Oncogene (2001) 20, 6418 ± 6434 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Cortactin: coupling membrane dynamics to cortical actin assembly

Scott A Weed*,1 and J Thomas Parsons2

1Department of Craniofacial Biology, University of Colorado Health Sciences Center, Denver, Colorado, CO 80262, USA; 2Department of Microbiology, Health Sciences Center, University of Virginia, Charlottesville, Virginia, VA 22903, USA

Exposure of cells to a variety of external signals causes consists of a highly organized meshwork of ®lamentous rapid changes in plasma membrane morphology. Plasma (F-) actin closely associated with the overlying plasma membrane dynamics, including membrane ru‚e and membrane (Small et al., 1995). In motile cells, F-actin microspike formation, fusion or ®ssion of intracellular underlies two main types of protrusive membranes; vesicles, and the spatial organization of transmembrane lamellipodia, which contain short ®laments of F-actin proteins, is directly controlled by the dynamic reorgani- linked into orthogonal arrays and ®lopodia, comprised zation of the underlying actin cytoskeleton. Two of bundled, crosslinked actin ®laments (Rinnerthaler et members of the Rho family of small GTPases, Cdc42 al., 1988). Work within the last decade has demon- and Rac, have been well established as mediators of strated that Rac and Cdc42, two members of the Rho extracellular signaling events that impact cortical actin family of small GTPases, govern lamellipodia and organization. Actin-based signaling through Cdc42 and ®lopodia formation (Hall, 1998). Cdc42 and Rac play a Rac ultimately results in activation of the actin-related pivotal role in the transmission of extracellular signals protein (Arp) 2/3 complex, which promotes the forma- that induce cortical cytoskeletal rearrangement (Zig- tion of branched actin networks. In addition, the activity mond, 1996), eliciting their e€ects through a large of both receptor and non-receptor protein tyrosine number of binding (or `e€ector') proteins (Aspenstrom, kinases along with numerous actin binding proteins 1999; Bishop and Hall, 2000). It is now well established works in concert with Arp2/3-mediated actin polymer- that members of the Wiskott ± Aldrich syndrome ization in regulating the formation of dynamic cortical protein (WASp) family serve as the e€ector proteins actin-associated structures. In this review we discuss the for Cdc42 and Rac-mediated cortical actin polymeriza- structure and role of the cortical actin binding protein tion by directly binding to and activating the actin cortactin in Rho GTPase and tyrosine kinase signaling nucleation activity of the actin related protein (Arp) 2/ events, with the emphasis on the roles cortactin plays in 3 complex (Higgs and Pollard, 1999). Arp2/3 complex tyrosine phosphorylation-based signal transduction, reg- activity is essential for the protrusion of the leading ulating cortical actin assembly, transmembrane receptor edge during cell migration (Borisy and Svitkina, 2000; organization and membrane dynamics. We also consider Pollard et al., 2000). how aberrant regulation of cortactin levels contributes to Activation of Cdc42 and Rac are also closely tumor cell invasion and metastasis. Oncogene (2001) 20, coordinated with tyrosine kinase signaling pathways. 6418 ± 6434. In the case of receptor tyrosine kinases such as the EGFR or PDGFR, ligand binding resulting in cortical Keywords: Rho GTPase; actin cytoskeleton; tyrosine actin rearrangements responsible for membrane ru‚ing kinase substrate; Arp2/3 complex; vesicle tracking; and motility are mediated by downstream activation invasion Cdc42 and Rac (Liu and Burridge, 2000). EGFR and PDGFR activation also result in activation of cellular Src (c-Src, or Src) family nonreceptor tyrosine kinases (Parsons and Parsons, 1997). The activity of Src and Introduction related kinases are responsible for the tyrosine phosphorylation of numerous actin-binding proteins Dynamic changes in the cortical actin cytoskeleton are that impact cortical actin cytoskeleton organization central to a wide variety of cellular events, including and cell shape (Brown and Cooper, 1996; Kellie et al., cell motility, adhesion, endo- and phagocytosis, 1991). This is highlighted in cells expressing oncogenic cytokinesis, movement of intracellular particles forms of Src, which causes dramatic alterations in the through the cytoplasm and organization of transmem- actin cytoskeleton resulting in aberrant cell adhesion brane proteins (Mitchison and Cramer, 1996; Ridley, and morphology (Jove and Hanafusa, 1987). While 2000; Qualmann et al., 2000; Chimini and Chavrier, Cdc42, Rac and Src are all activated during integrin- 2000; Mandato et al., 2000; Beckerle, 1998; Wheal et mediated cell adhesion, divergent pathways are utilized al., 1998). The actin cytoskeleton at the cell periphery in regulating GTPase and Src activity, suggesting that tyrosine phosphorylation of cytoskeletal Src substrates can occur independently of Cdc42 and Rac activation *Correspondence: SA Weed; E-mail: [email protected] (Clark et al., 1998). Src-mediated tyrosine phosphor- Cortactin and membrane dynamics SA Weed and JT Parsons 6419 ylation creates binding sites for proteins with Src followed by a series of six complete 37 amino acid homology (SH) 2 domains, allowing for phosphotyr- tandemly repeating segments and one incomplete osine-based assembly of macromolecular signaling segment 20 residues in length. The complete repeats complexes as well as directly in¯uencing substrate are predicted to form a helix-turn-helix structure (Wu activity (Parsons and Parsons, 1997; Brown and et al., 1991) and have been termed cortactin repeats Cooper, 1996). It is now clear that proteins involved (Sparks et al., 1996a) since they show no sequence in both Rho family GTPase and tyrosine kinase similarity to other known repeats except in HS1 (see signaling pathways play important cooperative and below). The repeats region is followed by a a-helical integrative roles in cortical actin cytoskeletal signaling domain 48 ± 52 amino acids in length, a proline-rich (Zigmond, 1996; Parsons, 1996). In this review we will domain of variable length abundant in tyrosine, serine consider the structure and role of the Src substrate and and threonine residues, and a Src homology (SH) 3 cortical actin-associated protein cortactin, with empha- domain at the distal carboxyl terminus. The cortactin sis on signaling pathways involved in cortactin tyrosine SH3 domain shares signi®cant homology to SH3 phosphorylation as well as the role of cortactin in domains from Src-family kinases, various adapter and dynamic membrane events involving Arp2/3-mediated cytoskeletal proteins (Wu et al., 1991; Sparks et al., cortical actin reorganization. 1996a). Cortactin genes have now been cloned from a number of diverse organisms. In addition to chicken, Structural organization of cortactin and related proteins complete cDNA sequences have been reported for human (Schuuring et al., 1993), rat (Ohoka and Takai, Cortactin was initially identi®ed as a tyrosine phos- 1998), mouse (Zhan et al., 1993; Miglarese et al., 1994), phorylated protein in v-Src infected chicken embryo Drosophila (Katsube et al., 1998) and the sea urchin ®broblasts (Kanner et al., 1990). Subsequent cloning of Strongylocentrouts purpuratus (Genebank accession the cDNA encoding cortactin revealed a novel protein #AF064260). All cortactins are structurally similar to with a unique domain structure (Wu et al., 1991). each other, with the SH3 domain showing the greatest Structural predictions based on the amino acid amount of evolutionary conservation while the proline- sequence indicate that cortactin is comprised of several rich domain the least (Figure 1). With the exception of domains (Figure 1). The ®rst 84 ± 94 amino-terminal the proline-rich domain, vertebrate cortactins show a residues are largely unstructured, containing a large particularly strong similarity, demonstrating greater number of acidic residues between amino acids 15 and than 87% identity in all other domains. Invertebrate 35 and is referred to as the amino terminal acidic cortactins show a similar pattern, with the SH3 domain domain (NTA; Weed et al., 2000). The NTA region is at least 71% identical while the proline-rich regions are less than 20% homologous to the mouse form. The large degree of conservation in the SH3 domain suggests that this domain binds ligands conserved across a broad evolutionary spectrum, performing a similar function(s) in diverse species. The proline-rich segments in invertebrate cortactins are considerably longer than the similar regions in the vertebrate proteins, accounting in part for the low degree of homology (Katsube et al., 1998). Only one cortactin gene has been identi®ed to date, and in most cell types is responsible for a single cortactin protein product that often migrates as two separate bands in SDS ± PAGE with relative molecular weights of 80 and 85 kDa (Wu et al., 1991). This is considerably larger than the predicted 61 ± 63 kDa molecular mass based on the primary sequence (Wu Figure 1 Structure of cortactin and comparison with related et al., 1991; Miglarese et al., 1994) and is presumably proteins. Domain organization of murine cortactin (top), HS1 due to the proline-rich domain in the carboxyl (middle) and Abp1 (bottom). Percent amino acid sequence similarities for homologous domains of cortactins from other terminus. The two observed cortactin bands likely species are shown compared to the murine protein. The percent represent di€erent conformational states, since both similarity of related domains in HS1 and Abp1 are indicated. forms are observed following expression of recombi- Boxed numbers denote complete cortactin repeats. D. melanoga- nant cortactin constructs in eukaryotic and prokaroytic ster contains four, S. purpuratus ®ve and HS1 three complete tandem repeats, and were therefore compared to the correspond- systems (Wu et al., 1991; Huang et al., 1997). This is ing repeats in cortactin. NTA, amino-terminal acidic region; helix, further supported by the observation of a single predicted a-helical region; P-rich, proline-rich region; SH3, Src cortactin band following denaturing SDS ± PAGE in homology 3 domain; ADF-H, actin-depolymerizing factor 5 M urea (Huang et al., 1997). However, cortactin homology domain. Genebank accession numbers for the analysed sequences are U03184 (murine), M98343 (human), M73705 isoforms have been recently identi®ed in rat brain that (chicken), AB009998 (D. melanogaster) and AF064260 (S. are products of alternative mRNA splicing (Ohoka and purpuratus) cortactin, D42120 (HS1) and NM013810 (mAbp) Takai, 1998). These shorter forms result from the

