ROLE OF RHO-FAMILY GUANOSINE TRIPHOSPHATASE EFFECTORS IN FILOPODIA DYNAMICS
Arpan De
A Thesis
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
December 2015
Committee:
Carol Heckman, Advisor
Paul Morris
Daniel Wiegmann © 2015
Arpan De
All Rights Reserved iii ABSTRACT
Carol Heckman, Advisor
Filopodia play sensory roles by acting like ‘antennae’ to sense the cell’s surroundings. In
nerve growth cone, they promote motility towards an attractive cue or away from a repulsive
one. Filopodia have been reported to be involved in wound healing, adhesion to the extracellular
matrix and embryonic development. A number of cytoskeletal regulatory proteins have been
implicated in regulating initiation, formation, maintenance and extension/retraction cycle of such
protrusions. Some of these proteins either bind to the Rho family GTPases, Cdc42 and Rac, as
effectors or function downstream of such effectors to regulate signaling pathways involved in
cytoskeletal reorganization.
The purpose of this study was to determine whether certain proteins, which had well-
defined binding interfaces with proteins downstream of GTPase-regulated proteins, were
implicated in filopodial dynamics. Synthetic binding peptides (BPs) were used to impede the
interaction of Cdc42 and Rac with the effectors and to interfere at protein-protein binding
interfaces downstream of the GTPases. Single cell peripheries were analyzed to determine the
levels of positive outgrowths of filopodia. This assay allowed for rapid determination of whether
the BPs had an effect on filopodia formation. BPs, namely IQGAP (132-140), ACK and Par6
showed a negative effect on filopodia, whereas IQGAP (84-93), PAK and WASP elevated
filopodia formation or had no effect compared to control. When interactions of PAK2 with Abl
and PAK4 with integrin-β5 were inhibited, filopodia prevalence increased.
Based on several previous findings, the tyrosine kinase ACK is thought to mediate
internalization, processing and trafficking pathways of cell surface receptors, especially iv epidermal growth factor receptors (EGFRs). ACK is an oncogene and its overexpression or mutation is implicated in several human cancers. During this study, when the E3 ubiquitin ligase, neural precursor cell expressed developmentally downregulated protein (Nedd4) was inhibited from binding to ACK, both cell periphery coverage with filopodia and number of cells showing filopodia, increased significantly compared to control. Since earlier findings have suggested that
ACK and RTK (especially EGFR) are co-processed, this study suggests that, ACK-mediated
RTK processing may play a role in regulating filopodial dynamics. v
To my Advisor, Dr. Carol Heckman, my beloved parents, Mrs. Kankan De and Mr. Asim Kumar
De and my beloved brother, Mr. Apratim De. vi
ACKNOWLEDGMENTS
I take the privilege to convey my sincerest and profound gratitude to Dr. Carol Heckman,
whose valuable guidance, inspiration, love and support have immensely helped me to understand
and learn the aspects of basic scientific research and gain insightful knowledge of the subject. I
am thankful to Dr. Paul Morris, Dr. Daniel Wiegmann, Dr. Michael Geusz and Dr. Scott Rogers
for constantly motivating me. I am indebted to Dr. Marilyn Cayer for her invaluable technical
support and help during the study. I would like to thank Tania Biswas, Pratima Pandey and
Francis Bugyei for their all along help. I am grateful to Pratima Pandey for sharing some data of
her study and immensely helping me during the course of research. I convey my sincere thanks
to Dr. Jeff Miner, Dr. Karen Root and the Department of Biological Sciences at Bowling Green
State University for their continuous support.
I have no words to express how much indebted I am to my beloved parents and family for
believing in me and for their constant encouragement and unconditional love. I convey a very
special note of thanks to my uncle, Mr. Arun Kumar De for being my invaluable support,
philosopher and guide. Thank you, Ms. Ananya Banerjee for being my best friend and
immensely helping me to stay strong, confident and focused to achieve my goal. I would like to
thank all my friends for being there beside me and supporting me all along.
I offer my heartfelt prayers to the Almighty for blessings. vii
TABLE OF CONTENTS Page
CHAPTER 1. INTRODUCTION ...... ……………………… 1
1.1 Specializations implicated in cell motility...... 1
1.2 Guanosine triphosphatase mediated regulation of protrusions...... 5
1.2.1 Cell division cycle 42 (Cdc42)...... 6 1.2.2 Ras related C3 botulinum toxin substrate (Rac)...... 9 1.3 Working model of filopodia initiation and elongation ...... 11 1.4 Adhesive proteins and regulatory kinases in filopodia...... 14 1.5 Proteins implicated in receptor trafficking...... 15
1.5.1 ACK and its role in receptor trafficking...... 15
1.5.2 ACK has complex effects on receptor trafficking...... 16
1.6 Eperimental model...... ̀ 21 CHAPTER 2. MATERIALS AND METHODS ………………………...... 24 2.1 Cell culture and chemicals ...... 24
2.2 Substrate Prepartion...... 25
2.3 Peptides designed to interrupt protein-protein interactions...... 26
2.4 Peptide delivery using BioPorter reagent...... 27 2.5 Analysis…………………………………………………………...... ……... 29 CHAPTER 3. RESULTS……………………………...... 31 3.1 Assay for effect of binding peptides on filopodia dynamics...... 31
3.2 Peptides blocking downstream of GTPase effectors...... 35
3.3 Membrane traffaicing ̀ and filopodia...... 43 viii
CHAPTER 4. DISCUSSION…………………………………………………………… .... 47 4.1 Role of Cdc42 effectors and downstream proteins in filopodia dynamics...... 47 4.2 ACK plays a critical role in receptor trafficking...... 50 4.2.1 ACK binds constitutively and conditionally to other proteins...... 50 4.2.2 Overexpressed ACK inhibits receptor internalization by sequestering its binding partners...... 52 4.2.3 Coordinate ACK and RTK trafficking and degradation...... 54 4.2.4 Role of ACK mediated EGFR processing in filopodia dynamics...... 57
REFERENCES...... 60
APPENDIX A. LIST OF ABBREVIATIONS……………………………………………. .. 72 ix
LIST OF FIGURES
Figure Page
1.1 Steps in filopodium initiation, elongation and retraction ...... 4
1.2 GTPases act as switch proteins ...... 5
1.3 Cdc42 pathway showing the different effectors and the cellular processes
they generate ...... 8
1.4 Rac1 pathway showing the different effectors and the cellular processes
they generate ...... 10
1.5 Formation of filopodia and lamellipodium showing the different proteins involved 12
1.6 The current working model for filopodia formation ...... 13
1.7 Graphical representation showing cyclical relationship predicted by the model for
hypothetical predator and prey populations ...... 22
2.1 Delivery of synthetic BPs into cells using BioPORTER reagent ...... 28
2.2 Method of estimating coverage with filopodia ...... 30
3.1 Percentage of cells showing filopodia after ACK-Nedd4 BP treatment versus control 38
3.2 Mean percent of cells showing filopodia after ACK-Nedd4 BP treatment in
combination with other BPs ...... 39
3.3 Percent coverage of cell periphery with filopodia after ACK-Nedd4 BP treatment
versus control ………...... 40
3.4 Mean percent coverage of cell periphery with filopodia after ACK-Nedd4 BP
treatment in combination with other BPs...... 41 x
3.5 Percentage of cells showing filopodia after cAbl-PAK2 BP treatment versus control 42
3.6 Percentage coverage of periphery with filopodia after cAbl-PAK2 BP treatment
versus control ...... 43
4.1 Schematic diagram showing ACK (1038 amino acid residues) and identifying
domains specific for binding its protein partners ...... 51
4.2 Mechanism of ubiquitination of target proteins by cooperative activity of E1, E2
and E3 ubiquitination enzymes ...... 53
4.3 Mechanisms of EGFR ubiquitination ...... 57
4.4 ACK1 sequence showing ten novel somatic and two germ line mutations ...... 58 xi
LIST OF TABLES
Table Page
1.1 Stages of ligand-induced EGFR trafficking in relation to ACK ...... 18
2.1 BPs designed to inhibit binding of effectors to Cdc42 and Rac GTPases ...... 26
2.2 BPs designed to inhibit downstream protein-protein interactions ...... 27
3.1 Effect of BPs representing GTPase sequences on mean percent of cells showing
filopodia in experimental sample versus paired control ...... 33
3.2 Effect of BPs representing GTPase sequences on mean percent coverage of
periphery with filopodia in experimental sample versus paired control ...... 34
3.3 Effect of BPs representing non-GTPase sequences on mean percent of cells showing
filopodia in experimental sample versus paired control ...... 36
3.4 Effect of BPs representing non-GTPase sequences on mean percent coverage of
periphery with filopodia in experimental sample versus paired control ...... 37
3.5 Effect of membrane trafficking modulation on filpodia ...... 44 1
CHAPTER 1. INTRODUCTION
1.1 Specializations implicated in cell motility
During the lifespan, a eukaryotic cell may face a number of different physiological
challenges that require it to migrate toward a specific locus. To accomplish this, the cell
coordinately balances different interconnected cellular processes at the physiological,
morphological and molecular level to respond to various environmental cues that reach it
through physical forces or proteins that bind to specific plasma membrane receptors and generate
signals. The cytoskeleton plays a pivotal role in processes such as morphogenesis, migration and
phagocytosis (1). Transmembrane proteins like integrins and cadherins are thought to be
involved in mechanotransduction. Integrins connect the cytoskeleton of the cell to the
extracellular matrix (ECM) and may transmit mechanical forces across the plasma membrane,
transducing the physical forces into chemical signals (2). Atomic force microscopy has shown that a cell forms actin-based stress fibers and is stiffened with the application of an external mechanical force. Such forces can also be transmitted by the integrins through biochemical pathways to reorganize the focal complexes (FCs) into large, elongated complexes called mature focal adhesions (3). Physiological processes like embryonic development, axon guidance, wound healing and immunological responses involve cellular migration. A major mechanism mediating such processes is the coordinated polymerization of actin filaments against cellular membranes to provide the required force.
An actively migrating cell forms protrusive structures at the plasma membrane where assembly of polar actin filaments results in a branched network embedded within a thin sheet- like protrusion called a ‘lamellipodium’ or in a tight parallel bundle of filamentous (F)-actin organized into a thin finger-like projection called a ‘filopodium’ (1). Evidence suggests the 2 existence of ‘lamellipodium’ and ‘lamella’ as two distinct dynamic actin modules organized centripetally in the cell (4). It is known that polymerization of the actin monomers, called globular (G)-actin, occurs at the ‘barbed end’ (plus end) and facilitates the protrusion of the lamellipodium. The subunits dissociate at the ‘pointed end’ (minus end). In addition, there are actin-depolymerizing proteins whose role in the lamellipodia movement is discussed below. The lamellipodia undergo cycles of extension and retraction. Lamellipodia expansion occurs some
20 seconds before maximal actin subunit addition (5), which supports previous suggestions that lamellipodia are driven by cycles of solation, osmotic expansion and re-gelation of the actin filament network (6). Nevertheless, the cyclical movement of the lamellipodium has been shown to depend on precise dynamic balance between the addition of actin monomers at the barbed end and anterograde flow of depolymerized monomers from the ‘pointed end’ (minus end) back to the opposite end. Depolymerization and release of actin monomers into the cytoplasm are required for continued polymerization at the plus end (7). Proteins like cofilin and actin-related protein-2/3 (Arp2/3) complex have been found to be localized in the lamellipodium and assumed to promote actin treadmilling (8). Because treadmilling ensures that filaments can be shortened and lengthened rapidly, this aspect of the actin filament has probably contributed to its uses in processes requiring motility throughout evolution. Nevertheless, experts disagree as to whether changes in filament length typically occur through cessation of polymerization activities. Behind the leading edge and more towards the interior of the cell, actin filaments are selectively recruited and polymerized to form a three-dimensional architecture called the lamella (7).
