Melanoma chondroitin sulphate proteoglycan regulates cell spreading through , ack-1 and p130cas. Nat Cell Biol 1:507-513. 152. Ellgaard, L., M. Molinari, and A. Helenius. 1999. Setting the standards: quality control in the secretory pathway. Science 286:1882-1888. 153. Emsley, J., S. L. King, J. M. Bergelson, and R. C. Liddington. 1997. Crystal structure of the I domain from integrin α2β1. J. Biol. Chem. 272:28512-28517. 154. Emsley, J., C. G. Knight, R. W. Farndale, M. J. Barnes, and R. C. Liddington. 2000. Structural basis of collagen recognition by integrin alpha2beta1. Cell 101:47-56. 155. Escalante, M., J. Courtney, W. G. Chin, K. K. Teng, J. I. Kim, J. E. Fajardo, B. J. Mayer, B. L. Hempstead, and R. B. Birge. 2000. Phosphorylation of c-Crk II on the negative regulatory tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis. J. Biol. Chem. 275:24787- 24797. 156. Fabbri, M., L. Fumagalli, G. Bossi, E. Bianchi, J. R. Bender, and R. Pardi. 1999. A tyrosine- based sorting signal in the beta2 integrin cytoplasmic domain mediates its recycling to the plasma membrane and is required for ligand-supported migration. EMBO J. 18:4915-4925. 157. Fadel, M. P., E. Dziak, C. M. Lo, J. Ferrier, N. Mesaeli, M. Michalak, and M. Opas. 1999. Calreticulin affects focal contact-dependent but not close contact- dependent cell-substratum adhesion. J. Biol. Chem. 274:15085-15094. 158. Fang, F., G. Orend, N. Watanabe, T. Hunter, and E. Ruoslahti. 1996. Dependence of cyclin E- CDK2 kinase activity on cell anchorage. Science 271:499-502.

70 159. Feller, S. M., B. Knudsen, and H. Hanafusa. 1994. c-Abl kinase regulates the protein binding activity of c-Crk. EMBO J. 13:2341-2351. 160. Feller, S. M., G. Posern, J. Voss, C. Kardinal, D. Sakkab, J. Zheng, and B. S. Knudsen. 1998. Physiological signals and oncogenesis mediated through Crk family adapter proteins. J. Cell Physiol. 177:535-552. 161. Fincham, V. J., M. James, M. C. Frame, and S. J. Winder. 2000. Active ERK/MAP kinase is targeted to newly forming cell-matrix adhesions by integrin engagement and v-Src. EMBO J. 19:2911- 2923. 162. Flinn, H. M., and A. J. Ridley. 1996. Rho stimulates tyrosine phosphorylation of focal adhesion kinase, p130 and paxillin. J. Cell Sci. 109:1133-1141. 163. Forman-Kay, J. D., and T. Pawson. 1999. Diversity in protein recognition by PTB domains. Curr Opin Struct Biol 9:690-695. 164. Fornaro, M., and L. R. Languino. 1997. Alternatively spliced variants: a new view of the integrin cytoplasmic domain. Matrix Biol. 16:185-193. 165. Fornaro, M., C. A. Steger, A. M. Bennett, J. J. Wu, and L. R. Languino. 2000. Differential role of beta(1C) and beta(1A) integrin cytoplasmic variants in modulating focal adhesion kinase, protein kinase B/AKT, and Ras/Mitogen-activated protein kinase pathways. Mol. Biol. Cell 11:2235-2249. 166. Fornaro, M., G. Tallini, C. J. Bofetiado, S. Bosari, and L. R. Languino. 1996. Down-regulation of β1C integrin, an inhibitor of cell proliferation, in prostate carcinoma. Am. J. Pathol. 149:765-773. 167. Fornaro, M., G. Tallini, D. Q. Zheng, W. M. Flanagan, M. Manzotti, and L. R. Languino. 1999. p27(kip1) acts as a downstream effector of and is coexpressed with the beta1C integrin in prostatic adenocarcinoma. J. Clin. Invest. 103:321-329. 168. Fornaro, M., D. Q. Zheng, and L. R. Languino. 1995. The novel structural motif Gln795-Gln802 in the integrin beta 1C cytoplasmic domain regulates cell proliferation. J. Biol. Chem. 270:24666- 24669. 169. Foster, D. B., L. H. Shen, J. Kelly, P. Thibault, J. E. Van Eyk, and A. S. Mak. 2000. Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction. J. Biol. Chem. 275:1959-1965. 170. Frank, S. R., J. C. Hatfield, and J. E. Casanova. 1998. Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO. Mol. Biol. Cell 9:3133-3146. 171. Frisch, S. M., K. Vuori, D. Kelaita, and S. Sicks. 1996. A role for Jun-N-terminal kinase in anoikis; suppression by bcl-2 and crmA. J. Cell Biol. 135:1377-1382. 172. Frisch, S. M., K. Vuori, E. Ruoslahti, and P. Y. Chan-Hui. 1996. Control of adhesion-dependent cell survival by focal adhesion kinase. J. Cell Biol. 134:793-799. 173. Gao, A. G., F. P. Lindberg, J. M. Dimitry, E. J. Brown, and W. A. Frazier. 1996. Thrombospondin modulates αvβ3 function through integrin- associated protein. J. Cell Biol. 135:533- 544. 174. Gao, A. G., F. P. Lindberg, M. B. Finn, S. D. Blystone, E. J. Brown, and W. A. Frazier. 1996. Integrin-associated protein is a receptor for the C-terminal domain of thrombospondin. J. Biol. Chem. 271:21-24. 175. Gao, J., K. E. Zoller, M. H. Ginsberg, J. S. Brugge, and S. J. Shattil. 1997. Regulation of the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 3. EMBO J. 16:6414-6425. 176. Garcia-Guzman, M., F. Dolfi, M. Russello, and K. Vuori. 1999. Cell adhesion regulates the interaction between the docking protein p130(Cas) and the 14-3-3 proteins. J. Biol. Chem. 274:5762- 5768. 177. Garton, A. J., M. R. Burnham, A. H. Bouton, and N. K. Tonks. 1997. Association of PTP-PEST with the SH3 domain of p130cas; a novel mechanism of protein tyrosine phosphatase substrate recognition. Oncogene 15:877-885. 178. Garton, A. J., A. J. Flint, and N. K. Tonks. 1996. Identification of p130(cas) as a substrate for the cytosolic protein tyrosine phosphatase PTP-PEST. Mol. Cell. Biol. 16:6408-6418. 179. Garton, A. J., and N. K. Tonks. 1999. Regulation of fibroblast motility by the protein tyrosine phosphatase PTP-PEST. J. Biol. Chem. 274:3811-3818. 180. Geiger, C., W. Nagel, T. Boehm, Y. van Kooyk, C. G. Figdor, E. Kremmer, N. Hogg, L. Zeitlmann, H. Dierks, K. S. Weber, and W. Kolanus. 2000. Cytohesin-1 regulates beta-2 integrin- mediated adhesion through both ARF-GEF function and interaction with LFA-1. EMBO J. 19:2525- 2536. 181. Genth, H., K. Aktories, and I. Just. 1999. Monoglucosylation of RhoA at threonine 37 blocks cytosol-membrane cycling. J. Biol. Chem. 274:29050-29056. 182. Gil, S. G., T. A. Brown, M. C. Ryan, and W. G. Carter. 1994. Junctional epidermolysis bullosis: defects in expression of epiligrin/nicein/kalinin and that inhibit hemidesmosome formation. J. Invest. Dermatol. 103:31S-38S.

71 183. Gilmore, A. P., and K. Burridge. 1996. Regulation of vinculin binding to talin and actin by phosphatidyl- inositol-4-5-bisphosphate. Nature 381:531-535. 184. Gilmore, A. P., A. D. Metcalfe, L. H. Romer, and C. H. Streuli. 2000. Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization. J. Cell Biol. 149:431-446. 185. Girault, J. A., G. Labesse, J. P. Mornon, and I. Callebaut. 1999. The N-termini of FAK and JAKs contain divergent band 4.1 domains. Trends Biochem. Sci. 24:54-57. 186. Giuriato, S., B. Payrastre, A. L. Drayer, M. Plantavid, R. Woscholski, P. Parker, C. Erneux, and H. Chap. 1997. Tyrosine phosphorylation and relocation of SHIP are integrin-mediated in thrombin-stimulated human blood platelets. J. Biol. Chem. 272:26857-26863. 187. Glenney, J. R., Jr., and L. Zokas. 1989. Novel tyrosine kinase substrates from Rous sarcoma virus- transformed cells are present in the membrane skeleton. J. Cell Biol. 108:2401-2408. 188. Gong, J., T. C. Ko, and M. G. Brattain. 1998. Disruption of fibronectin binding to the alpha 5 beta 1 integrin stimulates the expression of cyclin-dependent kinases and DNA synthesis through activation of extracellular signal-regulated kinase. J. Biol. Chem. 273:1662-1669. 189. Gotoh, A., H. Takahira, R. L. Geahlen, and H. E. Broxmeyer. 1997. Cross-linking of integrins induces tyrosine phosphorylation of the proto-oncogene product Vav and the protein tyrosine kinase Syk in human factor-dependent myeloid cells. Cell Growth Differ 8:721-729. 190. Gotoh, T., D. Cai, X. Tian, L. A. Feig, and A. Lerner. 2000. p130Cas regulates the activity of AND-34, a novel Ral, Rap1 and R-Ras guanine nucleotide exchange factor. J. Biol. Chem. in press. 191. Green, J. M., A. Zhelesnyak, J. Chung, F. P. Lindberg, M. Sarfati, W. A. Frazier, and E. J. Brown. 1999. Role of cholesterol in formation and function of a signaling complex involving alphavbeta3, integrin-associated protein (CD47), and heterotrimeric G proteins. J. Cell Biol. 146:673- 682. 192. Greenwood, J. A., A. B. Theibert, G. D. Prestwich, and J. E. Murphy-Ullrich. 2000. Restructuring of focal adhesion plaques by PI 3-kinase. Regulation By ptdins (3,4,5)-p(3) binding to alpha-actinin. J. Cell Biol. 150:627-642. 193. Gu, J., M. Tamura, R. Pankov, E. H. Danen, T. Takino, K. Matsumoto, and K. M. Yamada. 1999. Shc and FAK differentially regulate cell motility and directionality modulated by PTEN. J. Cell Biol. 146:389-403. 194. Gu, M., X. Xi, G. D. Englund, M. C. Berndt, and X. Du. 1999. Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX- mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model. J. Cell Biol. 147:1085-1096. 195. Guadagno, T. M., M. Ohtsubo, J. M. Roberts, and R. K. Assoian. 1993. A link between cyclin A expression and adhesion-dependent cell cycle progression [published erratum appears in Science 1994 Jan 28;263(5146):455]. Science 262:1572-1575. 196. Guan, J. L., and D. Shalloway. 1992. Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature 358:690-692. 197. Guilherme, A., and M. P. Czech. 1998. Stimulation of IRS-1-associated phosphatidylinositol 3- kinase and Akt/protein kinase B but not glucose transport by beta1-integrin signaling in rat adipocytes. J. Biol. Chem. 273:33119-33122. 198. Guilherme, A., K. Torres, and M. P. Czech. 1998. Cross-talk between insulin receptor and integrin alpha5 beta1 signaling pathways. J. Biol. Chem. 273:22899-22903. 199. Haas, T. A., and E. F. Plow. 1997. Development of a structural model for the cytoplasmic domain of an integrin. Protein Eng. 10:1395-1405. 200. Hagmann, J., M. Grob, A. Welman, G. van Willigen, and M. M. Burger. 1998. Recruitment of the LIM protein hic-5 to focal contacts of human platelets. J. Cell Sci. 111:2181-2188. 201. Haimovich, B., N. Kaneshiki, and P. Ji. 1996. Protein kinase C regulates tyrosine phosphorylation of pp125FAK in platelets adherent to fibrinogen. Blood 87:152-161. 202. Hakak, Y., and G. S. Martin. 1999. Cas mediates transcriptional activation of the serum response element by Src. Mol. Cell. Biol. 19:6953-6962. 203. Hall, A. 1998. Rho GTPases and the actin cytoskeleton. Science 279:509-514. 204. Haller, H., C. Lindschau, C. Maasch, H. Olthoff, D. Kurscheid, and F. C. Luft. 1998. Integrin- induced protein kinase Calpha and Cepsilon translocation to focal adhesions mediates vascular smooth muscle cell spreading. Circ. Res. 82:157-165. 205. Hamasaki, K., T. Mimura, N. Morino, H. Furuya, T. Nakamoto, S. Aizawa, C. Morimoto, Y. Yazaki, H. Hirai, and Y. Nojima. 1996. Src kinase plays an essential role in integrin-mediated tyrosine phosphorylation of Crk-associated substrate p130Cas. Biochem. Biophys. Res. Commun. 222:338-343. 206. Han, D. C., and J. L. Guan. 1999. Association of focal adhesion kinase with Grb7 and its role in cell migration. J. Biol. Chem. 274:24425-24430.