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6420 removal of exons from the more prevalent original uous stimulation of quiescent ®broblasts with FGF-1 form (termed cortactin-A) resulting in the translation (bFGF) or EGF leads to a rapid induction of cortactin of proteins with either ®ve (cortactin-B) or four tyrosine phosphorylation (within 5 min) that decreases (cortactin-C) complete tandem repeats. Currently it is within 1 h, increases again within 4 ± 7 h and is not clear if alternative spliced forms of cortactin are sustained for 12 ± 24 h (Maa et al., 1992; Zhan et al., present in other tissues. 1993). While the signi®cance of the biphasic nature of Northern blot analysis indicates that cortactin is cortactin tyrosine phosphorylation under these condi- expressed in nearly all mammalian tissues (Miglarese et tions is not clear, overexpression of Src enhances EGF- al., 1994; Du et al., 1998). One notable exception is in induced cortactin tyrosine phosphorylation (Maa et al., hematopoietic cells, where the related protein HS1 is 1992) while activation of Src and coimmunoprecipita- expressed in place of cortactin (Kitamura et al., 1989). tion with cortactin is achieved following FGF-1 HS1 is structurally related to cortactin, with the NTA, treatment (Zhan et al., 1994). These studies support a cortactin repeats and SH3 domain showing the greatest role for Src in growth factor-induced cortactin tyrosine homology. HS1 di€ers from cortactin in that it phosphorylation. In addition, Src has been implicated contains only three complete cortactin repeats (Figure as the kinase responsible for tyrosine phosphorylation 1), and that the helical and proline-rich domains are of cortactin in cell-matrix and cell-cell adhesion events. inverted with respect to cortactin (Kitamura et al., In NIH3T3 ®broblasts, a5b1 integrin-mediated cell 1995). In spite of these di€erences, HS1 shares adhesion and spreading on ®bronectin results in functional similarities with cortactin, serving as a Src- elevated cortactin tyrosine phosphorylation (Vuori family tyrosine kinase substrate (Takemoto et al., 1995; and Ruoslahti, 1995), a condition where Src is Brunati et al., 1999) as well as playing a unique and activated following binding to activated focal adhesion essential role in antigen-receptor induced B cell clonal kinase (FAK) (Schaller et al., 1994). In N18 neuro- expansion (Taniuchi et al., 1995). blastoma cells, cortactin is tyrosine phosphorylated Another protein that structurally resembles cortactin following binding of N-syndecan (syndecan 3) with is murine actin-binding protein 1 (mAbp; Kessels et al., heparin-binding growth-associated molecule (HB- 2000). The carboxyl terminus of mAbp1 contains a GAM), and both cortactin and Src copurify following helical, proline-rich and SH3 domain that shares anity chromotography using the N-syndecan cyto- moderate sequence similarity with the cognate domains plasmic domain (Kinnunen et al., 1998). Similarly, in cortactin (Figure 1). While mAbp1 does not contain binding of CD44 to hyaluronic acid (HA) in ovarian a NTA domain or cortactin repeats, the amino tumor cells leads to activation and association of Src terminus contains an actin depolymerizing factor with the CD44 cytoplasmic domain and subsequent homology (ADF-H) domain, an actin-binding domain cortactin tyrosine phosphorylation (Bourguignon et al., found in a number of cytoskeletal proteins (Lappalai- 2001). Engagement of avb3 integrin with soluble nen et al., 1998). mAbp1 and cortactin appear to be vitronectin, binding of ICAM-1 to aLb2 or a4b1 functionally similar in many respects (see below). integrin, as well as peroxide-mediated injury in endothelial cells also induces Src activation and subsequent cortactin tyrosine phosphorylation (Dur- Cortactin as a target for tyrosine and serine/threonine ieu-Trautmann et al., 1994; Bhattacharya et al., 1995; protein kinases Li et al., 2000). Finally, invasion of epithelial cells by either Shigella ¯exneri (Dehio et al., 1995) or Following the observation that cortactin is tyrosine Chlamydia trachomatis (Fawaz et al., 1997) leads to phosphorylated in cells transformed by v-Src, a wealth increased cortactin tyrosine phosphorylation, and in of data have indicated that cortactin tyrosine phos- the case of Shigella, Src overexpression enhances phorylation is closely correlated with Src activity in a cortactin tyrosine phosphorylation upon bacteria entry. number of signaling pathways (Table 1). Src kinase Collectively, the strong correlation between Src activa- activity is downregulated following phosphorylation of tion and cortactin tyrosine phosphorylation in these a carboxyl-terminal tyrosine (codon 527 in chicken) by studies suggest that cortactin is phosphorylated directly C-terminal Src Kinase (Csk), which creates an by Src (Table 1). intramolecular binding site for the Src SH2 domain Coimmunoprecipitation of Src with cortactin sug- that in turn holds the kinase in a conformationally gests that Src directly binds cortactin (Zhan et al., inactive state (Nada et al., 1991). While cells from 1994; Okamura and Resh, 1995; van Damme et al., Csk7/7 mouse embryos display elevated kinase activity 1997). In support of this, cortactin directly interacts of the Src family members Src, Fyn and Lyn (Imamoto with the recombinant SH2 domain of Src and other and Soriano, 1993; Nada et al., 1993), studies in Src7/7 SH2 domain-containing proteins, an interaction that is Csk7/7 and Fyn7/7 Csk7/7 ®broblasts indicate that Src prevented in the presence of a phosphopeptide contain- is primarily responsible for cortactin tyrosine phos- ing the optimal Src SH2 binding sequence (-pYEEI-; phorylation resulting from Csk knockout (Thomas et Songyang et al., 1993; Okamura and Resh, 1995). al., 1995). In accordance with these studies, it is now These results indicate that tyrosine phosphorylation of clear that activation of Src in response to diverse cell cortactin creates phospho-dependent binding site(s) for stimuli points to a direct physiological role for Src- Src and perhaps other SH2 domain-containing pro- mediated cortactin tyrosine phosphorylation. Contin- teins. Src directly phosphorylates cortactin in vitro