Proteins like myosin II and tropomyosin which regulate contractile machinery have been found to be localized in the lamella but not in lamellipodium (4, 9). The region of the cell where the 3
lamellar retrograde flow of F-actin merges with the anterograde flow of F-actin from the cell
center has been described as the ‘convergence zone’ (10).
As described in the review by Mattila and Lappalainen, two models have been proposed to understand the formation of filopodia (1). According to the ‘convergent elongation model’, filopodia are formed by extension of the lamellipodial F-actin network as a continuous bundle
(11). Arp2/3 complex-nucleated actin filaments are assembled by tip-complex proteins which include vasodilator-stimulated phosphoprotein (Ena/VASP), membrane diaphanous (mDia2), and myosin-X. These elongating filaments are then cross-linked by fascin and eventually converge into a tight parallel bundle to facilitate filopodia initiation (12). Although it was previously proposed that myosin-X mediated filopodia formation by its ability to deliver specific cargoes to the tip, recent studies have suggested that myosin-X may only act like a motor to initiate filopodia formation (13). On the other hand, a discontinuous F-actin bundle was observed in the filopodium core of Dictyostelium discoideum by cryo-electron tomography (14). The
evidence suggests that actin addition is precisely regulated and driven by Ena/VASP and a
Diaphanous (Dia) isoform(s), which act as processive motors for polymerization (15).
From studies on D. discoideum and mammalian cells, there is evidence that filopodia can
form in the absence of Arp2/3 and/or its activators (16). This gave rise to the alternative de novo
“filament nucleation model” which proposes that the actin filaments are nucleated by formins at
the tips of the filopodia (Figure 1.1). Further work is needed to elucidate the exact mechanisms
of filopodia formation, regulation, and dynamics. 4
Figure 1.1: Steps in filopodium initiation, elongation and retraction. Used by permission of (17).
According to this model, initiation of actin filament assembly occurs either by dissociation of capping proteins or by de novo nucleation of filaments. It is assumed that actin polymerization at the barbed end drives membrane protrusion. The activity of numerous proteins like insulin receptor tyrosine kinase substrate protein 53 (IRSp53), Ena/VASP, Wiskott-Aldrich 5
syndrome protein (WASP) and WASP –family verprolin-homologous protein (WAVE/Scar) is coordinated to promote actin assembly and enhance bundling of actin filaments by fascin.
However, how these and other cytoskeletal proteins function in a complex network to bring about initiation, regulation, maintenance and retraction of filopodium is yet to be completely understood. Disassembly of actin monomers from the other end (pointed or minus) and retrograde movement of the filaments is thought to drive filopodial retraction.
1.2 Guanosine triphosphatase(GTPase)-mediated regulation of protrusions
Rho-family of GTPases comprises around 20 mammalian different GTPase proteins.
They are all somehow implicated in balancing cytoskeletal reorganization. They act as switch
proteins and are regulated by GTPase-activating proteins (GAPs) and guanine exchange factors
(GEFs), as shown in Figure 1.2. 6
Figure 1.2: GTPases act as switch proteins. They cycle between two conformations that determine their activity: active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound states. A GTPase hydrolyzes the GTP moiety to GDP. The hydrolysis activity of GTPases can vary based on the modulation by other cellular factors, such as GTPase-activating proteins (GAPs). A guanine exchange factor (GEF) can catalyze the dissociation of GDP from the inactive GDP-bound state of a protein. Since GTP is in excess in the cytoplasm, this is thought to facilitate the addition of GTP, thus enabling GTP to reactivate the protein. Used by permission of (18).
1.2.1 Cell division cycle 42 (Cdc42)
As mentioned by Heasman and Ridley in their review, Cdc42 protein has a conserved role in regulating cell polarity and actin cytoskeletal dynamics in metazoans (19). Cdc42 belongs to the Rho family of guanosine triphosphatases (GTPases) and interacts with various proteins, as shown in Figure 1.3. The role of Cdc42 in formation of filopodia has already been established
(20). It has been shown that in many cell types, both constitutively active and dominant negative
Cdc42 affect the formation of filopodia (21). Cdc42-null embryonic stem cells have been shown to exhibit defects in actin cytoskeletal organization along with reduction in size and number of filopodia-like membrane protrusions (22). Cdc42-null neurons too have shown reduced filopodia
(23). However, one problem with the previous studies has been that filopodia are identified by subjective criteria. Since the variability among individual observers is substantial, the structures identified may include several different categories of protrusions.
As shown in Figure 1.2, GEFs activate one or more GTPases whereas GAPs repress them. Cdc42 interacts with a large number of different kinase and non-kinase effector proteins. 7
G-protein coupled receptors (GPCRs), integrins and receptor tyrosine kinases (RTKs) are the major upstream regulators of Cdc42. GTP-bound Cdc42 usually interacts with effector proteins containing the conserved binding motif called a Cdc42/Ras-related C3 botulinum substrate (Rac) interactive binding (CRIB) domain. However, some Cdc42 effector proteins such as IQ motif containing GTPase activating protein (IQGAP) do not contain CRIB domains. Six families of
CRIB domain-containing Cdc42 effector proteins have been identified: the Ser/Thr kinase p21- activated kinase (PAK), myotonic dystrophy related Cdc42-binding kinase (MRCK), activated
Cdc42-associated kinase (ACK), mixed-lineage kinase (MLK), WASP and binder of Rho
GTPases (BORG5/MSE55)/ Cdc42 effector protein (CEP). The last one is a nonkinase whose exact function is yet to be identified. ACK isoform 1(ACK1) has been recently identified as an inhibitor of the GTP-bound-Cdc42. Interaction between GTP-Cdc42 and ACK inhibited both the intrinsic and GAP-stimulated GTPase activity of Cdc42 (24). PAK has also been extensively studied. The substrates of PAK include LIMK, which induces actin polymerization by inactivating cofilin through phosphorylation. The LIM motif was first identified in three developmentally important transcription factors, Caenorhabditis elegans Lin-11, rat Isl-1 and C. elegans Met-3, from which the acronym LIM is derived (25, 26). WASP and neural homolog of
WASP (N-WASP) are CRIB-containing adaptor proteins that regulate actin polymerization (27,
28). Both WASP and N-WASP regulate the Arp2/3 protein complex. Cdc42 induces Arp2/3 complex-dependent nucleation and polymerization of actin filaments by binding to and activating WASP, or the related N-WASP.
Filopodia formation involves several additional Cdc42 effectors and other downstream
proteins. IRSp53 has also been found to be involved in dynamics of filopodia through IRSp53-
MIM homology domain (I-BAR)-mediated membrane tubulation and/or bundling of F-actin (29, 8
30). Arp is a seven-member protein complex containing two actin-related proteins along with five other proteins. This binds to pre-existing F-actin filaments and brings about nucleation of actin polymerization causing the growing barbed end to extend away from the mother filament.
Arp2/3 is activated by WASP/WAVE family proteins which act as nucleation promoting factors.
However, it has been shown that cells lacking both WASP isoforms exhibit filopodia (31).
Formin, a large multidomain protein, can directly nucleate polymerization of unbranched actin filaments by recruiting profilin-G-actin complexes at the barbed ends of a filament. Thus,
Arp2/3 complex and formins are the two major machineries of actin-nucleation in migrating cells. Cells may use one or both of these pathways to form filopodia. However, there are several other proteins that may work downstream of Cdc42 and thereby regulate filopodia dynamics (Figure 1.3).
Figure 1.3: Cdc42 pathway showing the different effectors and the cellular processes they
generate. Used by permission of Thomson Reuters, ©2014. 9
1.2.2 Ras related C3 botulinum toxin substrate (Rac)
The filopodia and lamellipodia rely on many of the same actin-associated proteins, because actin polymerization is essential to both of these structures. The homolog of Cdc42,
Rac, governs some aspects of lamellipodia formation as well as the activity of the leading edge.
Rac1 is a Rho-family GTPase that drives the formation of the lamellipodium. Rac1 too is activated by GEFs and repressed by GAPs. There is evidence of GEFs and GAPs showing a combinatorial effect upon activation and deactivation of Rac1 and Cdc42. Nectin (calcium independent cell-cell adhesion molecule) recruits c-Src (non-receptor protein tyrosine kinase) which then activates Cdc42 through activation of FRG (GEF for Cdc42). This activated Cdc42 then activates Vav2 (GEF) which activates Rac1 (32). RalA binding protein 1 (RalBP1) has shown GAP activity on both Rac1 and Cdc42 (33). As shown in Figure 1.4, a number of proteins which include PAKs and IQGAPs act as targets for activation by Rac1, which promotes actin polymerization in lamellipodium through activation of Arp2/3 complex. Rac1 binds to the protein complex containing partitioning defective 6 (Par6)-partitioning defective 3 (Par3)- atypical protein kinase C (aPKC), regulating cell polarity and actin cytoskeleton dynamics. Rac- mediated or Cdc42-mediated activation of PAK causes phosphorylation of LIMK which inhibits cofilin, thereby regulating actin filament turnover (19). 10
Figure 1.4: Rac1 pathway showing the different effectors and the cellular processes they
generate. Used by permission of Thomson Reuters, ©2014.
A second mechanism that drives filopodia dynamics is adhesion to the ECM via FCs. Such
adhesive plaques may form at the tip, shaft and base of the structure. Integrins, as mentioned
above, make the actual contact with proteins of the ECM and then form a platform or scaffold
interior to the plasma membrane. Numerous proteins are assembled on the scaffold and assure
its persistence or disappearance over time. Since PAK isoforms 1-3 affect both assembly and
disassembly of the FCs, this is one mechanism by which PAK may affect filopodia dynamics.
Indeed PAK2-mediated turnover of filopodia has been demonstrated previously in the Heckman laboratory but the mechanism underlying this effect is unknown. Another mechanism in which 11
PAK is implicated is the direct phosphorylation and regulation of integrin beta-5 (Iβ5). Human
Iβ5 is phosphorylated on both the serine residues 759 and 762 by PAK4. This portion of the
integrin molecule also forms a scaffold for binding the receptor for activated C kinase (RACK1)
(34).
In their review, Heasman and Ridley mention that cell polarization involves response to an
extracellular stimulus by maintenance and redistribution of proteins and organelles in an
asymmetrical pattern (19). In animal models, it has been found that Cdc42 binds to the polarity
protein Par6 and regulates its binding with Par3 and aPKC to induce cell polarity (35, 36).