72 207. Hanks, S. K., M. B. Calalb, M. C. Harper, and S. K. Patel. 1992. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin. Proc. Natl. Acad. Sci. U.S.A. 89:8487-8491. 208. Hannigan, G. E., C. Leung-Hagesteijn, L. Fitz-Gibbon, M. G. Coppolino, G. Radeva, J. Filmus, J. C. Bell, and S. Dedhar. 1996. Regulation of cell adhesion and anchorage-dependent growth by a new β1-integrin-linked protein kinase. Nature 379:91-96. 209. Harder, K. W., N. P. Moller, J. W. Peacock, and F. R. Jirik. 1998. Protein-tyrosine phosphatase α regulates Src family kinases and alters cell-substratum adhesion. J. Biol. Chem. 273:31890-31900. 210. Harris, E. S., T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 2000. The leukocyte integrins. J. Biol. Chem. 275:23409-23412. 211. Harte, M. T., J. D. Hildebrand, M. R. Burnham, A. H. Bouton, and J. T. Parsons. 1996. p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase. J. Biol. Chem. 271:13649-13655. 212. Hay, E. (ed.). 1991. Cell Biology of the Extracellular Matrix, Second ed. Plenum Press, New York. 213. Heino, J., R. A. Ignotz, M. E. Hemler, C. Crouse, and J. Massague. 1989. Regulation of cell adhesion receptors by transforming growth factor-β. Concomitant regulation of integrins that share a common beta 1 subunit. J. Biol. Chem. 264:380-388. 214. Helenius, A., E. S. Trombetta, D. N. Hebert, and J. F. Simons. 1997. Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol. 7:193-200. 215. Helfman, D. M., E. T. Levy, C. Berthier, M. Shtutman, D. Riveline, I. Grosheva, A. Lachish- Zalait, M. Elbaum, and A. D. Bershadsky. 1999. Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions. Mol. Biol. Cell 10:3097-3112. 216. Herreros, L., J. L. Rodriguez-Fernandez, M. C. Brown, J. L. Alonso-Lebrero, C. Cabanas, F. Sanchez-Madrid, N. Longo, C. E. Turner, and P. Sanchez-Mateos. 2000. Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton. J. Biol. Chem. 275:26436-26440. 217. Hibbs, M. L., S. Jakes, S. A. Stacker, R. W. Wallace, and T. A. Springer. 1991. The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 β subunit: sites required for binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated phosphorylation site. J. Exp. Med. 174:1227-1238. 218. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Regulation of adhesion of ICAM-1 by the cytoplasmic domain of LFA-1 integrin β subunit. Science 251:1611-1613. 219. Higgins, J. M., M. Cernadas, K. Tan, A. Irie, J. Wang, Y. Takada, and M. B. Brenner. 2000. The role of alpha and beta chains in ligand recognition by beta 7 integrins. J. Biol. Chem. 275:25652- 25664. 220. Higgs, H. N., and T. D. Pollard. 1999. Regulation of actin polymerization by Arp2/3 complex and WASp/Scar proteins. J. Biol. Chem. 274:32531-32534. 221. Hildebrand, J. D., M. D. Schaller, and J. T. Parsons. 1995. Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase. Mol. Biol. Cell 6:637-647. 222. Hildebrand, J. D., J. M. Taylor, and J. T. Parsons. 1996. An SH3 domain-containing GTPase- activating protein for Rho and Cdc42 associates with focal adhesion kinase. Mol. Cell. Biol. 16:3169- 3178. 223. Hildebrandt, F., E. Otto, C. Rensing, H. G. Nothwang, M. Vollmer, J. Adolphs, H. Hanusch, and M. Brandis. 1997. A novel gene encoding an SH3 domain protein is mutated in nephronophthisis type 1. Nat. Genet. 17:149-153. 224. Hinchliffe, K. A., R. F. Irvine, and N. Divecha. 1996. Aggregation-dependent, integrin-mediated increases in cytoskeletally associated PtdInsP2 (4,5) levels in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to the cytoskeleton. EMBO J. 15:6516-6524. 225. Hirsch, E., F. Balzac, C. Pastore, G. Tarone, L. Silengo, and F. Altruda. 1993. The beta 1 integrin distal promoter is developmentally regulated in transgenic mice. Cell Adhes. Commun. 1:203- 212. 226. Hmama, Z., K. L. Knutson, P. Herrera-Velit, D. Nandan, and N. E. Reiner. 1999. Monocyte adherence induced by lipopolysaccharide involves CD14, LFA-1, and cytohesin-1. Regulation by Rho and phosphatidylinositol 3-kinase. J. Biol. Chem. 274:1050-1057. 227. Hobert, O., D. G. Moerman, K. A. Clark, M. C. Beckerle, and G. Ruvkun. 1999. A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans. J. Cell Biol. 144:45-57. 228. Hobert, O., J. W. Schilling, M. C. Beckerle, A. Ullrich, and B. Jallal. 1996. SH3 domain- dependent interaction of the proto-oncogene product Vav with the focal contact protein zyxin. Oncogene 12:1577-1581. 229. Hogg, N., and P. A. Bates. 2000. Genetic analysis of integrin function in man: LAD-1 and other syndromes. Matrix Biol. 19:211-222.

73 230. Hogg, N., M. P. Stewart, S. L. Scarth, R. Newton, J. M. Shaw, S. K. Law, and N. Klein. 1999. A novel leukocyte adhesion deficiency caused by expressed but nonfunctional beta2 integrins Mac-1 and LFA-1. J. Clin. Invest. 103:97-106. 231. Holzmann, B., and I. L. Weissman. 1989. Peyer's patch-specific lymphocyte homing receptors consist of a VLA-4- like alpha chain associated with either of two integrin beta chains, one of which is novel. EMBO J. 8:1735-1741. 232. Honda, H., T. Nakamoto, R. Sakai, and H. Hirai. 1999. p130(Cas), an assembling molecule of actin filaments, promotes cell movement, cell migration, and cell spreading in fibroblasts. Biochem. Biophys. Res. Commun. 262:25-30. 233. Honda, H., H. Oda, T. Nakamoto, Z. Honda, R. Sakai, T. Suzuki, T. Saito, K. Nakamura, K. Nakao, T. Ishikawa, M. Katsuki, Y. Yazaki, and H. Hirai. 1998. Cardiovascular anomaly, impaired actin bundling and resistance to Src- induced transformation in mice lacking p130Cas. Nat. Genet. 19:361-365. 234. Horwitz, A., K. Duggan, C. Buck, M. C. Berkerle, and K. Burridge. 1986. Interaction of plasma membrane fibronectin receptor with talin: a transmembrane linkage. Nature 320:531-533. 235. Hotchin, N. A., and A. Hall. 1995. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/ GTPases. J. Cell Biol. 131:1857-1865. 236. Hotchin, N. A., and F. M. Watt. 1992. Transcriptional and post-translational regulation of beta 1 integrin expression during keratinocyte terminal differentiation. J. Biol. Chem. 267:14852-14858. 237. Hu, D. D., C. F. Barbas, and J. W. Smith. 1996. An allosteric Ca2+ binding site on the beta3- integrins that regulates the dissociation rate for RGD ligands. J. Biol. Chem. 271:21745-21751. 238. Huang, C., J. Liu, C. C. Haudenschild, and X. Zhan. 1998. The role of tyrosine phosphorylation of cortactin in the locomotion of endothelial cells. J. Biol. Chem. 273:25770-25776. 239. Huber, P. A. 1997. Caldesmon. Int. J. Biochem. Cell Biol. 29:1047-1051. 240. Hughes, P. E., F. Diaz-Gonzalez, L. Leong, C. Wu, J. A. McDonald, S. J. Shattil, and M. H. Ginsberg. 1996. Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J. Biol. Chem. 271:6571-6574. 241. Hughes, P. E., T. E. O'Toole, J. Ylanne, S. J. Shattil, and M. H. Ginsberg. 1995. The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand binding affinity. J. Biol. Chem. 270:12411-12417. 242. Hughes, P. E., M. W. Renshaw, M. Pfaff, J. Forsyth, V. M. Keivens, M. A. Schwartz, and M. H. Ginsberg. 1997. Suppression of integrin activation: a novel function of a Ras/Raf- initiated MAP kinase pathway. Cell 88:521-530. 243. Huhtala, P., M. J. Humphries, J. B. McCarthy, P. M. Tremble, Z. Werb, and C. H. Damsky. 1995. Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J. Cell Biol. 129:867-879. 244. Humphries, J. D., J. A. Askari, X. P. Zhang, Y. Takada, M. J. Humphries, and A. P. Mould. 2000. Molecular basis of ligand recognition by integrin alpha5beta 1. II. Specificity of arg-gly-Asp binding is determined by Trp157 OF THE alpha subunit. J. Biol. Chem. 275:20337-20345. 245. Hungerford, J. E., M. T. Compton, M. L. Matter, B. G. Hoffstrom, and C. A. Otey. 1996. Inhibition of pp125FAK in cultured fibroblasts results in apoptosis. J. Cell Biol. 135:1383-1390. 246. Huttelmaier, S., O. Mayboroda, B. Harbeck, T. Jarchau, B. M. Jockusch, and M. Rudiger. 1998. The interaction of the cell-contact proteins VASP and vinculin is regulated by phosphatidylinositol-4,5-bisphosphate. Curr. Biol. 8:479-488. 247. Huttenlocher, A., M. H. Ginsberg, and A. F. Horwitz. 1996. Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity. J. Cell Biol. 134:1551-1562. 248. Huttenlocher, A., S. P. Palecek, Q. Lu, W. Zhang, R. L. Mellgren, D. A. Lauffenburger, M. H. Ginsberg, and A. F. Horwitz. 1997. Regulation of cell migration by the calcium-dependent protease calpain. J. Biol. Chem. 272:32719-32722. 249. Huynh-Do, U., E. Stein, A. A. Lane, H. Liu, D. P. Cerre tti, and T. O. Daniel. 1999. Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins. EMBO J. 18:2165-2173. 250. Hyatt, S. L., L. Liao, C. Chapline, and S. Jaken. 1994. Identification and characterization of α- protein kinase C binding proteins in normal and transformed REF52 cells. Biochemistry 33:1223- 1228. 251. Igishi, T., S. Fukuhara, V. Patel, B. Z. Katz, K. M. Yamada, and J. S. Gutkind. 1999. Divergent signaling pathways link focal adhesion kinase to mitogen- activated protein kinase cascades. Evidence for a role of paxillin in c- Jun NH(2)-terminal kinase activation. J. Biol. Chem. 274:30738- 30746. 252. Ilic, D., E. A. Almeida, D. D. Schlaepfer, P. Dazin, S. Aizawa, and C. H. Damsky. 1998. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J. Cell Biol. 143:547-560.

74 253. Ilic, D., Y. Furuta, S. Kanazawa, N. Takeda, K. Sobue, N. Nakatsuji, S. Nomura, J. Fujimoto, M. Okada, T. Yamamoto, and et al. 1995. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377:539-544. 254. Ishiguro, K., K. Kadomatsu, T. Kojima, H. Muramatsu, S. Tsuzuki, E. Nakamura, K. Kusugami, H. Saito, and T. Muramatsu. 2000. Syndecan-4 deficiency impairs focal adhesion formation only under restricted conditions. J. Biol. Chem. 275:5249-5252. 255. Ivaska, J., H. Reunanen, J. Westermarck, L. Koivisto, V. M. Kahari, and J. Heino. 1999. Integrin alpha2beta1 mediates isoform-specific activation of p38 and upregulation of collagen gene transcription by a mechanism involving the alpha2 cytoplasmic tail. J. Cell Biol. 147:401-416. 256. Jaken, S., and P. J. Parker. 2000. Protein kinase C binding partners. Bioessays 22:245-254. 257. Jenkins, A. L., L. Nannizzi-Alaimo, D. Silver, J. R. Sellers, M. H. Ginsberg, D. A. Law, and D. R. Phillips. 1998. Tyrosine phosphorylation of the β3 cytoplasmic domain mediates integrin- cytoskeletal interactions. J. Biol. Chem. 273:13878-13885. 258. Johansson, M. W., E. Larsson, B. Luning, E. B. Pasquale, and E. Ruoslahti. 1994. Altered localization and cytoplasmic domain-binding properties of tyrosine- phosphorylated β1 integrin. J. Cell Biol. 126:1299-1309. 259. Jucker, M., K. McKenna, A. J. da Silva, C. E. Rudd, and R. A. Feldman. 1997. The Fes protein-tyrosine kinase phosphorylates a subset of macrophage proteins that are involved in cell adhesion and cell-cell signaling. J. Biol. Chem. 272:2104-2109. 260. Jung, S. M., and M. Moroi. 2000. Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1). J. Biol. Chem. 275:8016-8026. 261. Kaapa, A., K. Peter, and J. Ylanne. 1999. Effects of mutations in the cytoplasmic domain of integrin beta(1) to talin binding and cell spreading. Exp. Cell Res. 250:524-534. 262. Kamata, T., R. C. Liddington, and Y. Takada. 1999. Interaction between collagen and the alpha(2) I-domain of integrin alpha(2)beta(1). Critical role of conserved residues in the metal ion- dependent adhesion site (MIDAS) region. J. Biol. Chem. 274:32108-32111. 263. Kanner, S. B., A. B. Reynolds, R. R. Vines, and J. T. Parsons. 1990. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc. Natl. Acad. Sci. U.S.A. 87:3328-3332. 264. Kashiwagi, H., M. A. Schwartz, M. Eigenthaler, K. A. Davis, M. H. Ginsberg, and S. J. Shattil. 1997. Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail. J. Cell Biol. 137:1433-1443. 265. Kassner, P. D., and M. E. Hemler. 1993. Interchangeable alpha chain cytoplasmic domains play a positive role in control of cell adhesion mediated by VLA-4, a beta 1 integrin. J. Exp. Med. 178:649- 660. 266. Katagiri, K., M. Hattori, N. Minato, S. Irie, K. Takatsu, and T. Kinashi. 2000. Rap1 is a potent activation signal for leukocyte function-associated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase. Mol. Cell. Biol. 20:1956-1969. 267. Kaverina, I., O. Krylyshkina, and J. V. Small. 1999. Microtubule targeting of substrate contacts promotes their relaxation and dissociation. J. Cell Biol. 146:1033-1044. 268. Kawabuchi, M., Y. Satomi, T. Takao, Y. Shimonishi, S. Nada, K. Nagai, A. Tarakhovsky, and M. Okada. 2000. Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases. Nature 404:999-1003. 269. Kawaguchi, S., J. M. Bergelson, R. W. Finberg, and M. E. Hemler. 1994. cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions. Mol. Biol. Cell 5:977-988. 270. Kawaguchi, S., and M. E. Hemler. 1993. Role of the alpha subunit cytoplasmic domain in regulation of adhesive activity mediated by the integrin VLA-2. J. Biol. Chem. 268:16279-16285. 271. Kazanietz, M. G. 2000. Eyes wide shut: protein kinase C isozymes are not the only receptors for the phorbol ester tumor promoters. Mol. Carcinog. 28:5-11. 272. Kee, W. J., E. R. Li, and F. M. Watt. 2000. beta1B integrin subunit contains a double lysine motif that can cause accumulation within the endoplasmic reticulum. J. Cell Biochem. 78:97-111. 273. Keely, P. J., E. V. Rusyn, A. D. Cox, and L. V. Parise. 1999. R-Ras signals through specific integrin alpha cytoplasmic domains to promote migration and invasion of breast epithelial cells. J. Cell Biol. 145:1077-1088. 274. Keely, P. J., J. K. Westwick, I. P. Whitehead, C. J. Der, and L. V. Parise. 1997. Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature 390:632-636. 275. Kerr, J. R. 1999. Cell adhesion molecules in the pathogenesis of and host defence against microbial infection. Mol Pathol 52:220-230. 276. Kinashi, T., K. Katagiri, S. Watanabe, B. Vanhaesebroeck, J. Downward, and K. Takatsu. 2000. Distinct mechanisms of alpha 5beta 1 integrin activation by Ha-Ras and R-Ras. J. Biol. Chem. 275:22590-22596.