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6421 Table 1 Cellular conditions that promote cortactin tyrosine phosphorylation Type of stimulis Cell type Implicated kinase Comments Reference Growth factors FGF-1 fibroblast Src Zhan et al. (1993) EGF fibroblast Src Enhanced with Src expression Maa et al. (1992) PDGF fibroblast Fer Kim and Wong et al. (1998) CSF-1 fibroblast Fer Ectopically expressed Kim and Wong (1998) Thromboxane A2 platelet Syk Gallet et al. (1999) Adhesion receptors a5b1 integrin fibroblast Src Binding to FN Vuori and Ruoslahti (1995) aIIBb3 integrin platelet Syk Binding to thrombin or fibrinogen; Rosa et al. (1997); downstream of Src Gao et al. (1997) avb3 integrin endothelia Src Binding to VN Bhattachayra et al. (1995) syndecan 3 neuroblastoma Src Binding to HB-GAM Kinnunen et al. (1998) CD44 ovarian carcinoma Src Binding to HA Bourguignon et al. (2001) ICAM-1 endothelia Src Binding to aLb2 or a4b1 integrin Durieu-Trautmann et al. (1994) Bacterial invasion S. flexneri epithelia Src Dehio et al. (1995) C. trachomatis epithelia ? Fawaz et al. (1997) Oncogenic transformation V-Src infection fibroblast v-Src Wu et al. (1991) Csk7/7 fibroblast Src Nada et al. (1994) Others Ca2+ keratinocyte Fyn Increased during differentiation Calautti et al. (1995) m-opioids neuron ? Mangoura (1997) H2O2 endothelia Src Li et al. (2000) Cell shrinkage epithelia Fer Downstream of Fyn Kapus et al. (1999)

Table abbreviations: FGF-1, ®broblast growth factor-1; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; TxA2, thromboxane A2; FN, ®bronecton; VN, vitronectin; HB-GAM, heparin-binding growth-associated molecule; HA, hyaluronic acid

(Huang et al., 1997) on three tyrosine residues in comprise a small and distinct tyrosine kinase subfamily murine cortactin (tyrosine-421, tyrosine-466 and tyr- that is has unique structural characteristics, including osine-482) located within the proline-rich domain the presence of an amino terminal coiled-coil sequence (Huang et al., 1998) (Figure 2a). Expression of a proximal to a central SH2 domain and a carboxyl cortactin construct where tyrosines 421, 466 and 482 terminal catalytic domain (Hao et al., 1989). NIH3T3 were mutated to phenylalanine dramatically reduced cells treated with PDGF leads to Fer activation and cortactin tyrosine phosphorylation following expres- cortactin tyrosine phosphorylation (Downing and sion in v-Src transformed 3T3 ®broblasts, indicating Reynolds, 1991; Kim and Wong, 1998) which is that these residues are targeted by Src in vivo (Huang et markedly reduced in ®broblasts derived from Fer7/7 al., 1998). Although the sequential order by which Src mice (Craig et al., 2001). Fer immunoprecipitates with phosphorylates cortactin is unknown, the presence of a cortactin, and the Fer SH2 domain interacts with consensus Src SH2 binding sequence next to tyrosine cortactin in cell extracts, suggesting that Fer directly 421 (Figure 2c) raises the possibility that this residue interacts with and phosphorylates cortactin. This is may be initially phosphorylated by Src, thereby further supported by the suppression of CSF-1 allowing for the stabilization of Src with cortactin receptor-mediated cortactin tyrosine phosphorylation through an interaction between the Src SH2 domain by expression of kinase de®cient Fer (Kim and Wong, and phosphorylated tyrosine 421. Stabilization of Src 1998). Other evidence supporting a role for Fer in at tyrosine 421 may act to further facilitate the cortactin tyrosine phosphorylation has come from the phosphorylation of cortactin at tyrosines 466 and analysis of signaling events involved in cell shrinkage. 482. Evidence for the importance of Src SH2 domain Hyperosmolarity elevates tyrosine phosphorylation of binding in cortactin tyrosine phosphorylation is high- cortactin as well as the activity of the Fyn, while lighted by the marked reduction of tyrosine phosphor- suppressing Src activity (Kapus et al., 1999). The ylation of cortactin in Csk7/7 cells treated with Src requirement for Fyn in this process is further underscored SH2 inhibitory compounds (Violette et al., 2001). by the reduction of cortactin tyrosine phosphorylation in While phosphorylation of cortactin by Src appears to hyperosmotic Fyn7/7, but not Src7/7 cells (Kapus et be functionally linked to some aspects of actin al., 1999). A rapid reduction in cell volume also organization and cell motility, the full signi®cance of activates Fer, which is suppressed by pretreatment with Src mediated cortactin tyrosine phosphorylation is not the Src family inhibitor PP1 as well as in Fyn7/7 cells yet understood. (Kapus et al., 2000). Expression of kinase de®cient Fer A second nonreceptor tyrosine kinase that is also inhibits osmotic-induced cortactin tyrosine phos- involved in growth factor-mediated cortactin tyrosine phorylation, and hyperosmotic conditions fail to phosphorylation is Fer. Fer, along with Fps/Fes, signi®cantly increase the tyrosine phosphorylation level

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6422

Figure 2 Diagrammatic representation of cortactin-interacting proteins and sequence alignments of known and putative interaction sites. (a) Mapping of cortactin binding proteins and phosphorylation sites. Binding of Arp2/3 complex occurs through the three amino acid ± DDW -motif within the NTA domain. The actin-binding domain is located within the repeats region, requiring the fourth repeat and possibly adjacent sequences. Tyrosine phosphorylation by Src and Fer occurs within the proline-rich domain at Y421, Y466 and Y482, creating putative binding sites for SH2 domains. Putative serine phosphorylation by ERK1/2 occurs at S405 and S418. The SH3 domain interacts with numerous PDZ-containing sca€olding proteins as well with dynamin and CBP90. See text for details. (b) The cortactin Arp2/3 binding site is conserved among other Arp2/3 binding proteins. Sequence alignment of various Arp2/3 activating proteins with the cortactin Arp2/3 binding site reveals a conserved DDW- or -DEW- sequence (except for Scar-1) (boxed) required for Arp2/3 binding and activation. Displayed sequences are from Miglarese et al. (1994) (cortactin), Kitamura et al. (1995) (HSl), Drubin et al. (1990) (Abp1p), Dujon et al. (1994) (myo3p), Derry et al. (1995) (WASP), Fukuoka et al. (1997) (N-WASP) and Bear et al. (1998) (Scar-1). (c) Src tyrosine phosphorylation sites on cortactin conform to a consensus SH2-binding motif. Comparison of cortactin phosphorylation sites with known SH2 domain-binding motifs that mediate stable complex formation with activated Src. P0, position of Src-phosphorylated tyrosine; P1 ±P3, position of ¯anking residues; c, aliphatic residues. Sequence data is from Songyang et al. (1993) (consensus), Schaller et al. (1994) (FAK), Nakamoto et al. (1996) (CAS) and Guappone et al. (1998) (AFAP) and Huang et al. (1998) (cortactin). (d) Alignment of proline-rich sequences contained within cortactin SH3 domain-binding proteins. Proteins demonstrated or proposed to bind to the cortactin SH3 domain and their putative or con®rmed SH3 binding sites(s) are aligned with the consensus binding sequence (top) obtained by peptide phage display library screening (Sparks et al., 1996). Proteins with a consensus-conforming -KPPVPPKP- sequence are displayed in the upper half of the alignment (binding of mXin has yet to be demonstrated) while non-conforming proteins and their interaction sequences are shown underneath. Boxes indicate the positions of conserved proline residues. Sequences are from Du et al. (1998) (CortBP1), Naisbitt et al. (1999) (Shank 3), Wang et al. (1999) (mXin), Takahisa et al. (1996) (ZO-1), Ohoka and Takai (1998) (CBP90), and Cook et al. (1994) (Dynamin)