Cdc42-Par6 and Par complex (Par3-aPKC) have been proposed to mediate the capture and
stabilization of microtubules at the front of the cell and subsequent orientation of Golgi and
microtubule-organizing center (MTOC) during the establishment of polarity associated with
migration (37, 38). It has been recently established that Cdc42 and the Par complex might be
important for targeting recycling endosomes to specific intracellular sites (39).
1.3 Working model of filopodia initiation and elongation Branching of the actin filament by Arp2/3 complex initiation, as shown in Figure 1.5,
facilitates extension of the highly dynamic lamellipodium at the leading edge. This eventually
leads to the assembly of a dendritic network of branched actin filaments. Capping proteins bind
to the barbed ends and terminate further addition to the plus end of the actin filaments (Figure
1.5). Rac induces actin polymerization during lamellipodium formation specifically through
WAVE complex which activates Arp2/3. A scaffold component of the WAVE complex,
Abelson interactor-1(Abi1), is known to bind to both WASP and the formin mDia in the absence
of WAVE (40). Indeed, both Abi-1 and Abl have been localized to the tips of filopodial microspikes in fibroblasts (41). 12
Figure 1.5: Formation of filopodia and lamellipodium showing the different proteins involved.
Arp2/3 complex is essential for actin branching and extension of the highly dynamic lamellipodium. Here, filopodia are shown to form by de novo nucleation of actin filaments.
Lamellipodium forms the leading edge of the cell. The lamella (behind the lamellipodium) contains larger and unbranched actin filaments. Used by permission of (19).
According to the current working model of Mattila et al., filopodia formation is initiated by the assembly of uncapped or formin-nucleated actin filament barbed ends at the plasma membrane and is facilitated by activity of the motor protein myosin-X. Either myosin-X or myosin VII might act at the tip as a transient cross-linker (Figure 1.6a). However, the integrin- binding domain of myosin-X did not play any crucial role in induction of filopodia (42). Those actin filaments devoid of capping proteins are eventually targeted by Dia2 and/or Ena/VASP proteins for further elongation (Fig. 1.6b). The elongation of these protected barbed ends towards 13
the plasma membrane provides the force for membrane deformation during filopodial elongation
(Fig. 1.6c). I-BAR domain protein, IRSp53, might play a role in deforming the plasma
membrane to enhance the formation of tubular filopodial plasma membrane protrusion. A stiff
filopodial actin filament bundle is generated by subsequent crosslinking of the elongating actin
filaments by fascin and perhaps also by fimbrin or espin. Unlike Cdc42, another small GTPase
called RIF (Rho in filopodia) has been reported to induce formation of long filopodia from the
cell periphery, probably by directing or activating Dia2 protein (43). Lipid phosphatase-related protein-1 (LPR1) has also been shown to be involved in formation of filopodia but the mechanism has not yet been identified (44).
Figure 1.6: The current working model for filopodia formation. Used by permission of (1). 14
1.4 Adhesive proteins and regulatory kinases in filopodia
Integrins mediate cell adhesion thus playing a vital role in cell migration and also
participate in ‘outside-in’ and ‘inside-out’ signaling (45). They are heterodimers consisting of α
and β subunits which contain a large extracellular domain, a transmembrane domain and a small
cytoplasmic domain (46). Interactions with kinases, phosphatases and adaptor proteins regulate
the activation state of integrins. As mentioned earlier, PAK4 phosphorylates integrin-β5 and
induces integrin αvβ5-mediated cell migration (47). Recently, a unique SERS-motif (residues
759-762) within the membrane-proximal β5 domain of integrin was identified as a specific site for binding PAK4. Evidence suggest that PAK4-mediated phosphorylation at Ser-759 and Ser-
762 residues within the SERS motif are critical for regulating PAK4-induced cell motility (34).
The Abelson family of protein tyrosine kinases comprises c-Abl and c-Arg (48). The non- receptor tyrosine kinase c-Abl has been shown to be involved in the regulation of cell growth, differentiation, apoptosis and also in actin cytoskeletal reorganization (49). Mouse embryos with deletion mutation of c-Abl and c-Arg genes [abl/arg -/-] have been found to exhibit a significant defect in actin latticework (50). When cells are stimulated by epidermal growth factor (EGF) or platelet derived growth factor (PDGF), active Abelson tyrosine kinase (Abl) localizes at F-actin assembly sites where lateral ruffles are formed (51). The Crk (chicken tumor virus no.10 regulator kinase) adaptor protein Crk-associated substrate (p130cas) is one of the major substrates
of Abl. Phosphorylation of c-Abl at residues 593-730 by PAK2 has been shown to inhibit the
interaction between the SH3 domain of the Abl interactor protein-2 (Abi2) and the PxxP motif of
Abl (52). This phosphorylation enhances the association of Abl with Crk. A concomitant
enhancement in Abl mediated phosphorylation of Crk is a result of such association. This leads
to inhibition of Crk signaling (53) which usually involves signal transduction protein complexes 15
with guanine-nucleotide exchange factor C3G, p130cas, paxillin and insulin receptor substrate
(IRS) proteins (54-58). On the other hand, Abl interaction with the another adaptor protein Nck
(non catalytic region of tyrosine kinase adaptor protein) mediates its signaling. Abl mediated
inhibition of Crk signaling and signaling through Nck engagement inhibits lamelipodium and FA
formation but promotes filopodia formation during cell attachment (53).
1.5 Proteins implicated in receptor trafficking
1.5.1 ACK and its role in receptor trafficking
Protein tyrosine kinases (PTKs) regulate signaling pathways that mediate fundamental
cellular processes such as migration, proliferation, differentiation or cell survival. In cancer
initiation and progression, various PTKs are oncoproteins in wild type or mutated form (59).
ACK1 is a ubiquitously expressed cytoplasmic non-receptor tyrosine kinase which was first
identified to bind to activated Cdc42 by its CRIB domain (60). This interaction activates ACK1
and causes its phosphorylation , probably by c-Src which activates it (61). ACK1 subsequently recruits the adaptor protein p130cas (62) and activates the Rho guanine exchange factor Dbl (63).
Activated Dbl further activates Rho family GTPases causing cytoskeletal rearrangements (64).
Interestingly, unlike other dual specificity kinases which are mostly Ser/Thr kinases, ACK was
found to possess Ser/Tyr dual kinase activity towards WASP, promoting phosphorylation at Ser-
242 amd Tyr-256 (65). It is already established that WASP is a Cdc42 effector and also one of
the proteins regulating actin polymerization. 16
1.5.2 ACK has complex effects on receptor trafficking
The effects of ACK are not only receptor-specific but depend critically on the balance of binding partners available to it in the cell. Interaction of ACK with epidermal growth factor receptor (EGFR) was first reported by Yang and coworkers (66). They found that endogenous
ACK does not bind EGFR unless it is occupied by ligand, and so ACK was originally thought to mediate some steps of receptor internalization and/or processing. It now appears that ACK binds to EGFR after getting localized to clathrin-coated pit but before receptor internalization (67) by clathrin-mediated endocytosis (CME), a process involving cellular uptake of receptor-bound ligands and extracellular fluids through clathrin-coated vesicles. ACK is known to be essential for CME since its presence in the clathrin-coated pit becomes stable in dynamin-deficient mutants (67). However, it remains unclear how ACK affects receptor trafficking following internalization of the ACK-receptor complex. Using RNA interference and pharmacological methods, De camilli and coworkers have shown that ACK phosphorylation requires both clathrin assembly into endocytic clathrin-coated pits and active Cdc42 (67). ACK related tyrosine kinase
(ARK)-1, an orthologue of ACK in Caenorhabditis elegans, is an inhibitor of receptor tyrosine kinase (RTK) signals. Mutation of ARK-1 synergises with that of other negative regulators, resulting in partially penetrant embryonic lethality and hyperinduced vulva phenotypes (68).
Thus, it appears that ARK-1’s function is to negatively regulate signaling from the EGFR orthologue, Let-23. This role may have been expanded during evolution, because ACK now seems to act as a chaperone to regulate receptor sorting, recycling, and degradation. ACK also affects signaling from the EGFR, because some EGF-initiated signaling arises from receptor molecules within an endosomal compartment which they enter after internalization. Table 1.1 1.
below summarizes how ACK regulates stages of EGFR trafficking. Further details relating to
this table have been discussed later (see Discussion).
EGFR trafficking in cells is mediated by both clathrin-dependent (CME) and clathrin-
independent (clathrin-independent endocytosis [CIE]) pathways. Previous studies have shown
that, when a low dosage of EGF is administered to serum-free cells, EGFR is internalized along with continuous signaling from endosomal vesicles mediated by CME. Most of the internalized
EGFR molecules are recycled back to the plasma membrane. Under these conditions in HeLa cells, no colocalization with caveolin-1 is observed, indicating that caveolae are not implicated in such receptor trafficking pathway (69). On the other hand, when the EGF dosage is higher (~ 10 ng/ml), the receptor is internalized partly by clathrin-independent endocytosis (CIE) and more
EGFR molecules are sorted to late endosomes (LE) and subsequently degraded than are recycled back to the plasma membrane. However, this excess degradation is almost completely inhibited when cells are treated with 0.5-1 µg/ml of filipin (sterol binding agent that disrupts caveolae and caveolae-like structures, thus inhibiting CIE) for an hour (70). Interestingly, in cells where ACK had been knocked down by small interfering RNA (siRNA), the rate of 125I-EGF (radiolabeled
EGF) internalization was reduced, with increased recycling of internalized 125I-EGF and its
decreased degradation (71). Heath and coworkers found a similar effect while studying EGFR
localization to lysosomes in EGF-treated cells after ACK knockdown with siRNA (72).
However, the level of EGF used in their study was higher and thus, internalization of EGFRs could be assumed to take place via both CME and CIE-mediated mechanisms. The process of programmed cell death (known as apoptosis) in hematopoietic cells requires receptor clustering and formation of signaling complexes. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced translocation of TRAIL receptors (TRAIL-Rs) into lipid raft membrane 18 microdomains is essential for the normal apoptotic process. Linderoth and coworkers showed
TRAIL-induced transient increase in ACK levels and its recruitment in caveolin-1 containing lipid rafts along with TRAIL-Rs. ACK was shown to be required for EGFR-independent TRAIL induced apoptosis and TRAIL-R1 clustering through oligomerization. Interestingly, ACK depleted cells showed impaired localization of TRAIL-R1in lipid rafts (73).