75 277. Kioka, N., S. Sakata, T. Kawauchi, T. Amachi, S. K. Akiyama, K. Okazaki, C. Yaen, K. M. Yamada, and S. Aota. 1999. Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization. J. Cell Biol. 144:59-69. 278. Kiosses, W. B., R. H. Daniels, C. Otey, G. M. Bokoch, and M. A. Schwartz. 1999. A role for p21-activated kinase in endothelial cell migration. J. Cell Biol. 147:831-844. 279. Kirk, R. I., M. R. Sanderson, and K. M. Lerea. 2000. Threonine phosphorylation of the beta3 integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in vitro, regulates Shc binding. J. Biol. Chem. in press. 280. Kirsch, K. H., M. M. Georgescu, and H. Hanafusa. 1998. Direct binding of p130(Cas) to the guanine nucleotide exchange factor C3G. J. Biol. Chem. 273:25673-25679. 281. Kirsch, K. H., M. M. Georgescu, S. Ishimaru, and H. Hanafusa. 1999. CMS: an adapter molecule involved in cytoskeletal rearrangements. Proc. Natl. Acad. Sci. U.S.A. 96:6211-6216. 282. Kiyokawa, E., Y. Hashimoto, S. Kobayashi, H. Sugimura, T. Kurata, and M. Matsuda. 1998. Activation of Rac1 by a Crk SH3-binding protein, DOCK180. Genes Dev. 12:3331-3336. 283. Kiyokawa, E., Y. Hashimoto, T. Kurata, H. Sugimura, and M. Matsuda. 1998. Evidence that DOCK180 up-regulates signals from the CrkII-p130(Cas) complex. J. Biol. Chem. 273:24479-24484. 284. Kiyokawa, E., N. Mochizuki, T. Kurata, and M. Matsuda. 1997. Role of Crk oncogene product in physiologic signaling. Crit. Rev. Oncog. 8:329-342. 285. Klemke, R. L., S. Cai, A. L. Giannini, P. J. Gallagher, P. de Lanerolle, and D. A. Cheresh. 1997. Regulation of cell motility by mitogen-activated protein kinase. J. Cell Biol. 137:481-492. 286. Klemke, R. L., J. Leng, R. Molander, P. C. Brooks, K. Vuori, and D. A. Cheresh. 1998. CAS/Crk coupling serves as a "molecular switch" for induction of cell migration. J. Cell Biol. 140:961-972. 287. Klinghoffer, R. A., C. Sachsenmaier, J. A. Cooper, and P. Soriano. 1999. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J. 18:2459-2471. 288. Knezevic, I., T. M. Leisner, and S. C. T. Lam. 1996. Direct binding of the platelet integrin αIIbβ3 (GPIIb-IIIa) to talin. Evidence that interaction is mediated through the cytoplasmic domains of both αIIb and β3. J. Biol. Chem. 271:16416-16421. 289. Kolanus, W., W. Nagel, B. Schiller, L. Zeitlmann, S. Godar, H. Stockinger, and B. Seed. 1996. αLβ2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell 86:233-242. 290. Kondo, A., S. Hashimoto, H. Yano, K. Nagayama, Y. Mazaki, and H. Sabe. 2000. A new paxillin-binding protein, PAG3/Papalpha/KIAA0400, bearing an ADP- ribosylation factor GTPase- activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration. Mol. Biol. Cell 11:1315-1327. 291. Koyama, T., and R. C. Hughes. 1992. Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity. J. Biol. Chem. 267:25939-25944. 292. Ku, H., and K. E. Meier. 2000. Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells. J. Biol. Chem. 275:11333-11340. 293. Kuroda, S., C. Tokunaga, Y. Kiyohara, O. Higuchi, H. Konishi, K. Mizuno, G. N. Gill, and U. Kikkawa. 1996. Protein-protein interaction of zinc finger LIM domains with protein kinase C. J. Biol. Chem. 271:31029-31032. 294. Kuwada, S. K., and X. Li. 2000. Integrin alpha5/beta1 mediates fibronectin-dependent epithelial cell proliferation through epidermal growth factor receptor activation. Mol. Biol. Cell 11:2485-2496. 295. Kwon, M. S., C. S. Park, K. Choi, J. Ahnn, J. I. Kim, S. H. Eom, S. J. Kaufman, and W. K. Song. 2000. Calreticulin couples calcium release and calcium influx in integrin- mediated calcium signaling. Mol. Biol. Cell 11:1433-1443. 296. LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. Regulation of fibronectin receptor distribution. J. Cell Biol. 117:437-447. 297. LaFlamme, S. E., L. A. Thomas, S. S. Yamada, and K. M. Yamada. 1994. Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly. J. Cell Biol. 126:1287-1298. 298. Lamb, R. F., C. Roy, T. J. Diefenbach, H. V. Vinters, M. W. Johnson, D. G. Jay, and A. Hall. 2000. The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho. Nat Cell Biol 2:281-287. 299. Languino, L. R., and E. Ruoslahti. 1992. An alternative form of the integrin β1 subunit with a variant cytoplasmic domain. J. Biol. Chem. 267:7116-7120. 300. Law, S. F., Y. Z. Zhang, A. J. P. Klein-Szanto, and E. A. Golemis. 1998. Cell cycle-regulated processing of HEF1 to multiple protein forms differentially targeted to multiple subcellular compartments. Mol. Cell. Biol. 18:3540-3551. 301. Leahy, D. J., I. Aukhil, and H. P. Erickson. 1996. 2.0 A crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region. Cell 84:155-164.

76 302. Lee, J. O., P. Rieu, M. A. Arnaout, and R. Liddington. 1995. Crystal structure of the A domain from the α subunit of integrin CR3 (CD11b/CD18). Cell 80:631-638. 303. Lee, J. W., and R. L. Juliano. 2000. alpha5beta1 integrin protects intestinal epithelial cells from apoptosis through a phosphatidylinositol 3-kinase and protein kinase B- dependent pathway. Mol. Biol. Cell 11:1973-1987. 304. Lehmann, M., A. El Battari, B. Abadie, J. M. Martin, and J. Marvaldi. 1996. Role of alpha v beta 5 and alpha v beta 6 integrin glycosylation in the adhesion of a colonic adenocarcinoma cell line (HT29-D4). J. Cell Biochem. 61:266-277. 305. Lehmann, M., V. Rigot, N. G. Seidah, J. Marvaldi, and J. C. Lissitzky. 1996. Lack of integrin alpha-chain endoproteolytic cleavage in furin- deficient human colon adenocarcinoma cells LoVo. Biochem. J. 317:803-809. 306. Leisner, T. M., J. D. Wencel-Drake, W. Wang, and S. C. Lam. 1999. Bidirectional transmembrane modulation of integrin alphaIIbbeta3 conformations. J. Biol. Chem. 274:12945-12949. 307. Leitinger, B., and N. Hogg. 2000. Effects of I domain deletion on the function of the beta2 integrin lymphocyte function-associated antigen-1. Mol. Biol. Cell 11:677-690. 308. Lenter, M., and D. Vestweber. 1994. The integrin chains beta 1 and alpha 6 associate with the chaperone calnexin prior to integrin assembly. J. Biol. Chem. 269:12263-12268. 309. Leong, L., P. E. Hughes, M. A. Schwartz, M. H. Ginsberg, and S. J. Shattil. 1995. Integrin signaling: roles for the cytoplasmic tails of αIIbβ3 in the tyrosine phosphorylation of pp125FAK. J. Cell Sci. 108:3817-3825. 310. Lerea, K. M., K. P. Cordero, K. S. Sakariassen, R. I. Kirk, and V. A. Fried. 1999. Phosphorylation sites in the integrin beta3 cytoplasmic domain in intact platelets. J. Biol. Chem. 274:1914-1919. 311. Leung-Hagesteijn, C. Y., K. Milankov, M. Michalak, J. Wilkins, and S. Dedhar. 1994. Cell attachment to extracellular matrix substrates is inhibited upon downregulation of expression of calreticulin, an intracellular integrin α-subunit-binding protein. J. Cell Sci. 107:589-600. 312. Levkau, B., B. Herren, H. Koyama, R. Ross, and E. W. Raines. 1998. Caspase-mediated cleavage of focal adhesion kinase pp125FAK and disassembly of focal adhesions in human endothelial cell apoptosis. J. Exp. Med. 187:579-586. 313. Levy, L., S. Broad, D. Diekmann, R. D. Evans, and F. M. Watt. 2000. beta1 integrins regulate keratinocyte adhesion and differentiation by distinct mechanisms. Mol. Biol. Cell 11:453-466. 314. Lewis, J. M., R. Baskaran, S. Taagepera, M. A. Schwartz, and J. Y. Wang. 1996. Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic- nuclear transport. Proc. Natl. Acad. Sci. U.S.A. 93:15174-15179. 315. Lewis, J. M., D. A. Cheresh, and M. A. Schwartz. 1996. Protein kinase C regulates αvβ5- dependent cytoskeletal associations and focal adhesion kinase phosphorylation. J. Cell Biol. 134:1323- 1332. 316. Lewis, J. M., and M. A. Schwartz. 1998. Integrins regulate the association and phosphorylation of paxillin by c- Abl. J. Biol. Chem. 273:14225-14230. 317. Lewis, J. M., and M. A. Schwartz. 1995. Mapping in vivo associations of cytoplasmic proteins with integrin β1 cytoplasmic domain mutants. Mol. Biol. Cell 6:151-160. 318. Lewis, T. S., P. S. Shapiro, and N. G. Ahn. 1998. Signal transduction through MAP kinase cascades. Adv Cancer Res 74:49-139. 319. Li, E., D. G. Stupack, S. L. Brown, R. Klemke, D. D. Schlaepfer, and G. R. Nemerow. 2000. Association of p130CAS with phosphatidylinositol-3-OH kinase mediates adenovirus cell entry. J. Biol. Chem. 275:14729-14735. 320. Li, F., Y. Zhang, and C. Wu. 1999. Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats. J. Cell Sci. 112:4589-4599. 321. Li, J., R. Mayne, and C. Wu. 1999. A novel muscle-specific beta 1 integrin binding protein (MIBP) that modulates myogenic differentiation. J. Cell Biol. 147:1391-1398. 322. Li, X., and H. S. Earp. 1997. Paxillin is tyrosine-phosphorylated by and preferentially associates with the calcium-dependent tyrosine kinase in rat liver epithelial cells. J. Biol. Chem. 272:14341- 14348. 323. Liliental, J., and D. D. Chang. 1998. Rack1, a receptor for activated protein kinase C, interacts with integrin β subunit. J. Biol. Chem. 273:2379-2383. 324. Lin, E. C. K., B. I. Ratnikov, P. M. Tsai, E. R. Gonzalez, S. McDonald, A. J. Pelletier, and J. W. Smith. 1997. Evidence that the integrin beta3 and beta5 subunits contain a metal ion- dependent adhesion site-like motif but lack an I domain. J. Biol. Chem. 272:14236-14243. 325. Lin, T. H., A. E. Aplin, Y. Shen, Q. Chen, M. Schaller, L. Romer, I. Aukhil, and R. L. Juliano. 1997. Integrin-mediated activation of MAP kinase is independent of FAK: evidence for dual integrin signaling pathways in fibroblasts. J. Cell Biol. 136:1385-1395.

77 326. Lindberg, F. P., H. D. Gresham, M. I. Reinhold, and E. J. Brown. 1996. Integrin-associated protein immunoglobulin domain is necessary for efficient vitronectin bead binding. J. Cell Biol. 134:1313-1322. 327. Lindberg, F. P., H. D. Gresham, E. Schwarz, and E. J. Brown. 1993. Molecular cloning of integrin-associated protein: an immunoglobulin family member with multiple membrane-spanning domains implicated in alpha v beta 3-dependent ligand binding. J. Cell Biol. 123:485-496. 328. Lipsky, B. P., C. R. Beals, and D. E. Staunton. 1998. Leupaxin is a novel LIM domain protein that forms a complex with PYK2. J. Biol. Chem. 273:11709-11713. 329. Litvak, V., D. Tian, Y. D. Shaul, and S. Lev. 2000. Targeting of PYK2 to focal adhesions as a cellular mechanism for convergence between integrins and GPCR signaling cascades. J. Biol. Chem. in press. 330. Liu, B. P., M. Chrzanowska-Wodnicka, and K. Burridge. 1998. Microtubule depolymerization induces stress fibers, focal adhesions, and DNA synthesis via the GTP-binding protein Rho. Cell Adhes. Commun. 5:249-255. 331. Liu, F., D. E. Hill, and J. Chernoff. 1996. Direct binding of the proline-rich region of protein tyrosine phosphatase 1B to the Src homology 3 domain of p130(Cas). J. Biol. Chem. 271:31290- 31295. 332. Liu, F., M. A. Sells, and J. Chernoff. 1998. Transformation suppression by protein tyrosine phosphatase 1B requires a functional SH3 ligand. Mol. Cell. Biol. 18:250-259. 333. Liu, L., P. Moesner, N. L. Kovach, R. Bailey, A. D. Hamilton, S. M. Sebti, and J. M. Harlan. 1999. Integrin-dependent leukocyte adhesion involves geranylgeranylated protein(s). J. Biol. Chem. 274:33334-33340. 334. Liu, S., and M. H. Ginsberg. 2000. Paxillin binding to a conserved sequence motif in the alpha 4 integrin cytoplasmic domain. J. Biol. Chem. 275:22736-22742. 335. Liu, S., S. M. Thomas, D. G. Woodside, D. M. Rose, W. B. Kiosses, M. Pfaff, and M. H. Ginsberg. 1999. Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses. Nature 402:676-681. 336. Longley, R. L., A. Woods, A. Fleetwood, G. J. Cowling, J. T. Gallagher, and J. R. Couchman. 1999. Control of morphology, cytoskeleton and migration by syndecan-4. J. Cell Sci. 112:3421-3431. 337. Loo, D. T., S. B. Kanner, and A. Aruffo. 1998. Filamin binds to the cytoplasmic domain of the β1- integrin. Identification of amino acids responsible for this interaction. J. Biol. Chem. 273:23304- 23312. 338. Lu, C., C. Oxvig, and T. A. Springer. 1998. The structure of the beta-propeller domain and C- terminal region of the integrin alphaM subunit. Dependence on beta subunit association and prediction of domains. J. Biol. Chem. 273:15138-15147. 339. Lu, C. F., and T. A. Springer. 1997. The alpha subunit cytoplasmic domain regulates the assembly and adhesiveness of integrin lymphocyte function-associated antigen-1. J. Immunol. 159:268-278. 340. Lu, Y., J. Brush, and T. A. Stewart. 1999. NSP1 defines a novel family of adaptor proteins linking integrin and tyrosine kinase receptors to the c-Jun N-terminal kinase/stress- activated protein kinase signaling pathway. J. Biol. Chem. 274:10047-10052. 341. Lub, M., S. J. van Vliet, S. P. Oomen, R. A. Pieters, M. Robinson, C. G. Figdor, and Y. van Kooyk. 1997. Cytoplasmic tails of beta 1, beta 2, and beta 7 integrins differentially regulate LFA-1 function in K562 cells. Mol. Biol. Cell 8:719-728. 342. Lukashev, M. E., D. Sheppard, and R. Pytela. 1994. Disruption of integrin function and induction of tyrosine phosphorylation by the autonomously expressed β1 integrin cytoplasmic domain. J. Biol. Chem. 269:18311-18314. 343. Lyman, S., A. Gilmore, K. Burridge, S. Gidwitz, and G. C. White, 2nd. 1997. Integrin-mediated activation of focal adhesion kinase is independent of focal adhesion formation or integrin activation. Studies with activated and inhibitory beta3 cytoplasmic domain mutants. J. Biol. Chem. 272:22538- 22547. 344. Lynch, D. K., C. A. Ellis, P. A. Edwards, and I. D. Hiles. 1999. Integrin-linked kinase regulates phosphorylation of serine 473 of protein kinase B by an indirect mechanism. Oncogene 18:8024-8032. 345. Machesky, L. M. 2000. Putting on the brakes: a negative regulatory function for Ena/VASP proteins in cell migration. Cell 101:685-688. 346. Machesky, L. M., and A. Hall. 1997. Role of actin polymerization and adhesion to extracellular matrix in Rac- and Rho-induced cytoskeletal reorganization. J. Cell Biol. 138:913-926. 347. Mackay, D. J., and A. Hall. 1998. Rho GTPases. J. Biol. Chem. 273:20685-20688. 348. Mainiero, F., C. Murgia, K. K. Wary, A. M. Curatola, A. Pepe, M. Blumemberg, J. K. Westwick, C. J. Der, and F. G. Giancotti. 1997. The coupling of alpha6beta4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation. EMBO J. 16:2365-2375. 349. Manes, S., E. Mira, C. Gomez-Mouton, Z. J. Zhao, R. A. Lacalle, and A. C. Martinez. 1999. Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility. Mol. Cell. Biol. 19:3125-3135.