of a cortactin construct containing the Src-targeted induced keratinocyte di€erentiation (Calautti et al., point mutations at tyrosines 421, 466 and 482 (Kapus 1995). et al., 2000). Collectively these data indicate that Cortactin tyrosine phosphorylation is also observed osmotic-induced tyrosine phosphorylation of cortactin during platelet di€erentiation and activation. Although by Fer requires upstream activity of Fyn, and that Fer Src activation and cortactin tyrosine phosphorylation directly phosphorylates one or more of the same are coincident with thrombin-mediated platelet activa- tyrosine residues targeted by Src. The functional tion following binding to aIIBb3 integrin (Fox et al., signi®cance of Fer-mediated cortactin tyrosine phos- 1993), there is also evidence that the hematopoietic- phorylation is unclear. It is also not known if Fer is spec®c kinase Syk plays an important role in cortactin responsible for the Fyn-mediated increase in cortactin tyrosine phosphorylation during platelet maturation tyrosine phosphorylation observed during calcium- and activation. Syk is homologous to the lymphocyte-

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6423 speci®c ZAP-70 kinase, containing two amino-terminal proposed that ERK-mediated serine phosphorylation SH2 domains and a carboxyl-terminal catalytic domain may regulate an intramolecular interaction between the (Chan et al., 1992; Muller et al., 1994). Syk is activated cortactin SH3 domain and sequences within the following ®brinogen ligation to aIIBb3 integrin, and proline-rich domain. This would render cortactin in requires the intact cytoplasmic domains of both an `open' conformation, allowing binding of the SH3 integrin subunits (Gao et al., 1997). Defects in aIIBb3 domain to other ligands (Campbell et al., 1999). This integrin are found in individuals with Glanzmann model has been proposed for other proteins (Taylor et thrombasthenia, which experience excessive bleeding al., 1998), and is indirectly supported by the presence due to defective platelet activation and aggregation of a single 85 kDa band expressed from a cortactin (Nurden and Caen, 1974). Platelets from Glanzmann construct containing a disruptive point mutation within patients display reduced cortactin tyrosine phosphor- the SH3 domain (Du et al., 1998). Alternatively, serine/ ylation following ®brinogen stimulation (Rosa et al., threonine phosphorylation of cortactin within the 1997) indicating that aIIBb3 integrin-induced activation proline-rich domain may regulate SH3-mediated bind- of Syk is required for cortactin tyrosine phosphoryla- ing of heterologous proteins, as has been demonstrated tion. Syk autophosphorylation is inhibited in the for Sos and FAK (Corbalan-Garcia et al., 1996; Ma et presence of catalytic inactive Src, indicating that Src al., 2001). activity is required for Syk activation and subsequent cortactin tyrosine phosphorylation in a manner analogous to that of the Fyn/Fer pathway described The role of cortactin in cortical actin organization above. In addition, integrin-independent cortactin tyrosine phosphorylation attributable to Syk activation Localization and biochemical studies point to cortactin occurs following treatment of platelets with thrombox- playing an important role in regulating cortical actin ane analogues and in TPA-induced megakaryocyte assembly and organization. In most cell types cortactin di€erentiation of K562 leukemic cells (Maruyama et localizes in cytoplasmic punctate structures of un- al., 1996; Gallet et al., 1999). TPA treatment of K562 known composition concentrated at the perinuclear cells leads to Src activation in the presence of kinase region, and also with F-actin at sites of dynamic inactive Syk, further suggesting that Src acts upstream peripherial membrane activity (Figure 3a,b). Cortactin of Syk (Maruyama et al., 1996). Furthermore, translocates from the cytoplasm to the periphery in thromboxane stimulation of platelets does not elevate response to many of the same stimuli that induce its Src kinase activity, indicating that Syk can act tyrosine phosphorylation, including growth factor independent of Src in phosphorylating cortactin (Gallet treatment, integrin activation and bacterial entry et al., 1999). In these cases, cortactin forms complexes (Ozawa et al., 1995; Weed et al., 1998; Cantarelli et with Syk prior to simulus addition, suggesting that the al., 2000). These events also lead to activation of Rac interaction between Syk and cortactin is phosphotyr- (Hartwig et al., 1995; Clark et al., 1998; Mounier et al., osine independent. Although the role of Syk-induced 1999), which together with Cdc42 are responsible for cortactin phosphorylation is not known, it likely plays controlling the formation of cortical actin networks an important role in regulating platelet morphology (Ridley et al., 1992; Kozma et al., 1995). Rac activation (Gallet et al., 1999). is required for cortactin translocation to the cell In addition to serving as a tyrosine kinase substrate, periphery (Weed et al., 1998), and cortactin disperses cortactin is also phosphorylated on serine and from cortical actin networks following treatment with threonine residues (Wu et al., 1991). Serine and the F-actin disrupting drug cytochalasin D (Wu and threonine phosphorylation of cortactin is enhanced in Parsons, 1993). Cortical localization of cortactin is human squamous carcinoma cell lines following therefore closely associated with Rac-mediated actin stimulation with EGF, causing a shift from the 80 to assembly. the 85 kDa cortactin form (van Damme et al., 1997). In agreement with its localization to the cortical Treatment of 293 cells with EGF leads to a similar actin network, cortactin interacts directly with F-actin shift that is blocked by the MEK inhibitor PD98059, ®laments through sequences in the repeats domain (Wu indicating that EGF-induced activation of the Ras- and Parsons, 1993). Deletion mapping analysis has Raf-MEK-ERK pathway is responsible for cortactin determined that the fourth cortactin repeat is respon- serine and threonine phosphorylation (Campbell et al., sible for F-actin binding and cortical localization 1999). Based on peptide mapping data, ERK1/2 has (Weed et al., 2000). Cortactins -A, -B and -C all bind been proposed to phosphorylate cortactin at two ERK F-actin (Ohoka and Takai, 1998) whereas the ®rst consensus sites, serine-405 (-403PVSP) and serine-418 three cortactin repeats do not (He et al., 1998), (-416PSSP) within the proline-rich domain (Campbell con®rming the importance of the fourth cortactin et al., 1999). MEK activates a number of serine/ repeat in F-actin binding. Cortactin binds to the sides threonine kinases other than ERKs, which may in part of actin ®laments as determined by negative stained contribute to additional cortactin phosphorylation electron microscopy of recombinant cortactin/F-actin events (Kolch, 2000). Since serine 405 and 418 lie mixtures (H Wu and JT Parsons, unpublished observa- within putative SH3 binding sites, and although these tions) and by competition binding experiments with sequences do not conform to known cortactin SH3 myosin subfragment 1 (Ohoka and Takai, 1998). HS1 domain-binding sequences (Figure 2d), it has been also binds actin through the amino-terminal cortactin