Table 1.1: Stages of ligand-induced EGFR trafficking in relation to ACK a
Ligand Stage of Effect observed on concentration ACK expression Reference trafficking relevant stage of traffic (ng/ml)
Retention in EGF 60 ACK knockdown 30% increase on PM (71) PM
ACK-EGFR EGF 100 endogenous ≥ 30 minutes (66) binding
TNK2-EGFR EGF 100 endogenous ≥ by ~90 minutes (74) binding
ACK-EGFR EGF 100 exogenous ≥ 30 minutes (75) binding
ACK-EGFR ACK Ralt/MIG6 slightly > over 30 EGF 100 (66) binding domain (786-891) minutes
ACK-EGFR EGF 100 exogenous constitutively bound (66) binding
EGFR internalization rate by EGF 1.5 endogenous (70) internalization CME 0.315
EGFR << internalization rate EGF 20-100 endogenous (70) internalization CME ~0.060
TfR. exogenous NA CME inhibited (76) internalization 19
EGFR EGF 50 exogenous CME inhibited (71) internalization
EGFR EGF 1 Grb2 knockdown ~60% of normal rate (77) internalization
EGFR Cetuximab (1 endogenous CME inhibited (78) internalization nM)
EGFR clathrin reduced to ~20%, 6 EGF 1.5 (69) internalization knockdown minutes
EGFR clathrin reduced to ~50%, 6 EGF 20 (69) internalization knockdown minutes
EGFR reduced to ~70%, 10 EGF 1 exogenous (71) internalization minutes
EGFR reduced to ~70%, 6 EGF 1 ACK knockdown (71) internalization minutes
Retention in EE EGF 60 exogenous 5-fold increase in EE (71)
Retention in EE EGF 60 ACK knockdown 10-fold increase in EE (71)
EGFR recycling EGF pulse endogenous ~50% recycled/hour (70)
EGFR recycling EGF 100 endogenous <15-30% recycled/hour (70)
Cetuximab (1 2-fold recycling cf. EGFR recycling endogenous (78) nM) EGF
*EGFR ACK-Ralt/MIG6 EGF 100 inhibits degradation (79) degradation (727-915)
*EGFR half-life doubled to > EGF 100 ACK knockdown (66) degradation 60 minutes
EGFR half-life shortened from EGF 100 exogenous (66) degradation 3 to 1.5 hours
EGFR ACKΔUBA (970- inhibits degradation cf. EGF 100 (66) degradation 1055) exogenous
*EGFR EGF 100 ACK UBA half-life ~2x greater (59) 20 degradation mutant S985N
*EGFR EGF 100 ACK knockdown half-life increased (80) degradation
*EGFR half-life increased cf. EGF 50 ACK mutant Δ89 (81) degradation wtACK
*EGFR Nedd4-1 half-life increased EGF 50 (81) degradation knockdown many-fold
*EGFR Cetuximab (1 inhibits degradation cf. endogenous (78) degradation nM) EGF
EGFR ~25% of EGF 1.5 endogenous (70) degradation internalized/hour
EGFR ~50% of EGF 20-100 endogenous (69) degradation internalized/hour
*Axl half-life increased Gas6 400 ACK knockdown (82) degradation many-fold
a PM: Plasma membrane, TNK2: Tyrosine kinase, non-receptor, 2 (ACK1 is also known as
TNK2), Ralt/MIG6: Mitogen-inducible gene 6 (EGFR binding domain of ACK), TfR:
Transferrin receptor, Grb2: Growth factor receptor-bound protein 2 (an adaptor protein), EE:
Early endosome, UBA: Ubiquitin associated domain (domain of ACK), Nedd4-1: neural precursor cell expressed developmentally down-regulated protein 4 –isoform 1 (E3 ubiquitin ligase which binds to ACK), NBD: Nedd4 binding domain of ACK, Axl: receptor tyrosine kinase, Gas6: Growth arrest-specific gene-6, ACKΔUBA: UBA deletion mutant,ACKΔ89: ACK
SAM domain deletion mutant.
* Details of highlighted text are discussed later (see Discussion). 21
1.6 Experimental model
Filopodia play sensory roles by acting like ‘antennae’ to sense the cell’s surroundings. In
the nerve growth cone, they promote motility towards an attractive cue or away from a repulsive
one. In addition to this response to attractants or repellents, which regulates ‘nerve guidance’ or
neuronal growth-cone pathfinding, filopodia have also been reported to be involved in wound
healing, adhesion to ECM and embryonic development (17, 21). A number of cytoskeletal
regulatory proteins have been implicated in the extension/retraction cycle and its regulation. As
Mattila and Lappalainen mention in their review, although the basic mechanism of filopodia
formation can be elucidated through a ‘working model’, the role played by individual proteins
and their importance in the process may vary among organisms and cell types (1). This is a
possible explanation of the fact that morphology and dynamics of filopodia and associated
membrane protrusions vary in different cell lineages. Although a number of proteins which
include factors regulating actin filament nucleation and elongation, actin-crosslinking, motor
activity, signaling and also those responsible for protrusion-associated plasma membrane deformities have been identified, it is likely that other unidentified factors are also involved, thus requiring systematic identification and analysis of filopodial components in relation to filopodial dynamics.
All of these cytoskeletal regulatory proteins along with other associated ones function in a coordinated but complex network to bring about formation and regulation of filopodia. The effectors ACK, PAK, WASP and Par6 bind to Cdc42 through intermolecular interactions with the β2 strand. Cdc42 also binds to IRSp53 and mDia2. Similarly, IRSp53 was found to be present in an immunoprecipitable complex with WAVE2 and Abi1 in a Rac1-activation- dependent manner in RAW/LR5 cells (83). Importantly, reduction of endogenous IRSp53 or 22
expression of IRSp53 lacking the WAVE2-binding site resulted in a significant reduction in the
association of Rac1 with WAVE2 and Abi1, indicating that such an association is IRSp53
dependent. Such binding of effectors to the Cdc42 or Rac GTPases and subsequent recruitment
of Arp2/3, Ena/VASP, fascin, myosin-X, and structural proteins like actin and integrin, all
together form the complex structure.
The fundamental mechanism of filopodia dynamics is assumed to occur through cycles of
extension and retraction in a way reminiscent of motility of the lamellipodium. The process
might be considered in light of a predator-prey model. As shown in Figure 1.7 below, with an
increase in the population of prey, the predator population increases initially but after a certain time, when the predator population is high and depletes the prey population, both start decreasing. Similarly, during filopodial dynamics, we may postulate that when extension is upregulated, the retraction factors are co-upregulated. Eventually, retraction takes over and extension is downregulated. In this project, I have tried to alter the cycle of protrusion and retraction by introducing binding peptides (BPs) directed against specific protein-protein binding
interfaces.
Figure 1.7: Graphical representation showing cyclical relationship predicted by the model for
hypothetical predator and prey populations.
(Source:http://www.tiem.utk.edu/~gross/bioed/bealsmodules/predator-prey.html33TU )U 23
It will be interesting to find out how filopodia dynamics is regulated by Cdc42 and/or
Rac effectors and associated downstream protein interactions including those thought to be
involved in regulation of receptor trafficking. An epithelial cell line has been used since it is
already known that most cancers initiate as carcinomas i.e. cancer of epithelial cells. It has also
been shown that invasive colorectal cancer cells exhibit large amounts of filopodia (84). Since it seems that these effector proteins play crucial roles in the sensory function of filopodia, the objective is to inhibit their binding at specific domains by incorporating BPs to block the binding sites relevant to cyclical protrusion and retraction of the structures. Cell peripheries will be analyzed to determine the levels of filopodia prevalence in experimental and control treatments.
The functions of the proteins like Ena/VASP, fascin, actin, integrin and myosin-X in formation and regulation of filopodia have been somewhat determined and moreover, they have long binding domains and do not bind to GTPases. Thus, their binding interfaces have not been investigated in this study. Further work with the above purpose and idea will lead to understanding of the basic but complex underlying mechanisms of the filopodial dynamics. 24
CHAPTER 2. MATERIALS AND METHODS
2.1 Cell culture and chemicals
A heterotopic tracheal transplant from a Fisher rat was treated with 7, 12-dimethylbenz(a) anthracene to generate 1000W cell line which was maintained under routine conditions at 37oC
and 5%CO2 in 60mm tissue culture dishes (BD, Franklin Lakes, NJ) with Waymouth’s medium.
To make the medium, 14.0 g of Waymouth MB 752/1 medium (Sigma Aldrich, MO) powder with L-glutamine was dissolved in 900 ml of deionized water and sterilized by Millipore membrane filter with 0.2 µm pore size. The medium was finally supplemented with antibiotics, penicillin and streptomycin, 10% fetal bovine serum (Hyclone, UT or Atlanta Biologicals, GA),
2.24% of sterile 10% NaHCO3 solution (Matheson, OH), 0.1µg/ml insulin and 0.1 µg/ml hydrocortisone. The final formulated Waymouth’s medium (referred to as WIHC for the insulin and hydrocortisone supplements) was tested for sterility before use by incubating at 37oC and
5%CO2 for 48 hours. Also, WIHC was formulated from a commercial Waymouth’s medium
(Invitrogen, NY) following a similar protocol as above. For subsequent cell passages and maintenance of cell line, confluent 1000W cells were sub-cultured by removing them from tissue culture dishes using calcium and magnesium-free Hanks balanced salt solution (CMF-HBSS)
with ethylene diamine tetraacetate (EDTA) and trypsin. For its preparation, 50 ml of 10X CMF-
HBSS (Life Technologies, NY) and 0.875 g of Na-EDTA were dissolved in 100ml of deionized
water. 10 mg of phenol red (Matheson, OH) [pH indicator] and 175 mg of NaHCO3 (Matheson,
OH) were dissolved to a final volume of 400 ml and combined with CMF-HBSS EDTA solution. pH was adjusted by slow addition of 1N NaOH solution until the final color of the liquid turned light pinkish. Deionized water was further added to a final volume adjustment of 500 ml and the solution was finally sterilized by autoclaving. The final trypsin working solution was prepared by 25
addition of 12.5 ml of 0.5% trypsin-EDTA (Life Technologies, NY) to 87.5 ml of sterile CMF-
HBSS EDTA solution. This was tested for sterility before use by addition of an excess of sterile
WIHC medium supplemented with 10% fetal bovine serum followed by incubation at 37oC and
5%CO2 for 48 hours.
2.2 Substrate preparation
No. 1 round cover glasses (Electron Microscopy Sciences, Hatfield, PA) were made molecularly clean by keeping them immersed in 1N hydrochloric acid for 2.5 hours, rinsing them thoroughly with deionized water and keeping them immersed again in absolute ethanol for 1 hour. The coverglasses were turned frequently to ensure that they do not get stuck to each other.
Precautions were taken to touch the cover glasses only with forceps cleaned previously using hydrochloric acid and ethanol. The coverslips were coated with germanium in a high vacuum coater (Denton BTT-IV, NJ) to produce substrates favorable for attachment of 1000W cells. Six molecularly clean coverslips were completely air dried and mounted on a rotating table placed at a height of 5.5 cm from the base of the coating chamber. The tungsten wire basket (Style A,
Electron Microscopy Sciences, Hatfield, PA) was loaded with 0.025 g of germanium (Structure
Probe, Inc. West Chester, PA), connected to the respective electrodes and placed at a height of
11.5 cm from the base of the coating chamber and at a distance of 4.5 cm from the turntable. A vacuum of 2x10-5 torr, rotation of the turntable at 10% and resistive current of 9-10 amps for 2-
2.5 minutes were maintained until all the germanium in the tungsten wire basket evaporated and
the top surface of the cover glasses were coated. They were then placed in 35 mm petri dishes
(Celltreat Scientific Products) and both sides were rendered sterile by UV irradiation. 26
2.3 Peptides designed to interrupt protein-protein interactions
Cdc42 and Rac belong to the Rho family of GTPases and interact with a large and diverse array of effector proteins. Such interactions are critical for a number of downstream signaling pathways. As mentioned above, the effector proteins bind to Cdc42 and/or Rac at specific domains called CRIB. However, not all effectors may have such CRIB domains but still bind and interact with these GTPases through non-homologous domains. There are small stretches of amino acid residues that are critical for effective binding and interaction. Also, most of these effector proteins have their own binding partners and interact with more than one protein at the same time. These binding interfaces are also domain specific but critically depend on a particular sequence of amino acid residues. Such interactions are required for a number of cellular processes such as endocytosis and other receptor or non-receptor mediated signaling pathways including cell adhesion. Short stretches of amino acid residues were designed to block the binding of Cdc42 or Rac to their effectors and other proteins downstream of these GTPases to their binding partners. When such synthetic binding peptides (BPs) were delivered into the cells, it could be determined whether filopodia dynamics are regulated by the GTPase or other protein(s). Peptides were synthesized by Peptide 2.0 (Chantilly, VA). Table 2.1 shows how BPs with sequences that compete for corresponding effector binding, have been designed to represent
GTPase domains. Table 2.2 shows selected sequences that represent domains of other proteins.