78 350. Mangeat, P., C. Roy, and M. Martin. 1999. ERM proteins in cell adhesion and membrane dynamics. Trends Cell Biol. 9:187-192. 351. Mani, S. N., A. Astier, N. Haghayeghi, T. Canty, B. J. Druker, H. Hirai, and A. S. Freedman. 1997. Regulation of Integrin-mediated p130(Cas) Tyrosine Phosphorylation in Human B CellsA ROLE FOR p59(Fyn) AND SHP2. J. Biol. Chem. 272:15636-15641. 352. Manser, E., T. H. Loo, C. G. Koh, Z. S. Zhao, X. Q. Chen, L. Tan, I. Tan, T. Leung, and L. Lim. 1998. PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell 1:183-192. 353. Manzotti, M., P. Dell'Orto, P. Maisonneuve, M. Fornaro, L. R. Languino, and G. Viale. 2000. Down-regulation of beta(1C) integrin in breast carcinomas correlates with high proliferative fraction, high histological grade, and larger size. Am. J. Pathol. 156:169-174. 354. Martel, V., L. Vignoud, S. Dupe, P. Frachet, M. R. Block, and C. Albiges-Rizo. 2000. Talin controls the exit of the integrin alpha5 beta1 from an early compartment of the secretory pathway. J. Cell Sci. 113:1951-1961. 355. Matsuda, M., S. Ota, R. Tanimura, H. Nakamura, K. Matuoka, T. Takenawa, K. Nagashima, and T. Kurata. 1996. Interaction between the amino-terminal SH3 domain of CRK and its natural target proteins. J. Biol. Chem. 271:14468-14472. 356. Mazaki, Y., S. Hashimoto, and H. Sabe. 1997. Monocyte cells and cancer cells express novel paxillin isoforms with different binding properties to focal adhesion proteins. J. Biol. Chem. 272:7437-7444. 357. McGill, G., A. Shimamura, R. C. Bates, R. E. Savage, and D. E. Fisher. 1997. Loss of matrix adhesion triggers rapid transformation-selective apoptosis in fibroblasts. J. Cell Biol. 138:901-911. 358. McKay, B. S., D. S. Annis, S. Honda, D. Christie, and T. J. Kunicki. 1996. Molecular Requirements for Assembly and Function of a Minimized Human Integrin alphaIIbbeta3. J. Biol. Chem. 271:30544-30547. 359. McLean, G. W., V. J. Fincham, and M. C. Frame. 2000. v-Src induces tyrosine phosphorylation of focal adhesion kinase independently of tyrosine 397 and formation of a complex with Src. J. Biol. Chem. 275:23333-23339. 360. McNamee, H. P., H. G. Liley, and D. E. Ingber. 1996. Integrin-dependent control of inositol lipid synthesis in vascular endothelial cells and smooth muscle cells. Exp. Cell Res. 224:116-122. 361. Mehta, R. J., B. Diefenbach, A. Brown, E. Cullen, A. Jonczyk, D. Gussow, G. A. Luckenbach, and S. L. Goodman. 1998. Transmembrane-truncated alphavbeta3 integrin retains high affinity for ligand binding: evidence for an 'inside-out' suppressor? Biochem. J. 330:861-869. 362. Meredith, J., Jr., Z. Mu, T. Saido, and X. Du. 1998. Cleavage of the cytoplasmic domain of the integrin beta3 subunit during endothelial cell apoptosis. J. Biol. Chem. 273:19525-19531. 363. Meredith, J., Jr., Y. Takada, M. Fornaro, L. R. Languino, and M. A. Schwartz. 1995. Inhibition of cell cycle progression by the alternatively spliced integrin β1C. Science 269:1570-1572. 364. Miao, H., E. Burnett, M. Kinch, E. Simon, and B. Wang. 2000. Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation. Nat Cell Biol 2:62- 69. 365. Michalak, M., E. F. Corbett, N. Mesaeli, K. Nakamura, and M. Opas. 1999. Calreticulin: one protein, one gene, many functions. Biochem. J. 344 Pt 2:281-292. 366. Miki, H., T. Sasaki, Y. Takai, and T. Takenawa. 1998. Induction of filopodium formation by a WASP-related actin- depolymerizing protein N-WASP. Nature 391:93-96. 367. Miki, H., S. Suetsugu, and T. Takenawa. 1998. WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac. EMBO J. 17:6932-6941. 368. Minegishi, M., K. Tachibana, T. Sato, S. Iwata, Y. Nojima, and C. Morimoto. 1996. Structure and function of Cas-L, a 105-kD Crk-associated substrate- related protein that is involved in beta 1 integrin-mediated signaling in lymphocytes. J. Exp. Med. 184:1365-1375. 369. Miranti, C. K., S. Ohno, and J. S. Brugge. 1999. Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc. J. Biol. Chem. 274:10571- 10581. 370. Miyamoto, S., S. K. Akiyama, and K. M. Yamada. 1995. Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267:883-885. 371. Miyamoto, S., H. Teramoto, O. A. Coso, J. S. Gutkind, P. D. Burbelo, S. K. Akiyama, and K. M. Yamada. 1995. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J. Cell Biol. 131:791-805. 372. Miyamoto, S., H. Teramoto, J. S. Gutkind, and K. M. Yamada. 1996. Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors. J. Cell Biol. 135:1633-1642. 373. Mochizuki, N., Y. Ohba, S. Kobayashi, N. Otsuka, A. M. Graybiel, S. Tanaka, and M. Matsuda. 2000. Crk activation of JNK via C3G and R-Ras. J. Biol. Chem. 275:12667-12671.

79 374. Mochly-Rosen, D., and L. M. Kauvar. 2000. Pharmacological regulation of network kinetics by protein kinase C localization. Semin Immunol 12:55-61. 375. Mochly-Rosen, D., B. L. Smith, C. H. Chen, M. H. Disatnik, and D. Ron. 1995. Interaction of protein kinase C with RACK1, a receptor for activated C- kinase: a role in beta protein kinase C mediated signal transduction. Biochem. Soc. Trans. 23:596-600. 376. Moro, L., M. Venturino, C. Bozzo, L. Silengo, F. Altruda, L. Beguinot, G. Tarone, and P. Defilippi. 1998. Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival. EMBO J. 17:6622-6632. 377. Moss, J., and M. Vaughan. 1998. Molecules in the ARF orbit. J. Biol. Chem. 273:21431-21434. 378. Mould, A. P., S. K. Akiyama, and M. J. Humphries. 1995. Regulation of integrin α5β1- fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+. J. Biol. Chem. 270:26270-26277. 379. Mould, A. P., J. A. Askari, and M. J. Humphries. 2000. Molecular basis of ligand recognition by integrin alpha5 beta1. I. Specificity of ligand binding is determined by amino acid sequences in the second and third NH2-terminal repeats of the alpha subunit. J. Biol. Chem. 275:20324-20336. 380. Mould, A. P., L. Burrows, and M. J. Humphries. 1998. Identification of amino acid residues that form part of the ligand- binding pocket of integrin alpha5 beta1. J. Biol. Chem. 273:25664-25672. 381. Mould, A. P., A. N. Garratt, W. Puzon-McLaughlin, Y. Takada, and M. J. Humphries. 1998. Regulation of integrin function: evidence that bivalent-cation-induced conformational changes lead to the unmasking of ligand-binding sites within integrin alpha5 beta1. Biochem. J. 331:821-828. 382. Mueller, S. C., G. Ghersi, S. K. Akiyama, Q. X. Sang, L. Howard, M. Pineiro-Sanchez, H. Nakahara, Y. Yeh, and W. T. Chen. 1999. A novel protease-docking function of integrin at invadopodia. J. Biol. Chem. 274:24947-24952. 383. Munger, J. S., J. G. Harpel, F. G. Giancotti, and D. B. Rifkin. 1998. Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1. Mol. Biol. Cell 9:2627-2638. 384. Munger, J. S., X. Huang, H. Kawakatsu, M. J. Griffiths, S. L. Dalton, J. Wu, J. F. Pittet, N. Kaminski, C. Garat, M. A. Matthay, D. B. Rifkin, and D. Sheppard. 1999. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96:319-328. 385. Murakami, H., T. Iwashita, N. Asai, Y. Iwata, S. Narumiya, and M. Takahashi. 1999. Rho- dependent and -independent tyrosine phosphorylation of focal adhesion kinase, paxillin and p130Cas mediated by Ret kinase. Oncogene 18:1975-1982. 386. Nadal, F., S. Levy-Toledano, F. Grelac, J. P. Caen, J. P. Rosa, and M. Bryckaert. 1997. Negative regulation of mitogen-activated protein kinase activation by integrin alphaIIbbeta3 in platelets. J. Biol. Chem. 272:22381-22384. 387. Nagel, W., L. Zeitlmann, P. Schilcher, C. Geiger, J. Kolanus, and W. Kolanus. 1998. Phosphoinositide 3-OH kinase activates the β2 integrin adhesion pathway and induces membrane recruitment of cytohesin-1. J. Biol. Chem. 273:14853-14861. 388. Naik, U. P., P. M. Patel, and L. V. Parise. 1997. Identification of a novel calcium-binding protein that interacts with the integrin αIIb cytoplasmic domain. J. Biol. Chem. 272:4651-4654. 389. Nakahara, H., S. C. Mueller, M. Nomizu, Y. Yamada, Y. Yeh, and W. T. Chen. 1998. Activation of beta1 integrin signaling stimulates tyrosine phosphorylation of p190RhoGAP and membrane-protrusive activities at invadopodia. J. Biol. Chem. 273:9-12. 390. Nakamoto, T., R. Sakai, K. Ozawa, Y. Yazaki, and H. Hirai. 1996. Direct binding of C-terminal region of p130Cas to SH2 and SH3 domains of Src kinase. J. Biol. Chem. 271:8959-8965. 391. Nakamura, K., H. Yano, H. Uchida, S. Hashimoto, E. Schaefer, and H. Sabe. 2000. Tyrosine phosphorylation of paxillin alpha is involved in temporospatial regulation of paxillin-containing focal adhesion formation and F-actin organization in motile cells. J. Biol. Chem. 275:27155-27164. 392. Ng, T., D. Shima, A. Squire, P. I. Bastiaens, S. Gschmeissner, M. J. Humphries, and P. J. Parker. 1999. PKCalpha regulates beta1 integrin-dependent cell motility through association and control of integrin traffic. EMBO J. 18:3909-3923. 393. Ni, H., A. Li, N. Simonsen, and J. A. Wilkins. 1998. Integrin activation by dithiothreitol or Mn2+ induces a ligand-occupied conformation and exposure of a novel NH2-terminal regulatory site on the beta1 integrin chain. J. Biol. Chem. 273:7981-7987. 394. Nievers, M. G., R. B. Birge, H. Greulich, A. J. Verkleij, H. Hanafusa, and P. M. van Bergen en Henegouwen. 1997. v-Crk-induced cell transformation: changes in focal adhesion composition and signaling. J. Cell Sci. 110:389-399. 395. Nishiya, N., H. Sabe, K. Nose, and M. Shibanuma. 1998. The LIM domains of hic-5 protein recognize specific DNA fragments in a zinc-dependent manner in vitro. Nucleic Acids Res. 26:4267- 4273.