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6424 2001). Whereas binding of cortactin to F-actin is not in¯uenced by Src phosphorylation (Wu and Parsons, 1993), Src phosphorylation has been reported to inhibit the crosslinking ability of cortactin (Huang et al., 1997). In addition, PIP2 binding to cortactin also inhibits F-actin crosslinking (He et al., 1998), although in this case it is not known if this is due to an inability of the cortactin/PIP2 complex to bind actin. Since cortactin contains a single F-actin binding site (Weed et al., 2000) and multimeric forms of cortactin have not been detected (Ohoka and Takai, 1998; Wu and Parsons, unpublished observations) cortactin must crosslink F-actin by an undetermined mechanism that will require further elucidation. Activated (GTP-bound) Cdc42 and Rac interact with a number of proteins that regulate assembly of the cortical actin cytoskeleton (Aspenstrom, 1999). It is now well established that members of the Wiskott ± Aldrich syndrome protein family, or WASps, play a pivotal role in mediating Cdc42 and Rac e€ects on the actin cytoskeleton (Takenawa and Miki, 2001). The prototypic member, WASp, binds directly to Cdc42 and induces clusters of actin ®laments in cells dependent on the unmasking of a C-terminal acidic region (Machesky and Insall, 1998). Expression of WASp is restricted to lymphoid and hemopoietic cells while a second family member closely related to WASp, termed N-WASp, also directly binds Cdc42. N-WASp is ubiquitously expressed and contributes directly to Cdc42-induced ®lopodia formation in most cell types (Miki et al., 1998a). WASp and N-WASp are also activated by molecules other than Cdc42, Figure 3 Cortactin localizes at sites of dynamic actin assembly. including PIP (Higgs and Pollard, 2000; Rohatgi et Colocalization of cortactin (a, c, e) with F-actin-containing 2 cellular structures (b, d, f). (a) and (b) Localization of cortactin al., 2000), the proteins WISH (Fukuoka et al., 2001) and F-actin in Swiss 3T3 cells spreading on ®bronectin. Cortactin and WIP (Martinez-Quiles et al., 2001), indicating that selectively localizes with cortical F-actin within lamellipodia and multiple signaling pathways are utilized in WASp not with the stress ®ber network. A signi®cant pool of cortactin is activation. Another WASP-like protein, termed Scar1 also found in the perinuclear region. (c) and (d) Redistribution of or WAVE, plays a central role in Rac-induced cortical cortactin and F-actin into podosomes in v-Src transformed NIH3T3 cells. In addition to its cortical and perinuclear actin dynamics (Bear et al., 1998; Miki et al., 1998b). localization, cortactin is also present in `rosettes' or podosomes Scar1/WAVE is structurally similar to WASp, sharing resultant from transformation by oncogenic non-receptor tyrosine strong homology to the carboxy-terminal acidic kinases. (e) and (f) Cortactin localization is coincident with F- domain (Miki et al., 1998b). The activation of Scar1/ actin tails in Listeria monocytogenes-infected Swiss 3T3 cells. Arrowheads denote sites of cortactin and F-actin colocalization Wave by Rac is indirect and is mediated by IRSp53 (Miki et al., 2000). A Rac/IRSp53 complex directly binds and activates Scar1/WAVE, which in turn promotes lamellipodia and membrane ru‚ing. Scar1/ Wave colocalizes with cortical actin within membrane repeats domain, and the fourth repeat in cortactin as ru‚es induced by activated Rac (Miki et al., 1998b). well as the three repeats in HS1 bind to phosphatidy- These data indicate that Scar1/WAVE functions in a linositol 4,5 bisphosphate (PIP2)(Heet al., 1998), manner similar to N-WASP to promote actin poly- which has been shown to regulate the interaction of merization within lamellipodia. several actin-binding proteins with F-actin (Sechi and The Arp2/3 protein complex is now ®rmly estab- Wehland, 2000). Recombinant cortactin has been lished in directly mediating signals from the Cdc42/N- reported to crosslink F-actin (Huang et al., 1997), an WASP (Rohatgi et al., 1999) and Rac/IRSp53/Scar1/ activity attributed to the sixth cortactin repeat (Ohoka WAVE (Machesky and Insall, 1998) pathways to and Takai, 1998). This is somewhat controversial, since induce polymerization of the cortical actin cytoskele- recombinant constructs lacking the fourth but contain- ton. The Arp2/3 complex consists of the two ing the sixth repeat fail to bind F-actin (Weed et al., unconventional actins Arp (actin-related protein) 2 2000) and crosslinking has not been detected by other and 3 along with ®ve unrelated proteins (Pollard et al., groups under similar conditions (H Wu and JT 2000). Arp2/3 complex binds to the sides and pointed Parsons, unpublished observations; Weaver et al., (or slow-growing) ends of actin ®laments (Mullins et

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6425 al., 1998), nucleating de novo actin polymerization, suggests that cortactin can function cooperatively with producing barbed end (fast growing) `daughter' WASp proteins in addition to independently activating ®laments attached to the sides of preexisting `mother' Arp2/3 complex. Evidence for an alternative mechan- ®laments (Machesky et al., 1999; Amann and Pollard, ism of Arp2/3 activation by cortactin is supported by 2001). Binding of the carboxy-terminal acidic region of the lower anity of cortactin for Arp2/3 complex WASp family proteins enhances Arp2/3-mediated actin (Weed et al., 2000) and by the inability of cortactin to nucleation (Machesky et al., 1999) and in the case of interact with individual Arp2/3 subunits in a yeast two- Scar1/Wave, this region binds directly to the 21 kDa hybrid assay (LM Machesky, personal communica- Arp2/3 subunit (Machesky and Insall, 1998). This tion). In addition, N-WASP and cortactin are di€er- interaction forms a molecular complex indirectly entially localized in Vaccinia-induced actin tails (Zettl linking Rac to the actin cytoskeleton through IRSp53. and Way, 2001). Cortactin also inhibits debranching of For N-WASp, the interaction is direct since Cdc42 aged Arp2/3 networks (Weaver et al., 2001) suggesting directly binds N-WASp to stimulate ®lopodia (Miki et that cortactin also serves to stabilize the interaction al., 1998a). The observation that carboxy-terminal between Arp2/3 complex and mother/daughter ®lament fragments of WASP-family proteins are more active networks. The association of cortactin with mobile than the full-length protein at promoting actin cortical actin structures determined by live cell imaging nucleation coupled with the requirement for activated points to a functional role for cortactin in regulating Cdc42 or Rac to elicit actin polymerization has led to Arp2/3 activity (Dai et al., 2000; Kaksonen et al., the proposal that activation of N-WASp or Scar1/ 2000). WAVE is achieved by GTPase-mediated targeting to Activation of Cdc42 and Rac are required for cell the cortical membrane. This is followed by a migration (Nobes and Hall, 1999), with the resultant conformational change exposing the carboxy-terminal Arp2/3 activity widely believed to be responsible for acidic domain to allow binding of the Arp2/3 complex protrusive-based cell motility mediated by these and subsequent actin polymerization (Machesky et al., GTPases (Borisy and Svitkina, 2000). The ability of 1999; Rohatgi et al., 1999). The sidebinding, pointed cortactin to bind and activate Arp2/3 complex end capping and nucleation ability of Arp2/3 complex indicates a role for cortactin in actin-based motility results in the formation of Y-shaped branched net- events. Enhanced cell migration in NIH3T3 and works observed in vitro (Mullins et al., 1998; Blanchoin endothelial cells overexpressing cortactin supports this et al., 2000a) and in vivo (Svitkina and Borisy, 1999). notion (Patel et al., 1998; Huang et al., 1998). While Phosphate released from aged ADP-Pi actin ®laments cortactin stimulated cell motility can be attributed to leads to disassembly of Arp2/3-induced actin networks enhanced Arp2/3 activity, Src phosphorylation of at branch points (Blanchoin et al., 2000a,b). The cortactin also appears to play an important role in debranching process is enhanced by the activity of cell migration. Src activation and cortactin tyrosine actin depolymerizing factor (ADF)/co®lin, which also phosphorylation are also closely coordinated in ®bro- depolymerizies and severs F-actin ®laments (Maciver, blast cell motility induced by FGF-1 (LaVallee et al., 1998). ADF/co®lin localizes at sites distal to that of 1998; Liu et al., 1999) or in carcinoma cells by CD44 Arp2/3 complex, placing it in the proper position to binding to HA (Bourguignon et al., 2001). Rac is also breakdown Arp2/3-induced actin networks within activated under these conditions (Johnston et al., 1995; lamellipodia (Svitkina and Borisy, 1999). Co®lin Oliferenko et al., 2000), and is responsible for severing activity is abolished following phosphorylation recruiting Src to the cell periphery (Fincham et al., by the Rac e€ector LIM kinase (Yang et al., 1998), 1996). Collectively these data point to a role for which in turn is activated by a second Cdc42/Rac cortactin in integrating cell motility signaling pathways e€ector, PAK (Edwards et al., 1999). The coordinated involving activation of Arp2/3 complex and Src. activities of LIM kinase and PAK therefore serve to Furthermore, several intracellular pathogens utilize attenuate actin networks resultant from Arp2/3 activa- Arp2/3 complex-based actin polymerization for intra- tion. cellular movement (Frischknecht and Way, 2001) and In addition to WASp family proteins, cortactin also cortactin is found in the `rocketing' actin tails of many binds to Arp2/3 complex and stimulates actin nuclea- (if not all) of these microbes, including Listeria (Figure tion activity (Weed et al., 2000; Weaver et al., 2001; 3e,f), Shigella and Vaccinia (Zettl and Way, 2001). Uruno et al., 2001). Binding of cortactin to Arp2/3 is Pathogen-induced Arp2/3 actin networks are structu- mediated through a three amino acid motif in the NTA rally similar to those found in lamellipodia, suggesting domain (-20DDW) conserved in many Arp2/3 activat- a similar mechanism of assembly (Cameron et al., ing proteins (Figure 2b). Cortactin's ability to stimulate 2001). While in the case of Vaccinia, cellular infection Arp2/3 activity requires both the DDW motif and the results in Src activation and cortactin tyrosine actin binding domain (Weaver et al., 2001; Uruno et phosphorylation (Frischknecht et al., 1999a), it is not al., 2001), domains that are also required for Rac- likely that cortactin tyrosine phosphorylation contri- mediated localization of cortactin to cortical actin butes to Vaccinia motility since tyrosine phosphoryla- networks (Weed et al., 2000). While the ability of tion is restricted to the virus body and is not present in cortactin to stimulate Arp2/3 activity is weaker than actin tails (Frischknecht et al., 1999b). The lack of that of N-WASp, cortactin enhances N-WASp-induced tyrosine phosphorylation in Vaccinia (and endosome) Arp2/3 activation (Weaver et al., 2001). This synergy actin tails suggests that Src phosphorylation of