Table 2.1: BPs designed to inhibit binding of effectors to Cdc42 and Rac GTPases
Binding peptides Cdc42 domain Rac domain Residues
Cdc42-PAK VPTVFDNYA 33-41
Rac-PAK IPTVFDNYS 33-41 27
Cdc42-WASP KTVFDEAILAALEP 166-179
Rac-WASP KTVFDEAIRAVLCP 169-177
Cdc42-IQGAP1 VVSPSSFENV 84-93
Cdc42-IQGAP2 NKQKPITPE 132-140
Rac-IQGAP2 KKLTPITYP 132-140
Par6 inhibiting EDYDRLRPL 62-70
Cdc42-Ack VTVMIGGEPYTL 42-53
Table 2.2: BPs designed to inhibit downstream protein-protein interactions
Binding ACK sequences Integrin β5 cAbl sequences Residues peptides sequences
Ack-Clath1 NPMDPPDLLSVELSTSRP 489-506
Ack-Clath2 AEVTLIDFGEEPVVPALRP 566-584
Ack-Nedd4 ARPLPPPPAYDDVA 626-639
Ibeta5-PAK4 EFAKFQSERS 753-762 cAbl-PAK2 PTPPKRSSSFR 631-641
2.4 Peptide delivery using BioPorter Reagent
BioPORTER reagent was purchased from Genlantis (San Diego, CA) and prepared following manufacturer’s instructions. In vitro delivery of BioPORTER-BP complex into cells is shown in the Figure 2.1. 28
Figure 2.1: Delivery of synthetic BPs into cells using BioPORTER reagent. © 2011 Genlantis
Inc.
5 In each of the dishes prepared as described above (see Substrate Preparation), 1-2 x 10P P
o 1000W cells were plated and incubated overnight at 37P CP and 5% COR2R. Before the start of the
experiment, I checked to be sure that the cells attached to the germanium substrates. The WIHC
o medium in each dish (except untreated control) was replaced with 1.2 ml of pre-warmed (37P C,P
5%COR2R) serum-free WIHC, at least 30 minutes before the BioPORTER-peptide solution was added. 29
The binding peptides were previously reconstituted with sterile phosphate buffered saline
(PBS) (20mM Na-phosphate, 150mM NaCl, pH 7.4) at a final concentration of 50µg/ml.
A 5µl aliquot of BioPORTER reagent was previously dried down in a single sterile tube. A 7µl
aliquot of BP solution was now added to this tube, gently vortexed and allowed to stand at room
temperature for 5minutes. A 5µl aliquot of this solution was then added to the respective dishes.
When a combination of two or more peptides was used, the final total volume of peptide mixture added was also 5µl.
o The dishes were then incubated at 37 C and 5%CO2 for 8 hours. Then, 0.9ml of pre-
o warmed (37 C, 5%CO2) 20% serum supplemented WIHC was added to each of the dish and the
dishes were further incubated for 18-20 hours under routine conditions.
Dishes were finally fixed with pre-warmed (37oC) 3% paraformaldehyde (pH 7.2-7.4) for
10 minutes and rinsed thrice gently with sterile cold PBS.
The coverslips were stored in PBS at 4oC until analyzed.
2.5 Analysis
Each sample, treated with BP-BioPORTER solution or an untreated control sample
(FITC [fluorescein isothiocyanate]-tagged antibody-BioPORTER) was observed by phase
contrast microscopy. Only single cells with ≥75% of the periphery visible and which were not
rounded up were accepted for analysis. Each sample was observed in a raster pattern and every
single cell on each point of observation was scored. For every sample, 50-200 single cells were
recorded. The following two variables were measured:
(a) Mean percent of cells showing filopodia.
(b) Mean percent coverage of cell periphery with filopodia. 30
For the variable (b) above, the cell periphery was observed and the percent coverage with filopodia was assigned a value in the range of 0 to 100 in a multiple of 5. A value of 0% was assigned when the whole periphery was completely devoid of filopodia whereas a value of 100% meant that the periphery was completely covered with filopodia. Figure 2.2 shows an example of such analyses.
Figure 2.2: Method of estimating coverage with filopodia. Each single cell was arbitrarily divided into four quadrants during observation and the coverage of the periphery with filopodia was assigned a fractional value (see text).
The possibility of apoptosis and toxicity to the cells were taken into consideration but no signs of such effect were noted. It is to be noted that the observers did not consider the same cells since the observations were independently carried out. It is to be noted that each variable was measured by two or more observers who analyzed every sample independently. Thus, there is a possibility that for each sample, the variations could also arise from the variations in the sample.
Finally, all calculations and statistical analysis were done using Microsoft-Excel. 31
CHAPTER 3. RESULTS
3.1 Assay for effect of binding peptides on filopodia dynamics
The purpose of my studies was to investigate protein-protein binding interfaces downstream of Cdc42 effectors identified previously as engaged in filopodia regulation (85). To determine which of the candidate proteins, if any, were implicated in filopodia dynamics, BPs were introduced with BioPORTER reagent. As described in Materials and Methods, these samples were observed and analyzed in comparison to control samples which received only FITC-tagged antibody. The mean percent of cells showing filopodia and mean percent coverage of periphery with filopodia were determined for each sample. Because this assay was subjective, observations were made and recorded independently by two or more observers. For each BP treatment, the primary objective was to find out whether the proportion of cells showing filopodia and the percent coverage of periphery with filopodia were changed relative to the experimental control.
I used BPs with known effects on filopodia prevalence to find out whether the new assay described above (see Materials and Methods) could be used to determine effects on filopodia. The assay was not quantitatively and qualitatively exact like the assay used in previous studies by the laboratory. However, this subjective assay allowed for more rapid determination of whether BPs had a net effect on the two variables measured (see
Materials and Methods). To evaluate the two variables, the difference between experimental and control samples was used as an index (Tables 3.1 and 3.2). The means of differences with the control were also shown. I initially worked with BPs that were previously found to decrease the prevalence of filopodia, namely IQGAP2, Par 6 and ACK, in order to determine whether I could recognize the inhibitory effect. The range of values found was -3.04 to -18.69 and the average was -8.79 for mean percent of cells showing filopodia. For the mean percent coverage 32
of periphery with filopodia, the range was -18.76 to 3.99, the average being -14.24. Thus, the
overall effect on filopodia prevalence was negative in this assay as well as in the more precise
assay used in previous studies. On the other hand, BPs that were known to affect filopodia
positively or not at all, namely IQGAP1, PAK and WASP, gave a range of 0.95 to 40.20 and an
average of 12.30 for mean percent of cells showing filopodia. In case of the mean percent
coverage of periphery with filopodia, the BPs gave a range of -9.83 to 7.83, with an average of -
1.11. Thus, BPs which previously showed either a positive or no effect on filopodia prevalence
showed a positive effect on the mean percent of cells showing filopodia and a negligible effect
on mean percent coverage of periphery.
For analyzing the two variables, the difference from experimental control was used as an
index (Tables 3.1 and 3.2) to reveal the effect of BPs containing Cdc42 and Rac sequences (see
Table 2.1 in Materials and Methods). BPs interfering at Cdc42-IQGAP2, Cdc42-Par6 and
Cdc42-ACK interfaces gave negative numbers for both the variables (Tables 3.1 and 3.2). A
positive number in Table 3.1 for Cdc42-IQGAP1 BP treatment indicated an increase in the mean
percent of cells showing filopodia over the experimental control. However, the same BP did not
increase the mean percent coverage with filopodia as indicated by a negative number in Table
3.2. With repeated observations for treatments with Cdc42-IQGAP1, Cdc42-PAK and Cdc42-
WASP BPs, one could see whether the same cells are making more filopodia or there are more numerous cells making such protrusions. Unfortunately, it was not possible to aggregate the two variables into a single index and thereby attain a more robust variable. BPs for Rac-PAK and
Rac-WASP mostly showed positive numbers in both tables 3.1 and 3.2. 33
Table 3.1: Effect of BPs representing GTPase sequences on mean percent of cells showing
filopodia in experimental sample versus paired control a,b
Binding Expt.1 Expt.2 Expt.3 Expt.4 Expt.5 Expt.6 Expt.7
peptides
Cdc42- 8.51
IQGAP1
Cdc42- -10.19
IQGAP2
Par6 -3.25
inhibiting
Cdc42- -18.69 -3.04
ACK
Cdc42- 9.14 2.38 14.25 0.95 34.21
PAK
Cdc42- 2.60 40.20 6.79 3.96
WASP
Rac- 26.17 2.00 0.81
IQGAP2
Rac-PAK 6.41 3.32 6.48
Rac- -2.90 9.96
WASP
a Experiments 4-6 were done by Pratima Pandey. Amino acid sequences of the peptides are shown in Table 2.1 (see Materials and Methods). 34 b Amino acid sequences of the peptides are shown in Table 2.1 (see Materials and Methods)
Table 3.2: Effect of BPs representing GTPase sequences on mean percent coverage in experimental sample versus paired control a,b
Binding Expt.1 Expt.2 Expt.3 Expt.4 Expt.5 Expt.6 Expt.7
peptides
Cdc42- -9.83
IQGAP1
Cdc42- -18.76
IQGAP2
Par6 -17.07
inhibiting
Cdc42- -17.14 3.99
ACK
Cdc42- -16.24 -4.05 4.47 0.22 7.83
PAK
Cdc42- -12.33 7.23 1.95 1.54
WASP
Rac- 12.08 1.80 -1.67
IQGAP2
Rac-PAK 4.53 3.08 -0.94
Rac- 9.16 5.91
WASP 35
a Experiments 4-6 were done by Pratima Pandey.
bAmino acid sequences of the peptides are shown in Table 2.1 (see Materials and Methods).