80 396. Nix, D. A., and M. C. Beckerle. 1997. Nuclear-cytoplasmic shuttling of the focal contact protein, zyxin: a potential mechanism for communication between sites of cell adhesion and the nucleus. J. Cell Biol. 138:1139-1147. 397. Nobes, C. D., and A. Hall. 1999. Rho GTPases control polarity, protrusion, and adhesion during cell movement. J. Cell Biol. 144:1235-1244. 398. Nobes, C. D., and A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53-62. 399. Nobes, C. D., I. Lauritzen, M. G. Mattei, S. Paris, A. Hall, and P. Chardin. 1998. A new member of the Rho family, Rnd1, promotes disassembly of actin filament structures and loss of cell adhesion. J. Cell Biol. 141:187-197. 400. Nojima, Y., N. Morino, T. Mimura, K. Hamasaki, H. Furuya, R. Sakai, T. Sato, K. Tachibana, C. M orimoto, Y. Yazaki, and et al. 1995. Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130Cas, a Src homology 3-containing molecule having multiple Src homology 2-binding motifs. J. Biol. Chem. 270:15398-15402. 401. Nolan, K., J. Lacoste, and J. T. Parsons. 1999. Regulated expression of focal adhesion kinase- related nonkinase, the autonomously expressed C-terminal domain of focal adhesion kinase. Mol. Cell. Biol. 19:6120-6129. 402. Nolan, K. M., K. Barrett, Y. Lu, K. Q. Hu, S. Vincent, and J. Settleman. 1998. Myoblast city, the Drosophila homolog of DOCK180/CED-5, is required in a Rac signaling pathway utilized for multiple developmental processes. Genes Dev. 12:3337-3342. 403. Nolte, M., R. B. Pepinsky, S. Venyaminov, V. Koteliansky, P. J. Gotwals, and M. Karpusas. 1999. Crystal structure of the alpha1beta1 integrin I-domain: insights into integrin I-domain function. FEBS Lett. 452:379-385. 404. Norman, J. C., D. Jones, S. T. Barry, M. R. Holt, S. Cockcroft, and D. R. Critchley. 1998. ARF1 mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts. J. Cell Biol. 143:1981-1995. 405. Norris, F. A., R. C. Atkins, and P. W. Majerus. 1997. Inositol polyphosphate 4-phosphatase is inactivated by calpain-mediated proteolysis in stimulated human platelets. J. Biol. Chem. 272:10987- 10989. 406. Noti, J. D., B. C. Reinemann, and M. N. Petrus. 1996. Sp1 binds two sites in the CD11c promoter in vivo specifically in myeloid cells and cooperates with AP1 to activate transcription. Mol. Cell. Biol. 16:2940-2950. 407. O'Neill, G. M., S. J. Fashena, and E. A. Golemis. 2000. Integrin signalling: a new Cas(t) of characters enters the stage. Trends Cell Biol. 10:111-119. 408. O'Neill, S., A. Robinson, A. Deering, M. Ryan, D. F. Fitzgerald, and N. Moran. 2000. The platelet integrin alphaIIbbeta3 has an endogenous thiol isomerase activity. J. Biol. Chem. in press. 409. O'Toole, T. E., Y. Katagiri, R. J. Faull, K. Peter, R. Tamura, V. Quaranta, J. C. Loftus, S. J. Shattil, and M. H. Ginsberg. 1994. Integrin cytoplasmic domains mediate inside-out signal transduction. J. Cell Biol. 124:1047-1059. 410. O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Modulation of the affinity of integrin αIIbβ3 (GPIIb-IIIa) by the cytoplasmic domain of αIIb. Science 254:845-847. 411. Obermeier, A., S. Ahmed, E. Manser, S. C. Yen, C. Hall, and L. Lim. 1998. PAK promotes morphological changes by acting upstream of Rac. EMBO J. 17:4328-4339. 412. Oh, E. S., H. Gu, T. M. Saxton, J. F. Timms, S. Hausdorff, E. U. Frevert, B. B. Kahn, T. Pawson, B. G. Neel, and S. M. Thomas. 1999. Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2. Mol. Cell. Biol. 19:3205-3215. 413. Oh, W. K., J. C. Yoo, D. Jo, Y. H. Song, M. G. Kim, and D. Park. 1997. Cloning of a SH3 domain-containing proline-rich protein, p85SPR, and its localization in focal adhesion. Biochem. Biophys. Res. Commun. 235:794-798. 414. Ohmori, T., Y. Yatomi, K. Inoue, K. Satoh, and Y. Ozaki. 2000. Tyrosine dephosphorylation, but not phosphorylation, of p130Cas is dependent on integrin alpha IIb beta 3-mediated aggregation in platelets: implication of p130Cas involvement in pathways unrelated to cytoskeletal reorganization. Biochemistry 39:5797-5807. 415. Ohtoshi, A., T. Maeda, H. Higashi, S. Ashizawa, M. Yamada, and M. Hatakeyama. 2000. beta3-endonexin as a novel inhibitor of cyclin A-associated kinase. Biochem. Biophys. Res. Commun. 267:947-952. 416. Ojaniemi, M., S. S. Martin, F. Dolfi, J. M. Olefsky, and K. Vuori. 1997. The proto-oncogene product p120(cbl) links c-Src and phosphatidylinositol 3'-kinase to the integrin signaling pathway. J. Biol. Chem. 272:3780-3787.

81 417. Okada, S., M. Matsuda, M. Anafi, T. Pawson, and J. E. Pessin. 1998. Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes. EMBO J. 17:2554-2565. 418. Oktay, M., K. K. Wary, M. Dans, R. B. Birge, and F. G. Giancotti. 1999. Integrin-mediated activation of focal adhesion kinase is required for signaling to Jun NH2-terminal kinase and progression through the G1 phase of the cell cycle. J. Cell Biol. 145:1461-1469. 419. Olson, M. F., A. Ashworth, and A. Hall. 1995. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science 269:1270-1272. 420. Ostergaard, H. L., O. Lou, C. W. Arendt, and N. N. Berg. 1998. Paxillin phosphorylation and association with Lck and Pyk2 in anti-CD3- or anti-CD45-stimulated T cells. J. Biol. Chem. 273:5692-5696. 421. Otey, C. A., F. M. Palavalko, and K. Burridge. 1990. An interaction between α−actinin and the β1 integrin subunit in vitro. J. Cell Biol. 111:721-729. 422. Otey, C. A., G. B. Vasquez, K. Burridge, and B. W. Erickson. 1993. Mapping of the α-actinin binding site within the β1 integrin cytoplasmic domain. J. Biol. Chem. 268:21193-21197. 423. Owen, J. D., P. J. Ruest, D. W. Fry, and S. K. Hanks. 1999. Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2. Mol. Cell. Biol. 19:4806-4818. 424. Oxvig, C., and T. A. Springer. 1998. Experimental support for a beta-propeller domain in integrin alpha- subunits and a calcium binding site on its lower surface. Proc. Natl. Acad. Sci. U.S.A. 95:4870- 4875. 425. Pardi, R., G. Bossi, L. Inverardi, E. Rovida, and J. R. Bender. 1995. Conserved regions in the cytoplasmic domains of the leukocyte beta 2 are involved in endoplasmic reticulum retention, dimerization, and cytoskeletal association. J. Immunol. 155:1252-1263. 426. Parise, L. V., A. B. Criss, L. Nannizzi, and M. R. Wardell. 1990. Glycoprotein IIIa is phosphorylated in intact human platelets. Blood 75:2363-2368. 427. Parodi, A. J. 2000. Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation. Biochem. J. 348 Pt 1:1-13. 428. Pasqualini, R., and M. E. Hemler. 1994. Contrasting roles for and beta 5 cytoplasmic domains in subcellular localization, cell proliferation, and cell migration. J. Cell Biol. 125:447-460. 429. Patel, A. S., G. L. Schechter, W. J. Wasilenko, and K. D. Somers. 1998. Overexpression of EMS1/cortactin in NIH3T3 fibroblasts causes increased cell motility and invasion in vitro. Oncogene 16:3227-3232. 430. Patil, S., A. Jedsadayanmata, J. D. Wencel-Drake, W. Wang, I. Knezevic, and S. C. Lam. 1999. Identification of a talin-binding site in the integrin beta(3) subunit distinct from the NPLY regulatory motif of post-ligand binding functions. The talin n-terminal head domain interacts with the membrane- proximal region of the beta(3) cytoplasmic tail. J. Biol. Chem. 274:28575-28583. 431. Pelletier, A. J., T. Kunicki, and V. Quaranta. 1996. Activation of the integrin alpha v beta 3 involves a discrete cation- binding site that regulates conformation. J. Biol. Chem. in press. 432. Penninger, J. M., and G. R. Crabtree. 1999. The actin cytoskeleton and lymphocyte activation. Cell 96:9-12. 433. Persad, S., S. Attwell, V. Gray, M. Delcommenne, A. Troussard, J. Sanghera, and S. Dedhar. 2000. Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells. Proc. Natl. Acad. Sci. U.S.A. 97:3207-3212. 434. Peter, K., and T. E. O'Toole. 1995. Modulation of cell adhesion by changes in alpha L beta 2 (LFA- 1, CD11a/CD18) cytoplasmic domain/cytoskeleton interaction. J. Exp. Med. 181:315-326. 435. Petit, M. M., J. Fradelizi, R. M. Golsteyn, T. A. Ayoubi, B. Menichi, D. Louvard, W. J. Van de Ven, and E. Friederich. 2000. LPP, an actin cytoskeleton protein related to zyxin, harbors a nuclear export signal and transcriptional activation capacity. Mol. Biol. Cell 11:117-129. 436. Petit, V., B. Boyer, D. Lentz, C. E. Turner, J. P. Thiery, and A. M. Valles. 2000. Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells. J. Cell Biol. 148:957-970. 437. Pfaff, M., X. Du, and M. H. Ginsberg. 1999. Calpain cleavage of integrin beta cytoplasmic domains. FEBS Lett. 460:17-22. 438. Pfaff, M., S. Liu, D. J. Erle, and M. H. Ginsberg. 1998. Integrin β cytoplasmic domains differentially bind to cytoskeletal proteins. J. Biol. Chem. 273:6104-6109. 439. Plow, E. F., T. A. Haas, L. Zhang, J. Loftus, and J. W. Smith. 2000. Ligand binding to integrins. J. Biol. Chem. 275:21785-21788. 440. Polte, T. R., and S. K. Hanks. 1997. Complexes of focal adhesion kinase (FAK) and Crk-associated substrate (p130(Cas)) are elevated in cytoskeleton-associated fractions following adhesion and Src

82 transformation. Requirements for Src kinase activity and FAK proline-rich motifs. J. Biol. Chem. 272:5501-5509. 441. Polte, T. R., and S. K. Hanks. 1995. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas. Proc. Natl. Acad. Sci. U.S.A. 92:10678-10682. 442. Pontremoli, S., E. Melloni, B. Sparatore, M. Michetti, F. Salamino, and B. L. Horecker. 1990. Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases. J. Biol. Chem. 265:706-712. 443. Pontremoli, S., M. Michetti, E. Melloni, B. Sparatore, F. Salamino, and B. L. Horecker. 1990. Identification of the proteolytically activated form of protein kinase C in stimulated human neutrophils. Proc. Natl. Acad. Sci. U.S.A. 87:3705-3707. 444. Porter, J. C., and N. Hogg. 1997. Integrin cross talk: activation of lymphocyte function-associated antigen-1 on human T cells alters alpha4beta1- and alpha5beta1-mediated function. J. Cell Biol. 138:1437-1447. 445. Posern, G., C. K. Weber, U. R. Rapp, and S. M. Feller. 1998. Activity of Rap1 is regulated by bombesin, cell adhesion, and cell density in NIH3T3 fibroblasts. J. Biol. Chem. 273:24297-24300. 446. Price, L. S., J. Leng, M. A. Schwartz, and G. M. Bokoch. 1998. Activation of rac and cdc42 by integrins mediates cell spreading. Mol. Biol. Cell 9:1863-1871. 447. Priddle, H., L. Hemmings, S. Monkley, A. Woods, B. Patel, D. Sutton, G. A. Dunn, D. Zicha, and D. R. Critchley. 1998. Disruption of the talin gene compromises focal adhesion assembly in undifferentiated but not differentiated embryonic stem cells. J. Cell Biol. 142:1121-1133. 448. Puzon-McLaughlin, W., and Y. Takada. 1996. Critical residues for ligand binding in an I domain- like structure of the integrin beta1 subunit. J. Biol. Chem. 271:20438-20443. 449. Qi, J. H., N. Ito, and L. Claesson-Welsh. 1999. Tyrosine phosphatase SHP-2 is involved in regulation of platelet- derived growth factor-induced migration. J. Biol. Chem. 274:14455-14463. 450. Qu, A., and D. J. Leahy. 1995. Crystal structure of the I-domain from the CD11a/CD18 (LFA-1, αLβ2) integrin. Proc. Natl. Acad. Sci. U.S.A. 92:10277-10281. 451. Radeva, G., T. Petrocelli, E. Behrend, C. Leung-Hagesteijn, J. Filmus, J. Slingerland, and S. Dedhar. 1997. Overexpression of the integrin-linked kinase promotes anchorage-independent cell cycle progression. J. Biol. Chem. 272:13937-13944. 452. Rameh, L. E., and L. C. Cantley. 1999. The role of phosphoinositide 3-kinase lipid products in cell function. J. Biol. Chem. 274:8347-8350. 453. Ramesh, N., I. M. Anton, N. Martinez-Quiles, and R. S. Geha. 1999. Waltzing with WASP. Trends Cell Biol. 9:15-19. 454. Ramos, J. W., T. K. Kojima, P. E. Hughes, C. A. Fenczik, and M. H. Ginsberg. 1998. The death effector domain of PEA-15 is involved in its regulation of integrin activation. J. Biol. Chem. 273:33897-33900. 455. Reddien, P. W., and H. R. Horvitz. 2000. CED-2/CrkII and CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans. Nat Cell Biol 2:131-136. 456. Reddy, K. B., P. Gascard, M. G. Price, E. V. Negrescu, and J. E. B. Fox. 1998. Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells. J. Biol. Chem. 273:35039- 35047. 457. Redick, S. D., D. L. Settles, G. Briscoe, and H. P. Erickson. 2000. Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis. J. Cell Biol. 149:521-527. 458. Reedquist, K. A., E. Ross, E. A. Koop, R. M. Wolthuis, F. J. Zwartkruis, Y. van Kooyk, M. Salmon, C. D. Buckley, and J. L. Bos. 2000. The small GTPase, Rap1, mediates CD31-induced integrin adhesion. J. Cell Biol. 148:1151-1158. 459. Reinhard, M., J. Zumbrunn, D. Jaquemar, M. Kuhn, U. Walter, and B. Trueb. 1999. An alpha-actinin binding site of zyxin is essential for subcellular zyxin localization and alpha-actinin recruitment. J. Biol. Chem. 274:13410-13418. 460. Reiske, H. R., S. C. Kao, L. A. Cary, J. L. Guan, J. F. Lai, and H. C. Chen. 1999. Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase- promoted cell migration. J. Biol. Chem. 274:12361-12366. 461. Ren, X. D., W. B. Kiosses, and M. A. Schwartz. 1999. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18:578-585. 462. Renshaw, M. W., L. S. Price, and M. A. Schwartz. 1999. Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase. J. Cell Biol. 147:611-618. 463. Renshaw, M. W., D. Toksoz, and M. A. Schwartz. 1996. Involvement of the small GTPase rho in integrin-mediated activation of mitogen-activated protein kinase. J. Biol. Chem. 271:21691-21694. 464. Retta, S. F., F. Balzac, P. Ferraris, A. M. Belkin, R. Fässler, M. J. Humphries, G. De Leo, L. Silengo, and G. Tarone. 1998. β1-integrin cytoplasmic subdomains involved in dominant negative function. Mol. Biol. Cell 9:715-731.