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6426 cortactin does not in¯uence cortactin's ability to mGluR receptors (Xiao et al., 1998) or with the activate and stabilize Arp2/3-mediated networks. cytoplasmic amino terminus of inositol trisphosphate receptors (IP3R) (Tu et al., 1998). Homer therefore serves as a sca€old for coupling mGluR and IP3 Function of cortactin SH3-domain binding proteins receptors to Shank3, and links these receptors to NMDA and Shaker K+ signaling pathways. CortBP1/ Cortactin interacts with several proteins though its Shank proteins also contain a sterile alpha motif carboxyl terminal SH3 domain. The binding of SH3 (SAM) domain at the extreme carboxyl terminus that domains to proline-rich sequence motifs has been well serves as a homo- and/or heterodimerization domain established (Kay et al., 2000) and based on screening of (Naisbitt et al., 1999). The carboxyl terminus of Homer phage display peptide libraries, the cortactin SH3 proteins contains a coiled-coil domain that functions in domain binds to a consensus sequence of +PPCPXKP an analogous manner to SAM domains (Xiao et al., (Sparks et al., 1996b). Yeast two hybrid screening of 1998). The ability of Shank and Homer proteins to brain libraries with the cortactin SH3 domain led to dimerize in various combinations contributes to the the identi®cation of ®rst cortactin SH3 domain-binding overall complexity of receptor organization at the PSD. protein, termed cortactin binding protein 1 (CortBP1) The precise level of bridging between various receptors (Du et al., 1998). Subsequent studies determined that within the PSD is likely to be further complicated given CortBP1 is identical ProSAP1 (Boeckers et al., 1999a) the abundant alternative splicing that occurs in both and to a splice variant of Shank2 (Naisbitt et al., Shank and Homer gene products (Lim et al., 1999; 1999), which is a member of the Shank family of Soloviev et al., 2000). Shanks, cortactin and Arp2/3 sca€olding proteins (Sheng and Kim, 2000). Abun- complex are present in neuronal growth cones (Du et dantly expressed in neurons, CortBP1 and the related al., 1998; Goldberg et al., 2000) and given the ability to protein Shank3 contain a canonical cortactin SH3 bind to both CortBP1 and Shank 3 molecular `bridges', domain-binding sequence (KPPVPPKP) (Figure 2d) cortactin can serve to link multiple postsynaptic which mediates binding to cortactin (Du et al., 1998). receptors to sites of Arp2/3 mediated actin assembly Shank proteins serve as molecular sca€olds at post- (Figure 4). Therefore, clustering and organization of synaptic sites (PSD) of excitatory synapses, and are receptor components at the synapse may in part be characterized by the presence of an amino-terminal attributable to Arp2/3 mediated actin polymerization PSD95/dlg/ZO-1 (PDZ) domain. The CortBP1 PDZ regulated by cortactin in a manner similar to that domain directly binds to the carboxyl terminus of the described for the movement and formation of acet- transmembrane type 2 somatostatin receptor (Zitzer et ylcholine receptor clusters (Dai et al., 2000). Shank al., 1999) and the a-latrotoxin receptor CL1 (Kreien- proteins have been recently observed in non-neuronal kamp et al., 2000; Tobaben et al., 2000). In addition, tissues (Redecker et al., 2001), indicating that they may Shank1 and Shank3 contain a set of seven ankyrin also participate in receptor organization at sites other repeats and an SH3 domain proximal to the PDZ than the PSD. It of interest to note that the cortactin domain (Sheng and Kim, 2000). While a ligand for the SH3 domain-binding sequence found in Shanks is also Shank SH3 domain has not been reported, the ankyrin present in the murine form of Xin, a muscle-speci®c repeats interact with a novel protein of unknown protein implicated in cardiac morphogenesis (Wang et function termed sharpin, which can multmerize al., 1999). The cortactin SH3 domain also interacts through an amino terminal coiled-coil region (Lim et with a novel brain-speci®c protein of unknown al., 2001). The PDZ domain of CortBP1 and Shank3 function termed CBP90 though a proline-rich sequence also interacts with the carboxyl terminus of GKAP that di€ers from the binding sequence in Shank (Naisbitt et al., 1999; Boeckers et al., 1999b), a proteins (Figure 2d; Ohoka and Takai, 1998). cytosolic PSD protein that binds through its amino The epithelial tight junction is another region of terminal region to the C-terminal guanylate kinase-like subcellular specialization where cortactin may partici- domain of PSD-95 (Kim et al., 1997). The amino pate in organizing transmembrane receptor networks terminal region of PSD-95 contains three tandem PDZ (Figure 4). Tight junctions form a barrier between motifs, the ®rst two which are capable of directly adjacent polarized epithelia that restricts the movement interacting with the carboxyl terminus of both the of immune cells and solutes between cells. In addition, NMDA receptor (Kornau et al., 1995) and the Shaker they establish a di€usion barrier for integral membrane K+ channel (Kim et al., 1995). Additionally, the PDZ proteins within individual cells by dividing the plasma domain of Shank3 directly interacts with the carboxyl membrane into apical and basolateral domains terminus of the group 1 metabotropic receptors (Tsukita et al., 1999). Tight junctions are associated mGluR1a and mGluR5 as well as indirectly binding with an apical cytoskeletal structure known as the to these receptors through Homer (Tu et al., 1999). terminal web (Fath et al., 1993). Cortactin is localized Homer is a protein localized in PSDs that posses an at the terminal web in human small bowel epithelium Enabled/VASP homology domain (EVH) that interacts (Wu and Montone, 1998) and the SH3 domain with a proline-rich segment on Shank3 proximal to the associates with a proline-rich motif in ZO-1 (Katsube cortactin binding site (Tu et al., 1999). The Homer et al., 1998), a protein selectively localized to the EVH domain is capable of simultaneously interacting epithelial tight junction (Stevenson et al., 1986). The with Shank3 and either the carboxyl terminus of ZO-1 proline-rich domain contains a homologous