3.2 Peptides blocking downstream of GTPase effectors
The effect of BPs representing ACK, cAbl and integrinβ5 sequences (see Table 2.2 in
Materials and Methods) were assessed by the above assay to determine whether their interaction with their respective binding partner(s) played any role in governing filopodial dynamics. For each BP treatment, the difference relative to the control has been considered as an index of the effect (Tables 3.3 and 3.4). When ACK-Nedd4 binding was interrupted by ACK-Nedd4 BP, the mean percent of cells showing filopodia increased over the control in repeated measures, as evident from the consistent positive numbers in Table 3.3. The mean percent coverage with filopodia also showed positive differences over the control in repeated experiments (Table 3.4) with this BP. A BP designed to block the interaction of clathrin at the clathrin-binding domain of
ACK, containing the residues LLSVE, mostly showed positive numbers, indicating increases over the control for both the variables measured. Interestingly, when cAbl-PAK2 and Iβ5-PAK4 binding interfaces were competed by introducing cAbl-PAK2 and Iβ5-PAK4 BPs, both the mean percent of cells showing filopodia and mean percent coverage with filopodia revealed inconsistent differences with the control (Tables 3.3 & 3.4). However, the average overall trends suggest an elevation in values, at least for cAbl-PAK2. 36
Table 3.3: Effect of BPs representing non-GTPase sequences on percent of cells showing filopodia in experimental sample versus paired control a,b
Binding Expt. Expt. Expt. Expt. Expt. Expt. Expt. Mean
peptides 1 2 3 4 5 6 7 difference
with paired
control
Ack- 22.73 16.99 11.95 9.32 5.02 10.05 12.68 c Nedd4
Ack- 12.41 5.48 5.55 -0.33 5.78 Clath1 cAbl- 7.07 9.64 1.85 -1.55 4.25 d PAK2
Ibeta5- 20.14 -10.2 4.10 4.68 PAK4 a Experiments 4-6 were done by Pratima Pandey. bAmino acid sequences of the peptides are shown in Table 2.2 (see Materials and Methods). c Significant upon t-test, degrees of freedom=5, t-value=4.9486 and p-value (two-tailed)= 0.004. d Insignificant upon t-test, degrees of freedom=3, t-value=1.3937 and p-value (two-tailed)=0.258. 37
Table 3.4: Effect of BPs representing non-GTPase sequences on mean percent coverage in
experimental versus paired control a,b
Binding Expt.1 Expt.2 Expt.3 Expt.4 Expt.5 Expt.6 Expt.7 Mean
peptides difference
with
paired
control
Ack- 1.25 5.05 5.78 10.33 0.92 2.43 4.29 c Nedd4
Ack- 7.30 5.48 -2.11 0.79 2.87 Clath1 cAbl- 12.01 4.68 -0.27 0.42 4.21 d PAK2
Ibeta5- 14.28 -2.60 1.49 3.29 PAK4 aExperiments 4-6 were done by Pratima Pandey.
bAmino acid sequences of the peptides are shown in Table 2.2 (see Materials and Methods).
c Significant upon t-test, degrees of freedom=5, t-value=2.9965 and p-value (two-tailed)= 0.030.
d Insignificant upon t-test, degrees of freedom=3, t-value=1.3541 and p-value (two-tailed)=0.269.
For certain BPs, the results of replicated experiments suggested a possibility that
filopodia prevalence was elevated over the control. The results for ACK-Nedd4 BP treatment are shown graphically in Figures 3.1 and 3.3. The mean differences with paired control for both the measured variables were statistically significant as shown in Tables 3.3 and 3.4. 38
In another experiment, ACK-Nedd4 BP was used in combination with other BPs namely,
ACK-Clath1, ACK-Clath2 (competing the binding of clathrin to another clathrin binding domain
representing residues LIDFG of ACK) and Cdc42-WASP. The aim was to see any combinations
of BPs showed synergistic effects on filopodia prevalence. Both the variables were measured and
the results represented graphically (Figures 3.2 and 3.4). When both the clathrin binding domains
of ACK were blocked independently along with ACK-Nedd4 BP, mean percentage of cells
showing filopodia did increase over the control (Fig. 3.2). When both clathrin binding domains
of ACK and WASP binding domain of Cdc42 were blocked simultaneously along with Nedd4
binding motif of ACK, the variable revealed an increase over the control. All the combinations
of BPs revealed increase over the control in case of mean percent coverage of cell periphery with filopodia (Figure 3.4). However, in general, the differences were so small as to be insignificant, suggesting a lack of synergy.
90.00
60.00
filopodia 30.00 Percentage ofcells showing
0.00 ACK-Nedd4 Control
Figure 3.1: Percentage of cells showing filopodia after ACK-Nedd4 BP treatment versus control.
Mean value of six repeated ACK-Nedd4 BP treatments compared to controls. Bars represent 39
±standard error (SE) of the mean. The statistical significance of the difference with paired control is shown in Table 3.3.
90.00
60.00
30.00
Percentage of cells showing filopodia showing cells of Percentage 0.00
Figure 3.2: Mean percent of cells showing filopodia after ACK-Nedd4 BP treatment in
combination with other BPs (see text for details). The standard error represents the variation in observations by two or more observers. 40
30.00
20.00
filopodia 10.00 Percent coverage ofperiphery with
0.00 ACK-Nedd4 Control
Figure 3.3: Percent coverage of periphery with filopodia after ACK-Nedd4 BP treatment versus control. Mean value of six repeated ACK-Nedd4 BP treatments compared to controls. Bars represent ±standard error (SE) of the mean. The statistical significance of the difference with paired control is shown in Table 3.4. 41
10.00
5.00
Percent coverage of periphery with filopodia with periphery of coverage Percent 0.00
Figure 3.4: Mean percent coverage of periphery with filopodia after ACK-Nedd4 BP treatment in
combination with other BPs (see text for details). The standard error represents the level of
variation in observations by two or more observers.
The cAbl-PAK2 BP inhibited the binding of PAK2 to a specific cAbl domain
representing its substrate. The results of cAbl-PAK2 BP treatment are represented graphically in
Figures 3.5 and 3.6. Both mean percentage of cells showing filopodia and mean percent coverage 42
of periphery with filopodia showed an increase over the control but the variables were not
significantly different from the control (Tables 3.3 and 3.4).
100.00
50.00 Percentage of cells showing filopodia showing cells of Percentage 0.00 cAbl-PAK2 Control
Figure 3.5: Percentage of cells showing filopodia after cAbl-PAK2 BP treatment versus control.
Mean value of four repeated cAbl-PAK2 BP treatments compared to controls. Bars represent
±standard error (SE) of the mean. The statistical significance of the difference with paired control is shown in Table 3.3. 43
16.00
filopodia 8.00 Percent coverage ofperiphery with
0.00 cAbl-PAK2 Control
Figure 3.6: Percentage coverage of periphery with filopodia after cAbl-PAK2 BP treatment versus control. Mean value of four repeated cAbl-PAK2 BP treatments compared to controls.
Bars represent ±standard error (SE) of the mean. The statistical significance of the difference with paired control is shown in Table 3.4.
3.3 Membrane trafficking and filopodia
During my study, I investigated the effect of modulating membrane trafficking on filopodia formation. The cells were grown under routine conditions on favorable adhesive substrates (see
Materials and Methods) and treated with a number of chemical compounds, which have been reported to affect the stages of receptor trafficking. Samples were analyzed to determine whether cells were able to make more filopodia compared to the control. The mean difference with paired control was used as an index to determine the effect of the compounds on filopodia coverage of periphery. The results of this assay are summarized in Table 3.5. 44
Table 3.5: Effect of membrane trafficking modulation on mean coverage of periphery with filopodia a
Compounds Function Concentration Time of Difference
treatment with paired
control
Filipin Sterol binding agent that 1 μg/ml 30 +1.29
disrupts caveolae and minutes
caveolae-like structures, 1 μg/ml 15 -2.56
thus inhibits CIE (86) minutes
5 μg/ml 30 -4.15
minutes
Brefeldin A Prevents assembly of non- 1 μg/ml 1 hour -1.81
clathrin coated buds on 1 μg/ml 30 -3.29
Golgi cisternae, thus minutes
prevents transport from 5 μg/ml 1 hour +13.59
Golgi to endoplasmic
reticulum (87)
K252a A structural analogue of 6 nM 2 hours -0.31
staurosporin, acts as a 24 nM 2 hours +1.08
protein tyrosine kinase
inhibitor , thus inhibits
receptor phosphorylation 45
(88)
Neomycin Inhibits CME by probably 10 mM 30 -2.74
binding to minutes
phosphatidylinositol-4,5-
bisphosphate which is
required for endocytic
coated vesicle formation
(89)
Sodium Inhibits protein tyrosine 10-3 M 30 +9.51
orthovanadate phosphatases (90) and minutes
+ hydrogen induces translocation of
peroxide receptors to plasma
membrane (91)
Phenyl arsine Reduces rate of receptor 100 nM 30 +3.37
oxide internalization /endocytosis minutes
(92) by inhibiting protein
tyrosine phosphatase
activity (93)
a A positive difference indicates an effect higher than the control whereas, a negative difference indicates an effect lower than the control.
The only compound which showed a large effect on filopodia formation was Brefeldin A at the highest concentration and longest treatment duration, as shown in Table 3.5 above. Cells were 46 able to make more filopodia compared to the control when Golgi-dependent retrograde trafficking of receptors was inhibited for as long as one hour and with a higher dosage of the inhibiting compound. Possibly, there was enhanced exocytosis at the plasma membrane which could have increased filopodia formation. The combination of sodium orthovanadate and hydrogen peroxide inhibits protein tyrosine phosphatases and thus probably disrupts endocytic processing of the receptors. I speculate that such retarded endocytosis might have resulted in increased filopodia formation. 47
CHAPTER 4. DISCUSSION 4.1 Role of Cdc42 effectors and downstream proteins in filopodia adynmics
The aim of my investigation was to determine which proteins regulate filopodia dynamics. A number of Cdc42 effectors and downstream proteins which were previously reported to be involved in cytoskeletal remodeling and filopodia formation were explored.
IQGAP functions as a scaffold protein for the assembly of a multicomponent system, binds to paxillin in focal adhesions and cross-links actin and microtubule cytoskeletal structures (85).
It localizes in the leading edge of the cell by binding to the tumor suppressor protein APC
(adenomatous polyposis coli) has been implicated in the processing of epidermal growth factor
(EGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) (94).
IQGAP has been shown to interact with EGFR and EGF-stimulated activation of EGFR catalyzes phosphorylation of IQGAP. This in turn, enhances EGFR phosphorylation and thus,
IQGAP has been suggested to modulate EGFR activation (95). IQGAP interacts with the Cdc42 alpha3 helix and requires residues in the sequence 83-120 for binding. Previous studies assessing prevalence of filopodia in cells grown on different zones [high (H), medium (M) and low (L)] of an adhesive gradient have reported dissimilar effects of the two BPs targeting two different
Cdc42 domains specific for IQGAP binding. Cells treated with Cdc42-IQGAP1 (residues 84-93) showed numerous filopodia whereas those treated with Cdc42-IQGAP2 (residues 132-140) lacked filopodia in all the zones of the gradient (85). As shown in Tables 3.1 and 3.2 (see
Results), treatment with Cdc42-IQGAP1 BP caused an increased mean percent of cells showing filopodia but a decreased mean percent coverage of periphery with filopodia over the experimental control. For Cdc42-IQGAP2, both the variables showed decreased values compared to the control. Interestingly, when the BP Rac-IQGAP2 (residues 132-140 but 48 differing from the Cdc42 sequence by five residues) blocked the binding of IQGAP to Rac, both variables exhibited increases over the control (Tables 3.1 and 3.2 in Results).