83 465. Reuther, G. W., and C. J. Der. 2000. The Ras branch of small GTPases: Ras family members don't fall far from the tree. Curr. Opin. Cell Biol. 12:157-165. 466. Rezniczek, G. A., J. M. de Pereda, S. Reipert, and G. Wiche. 1998. Linking integrin alpha6beta4- based cell adhesion to the intermediate filament cytoskeleton: direct interaction between the beta4 subunit and plectin at multiple molecular sites. J. Cell Biol. 141:209-225. 467. Richardson, A., R. K. Malik, J. D. Hildebrand, and J. T. Parsons. 1997. Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation. Mol. Cell. Biol. 17:6906-6914. 468. Richardson, A., and T. Parsons. 1996. A mechanism for regulation of the adhesion-associated proteintyrosine kinase pp125FAK. Nature 380:538-540. 469. Richardson, A., J. D. Shannon, R. B. Adams, M. D. Schaller, and J. Parsons. 1997. Identification of integrin-stimulated sites of serine phosphorylation in FRNK, the separately expressed C-terminal domain of focal adhesion kinase: a potential role for protein kinase A. Biochem. J. 324:141- 149. 470. Ridley, A. J., and A. Hall. 1994. Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase. EMBO J. 13:2600-2610. 471. Rietzler, M., M. Bittner, W. Kolanus, A. Schuster, and B. Holzmann. 1998. The human WD repeat protein WAIT-1 specifically interacts with the cytoplasmic tails of beta7-integrins. J. Biol. Chem. 273:27459-27466. 472. Rigot, V., F. Andre, M. Lehmann, J. C. Lissitzky, J. Marvaldi, and J. Luis. 1999. Biogenesis of alpha6beta4 integrin in a human colonic adenocarcinoma cell line involvement of calnexin. Eur. J. Biochem. 261:659-666. 473. Rigot, V., M. Lehmann, F. Andre, N. Daemi, J. Marvaldi, and J. Luis. 1998. Integrin ligation and PKC activation are required for migration of colon carcinoma cells. J. Cell Sci. 111:3119-3127. 474. Rock, M. T., W. H. Brooks, and T. L. Roszman. 1997. Calcium-dependent signaling pathways in T cells. Potential role of calpain, protein tyrosine phosphatase 1b, and p130Cas in integrin- mediated signaling events. J. Biol. Chem. 272:33377-33383. 475. Rodriguez-Fernandez, J. L. 1999. Why do so many stimuli induce tyrosine phosphorylation of FAK? Bioessays 21:1069-1075. 476. Rodriguez-Fernandez, J. L., M. Gomez, A. Luque, N. Hogg, F. Sanchez-Madrid, and C. Cabanas. 1999. The interaction of activated integrin lymphocyte function-associated antigen 1 with ligand intercellular adhesion molecule 1 induces activation and redistribution of focal adhesion kinase and proline-rich tyrosine kinase 2 in T lymphocytes. Mol. Biol. Cell 10:1891-1907. 477. Ron, D., Z. Jiang, L. Yao, A. Vagts, I. Diamond, and A. Gordon. 1999. Coordinated movement of RACK1 with activated betaIIPKC. J. Biol. Chem. 274:27039-27046. 478. Ron, D., J. Luo, and D. Mochly-Rosen. 1995. C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo. J. Biol. Chem. 270:24180-24187. 479. Rosato, R., J. M. Veltmaat, J. Groffen, and N. Heisterkamp. 1998. Involvement of the tyrosine kinase fer in cell adhesion. Mol. Cell. Biol. 18:5762-5770. 480. Rosfjord, E. C., M. Maemura, M. D. Johnson, J. A. Torri, S. K. Akiyama, V. L. Woods, Jr., and R. B. Dickson. 1999. Activation of protein kinase C by phorbol esters modulates alpha2beta1 integrin on MCF-7 breast cancer cells. Exp. Cell Res. 248:260-271. 481. Rosmarin, A. G., R. Levy, and D. G. Tenen. 1992. Cloning and analysis of the CD18 promoter. Blood 79:2598-2604. 482. Rubinstein, E., V. Poindessous-Jazat, F. Le Naour, M. Billard, and C. Boucheix. 1997. CD9, but not other tetraspans, associates with the beta1 integrin precursor. Eur. J. Immunol. 27:1919-1927. 483. Saher, G., and E. Hildt. 1999. Activation of c-Raf-1 kinase signal transduction pathway in alpha7 integrin-deficient mice. J. Biol. Chem. 274:27651-27657. 484. Sakai, R., A. Iwamatsu, N. Hirano, S. Ogawa, T. Tanaka, H. Mano, Y. Yazaki, and H. Hirai. 1994. A novel signaling molecule, p130, forms stable complexes in vivo with v- Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J. 13:3748-3756. 485. Sakai, T., Q. Zhang, R. Fässler, and D. F. Mosher. 1998. Modulation of β1A integrin functions by tyrosine residues in the β1 cytoplasmic domain. J. Cell Biol. 141:527-538. 486. Sakakibara, A., and S. Hattori. 2000. Chat, a Cas/HEF1-associated adaptor protein that integrates multiple signaling pathways. J. Biol. Chem. 275:6404-6410. 487. Salgia, R., S. Avraham, E. Pisick, J. L. Li, S. Raja, E. A. Greenfield, M. Sattler, H. Avraham, and J. D. Griffin. 1996. The related adhesion focal tyrosine kinase forms a complex with paxillin in hematopoietic cells. J. Biol. Chem. 271:31222-31226. 488. Salgia, R., J. L. Li, S. H. Lo, B. Brunkhorst, G. S. Kansas, E. S. Sobhany, Y. Sun, E. Pisick, M. Hallek, T. Ernst, and et al. 1995. Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL. J. Biol. Chem. 270:5039-5047.

84 489. Sampath, R., P. J. Gallagher, and F. M. Pavalko. 1998. Cytoskeletal interactions with the leukocyte integrin β2 cytoplasmic tail. Activation-dependent regulation of associations with talin and α-actinin. J. Biol. Chem. 273:33588-33594. 490. Sander, E. E., J. P. ten Klooster, S. van Delft, R. A. van der Kammen, and J. G. Collard. 1999. Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J. Cell Biol. 147:1009-1022. 491. Saoncella, S., F. Echtermeyer, F. Denhez, J. K. Nowlen, D. F. Mosher, S. D. Robinson, R. O. Hynes, and P. F. Goetinck. 1999. Syndecan-4 signals cooperatively with integrins in a Rho- dependent manner in the assembly of focal adhesions and actin stress fibers. Proc. Natl. Acad. Sci. U.S.A. 96:2805-2810. 492. Sarkar, S., M. M. Rooney, and S. T. Lord. 1999. Activation of integrin-beta3-associated syk in platelets. Biochem. J. 338:677-680. 493. Sasaki, H., K. Nagura, M. Ishino, H. Tobioka, K. Kotani, and T. Sasaki. 1995. Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. J. Biol. Chem. 270:21206-21219. 494. Sasaki, T., and Y. Takai. 1998. The Rho small G protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control. Biochem. Biophys. Res. Commun. 245:641-645. 495. Sastry, S. K., M. Lakonishok, D. A. Thomas, J. Muschler, and A. F. Horwitz. 1996. Integrin α subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation. J. Cell Biol. 133:169-184. 496. Schaapveld, R. Q., L. Borradori, D. Geerts, M. R. van Leusden, I. Kuikman, M. G. Nievers, C. M. Niessen, R. D. Steenbergen, P. J. Snijders, and A. Sonnenberg. 1998. Hemidesmosome formation is initiated by the beta4 integrin subunit, requires complex formation of beta4 and HD1/plectin, and involves a direct interaction between beta4 and the bullous pemphigoid antigen 180. J. Cell Biol. 142:271-284. 497. Schaller, M. D., C. A. Borgman, B. S. Cobb, R. R. Vines, A. B. Reynolds, and J. T. Parsons. 1992. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc. Natl. Acad. Sci. U.S.A. 89:5192-5196. 498. Schaller, M. D., J. D. Hildebrand, and J. T. Parsons. 1999. Complex formation with focal adhesion kinase: A mechanism to regulate activity and subcellular localization of Src kinases. Mol. Biol. Cell 10:3489-3505. 499. Schaller, M. D., J. D. Hildebrand, J. D. Shannon, J. W. Fox, R. R. Vines, and J. T. Parsons. 1994. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2- dependent binding of pp60src. Mol. Cell. Biol. 14:1680-1688. 500. Schaller, M. D., C. A. Otey, J. D. Hildebrand, and J. T. Parsons. 1995. Focal adhesion kinase and paxillin bind to peptides mimicking β integrin cytoplasmic domains. J. Cell Biol. 130:1181-1187. 501. Schlaepfer, D. D., and T. Hunter. 1996. Evidence for in vivo phosphorylation of the Grb2 SH2- domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases. Mol. Cell. Biol. 16:5623-5633. 502. Schlaepfer, D. D., and T. Hunter. 1997. Focal adhesion kinase overexpression enhances ras- dependent integrin signaling to ERK2/Mitogen-activated protein kinase through interactions with and activation of c-Src. J. Biol. Chem. 272:13189-13195. 503. Schlaepfer, D. D., K. C. Jones, and T. Hunter. 1998. Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol. Cell. Biol. 18:2571-2585. 504. Schneller, M., K. Vuori, and E. Ruoslahti. 1997. αvβ3 integrin associates with activated insulin and PDGFbeta receptors and potentiates the biological activity of PDGF. EMBO J. 16:5600-5607. 505. Schoenwaelder, S. M., and K. Burridge. 1999. Bidirectional signaling between the cytoskeleton and integrins. Curr. Opin. Cell Biol. 11:274-286. 506. Schoenwaelder, S. M., Y. Yuan, P. Cooray, H. H. Salem, and S. P. Jackson. 1997. Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin alphaIIbbeta3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots. J. Biol. Chem. 272:1694-1702. 507. Schuebel, K. E., N. Movilla, J. L. Rosa, and X. R. Bustelo. 1998. Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2. EMBO J. 17:6608-6621. 508. Schwartz, M. A., D. Toksoz, and R. Khosravi-Far. 1996. Transformation by Rho exchange factor oncogenes is mediated by activation of an integrin-dependent pathway. EMBO J. 15:6525-6530. 509. Sells, M. A., J. T. Boyd, and J. Chernoff. 1999. p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts. J. Cell Biol. 145:837-849. 510. Serru, V., F. Le Naour, M. Billard, D. O. Azorsa, F. Lanza, C. Boucheix, and E. Rubinstein. 1999. Selective tetraspan-integrin complexes (CD81/alpha4beta1, CD151/alpha3beta1, CD151/alpha6beta1) under conditions disrupting tetraspan interactions. Biochem. J. 340:103-111.

85 511. Sethi, T., M. H. Ginsberg, J. Downward, and P. E. Hughes. 1999. The small GTP-binding protein R-Ras can influence integrin activation by antagonizing a Ras/Raf-initiated integrin suppression pathway. Mol. Biol. Cell 10:1799-1809. 512. Shattil, S. J., T. O'Toole, M. Eigenthaler, V. Thon, M. Williams, B. M. Babior, and M. H. Ginsberg. 1995. β3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin β3 subunit. J. Cell Biol. 131:807-816. 513. Shaw, L. M., and A. M. Mercurio. 1993. Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit. J. Cell Biol. 123:1017-1025. 514. Shen, Y., P. Lyons, M. Cooley, D. Davidson, A. Veillette, R. Salgia, J. D. Griffin, and M. D. Schaller. 2000. The noncatalytic domain of protein-tyrosine phosphatase-PEST targets paxillin for dephosphorylation in vivo. J. Biol. Chem. 275:1405-1413. 515. Shen, Y., and M. D. Schaller. 1999. Focal adhesion targeting: the critical determinant of FAK regulation and substrate phosphorylation. Mol. Biol. Cell 10:2507-2518. 516. Shen, Y., G. Schneider, J. F. Cloutier, A. Veillette, and M. D. Schaller. 1998. Direct association of protein-tyrosine phosphatase PTP-PEST with paxillin. J. Biol. Chem. 273:6474-6481. 517. Shibanuma, M., K. Ishino, N. Sakamoto, J. Kim-Kaneyama, and K. Nose. 2000. Accumulation of Hic-5, a focal adhesion protein, in the nucleus in response to hydrogen peroxide and its is involvement in gene expression., p. pp 75. Keystone Symposium, Joint Regulation of Signaling Pathways by Integrins and Growth Factors, Breckenridge, Colorado. 518. Shibayama, H., N. Anzai, S. E. Braun, S. Fukuda, C. Mantel, and H. E. Broxmeyer. 1999. H- Ras is involved in the inside-out signaling pathway of interleukin-3- induced integrin activation. Blood 93:1540-1548. 519. Shields, J. M., K. Pruitt, A. McFall, A. Shaub, and C. J. Der. 2000. Understanding Ras: 'it ain't over 'til it's over'. Trends Cell Biol. 10:147-154. 520. Shimizu, Y., G. A. Van Seventer, K. J. Horgan, and S. Shaw. 1990. Regulated expression and binding of three VLA (beta 1) integrin receptors on T cells. Nature 345:250-253. 521. Shock, D. D., U. P. Naik, J. E. Brittain, S. K. Alahari, J. Sondek, and L. V. Parise. 1999. Calcium-dependent properties of CIB binding to the integrin alphaIIb cytoplasmic domain and translocation to the platelet cytoskeleton. Biochem. J. 342 Pt 3:729-735. 522. Short, S. M., J. L. Boyer, and R. L. Juliano. 2000. Integrins regulate the linkage between upstream and downstream events in G protein-coupled receptor signaling to mitogen-activated protein kinase. J. Biol. Chem. 275:12970-12977. 523. Short, S. M., G. A. Talbott, and R. L. Juliano. 1998. Integrin-mediated signaling events in human endothelial cells. Mol. Biol. Cell 9:1969-1980. 524. Shou, Y., S. Baron, and M. Poncz. 1998. An Sp1-binding silencer element is a critical negative regulator of the megakaryocyte-specific alphaIIb gene. J. Biol. Chem. 273:5716-5726. 525. Sieg, D. J., C. R. Hauck, D. Ilic, C. K. Klingbeil, E. Schaefer, C. H. Damsky, and D. D. Schlaepfer. 2000. FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2:249-256. 526. Sieg, D. J., C. R. Hauck, and D. D. Schlaepfer. 1999. Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J. Cell Sci. 112:2677-2691. 527. Sieg, D. J., D. Ilic, K. C. Jones, C. H. Damsky, T. Hunter, and D. D. Schlaepfer. 1998. Pyk2 and src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but pyk2 does not fully function to enhance FAK- cell migration. EMBO J. 17:5933- 5947. 528. Simon, D. I., Y. Wei, L. Zhang, N. K. Rao, H. Xu, Z. Chen, Q. Liu, S. Rosenberg, and H. A. Chapman. 2000. Identification of a -integrin interaction site. Promiscuous regulator of integrin function. J. Biol. Chem. 275:10228-10234. 529. Simon, K. O., E. M. Nutt, D. G. Abraham, G. A. Rodan, and L. T. Duong. 1997. The alphavbeta3 integrin regulates alpha5beta1-mediated cell migration toward fibronectin. J. Biol. Chem. 272:29380-29389. 530. Sjaastad, M. D., R. S. Lewis, and W. J. Nelson. 1996. Mechanisms of integrin-mediated calcium signaling in MDCK cells: regulation of adhesion by IP3- and store-independent calcium influx. Mol. Biol. Cell 7:1025-1041. 531. Skinner, M. A., and A. G. Wildeman. 1999. beta(1) integrin binds the 16-kDa subunit of vacuolar H(+)-ATPase at a site important for human papillomavirus E5 and platelet-derived growth factor signaling. J. Biol. Chem. 274:23119-23127. 532. Smilenov, L. B., A. Mikhailov, R. J. Pelham, E. E. Marcantonio, and G. G. Gundersen. 1999. Focal adhesion motility revealed in stationary fibroblasts. Science 286:1172-1174. 533. Smith, J. W., R. S. Piotrowicz, and D. Mathis. 1994. A mechanism for divalent cation regulation of beta 3-integrins. J. Biol. Chem. 269:960-967.