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6427

Figure 4 Cortactin serves to link diverse sca€olding protein complexes to Arp2/3-driven actin polymerization. Schematic representation of the proposed molecular interactions within the neuronal post-synaptic density (top), epithelial tight junction (bottom left) and in receptor mediated endocytosis (bottom right). In all cases, cortactin (red) is proposed to interact through its SH3 domain with proline-rich segments within numerous sca€olding proteins, including Shank proteins, ZO-1 and dynamin 2. These multiprotein sca€olds are in turn linked to the Arp2/3 complex (dark green) and associated actin cytoskeleton through sequences within the cortactin NTA and repeat domains. Protein domains are: a, cortactin NTA; rep, cortactin repeats; 3, SH3; PZ, PDZ; PP, proline-rich; SM, SAM; GK, guanylate kinase-like domain; ANK, ankyrin-like repeats; EVH, Enabled/VASP homology; CC, coiled-coil, RH, RBCK1 homology; PH, pleckstrin homolgy; GED, GTPase e€ector domain; ADF, actin depolymerizing factor homology; VC, verprolin/co®lin and acidic region in N-WASP

sequence to the consensus cortactin SH3 domain- 2 binding mediated through the second PDZ domains binding motif (Figure 2d). The ZO-1 domain structure of both proteins (Fanning et al., 1998; Itoh et al., is similar to that of PSD-95, containing three amino 1999b). The ®rst PDZ domain of ZO-2 and ZO-3 also terminal PDZ domains, an SH3 domain, a guanylate bind to the carboxyl terminus of claudin (Itoh et al., kinase-like domain and a carboxyl-terminal proline 1999a) and the amino-terminal region of both proteins rich motif (Willott et al., 1993; Itoh et al., 1993). Based also associate with occludin (Itoh et al., 1999b; on its localization and domain structure, ZO-1 is Haskins et al., 1998). All three ZO proteins also bind thought to participate in the biogenesis and regulation F-actin (Wittchen et al., 1999), which in ZO-1 is of tight junction assembly (Sheth et al., 1997). mediated through sequences in the proline-rich domain Consistent with this, the guanylate kinase-like domain (Fanning et al., 1998), thus linking and organizing of ZO-1 interacts with cytoplasmic motifs of occludin occludin and claudin clusters within the tight junction (Furuse et al., 1994; Fanning et al., 1998) while the membrane. The ZO-1 proline-rich domain also ®rst PDZ domain binds the carboxyl terminus of mediates interaction with the Ras e€ector AF-6 claudin (Itoh et al., 1999a), two transmembrane (Yamamoto et al., 1997). While the interactions of proteins that are implicated in forming the paracellular the various ZO proteins are more complex than PSD seal between adjacent cells (Furuse et al., 1993, sca€olding proteins in terms of their association with 1998a,b). ZO-1 contains a centrally located SH3 the actin cytoskeleton, the binding of cortactin to ZO- domain that interacts with the transcription factor 1 suggests that the tight junction complex in part is ZONAB (Balda and Matter, 2000). ZO-1 is homo- linked to Arp2/3 complex, and further suggests that logous to two other tight junction associated proteins, indirect regulation of transmembrane complexes by ZO-2 and ZO-3 (Jesaitis and Goodenough, 1994; attachment to sites of cortical actin assembly is a Haskins et al., 1998) which share the same domain hallmark of cortactin function. How Arp2/3 driven structure as ZO-1. ZO-1 forms binary complexes with actin polymerization contributes to tight junction ZO-2 and ZO-3 (Wittchen et al., 1999), with ZO-1/ZO- organization remains unexplored.

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6428 Besides transmembrane receptor complex organiza- been proposed that Arp2/3 activity is involved in tion, cortactin also plays a role in coupling Arp2/3 regulating membrane morphology and movement in complex-mediated actin dynamics to endocytosis and pre- and post-endocytic events (Qualmann et al., 2000). vesicle movement by binding to dynamin 2 (Figure 4). Support for the involvement of cortactin in post Dynamins comprise a family of a large GTPases that endocytic vesicle dynamics comes from the localization play a fundamental role in the ®ssion and recycling of of cortactin to actin tails of cytoplasmic rocketing membrane vesicles during receptor mediated endocy- endosomes (Kaksonen et al., 2000; Schafer et al., 2000), tosis (Schmid, 1997). Dynamins contain a GTP an activity that is dependent on Arp2/3 activation hydrolysis domain within the amino terminus, followed (Taunton et al., 2000). Actin tails on endosomes are by a linker region, a phospholipid binding pleckstrin strikingly similar to tails produced by intracellular homology (PH) domain, a GTPase e€ector domain and pathogens, suggesting that cortactin plays a similar role a proline-rich domain at the carboxyl terminus (Schmid by the simultaneous stimulation and stabilization of et al., 1998). The SH3 domain of cortactin can interact Arp2/3-induced actin networks. Taken together, these with two di€erent proline motifs within the dynamin 2 results suggest that Arp2/3 activation and actin network proline-rich domain as determined by peptide competi- stabilization is a conserved cellular function of tion studies (McNiven et al., 2000). Like cortactin, cortactin, and this activity is utilized directly for actin- dynamin 2 translocates into membrane ru‚es following based motility as well as indirectly through the PDGF stimulation of NIH3T3 cells. The growth factor- interaction of SH3 binding proteins to dynamically induced tranlocation of dynamin 2 to lamellipodia is regulate transmembrane protein organization. inhibited in cells expressing cortactin constructs lacking the SH3 domain, pointing to a functional requirement for cortactin in targeting dynamin 2 to sites of Overexpression and role of cortactin in human tumors membrane ru‚ing (McNiven et al., 2000). A second protein that may participate in localizing dynamin to A large body of evidence indicates that cortactin plays lamellipodia in a similar manner is mAbp1. The SH3 an important role in human neoplasia. The human domain of mAbp1 also binds to the dynamin proline- cDNA encoding cortactin (termed EMS1) was isolated rich region and is localized with dynamin at sites of by the cloning of genes found in human carcinoma cell endocytosis (Kessels et al., 2001). mAbp1 is a Src lines containing ampli®ed copies of the chromosome substrate (Lock et al., 1998), binds F-actin at two sites 11q13 region (Schuuring et al., 1991). The ampli®ed through its ADF-H domain, and is translocated to the region typically ranges from 3 ± 5 Mb in size and cortical cytoskeleton following Rac activation (Kessels contains several DNA markers, including the onco- et al., 2000). While the yeast homolog of mAbp1, genes cyclin D1 (PRAD1), INT2/FGF3, HSTF1/ Abp1p, interacts with and activates Arp2/3 complex FGF4 and BCL1 that are often found in a variety through two DDW motifs (Goode et al., 2001), mAbp1 of human tumors (Lammie and Peters, 1991). does not contain a DDW motif and is therefore not Ampli®cation of 11q13 occurs most frequently in likely to participate in Arp2/3 complex activation. A carcinomas of the head and neck (29%), ovary third SH3 domain containing protein that couples the (20%), bladder (15%), breast (13%) and lung (13%) actin cytoskeleton to dyamin is syndapin, which also (Schuuring, 1995). Individuals with carcinomas con- localizes with dynamin at sites of endocytosis (Qual- taining chromosome 11q13 ampli®cations have a poor mann et al., 1999). Syndapin directly binds N-WASp, prognosis (Schuuring et al., 1992; Meredith et al., and overepxression of syndapin leads to ®lopodia 1995). While overexpression of FGF3, FGF4 and formation dependent on Arp2/3 complex activity BCL1 is rarely observed in the above listed cancers (Qualmann and Kelly, 2000) providing a second (Liscia et al., 1989; Fantl et al., 1990), in the majority molecular link between dynamin and Arp2/3 complex of cases expression levels of cyclin D1 and cortactin in mammalian cells. Overexpression of the mAbp1 or are dramatically increased (Bringuier et al., 1996; Hui syndapin SH3 domain inhibits endocytosis (Kessels et et al., 1998). Cyclin D1 is a cell cycle regulatory al., 2001; Qualmann and Kelly, 2000), further high- protein that is essential for passage through G1 lighting the functional importance of linking dynamin (Baldin et al., 1993), and overexpression in human to the cortical actin skeleton in endocytic events. The tumors is thought to lead to a loss of cell cycle association of dynamin with clathrin-coated pits is regulation (Schuuring, 1995). Ampli®cation of the actin-independent, and is mediated through amphiphy- cortactin locus can occur with or without cyclin D1 sins I and II (Wigge and McMahon, 1998). The ampli®cation, and elevated cortactin gene levels amphiphysin SH3 domain binds to sequences in the correlate with poor patient prognosis (Rodrigo et al., dynamin proline-rich region (David et al., 1996) while 2000), suggesting that ampli®cation of the cortactin the amino terminal region of amphiphysins interact with gene directly contributes to tumor severity. Cell lines clathrin (Ramjaun et al., 1997) and the clathrin-binding isolated from patients with 11q13 ampli®cation exhibit adaptor protein AP2 (David et al., 1996). How the increased cortactin mRNA and protein levels (Patel et activities of cortactin, mAbp1, syndapin and amphipy- al., 1996; Campbell et al., 1996), providing strong sin regulate the cortical actin cytoskeleton and circumstantial evidence that cortactin overexpression coordinate with dynamin GTPase activity during plays an important role in tumors with 11q13 endocytic membrane ®ssion is highly complex. It has ampli®cation.