Several Cdc42 binding proteins like IRSp53, Toca-1 and/or formins have reportedly been involved in filopodia formation, but the exact role played by Cdc42 in regulating filopodia is still unclear (96). Previously, it was thought that WASP binding to Cdc42 activates Arp2/3 mediated filament formation in filopodia (97, 98). However, recent studies have indicated that such a pathway of filopodia formation is uncommon (96). WASP binds to β2 strand of Cdc42 (99), closer to the Switch II region. Earlier studies have shown that introduction of Cdc42-WASP BP
(residues 166-179) had no effect on filopodia prevalence in cells grown on a gradient of adhesive metallic substrate (85). Results shown in Tables 3.1 and 3.2 also suggest that Cdc42 domain specific for WASP has no significant effect on filopodia.
Cells adhere to substrates through dynamic structures called focal contacts (FCs) composed of numerous proteins assembled on integrins (100). Actin polymerization on FCs is mediated by molecules that are recruited by integrins. PAK is recruited to FC by PAK-interacting exchange factor (PIX) which also activates it (101, 102) by supplying active Cdc42 or Rac (103). PAK turns over stable FCs and mediates cell motility (104). Interestingly, there is evidence that expression of PAK mutated to eliminate GTPase binding did not affect FCs but prevented cell orientation during directional migration suggesting that persistent cellular motility required interaction of PAK with Cdc42 and/or Rac (105). Also, PAK along with another adaptor Nck regulate oriented movement during photoreceptor axon guidance in D. melanogaster (106).
Previous studies have shown that when PAK recruitment by PIX or residence time of PAK in
FCs were restricted by exogenous expression of genetic constructs, filopodia decreased in tracheal epithelial cells, suggesting PAK turns over filopodia (107). In the current study, when 49
the binding of PAK to Cdc42 or Rac was blocked by using the BPs Cdc42-PAK (residues 33-41) and Rac-PAK (residues 33-41 but differing from Cdc42 sequence by two residues) respectively, cells had slightly more or the same cell periphery coverage with filopodia compared to control
(Table 3.2 in Results).
As discussed above, PAK4-mediated phosphorylation at specific sites within the integrin-β5 domain is critical for regulating PAK4-mediated cell motility. In contrast to other isoforms of
PAK, PAK4, activated by constitutively active Cdc42, is localized to the Golgi apparatus by
Cdc42 with concomitant effects on filopodia formation (108). Continuous and sequential generation of filopodial structures containing integrins and focal adhesion proteins has been implicated in fibroblast spreading (109). Patridge and co-workers have shown that cell spreading proceeds through assembly of focal adhesion components into filopodia. Based on their findings,
Cdc42 is implicated in the formation of integrin-containing filopodia that serve as adhesion precursors in spreading cells. Interestingly, they found that PAK4 acts as a mediator during such an event and thus, has an effect on filopodia formation. In the current study, when phosphorylation of integrin-β5 by PAK4 was inhibited by introducing Iβ5-PAK4 BP, the
percentage of cells showing filopodia and cell periphery coverage with such protrusions
increased, compared to the control. Although the differences fell short of statistical significance,
these results do suggest that PAK4 and its interaction with integrin-β5 domain is important in
regulating filopodial dynamics.
As mentioned earlier (see Introduction), PAK2 binding and phosphorylation of cAbl,
inhibited the interaction of Abi and c-Abl. This resulted in phosphorylation of Crk by c-Abl and
subsequent inhibition of Crk signaling. Inhibition of Crk signaling along with Nck engagement
was shown to promote filopodia formation during cell attachment (53). PAK2 is also thought to 50 affect filopodia dynamics by affecting assembly and disassembly of FCs. In fact, Heckman and coworkers have shown that a peptide blocking PAK2-Nck interaction increased filopodia although the underlying mechanism is still unclear (107).
4.2 ACK plays a critical role in receptor trafficking
4.2.1 ACK binds constitutively and conditionally to other proteins
Endogenous ACK has been shown to interact with EGFR. However, ACK appears to bind to EGFR after getting localized to clathrin-coated pit but before receptor internalization
(67). Two domains of ACK are implicated in its colocalization with the EGFR; MIG6-homology domain and the clathrin-binding domain (66, 72). MIG6 is an inducible Cdc42-binding protein that inhibits EGFR signaling (110). Teo and coworkers found that clathrin heavy chain (CHC) can be pulled down with GST-ACK (residues 443-646) (111). Two domains of ACK are implicated in binding CHC; LLSVE (residues 496-500) and LIDFG (residues 584-588) (67).
Since ACK exhibits a multi-domain structure (Figure 4.1), additional proteins associate with
ACK, including amphiphysin and adaptor protein 2 (AP2) (79). These proteins are involved in trafficking but may not bind directly to ACK. Manser and coworkers showed that a third protein mediating sorting, sorting nexin 9 (SNX9/SH3PX1) binds to inactive ACK at its fifth proline rich (PR) motif (residues 920-955) (112). 51
Figure 4.1: Schematic diagram showing ACK (1038 amino acid residues) and identifying
domains specific for binding its protein partners. SAM (1-110 residues): sterile α-domain, PK
(126-385): Protein kinase domain, CRIB (126-135): Cdc42/Rac interactive binding, CBD:
Clathrin heavy chain binding domains: LLSVE (496-500) & LIDFG (584-588), Src (623-652):
Src binding domain, Nedd (632-635): Nedd4-2 binding domain, EGFR (733-876): EGFR
binding domain, UBA (958-996): Ubiquitin association domain. PR (577-958) represents a
proline rich region (113, 114). (Source: http://www.uniprot.org/uniprot/Q0791233TU ).U33T Nck adaptor
protein binding domain Nck is within the SBD (623-652) and the other adaptor protein Grb2 binds at the two PR motifs (723-761 and 771-800) indicated as Grb2 (61). SNX9: SNX9 binding
domain (920-955) (112).
Interestingly, ACK’s effects on receptor trafficking are not only receptor-specific but also
depend on ACK phosphorylation by other kinases. Phosphorylation in turn regulates the binding
partners available to ACK. Drosophila ACK was pulled down from lysates via the SH2 domain
of Dock, a Nck homologue (115). Nck isoforms are constitutively bound to ACK via the Nck
SH2 domain and thus the mechanism of such interaction is likely to be phosphorylation-specific. 52
ACK was also found in a stable complex with Nck in various mammalian cell lines, such as
PC12, NIH3T3, Swiss-3T3 and L6. However, it is unlikely that Nck mediates the interaction
between ACK and EGFR since the SH2 domain of Nck has been shown to bind phosphorylated
ACK (114). On the other hand, another adaptor protein, growth factor receptor binding-2 (Grb2)
possibly mediates ACK and EGFR interaction. Grb2 binds to the two PR motifs (61) within the
EGFR binding domain, as shown in Figure 4.1. Grb2 regulates EGFR internalization through
clathrin-coated pits. Sorkin and coworkers found that overexpression of Grb2 mutants, in which
SH3 domains were either deleted or inactivated by point mutations, inhibited EGFR
internalization. Also, EGFR mutants lacking Grb2 binding sites failed to get recruited to clathrin-
coated pits (116). Additional proteins like WASP, Dbl and Ras-guanine-nucleotide-releasing factor-1 (Ras-GRF1) are thought to interact with ACK (117).
4.2.2 Overexpressed ACK inhibits receptor internalization by sequestering its binding partners
ACK overexpression is thought to inhibit EGFR internalization since ACK binds up its
binding partners, especially clathrin. Cells with highly expressed ACK exhibited clathrin
trapping on membranes in a tubulo-reticular compartment (71). Wild type ACK overexpression inhibited transferrin endocytosis (76) and resulted in enhanced EGFR retention at early endosomes in cells (71). Because transferrin receptors are internalized solely by CME, endogenous clathrin possibly forms aggregates with overexpressed ACK. Such aggregates inhibit CME since ACK sequesters clathrin and prevent assembly of clathrin coated vesicles. The same may be true of other protein partners of ACK, for example Grb2 (113). As discussed earlier, EGFR molecules with double point mutations to inhibit Grb2 binding, failed to get internalized following EGF binding (116). Since endogenous ACK is phosphorylated and 53 activated by Src-family kinases (61), overexpression of ACK may change the balance between inactive and active enzyme, thereby inhibiting pathways that it normally mediates. Thus, overexpressed ACK seems to function as a dominant negative protein.
Chan and coworkers (2009) found that isoforms of E3 ubiquitin ligase, Nedd4-1 and
Nedd4-2, bind ACK and redistribute into clathrin-coated vesicles along with ACK (79).
Ubiquitination is a cascade of events mediated by three ligase enzymes, E1, E2 and E3 that catalyze the covalent attachment of ubiquitin to lysine residues of target proteins (118). This directs target proteins to varied fates, depending on how they are trafficked, as shown in Figure
4.2. Nedd4-2 has been found to catalyze ubiquitination of the UBA domain of ACK (79).
Overexpression of ACK mutants deficient in either binding to or ubiquitination by HECT
(Homologous to the E6-AP carboxyl terminus) family E3 ubiquitin ligase, Nedd4-1, also blocked
EGF-induced degradation of EGFR (81).
Fig. 4.2: Mechanism of ubiquitination of target proteins by cooperative activity of E1, E2 and E3 ubiquitination enzymes. Used by permission of (119). 54
4.2.3 Coordinate ACK and RTK trafficking and degradation
Previous work on steady-state kinetics of marker uptake and trafficking in cells have
shown that marker internalization temporally precedes further trafficking. With regard to
receptor processing, rates of downstream processes are limited by the rate of receptor
internalization (120). Therefore, it is not surprising to observe that rate of receptor degradation
should be inhibited upon inhibition of the receptors’ internalization. This principle was not obvious to authors working in the field of biochemistry. To avoid misinterpretation of results, the related details of findings have been highlighted in Table 1.1 (see Introduction).
ACK associates with EGFR through the MIG6 domain, although adaptor-mediated mechanisms of binding also seem to be possible. The MIG6 domain of ACK (corresponding to residues 334-363 of MIG6) may play a role in EGFR stabilization at the plasma membrane. This domain blocks the formation of an active EGFR dimer, thereby attenuating receptor signaling
(121). Stabilization of EGFR at the plasma membrane may in turn slow the rate of receptor processing, thereby decreasing the rates of receptor internalization and ubiquitination. ACK may also associate with its binding partners, including EGFR, through UBA (see Figure 4.1). The
UBA is implicated in ACK colocalization in the pre-autophagosome with sequestosome 1
(p62/SQSTM1), a receptor for selective autophagy (72). A mutation in the UBA domain of ACK decreases its binding to EGFR, stabilizing the EGFR at the plasma membrane and inhibiting its internalization. A mutant ACK with deletion of UBA was found to inhibit EGFR degradation compared to ACK overexpression alone (66). Similarly, a mutation in UBA (S985N) also prevented degradation of EGFR but had little effect on ACK protein half-life. A similar deletion of UBA (residues 970-1055) enhanced ACK ubiquitination, possibly by facilitating Nedd4-1 binding (59). These studies suggested that ACK ubiquitination site was both on UBA and 55
possibly on SAM domain. In fact, deletion of the SAM domain at the N-terminus which
mediates interactions with the membrane, dramatically reduced ACK ubiquitination by Nedd4-1
(81). This process (ACK ubiquitination by Nedd4), like receptor processing, internalization and
ubiquitination itself occurs far upstream of degradation. Therefore, all the results that are
highlighted in gray in Table 1.1 (see Introduction) must be discounted because they pertain to
events far upstream and not to ACK’s direct role in degradation.