86 534. Soldi, R., S. Mitola, M. Strasly, P. Defilippi, G. Tarone, and F. Bussolino. 1999. Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J. 18:882-892. 535. Solowska, J., J. M. Edelman, S. M. Albelda, and C. A. Buck. 1991. Cytoplasmic and transmembrane domains of integrin beta 1 and beta 3 subunits are functionally interchangeable. J. Cell Biol. 114:1079-1088. 536. Somani, A. K., J. S. Bignon, G. B. Mills, K. A. Siminovitch, and D. R. Branch. 1997. Src kinase activity is regulated by the SHP-1 protein-tyrosine phosphatase. J. Biol. Chem. 272:21113- 21119. 537. Sonoda, Y., S. Watanabe, Y. Matsumoto, E. Aizu-Yokota, and T. Kasahara. 1999. FAK is the upstream signal protein of the phosphatidylinositol 3-kinase- Akt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line. J. Biol. Chem. 274:10566-10570. 538. Spong, M. S., J. Askins, and D. Sheppard. 2000. Beta6 integrin interaction with PICK1, a PKCalpha binding protein, p. 99. Keystone Symposium, Joint Regulation of Signaling Pathways by Integrins and Growth Factors, Breckenridge, Colorado. 539. Staudinger, J., J. Lu, and E. N. Olson. 1997. Specific interaction of the PDZ domain protein PICK1 with the COOH terminus of protein kinase C-alpha. J. Biol. Chem. 272:32019-32024. 540. Staudinger, J., J. Zhou, R. Burgess, S. J. Elledge, and E. N. Olson. 1995. PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J. Cell Biol. 128:263-271. 541. Steimle, P. A., J. D. Hoffert, N. B. Adey, and S. W. Craig. 1999. Polyphosphoinositides inhibit the interaction of vinculin with actin filaments. J. Biol. Chem. 274:18414-18420. 542. Stephens, G., N. O'Luanaigh, D. Reilly, P. Harriott, B. Walker, D. Fitzgerald, and N. Moran. 1998. A sequence within the cytoplasmic tail of GpIIb independently activates platelet aggregation and thromboxane synthesis. J. Biol. Chem. 273:20317-20322. 543. Sterk, L. M., C. A. Geuijen, L. C. Oomen, J. Calafat, H. Janssen, and A. Sonnenberg. 2000. The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes. J. Cell Biol. 149:969-982. 544. Stewart, M. P., A. McDowall, and N. Hogg. 1998. LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain. J. Cell Biol. 140:699-707. 545. Sun, H., R. Lesche, D. M. Li, J. Liliental, H. Zhang, J. Gao, N. Gavrilova, B. Mueller, X. Liu, and H. Wu. 1999. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 96:6199-6204. 546. Sundberg, C., and K. Rubin. 1996. Stimulation of β1 integrins on fibroblasts induces PDGF independent tyrosine phosphorylation of PDGF beta-receptors. J. Cell Biol. 132:741-752. 547. Svineng, G., R. Fassler, and S. Johansson. 1998. Identification of β1C-2, a novel variant of the integrin β1 subunit generated by utilization of an alternative splice acceptor site in exon C. Biochem. J. 330:1255-1263. 548. Svineng, G., and S. Johansson. 1999. Integrin subunits beta1C-1 and beta1C-2 expressed in GD25T cells are retained and degraded intracellularly rather than localised to the cell surface. J. Cell Sci. 112:4751-4761. 549. Szaszi, K., K. Kurashima, A. Kapus, A. Paulsen, K. Kaibuchi, S. Grinstein, and J. Orlowski. 2000. RhoA and Rho Kinase Regulate the Epithelial Na+/H+ Exchanger NHE3. ROLE OF MYOSIN LIGHT CHAIN PHOSPHORYLATION. J. Biol. Chem. 275:28599-28606. 550. Tachibana, I., J. Bodorova, F. Berditchevski, M. M. Zutter, and M. E. Hemler. 1997. NAG-2, a novel transmembrane-4 superfamily (TM4SF) protein that complexes with integrins and other TM4SF proteins. J. Biol. Chem. 272:29181-29189. 551. Tachibana, K., T. Urano, H. Fujita, Y. Ohashi, K. Kamiguchi, S. Iwata, H. Hirai, and C. Morimoto. 1997. Tyrosine phosphorylation of Crk-associated substrates by focal adhesion kinase. A putative mechanism for the integrin-mediated tyrosine phosphorylation of Crk-associated substrates. J. Biol. Chem. 272:29083-29090. 552. Tadokoro, S., Y. Tomiyama, S. Honda, M. Arai, N. Yamamoto, M. Shiraga, S. Kosugi, Y. Kanakura, Y. Kurata, and Y. Matsuzawa. 1998. A Gln747-->Pro substitution in the IIb subunit is responsible for a moderate IIbbeta3 deficiency in Glanzmann thrombasthenia. Blood 92:2750-2758. 553. Takagi, J., T. Kamata, J. Meredith, W. Puzon-McLaughlin, and Y. Takada. 1997. Changing ligand specificities of alphavbeta1 and alphavbeta3 integrins by swapping a short diverse sequence of the beta subunit. J. Biol. Chem. 272:19794-19800. 554. Takaoka, A. S., T. Yamada, M. Gotoh, Y. Kanai, K. Imai, and S. Hirohashi. 1998. Cloning and characterization of the human beta4-integrin gene promoter and enhancers. J. Biol. Chem. 273:33848-33855. 555. Takayama, Y., S. Tanaka, K. Nagai, and M. Okada. 1999. Adenovirus-mediated overexpression of C-terminal Src kinase (Csk) in type I astrocytes interferes with cell spreading and attachment to

87 fibronectin. Correlation with tyrosine of paxillin and FAK. J. Biol. Chem. 274:2291- 2297. 556. Takeya, R., K. Takeshige, and H. Sumimoto. 2000. Interaction of the PDZ domain of human PICK1 with class I ADP- ribosylation factors. Biochem. Biophys. Res. Commun. 267:149-155. 557. Tamkun, J. W., D. W. DeSimone, D. Fonda, R. S. Patel, C. Buck, A. F. Horwitz, and R. O. Hynes. 1986. Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell 46:271-282. 558. Tamura, M., J. Gu, E. H. Danen, T. Takino, S. Miyamoto, and K. M. Yamada. 1999. PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway. J. Biol. Chem. 274:20693-20703. 559. Tanaka, T., R. Yamaguchi, H. Sabe, K. Sekiguchi, and J. M. Healy. 1996. Paxillin association in vitro with integrin cytoplasmic domain peptides. FEBS Lett. 399:53-58. 560. Tang, S., Y. Gao, and J. A. Ware. 1999. Enhancement of endothelial cell migration and in vitro tube formation by TAP20, a novel beta 5 integrin-modulating, PKC theta-dependent protein. J. Cell Biol. 147:1073-1084. 561. Tang, S., K. G. Morgan, C. Parker, and J. A. Ware. 1997. Requirement for protein kinase C theta for cell cycle progression and formation of actin stress fibers and filopodia in vascular endothelial cells. J. Biol. Chem. 272:28704-28711. 562. Tapia, J. A., H. A. Ferris, R. T. Jensen, and L. J. Garcia. 1999. Cholecystokinin activates PYK2/CAKbeta by a phospholipase C-dependent mechanism and its association with the mitogen- activated protein kinase signaling pathway in pancreatic acinar cells. J. Biol. Chem. 274:31261-31271. 563. Tarone, G., S. Degani, F. Balzac, M. Brancaccio, S. Guazzone, C. Bozzo, and L. Silengo. 2000. Role of ICAP-1 in cell adhesion and integrin signaling, p. 76. Keystone Symposium, Joint Regulation of Signaling Pathways by Integrins and Growth Factors, Breckenridge, Colorado. 564. Thomas, J. W., M. A. Cooley, J. M. Broome, R. Salgia, J. D. Griffin, C. R. Lombardo, and M. D. Schaller. 1999. The role of focal adhesion kinase binding in the regulation of tyrosine phosphorylation of paxillin. J. Biol. Chem. 274:36684-36692. 565. Thomas, J. W., B. Ellis, R. J. Boerner, W. B. Knight, G. C. White, 2nd, and M. D. Schaller. 1998. SH2- and SH3-mediated interactions between focal adhesion kinase and Src. J. Biol. Chem. 273:577-583. 566. Thomas, S. M., and J. S. Brugge. 1997. Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol. 13:513-609. 567. Thomas, S. M., M. Hagel, and C. E. Turner. 1999. Characterization of a focal adhesion protein, Hic-5, that shares extensive homology with paxillin. J. Cell Sci. 112:181-190. 568. Tominaga, T., and D. L. Barber. 1998. Na-H exchange acts downstream of RhoA to regulate integrin-induced cell adhesion and spreading. Mol. Biol. Cell 9:2287-2303. 569. Tonks, N. K., and M. P. Myers. 1999. Structural assets of a tumor suppressor. Science 286:2096- 2097. 570. Tozer, E. C., R. C. Liddington, M. J. Sutcliffe, A. H. Smeeton, and J. C. Loftus. 1996. Ligand binding to integrin alphaIIbbeta3 is dependent on a MIDAS-like domain in the beta3 subunit. J. Biol. Chem. 271:21978-21984. 571. Tsukamoto, N., M. Hattori, H. Yang, J. L. Bos, and N. Minato. 1999. Rap1 GTPase-activating protein SPA-1 negatively regulates cell adhesion. J. Biol. Chem. 274:18463-18469. 572. Tu, Y., F. Li, S. Goicoechea, and C. Wu. 1999. The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells. Mol. Cell. Biol. 19:2425-2434. 573. Tu, Y., F. Li, and C. Wu. 1998. Nck-2, a novel Src homology2/3-containing adaptor protein that interacts with the LIM-only protein PINCH and components of growth factor receptor kinase-signaling pathways. Mol. Biol. Cell 9:3367-3382. 574. Tuckwell, D. S., K. B. Reid, M. J. Barnes, and M. J. Humphries. 1996. The A-domain of integrin alpha 2 binds specifically to a range of collagens but is not a general receptor for the collagenous motif. Eur. J. Biochem. 241:732-739. 575. Turner, C. E. 1998. Paxillin. Int. J. Biochem. Cell Biol. 30:955-959. 576. Turner, C. E., M. C. Brown, J. A. Perrotta, M. C. Riedy, S. N. Nikolopoulos, A. R. McDonald, S. Bagrodia, S. Thomas, and P. S. Leventhal. 1999. Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling. J. Cell Biol. 145:851-863. 577. Ueda, H., S. Abbi, C. Zheng, and J. L. Guan. 2000. Suppression of Pyk2 kinase and cellular activities by FIP200. J. Cell Biol. 149:423-430. 578. Vallar, L., C. Melchior, S. Plancon, H. Drobecq, G. Lippens, V. Regnault, and N. Kieffer. 1999. Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein. J. Biol. Chem. 274:17257-17266.

88 579. Valmu, L., and C. G. Gahmberg. 1995. Treatment with okadaic acid reveals strong threonine phosphorylation of CD18 after activation of CD11/CD18 leukocyte integrins with phorbol esters or CD3 antibodies. J. Immunol. 155:1175-1183. 580. van Agthoven, T., T. L. van Agthoven, A. Dekker, P. J. van der Spek, L. Vreede, and L. C. Dorssers. 1998. Identification of BCAR3 by a random search for genes involved in antiestrogen resistance of human breast cancer cells. EMBO J. 17:2799-2808. 581. van de Water, B., J. F. Nagelkerke, and J. L. Stevens. 1999. Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells. J. Biol. Chem. 274:13328-13337. 582. van der Flier, A., I. Kuikman, C. Baudoin, R. van der Neut, and A. Sonnenberg. 1995. A novel β1 integrin isoform produced by alternative splicing: unique expression in cardiac and skeletal muscle. FEBS Lett. 369:340-344. 583. van Kooyk, Y., S. J. van Vliet, and C. G. Figdor. 1999. The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes. J. Biol. Chem. 274:26869-26877. 584. Vanhaesebroeck, B., and D. R. Alessi. 2000. The PI3K-PDK1 connection: more than just a road to PKB. Biochem. J. 346 Pt 3:561-576. 585. Vanhaesebroeck, B., S. J. Leevers, G. Panayotou, and M. D. Waterfield. 1997. Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem. Sci. 22:267-272. 586. Vazquez, F., and W. R. Sellers. 2000. The PTEN tumor suppressor protein: an antagonist of phosphoinositide 3- kinase signaling. Biochim. Biophys. Acta 1470:M21-35. 587. Vidal, F., D. Aberdam, C. Miquel, A. M. Christiano, L. Pulkkinen, J. Uitto, J. P. Ortonne, and G. Meneguzzi. 1995. Integrin beta 4 mutations associated with junctional epidermolysis bullosa with pyloric atresia. Nat. Genet. 10:229-234. 588. Vignoud, L., C. Albiges-Rizo, P. Frachet, and M. R. Block. 1997. NPXY motifs control the recruitment of the alpha5beta1 integrin in focal adhesions independently of the association of talin with the beta1 chain. J. Cell Sci. 110:1421-1430. 589. Vinogradova, O., T. Haas, E. F. Plow, and J. Qin. 2000. A structural basis for integrin activation by the cytoplasmic tail of the alpha IIb-subunit. Proc. Natl. Acad. Sci. U.S.A. 97:1450-1455. 590. Volberg, T., B. Geiger, Z. Kam, R. Pankov, I. Simcha, H. Sabanay, J. L. Coll, E. Adamson, and A. Ben-Ze'ev. 1995. Focal adhesion formation by F9 embryonal carcinoma cells after vinculin gene disruption. J. Cell Sci. 108:2253-2260. 591. Vuori, K., H. Hirai, S. Aizawa, and E. Ruoslahti. 1996. Introduction of p130cas signaling complex formation upon integrin- mediated cell adhesion: a role for Src family kinases. Mol. Cell. Biol. 16:2606-2613. 592. Vuori, K., and E. Ruoslahti. 1993. Activation of protein kinase C precedes α5β1 integrin-mediated cell spreading on fibronectin. J. Biol. Chem. 268:21459-21462. 593. Vuori, K., and E. Ruoslahti. 1994. Association of insulin receptor substrate-1 with integrins. Science 266:1576-1578. 594. Vuori, K., and E. Ruoslahti. 1995. Tyrosine phosphorylation of p130Cas and cortactin accompanies integrin- mediated cell adhesion to extracellular matrix. J. Biol. Chem. 270:22259-22262. 595. Wang, B., J. X. Zou, B. Ek-Rylander, and E. Ruoslahti. 2000. R-Ras contains a proline-rich site that binds to SH3 domains and is required for integrin activation by R-Ras. J. Biol. Chem. 275:5222- 5227. 596. Wang, X. Q., and W. A. Frazier. 1998. The thrombospondin receptor CD47 (IAP) modulates and associates with α2β1 integrin in vascular smooth muscle cells. Mol. Biol. Cell 9:865-874. 597. Wang, X. Q., F. P. Lindberg, and W. A. Frazier. 1999. Integrin-associated protein stimulates alpha2beta1-dependent chemotaxis via Gi-mediated inhibition of adenylate cyclase and extracellular- regulated kinases. J. Cell Biol. 147:389-400. 598. Wary, K. K., F. Mainiero, S. J. Isakoff, E. E. Marcantonio, and F. G. Giancotti. 1996. The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell 87:733- 743. 599. Wary, K. K., A. Mariotti, C. Zurzolo, and F. G. Giancotti. 1998. A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell 94:625-634. 600. Waterman-Storer, C. M., R. A. Worthylake, B. P. Liu, K. Burridge, and E. D. Salmon. 1999. Microtubule growth activates Rac1 to promote lamellipodial protrusion in fibroblasts. Nat Cell Biol 1:45-50. 601. Watt, F. M., M. D. Kubler, N. A. Hotchin, L. J. Nicholson, and J. C. Adams. 1993. Regulation of keratinocyte terminal differentiation by integrin- extracellular matrix interactions. J. Cell Sci. 106:175-182. 602. Weber, C., R. Alon, B. Moser, and T. A. Springer. 1996. Sequential regulation of alpha 4 beta 1 and alpha5 beta1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis. J. Cell Biol. 134:1063-1073.