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6429 Based on observations of cortactin localization and the ability of cortactin to regulate the Arp2/3- function in non-transformed cells, overexpression of mediated assembly of the cortical actin meshwork, a cortactin in human tumors has been proposed to result process that would be further attenuated in tumor in increased cell migration and metastatic potential cells with elevated cortactin levels. The observation (Schuuring, 1995). Support for this comes from that overexpression of the cortactin NTA domain in increased cell migration but not growth rates in invasive MDA-MB-231 breast cancer cells disrupts NIH3T3 ®broblasts overexpressing cortactin (Patel et large cytoplasmic actin aggregates (which may al., 1998). Although not directly addressed, the increase provide the structural precurser elements for invado- in cell motility is likely due to increased Arp2/3 podia formation) (Uruno et al., 2001) also supports a activation and lamellipodia extension as described role for cortactin in tumor cell adhesion and above. In addition, cortactin redistributes into aberrant invasion. cell-matrix contact sites in v-Src transformed cells and in head and neck squamous cell carcinoma lines overexpressing cortactin (Figure 3c,d; Wu et al., 1991; Perspectives Schuuring et al., 1993). These contact sites, known as podosomes or rosettes, consist of invaginations of Regulation of the cortical actin cytoskeleton is ventral plasma membrane ringed by circular actin-rich fundamental to a wide range of dynamic cellular structures abundant in tyrosine phosphorylated cytos- events. Signaling pathways utilized in the control of keletal adhesion proteins (Tarone et al., 1985). plasma membrane dynamics and organization require Podosomes are also found in osteoclasts (Marchisio reorganization of the cortical actin cytoskeleton. et al., 1984) where they contribute to bone resorption Dynamic changes in the actin skeleton require the (Tanaka et al., 1996) and in macrophages (Davies and coordinated activity of tyrosine kinase and Rho Stossel, 1977) where they are involved in cell motility GTPase signaling pathways. Molecules at the `cross- (Linder et al., 1999). In v-Src transformed cells, roads' of these two pathways, such as cortactin, play podosomes are sites of increased extracellular matrix an important role in integrating and mediating degradation (Chen et al., 1985) caused by elevated cellular responses. As discussed above, cortactin plays membrane metalloproteinase activity (Chen and Wang, important roles in tyrosine kinase signaling pathways 1999). The resulting decrease in cell-matrix adhesion that involve alterations in cell morphology. Regula- likely contributes to the rounded transformed pheno- tion of the cortical cytoskeleton is central to changes type observed in v-Src transformed cells (Kellie et al., in cell morphology, and the direct role of cortactin in 1991) and therefore may lead to increased metastatic Arp2/3-mediated actin dynamics indicates that an potential. The actin cytoskeleton surrounding podo- important function of cortactin is to regulate actin- somes is highly labile (Ochoa et al., 2000), sensitive to based motility. The interaction of cortactin with PDZ changes in Rac activity (Ory et al., 2000), and is sca€olding proteins and with dynamin indicates that regulated in macrophages by WASp-mediated Arp2/3 cortactin serves to link transmembrane receptor activation (Linder et al., 1999, 2000). The presence of complexes and membrane vesicle dynamics to areas cortactin in podosomes of Src transformed cells of active actin polymerization. This linkage is likely coupled with the appearance of podosomes in tumor to be critical for the organization of receptor cells that overexpress cortactin strongly suggests that complexes and for vesicle motility. Increased cortactin cortactin plays a role in regulating podosome actin and levels in human cancers augments tumor cell membrane dynamics. This is supported by the adhesion, motility and invasion in a manner that coincident localization of dynamin 2 with cortactin in likely involves changes in Arp2/3 dynamics. In spite the podosomes of multiple cell types (Ochoa et al., of recent advances in understanding cortactin func- 2000). tion, much remains to be discovered. Although A role for cortactin in tumor cell invasion is tyrosine phosphorylation of cortactin is associated further borne out by the localization of cortactin with cell motility and morphological changes, the within invadopodia of invasive breast cancer cells molecular consequences of tyrosine and/or serine/ (Bowden et al., 1999). Invadopodia are unique to threonine phosphorylation remain unclear. Does transformed cells, occurring in the center of the cell tyrosine phosphorylation of cortactin by kinases such and extending well into the neighboring substratum as Src, Fer or Syk create binding sites for SH2- (Chen and Wang, 1999). Invadopodia exhibit robust containing proteins and if so, what is their contribu- membrane bound matrix metaloproteinase activity tion to cortactin function? Do other tyrosine kinases (Kelly et al., 1994) which facilitates the invasion of phosphorylate cortactin? How does MEK-meditated human breast cancer cells (Kelly et al., 1998). Tumor serine/threonine phosphorylation impact cortactin cell motility and invasion also requires the activity of function, and are other kinases involved? While the Cdc42 and Rac (Keely et al., 1997; Banyard et al., identi®cation of cortactin SH3 domain-binding pro- 2000), which in part contributes to endocytosis and teins such as CortBP1, ZO-1 and dynamin point to a proteolysis of extracellular matrix components (Kher- indirect role for cortactin in membrane organization, admand et al., 1998; Banyard et al., 2000). Rac- how cortactin binding ultimately in¯uences synaptic mediated recruitment of cortactin into podosomes organization, tight junction assembly and vesicle and invadopodia would therefore serve to enhance biogenesis is unknown. The existence of Shank

Oncogene Cortactin and membrane dynamics SA Weed and JT Parsons 6430 proteins in non-neuronal tissues also raises the Acknowledgments likelihood that additional transmembrane protein We wish to thank Drs Helene Marquis and Steve Anderson complexes are regulated by cortactin. What are the (University of Colorado Health Sciences Center) for events leading to chromosome 11q13 ampli®cation providing Listeria infected and v-Src transformed cells, and cortactin overexpression? Finally, how does Dr Alan Fanning (Yale University) for helpful discussions and Julie Head for technical support. JT Parsons acknowl- cortactin in¯uence podosome and invadopodia activ- edges the support of NIH grants CA40042 and CA29243. ity? These questions and others will likely be the SA Weed is a member of the University of Colorado subject of much investigation in the years to come. Cancer Center.

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