Interestingly, RNA interference (RNAi) knockdown of Nedd4-1, but not Nedd4-2,
inhibited degradation of both ACK and EGFR (81), suggesting that ACK and EGFR are
processed in tandem. To date, UBA domains are known to function as ubiquitination target sites,
binding both mono- and multi-ubiquitin chains to block further chain elongation. There is a
possibility that ACK UBA domains act in both of these ways. Moreover, findings suggest that
the two Nedd4 isoforms may add ubiquitin onto different domains of proteins, including ACK.
However, the discrepancy in results remains to be explained. During processing RTKs, ACK binds to various Nedd isoforms within the clathrin-coated vesicles. So, the results suggest that processing is retarded in absence of ubiquitination.
The above findings suggested that ACK conducts EGFR into a branched pathway which
can lead to either recycling or degradation. However, based on accumulated evidence, it is
unlikely that ACK itself is processed by the same mechanism as EGFR. Thus, it gives rise to the
intriguing question, where does the route of processing of receptor and ACK coincide? Early
work by Levkowitz and coworkers (1998) showed that the E3 ubiquitin ligase c-Cbl (a
mammalian ortholog of Sli-1 protein of C. elegans) induces negative signaling by
downregulating the ErbB-1 EGF receptors. c-Cbl mediates ligand-induced ubiquitination of
ErbB-1 receptor and commits it to lysosomal degradation. Overexpression of c-Cbl caused an
increased amount of ubiquitin covalently attached to the receptor (122). In 1999, Waterman and 56
coworkers demonstrated the absolute necessity of the RING type zinc finger motif of c-Cbl for ubiquitination and desensitization of EGFRs (123). They created mutations in the RING finger and showed that such mutations impaired c-Cbl mediated degradation of EGFRs and negative signaling. Cbl binding to EGFR via Grb2 was later found to be essential for receptor internalization. Since, Grb2 knockdown inhibits EGFR endocytosis; the results suggested that the prime role of Grb2 in EGFR internalization was recruitment of Cbl (124).
Recruitment of Cbl at the Y1045 site of EGFR leads to mono-ubiquitination at multiple lysine sites, thus targeting it to endosomal sorting and eventually for degradation (125, 126), as illustrated in Figure 4.3. There is also a ubiquitin-specific protease, Ub-specific protease Y
(UBPY) also designated as Ub-specific protease 8 (Usp8), which is essential for deubiquitination of monoubiquitinated RTKs (127). Interestingly, UBPY/Usp8 undergoes activation by tyrosine phosphorylation upon EGF-induced EGFR activation (128). It may reverse monoubiquitinated sites and allow RTKs to be targeted for downregulation (129). Constitutive monoubiquitination of EGFR by a Tyr(P)-1045 independent mechanism was shown to be counteracted by the de- ubiquitinating catalytic activity of UBPY/Usp8. UBPY deubiquitinates ligand-activated EGFR on endosomes and negatively regulates its downregulation. Thus, there are several known mechanisms of re-routing EGFR molecules during their voyage through the internal membrane- bound compartments of the cell. 57
Figure 4.3: Mechanisms of EGFR ubiquitination Used by permission of (130)
4.2.4 Role of ACK-mediated EGFR processing in filopodia dynamics
There are undoubtedly dozens of different receptor types displayed on the surface of a
typical mammalian cell and the number of receptors of each type is maintained constant by
endocytic and exocytic trafficking. In my discussion, I use EGFR to illustrate receptor
processing pathways, because it is the receptor studied most often. Cells were maintained under conditions that favored steady-state display of receptors on the plasma membrane. Several receptors are associated with ACK1, including EGFR and PDGFR (PDGF receptor) (114), whereas others are known to show ACK-independent trafficking. Fibroblast growth factor receptor (FGFR) is an example of such receptor type. The trafficking pathway is not only affected by the receptor type but also by the chemistry of the ligand bound to the receptor. For example, cetuximab-bound EGFR undergoes CIE, which favors receptor degradation, compared to CME, which promotes EGFR recycling to the plasma membrane (70, 78). 58
Recent findings suggest the role of ACK1 in cancer progression. ACK1 gene
amplification has been found in a number of cancer types including lung, prostate and ovarian
tumors (131). The tumor suppressor Wwox has been shown to be negatively regulated by ACK1
induced enhanced phosphorylation at Tyr-287 in primary androgen independent prostrate tumors
(132). Interestingly, a number of somatic and germ line mutations in ACK1 sequence have been
identified in diverse cancer cell lines (Fig. 4.4). Though specific functions of such mutations
have not yet been completely understood, such mutations possibly render the oncogenic property
of ACK.
Figure 4.4: ACK1 sequence showing ten novel somatic and two germ line mutations. The
mutations are identified in 261 cancer cell lines of diverse origins along with 15 control tissues.
A specific mutation locus has been indicated with an arrow on the ACK1 sequence (59).
During my study, when ACK was inhibited from binding to the specific Cdc42 domain by the introduction of the corresponding BP, both percentage of cells showing filopodia and the percent coverage of periphery with such protrusions showed decrease from the control, as indicated in Tables 3.1 amd 3.2 respectively. These results support the previous findings by 59
Surya Amarachintha (85) and suggest that, ACK binding to Cdc42 is essential for regulating filopodial formation and maintenance. However, Heckman and coworkers (2009) have shown that ACK overexpression had no effect on filopodia prevalence (107). Amplification of the ACK gene and subsequent overexpression of ACK resulted in its enhanced oncogenic property in cells. It is possible that, under such conditions, cells might have started behaving like cancer cells. It is generally attributed to changes in RTK processing that prolong signaling from these receptors while they are in the plasma membrane or in endocytic compartments. On the other hand, in my experiments, I used epithelial cells grown and maintained under normal conditions.
As discussed above, the E3 ubiquitin ligase Nedd4 binds ACK at a specific domain and catalyzes covalent attachment of ubiquitin on ACK. There is a possibility that Nedd4 may also add ubiquitin on EGFR when ACK binds EGFR in clathrin-coated vesicles during CME, since
previous findings have suggested that ACK and EGFR might be co-processed. Interestingly,
during my experiments, when ACK-Nedd4-2 binding was inhibited by introducing the
corresponding BP, the number of cells showing filopodia and periphery coverage with such
protrusions increased significantly compared to control (see Tables 3.3 and 3.4 in Results). By
inhibiting the interaction of ACK with Nedd4, the rate of ACK ubiquitination would be retarded
which in turn might have resulted in reduced rate of EGFR processing, since earlier works have
suggested that ACK is crucial for EGFR trafficking. Interestingly, my studies have shown that
reduced ACK and RTK processing appears to enhance filopdoia prevalence in cells. Therefore, I
hypothesize that, ACK-mediated RTK trafficking may have a role in regulating filopodia
dynamics. 60
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APPENDIX A. LIST OF ABBREVIATIONS
Abi1 Abl interactor protein-1
Abi2 Abl interactor protein-2
Abl Abelson tyrosine kinase
ACK Activated Cdc42-associated kinase
AP2 Adaptor protein-2
APC Adenomatous polyposis coli aPKC Atypical protein kinase C
ARK1 ACK-related tyrosine kinase -1
Arp2/3 Actin-related protein-2/3
Axl Receptor tyrosine kinase
BP Binding peptide
CBD Clathrin heavy chain binding domain
Cdc42 Cell division cycle 42
CEP Cdc42 effector protein
CHC Clathrin heavy chain
CIE Clathrin-independent endocytosis
CME Clathrin-mediated endocytosis
CMF-HBSS Calcium and magnesium-free Hanks balanced salt solution
CRIB Cdc42/Rac interactive binding
Crk Chicken tumor virus no. 10 regulator kinase
Dia Diaphanous
ECM Extracellular matrix 73
EDTA Ethylene diamine tetraacetic acid
EE Early endosome
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
Ena/VASP Vasodilator-stimulated phosphoprotein
FA Focal adhesion
FC Focal contact
FGF FDG1-related Cdc42-GEF
FGF Fibroblast growth factor
FITC Fluoroscein isothiocyanate
GAP GTPase activating protein
Gas6 Growth arrest-specific gene-6
GDP Guanosine diphosphate
GEF Guanine exchange factor
GPCR G-protein coupled receptor
Grb2 Growth factor receptor-bound protein-2
GST Glutathione s-transferase
GTPase Guanosine triphosphate binding proteins
HECT Homologous to the E6-AP carboxyl terminus
IQGAP IQ motif containing GTPase activating protein
IRS Insulin receptor substrate
IRSp53 Insulin receptor tyrosine kinase substrate protein-53
Iβ5 Integrin beta-5 74
LE Late endosome
LIMK Lin-11, rat Isl-1 and C. elegans Met-3 kinase
LPR1 Lipid phosphatase-related protein-1 mDia2 Membrane diaphanous 2
MLK Mixed-lineage kinase
MRCK Myotonic dystrophy related Cdc42-binding kinase
MTOC Microtubule -organizing center
NBD Nedd4 binding domain
Nck Non-catalytic region of tyrosine kinase adaptor protein
Neural precursor cell expressed developmentally downregulated protein 4-
Nedd4-1 isoform-1
N-WASP Neural homolog of WASP p130cas Crk-associated substrate
PAK p21-activated kinase
Par3 Partitioning defective 3
Par6 Partitioning defective 6
PBS Phosphate buffered saline
PDGF Platelet derived growth factor
PK Protein kinase
PM Plasma membrane
PR Proline rich
PTK Protein tyrosine kinase
Rac Ras-related C3 botulinum toxin substrate 75
RACK1 Receptor for activated C kinase
RalBP1 RalA binding protein-1
Ras-GRF1 Ras-guanine nucleotide-releasing factor-1
RIF Rho in filopodia
RTK Receptor tyrosine kinase
SAM Sterile alpha domain siRNA Small interefering RNA
SNX9 Sorting nexin-9
Src "Sarcoma" (Proto-oncogene tyrosine-protein kinase)
Tfr Transferrin receptor
TNF Tumor necrosis factor
TNK2 Tyrosine kinase non-receptor-2
TRAIL TNF-related apoptosis inducing ligand
TRAIL-R TRAIL-receptor
Ub Ubiquitin
UBA Ubiquitin associated domain
UBPY Ubiquitin-specific protease Y
Usp8 Ub-specific protease-8
VEGF Vascular endothelial growth factor
WASP Wiskott-Aldrich syndrome protein
WAVE/Scar WASP-family verprolin-homologous protein