89 603. Weber, K. S., L. B. Klickstein, and C. Weber. 1999. Specific activation of leukocyte beta2 integrins lymphocyte function- associated antigen-1 and Mac-1 by chemokines mediated by distinct pathways via the alpha subunit cytoplasmic domains. Mol. Biol. Cell 10:861-873. 604. Weber, K. S., M. R. York, T. A. Springer, and L. B. Klickstein. 1997. Characterization of lymphocyte function-associated antigen 1 (LFA-1)- deficient T cell lines: the alphaL and beta2 subunits are interdependent for cell surface expression. J. Immunol. 158:273-279. 605. Weed, S. A., Y. Du, and J. T. Parsons. 1998. Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1. J. Cell Sci. 111:2433-2443. 606. Wei, J., L. M. Shaw, and A. M. Mercurio. 1998. Regulation of mitogen-activated protein kinase activation by the cytoplasmic domain of the α6 integrin subunit. J. Biol. Chem. 273:5903-5907. 607. Wei, Y., X. Yang, Q. Liu, J. A. Wilkins, and H. A. Chapman. 1999. A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling. J. Cell Biol. 144:1285-1294. 608. Welch, M. D. 1999. The world according to Arp: regulation of actin nucleation by the Arp2/3 complex. Trends Cell Biol. 9:423-427. 609. Wen, L. P., J. A. Fahrni, S. Troie, J. L. Guan, K. Orth, and G. D. Rosen. 1997. Cleavage of focal adhesion kinase by caspases during apoptosis. J. Biol. Chem. 272:26056-26061. 610. Weng, L. P., X. Wang, and Q. Yu. 1999. Transmembrane tyrosine phosphatase LAR induces apoptosis by dephosphorylating and destabilizing p130Cas. Genes Cells 4:185-196. 611. Wennerberg, K., A. Armulik, T. Sakai, M. Karlsson, R. Fassler, E. M. Schaefer, D. F. Mosher, and S. Johansson. 2000. The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation. Mol. Cell. Biol. 20:5758-5765. 612. Wennerberg, K., R. Fässler, B. Wärmegård, and S. Johansson. 1998. Mutational analysis of the potential phosphorylation sites in the cytoplasmic domain of integrin β1A. Requirement for threonines 788- 789 in receptor activation. J. Cell Sci. 111:1117-1126. 613. Wennerberg, K., L. Lohikangas, D. Gullberg, M. Pfaff, S. Johansson, and R. Fässler. 1996. β1 integrin-dependent and -independent polymerization of fibronectin. J. Cell Biol. 132:227-238. 614. White, T. K., Q. Zhu, and M. L. Tanzer. 1995. Cell surface calreticulin is a putative mannoside lectin which triggers mouse melanoma cell spreading. J. Biol. Chem. 270:15926-15929. 615. Williams, M. J., P. E. Hughes, T. E. O'Toole, and M. H. Ginsberg. 1994. The inner world of cell adhesion:integrin cytoplasmic domains. Trends Cell Biol. 4:109-112. 616. Wixler, V., E. Laplantine, D. Geerts, A. Sonnenberg, D. Petersohn, B. Eckes, M. Paulsson, and M. Aumailley. 1999. Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains. FEBS Lett. 445:351-355. 617. Woodard, A., G. Garcia-Cardeña, M. Leong, J. Madri, W. Sessa, and L. Languino. 1998. The synergistic activity of (alpha)vß3 integrin and PDGF receptor increases cell migration. J. Cell Sci. 111:469-478. 618. Woods, A., and J. R. Couchman. 1992. Protein kinase C involvement in focal adhesion formation. J. Cell Sci. 101:277-290. 619. Woods, A., and J. R. Couchman. 1994. Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component. Mol. Biol. Cell 5:183-192. 620. Wu, C., S. Y. Keightley, C. Leung-Hagesteijn, G. Radeva, M. Coppolino, S. Goicoechea, J. A. McDonald, and S. Dedhar. 1998. Integrin-linked protein kinase regulates fibronectin matrix assembly, E- cadherin expression, and tumorigenicity. J. Biol. Chem. 273:528-536. 621. Xiao, G., D. Wang, M. D. Benson, G. Karsenty, and R. T. Franceschi. 1998. Role of the alpha2- integrin in osteoblast-specific gene expression and activation of the Osf2 transcription factor. J. Biol. Chem. 273:32988-32994. 622. Xie, J., R. Li, P. Kotovuori, C. Vermot-Desroches, J. Wijdenes, M. A. Arnaout, P. Nortamo, and C. G. Gahmberg. 1995. Intercellular adhesion molecule-2 (CD102) binds to the leukocyte integrin CD11b/CD18 through the A domain. J. Immunol. 155:3619-3628. 623. Xie, W., F. Li, J. E. Kudlow, and C. Wu. 1998. Expression of the integrin-linked kinase (ILK) in mouse skin: loss of expression in suprabasal layers of the epidermis and up-regulation by erbB-2. Am. J. Pathol. 153:367-372. 624. Yalamanchili, P., C. Lu, C. Oxvig, and T. A. Springer. 2000. Folding and function of I domain- deleted Mac-1 and lymphocyte function- associated antigen-1. J. Biol. Chem. 275:21877-21882. 625. Yamakita, Y., G. Totsukawa, S. Yamashiro, D. Fry, X. Zhang, S. K. Hanks, and F. Matsumura. 1999. Dissociation of FAK/p130(CAS)/c-Src complex during mitosis: role of mitosis- specific serine phosphorylation of FAK. J. Cell Biol. 144:315-324. 626. Yan, B., D. D. Hu, S. K. Knowles, and J. W. Smith. 2000. Probing chemical and conformational differences in the resting and active conformers of platelet integrin alpha(IIb)beta(3). J. Biol. Chem. 275:7249-7260. 627. Yan, S. R., and G. Berton. 1998. Antibody-induced engagement of beta2 integrins in human neutrophils causes a rapid redistribution of cytoskeletal proteins, Src-family tyrosine kinases, and p72syk that precedes de novo actin polymerization. J. Leukoc. Biol. 64:401-408.

90 628. Yang, N., O. Higuchi, K. Ohashi, K. Nagata, A. Wada, K. Kangawa, E. Nishida, and K. Mizuno. 1998. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin. Nature 393:809-812. 629. Yang, W., and R. A. Cerione. 1997. Cloning and characterization of a novel Cdc42-associated tyrosine kinase, ACK-2, from bovine brain. J. Biol. Chem. 272:24819-24824. 630. Yang, W., Q. Lin, J. L. Guan, and R. A. Cerione. 1999. Activation of the Cdc42-associated tyrosine kinase-2 (ACK-2) by cell adhesion via integrin beta1. J. Biol. Chem. 274:8524-8530. 631. Yano, H., H. Uchida, T. Iwasaki, M. Mukai, H. Akedo, K. Nakamura, S. Hashimoto, and H. Sabe. 2000. Paxillin alpha and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 97:9076- 9081. 632. Yauch, R. L., F. Berditchevski, M. B. Harler, J. Reichner, and M. E. Hemler. 1998. Highly stoichiometric, stable, and specific association of integrin α3β1 with CD151 provides a major link to phosphatidylinositol 4- kinase, and may regulate cell migration. Mol. Biol. Cell 9:2751-2765. 633. Yauch, R. L., D. P. Felsenfeld, S. K. Kraeft, L. B. Chen, M. P. Sheetz, and M. E. Hemler. 1997. Mutational evidence for control of cell adhesion through integrin diffusion/clustering, independent of ligand binding. J. Exp. Med. 186:1347-1355. 634. Yauch, R. L., A. R. Kazarov, B. Desai, R. T. Lee, and M. E. Hemler. 2000. Direct extracellular contact between integrin alpha3 beta1 and TM4SF protein CD151. J. Biol. Chem. 275:9230-9238. 635. Yedovitzky, M., D. Mochly-Rosen, J. A. Johnson, M. O. Gray, D. Ron, E. Abramovitch, E. Cerasi, and R. Nesher. 1997. Translocation inhibitors define specificity of protein kinase C isoenzymes in pancreatic beta-cells. J. Biol. Chem. 272:1417-1420. 636. Ylänne, J., Y. Chen, T. E. O'Toole, J. C. Loftus, Y. Takada, and M. H. Ginsberg. 1993. Distinct functions of integrin α and β subunit cytoplasmic domains in cell spreading and formation of focal adhesions. J. Cell Biol. 122:223-233. 637. Yu, D. H., C. K. Qu, O. Henegariu, X. Lu, and G. S. Feng. 1998. Protein-tyrosine phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion. J. Biol. Chem. 273:21125-21131. 638. Yuan, Y., S. M. Dopheide, C. Ivanidis, H. H. Salem, and S. P. Jackson. 1997. Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets. Distinct roles for glycoprotein Ib-V-IX and glycoprotein IIb-IIIa (integrin alphaIIbbeta3) in von Willebrand factor-induced signal transduction. J. Biol. Chem. 272:21847-21854. 639. Zalcman, G., V. Closson, J. Camonis, N. Honore, M. F. Rousseau-Merck, A. Tavitian, and B. Olofsson. 1996. RhoGDI-3 is a new GDP dissociation inhibitor (GDI). Identification of a non- cytosolic GDI protein interacting with the small GTP-binding proteins RhoB and RhoG. J. Biol. Chem. 271:30366-30374. 640. Zhang, H., Z. Li, E. Viklund, and S. Strömblad. 2000. Integrin beta5 as a novel substrate for p21- activated kinase4: Potental role in inside-out signaling, p. 89. Keystone Symposium, Joint Regulation of Signaling Pathways by Integrins and Growth Factors, Breckenridge, Colorado. 641. Zhang, J., H. Banfic, F. Straforini, L. Tosi, S. Volinia, and S. E. Rittenhouse. 1998. A type II phosphoinositide 3-kinase is stimulated via activated integrin in platelets. A source of phosphatidylinositol 3-phosphate. J. Biol. Chem. 273:14081-14084. 642. Zhang, X., A. Chattopadhyay, Q. S. Ji, J. D. Owen, P. J. Ruest, G. Carpenter, and S. K. Hanks. 1999. Focal adhesion kinase promotes phospholipase C-gamma1 activity. Proc. Natl. Acad. Sci. U.S.A. 96:9021-9026. 643. Zhang, X. A., and M. E. Hemler. 1999. Interaction of the integrin β1 cytoplasmic domain with ICAP-1 protein. J. Biol. Chem. 274:11-19. 644. Zhang, Z., K. Vuori, J. C. Reed, and E. Ruoslahti. 1995. The α5β1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc. Natl. Acad. Sci. U.S.A. 92:6161-6165. 645. Zhang, Z., K. Vuori, H. Wang, J. C. Reed, and E. Ruoslahti. 1996. Integrin activation by R-ras. Cell 85:61-69. 646. Zheng, D. Q., A. S. Woodard, G. Tallini, and L. R. Languino. 2000. Substrate specificity of alphav beta3 integrin-mediated cell migration and phosphatidylinositol 3-Kinase/AKT pathway activation. J. Biol. Chem. 275:24565-24574. 647. Zheng, M., H. Fang, and S. Hakomori. 1994. Functional role of N-glycosylation in alpha 5 beta 1 integrin receptor. De-N-glycosylation induces dissociation or altered association of alpha 5 and beta 1 subunits and concomitant loss of fibronectin binding activity. J. Biol. Chem. 269:12325-12331. 648. Zheng, X. M., R. J. Resnick, and D. Shalloway. 2000. A phosphotyrosine displacement mechanism for activation of Src by PTPalpha. EMBO J. 19:964-978. 649. Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Novel isoform of β1 integrin expressed in skeletal and . Biochem. Biophys. Res. Commun. 214:279-285. 650. Zhong, C., M. Chrzanowska-Wodnicka, J. Brown, A. Shaub, A. M. Belkin, and K. Burridge. 1998. Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly. J. Cell Biol. 141:539-551.

91 651. Zhou, X., and J. Li. 2000. Macrophage-enriched myristoylated alanine-rich C kinase substrate and its phosphorylation is required for the phorbol ester-stimulated diffusion of beta 2 integrin molecules. J. Biol. Chem. 275:20217-20222. 652. Zhu, Q., P. Zelinka, T. White, and M. L. Tanzer. 1997. Calreticulin-integrin bidirectional signaling complex. Biochem. Biophys. Res. Commun. 232:354-358.

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