Functions of the Adapter Protein Cas: Signal Convergence and the Determination of Cellular Responses

Functions of the adapter protein Cas: signal convergence and the determination of cellular responses

Amy H Bouton*,1, Rebecca B Riggins1 and Pamela J Bruce-Staskal1

1Department of Microbiology, University of Virginia School of Medicine, Box 800734, Charlottesville, Virginia VA 22908, USA

Since Cas was ®rst identi®ed as a highly phosphorylated Cas and its family members 130 kilodalton protein that associated with the v-Src and v-Crk-oncoproteins, considerable eort has been made to Cas was ®rst identi®ed as a pTyr-containing 130 kDa determine its function. Its predicted role as a scaolding protein in cells transformed by the oncogenes v-src and molecule based on its domain structure has been largely v-crk (Matsuda et al., 1990; Reynolds et al., 1989). con®rmed. Through its ability to undergo rapid changes Transformation by both of these oncogenes requires in phosphorylation, subcellular localization and associa- protein tyrosine kinase (PTK) activity; in the case of v- tion with heterologous proteins, Cas may spatially and Src, PTK activity is provided by its own intrinsic temporally regulate the function of its binding partners. catalytic activity (Jove and Hanafusa, 1987), whereas Numerous proteins have been identi®ed that bind to Cas expression of v-Crk induces the PTK activity of a in vitro and/or in vivo, but in only a few cases is there an heterologous kinase (Mayer et al., 1988; Mayer and understanding of how Cas may function in these protein Hanafusa, 1990a). The 130 kDa protein (p130), which complexes. To date, Cas-Crk and Cas-Src complexes was later identi®ed as Cas, was found to associate with have been most frequently implicated in Cas function, activated variants of cellular Src (c-Src, Src) and v-Crk particularly in regards to processes involving regulation (Matsuda et al., 1990, 1991; Mayer and Hanafusa, of the actin cytoskeleton and proliferation. These and 1990a; Reynolds et al., 1989). Mutations in v-Src and other Cas protein complexes contribute to the critical v-Crk that abrogated binding of this 130 kDa protein role of Cas in cell adhesion, migration, proliferation and also abolished the transforming activity of these survival of normal cycling cells. However, under oncoproteins, suggesting that p130 played a critical conditions in which these processes are deregulated, role in the transformation process (Kanner et al., 1991; Cas appears to play a role in oncogenic transformation Mayer and Hanafusa, 1990b). and perhaps metastasis. Therefore, in its capacity as an A cDNA clone encoding Cas was isolated in 1994 adapter protein, Cas serves as a point of convergence for (Sakai et al., 1994a,b) and its predicted domain many distinct signaling inputs, ultimately contributing to structure suggested that it functioned as an adapter the generation of speci®c cellular responses. Oncogene or scaolding molecule (Figure 1). Cas contains an (2001) 20, 6448 ± 6458. amino-terminal src-homology 3 (SH3) domain, followed by a short proline-rich segment, a large Keywords: Cas; Src; Crk; Rac1; migration; adapter `substrate-binding' domain containing ®fteen repeats of a four amino acid sequence (tyrosine-any two amino acids-proline; YXXP), a serine-rich region and Cas (p130Cas; Crk-associated substrate) was ®rst a carboxy-terminal domain. The YXXP motif found recognized over 10 years ago as a 130 kilodalton in the substrate-binding domain can serve as a PTK (kDa) phosphotyrosine (pTyr)-containing protein that substrate (Songyang, 2001; Songyang et al., 1994; associated with two oncoproteins, pp60v-src (v-Src) and Songyang and Cantley, 1998), leading to phosphor- p47gag-crk (v-Crk) (Matsuda et al., 1990; Reynolds et al., ylation of one or more tyrosine residues. Once 1989). Following cloning of the Rat cDNA in 1994 phosphorylated, these tyrosine residues can then (Sakai et al., 1994a,b), tremendous eorts have been serve as ligands for src-homology 2 (SH2) or pTyr placed on determining the function of Cas in both binding (PTB) domains contained in many dierent oncogenic and normal cellular processes. This review cellular proteins. Within the carboxy-terminal half of will begin with a short perspective on Cas and its Cas, there are several additional protein interaction family members. It will then discuss some of the known sites (Burnham et al., 1996). The predicted function functions of Cas and explore possible mechanisms of Cas as an adapter protein has been borne out through which Cas may perform these functions. through the identi®cation of numerous binding Finally, it will investigate potential roles for Cas in partners (Figure 1). While several of these interac- the development and/or progression of oncogenesis. tions have been detected in vivo following activation of speci®c signaling pathways, the regulation and function of many Cas protein complexes remain *Correspondence: AH Bouton; E-mail: [email protected] unresolved. Functions of the adapter protein Cas AH Bouton et al 6449

Figure 1 Cas structure and its binding partners. A graphic depiction of the domain structure of Cas is shown, including the SH3 domain (SH3), proline-rich region (PRO), substrate-binding YXXP domain (YXXP15), serine-rich region (SER) and carboxy- terminus (C-terminus). The bipartite Src binding sequence is indicated by single letter amino acid codes; the Src SH3 domain binds to the sequence RPLPSPP beginning at residue 639 and the Src SH2 domain binds to the sequence motif pYDYV beginning at residue 668 (Nakamoto et al., 1996). Proteins that have been shown to bind to the domains of Cas, either in vitro or in vivo, are presented below. A partial list of references that address binding of these proteins to Cas includes: Fak (Burnham et al., 1996; Harte et al., 1996; Polte and Hanks, 1995), Pyk2 (Astier et al., 1997b; Lakkakorpi et al., 1999), FRNK (Harte et al., 1996), PTP1B (Liu et al., 1996), PTP-PEST (Garton et al., 1996), C3G (Kirsch et al., 1998), PR-39 (Chan and Gallo, 1998), CMS (Kirsch et al., 1999; Nakamoto et al., 2000), Crk (Burnham et al., 1996; Sakai et al., 1994a), Nck (Schlaepfer et al., 1997), PI3K (Li et al., 2000), SHIP2 (Prasad et al., 2001), 14-3-3 (Garcia-Guzman et al., 1999), Src family kinases (Burnham et al., 1996; Nakamoto et al., 1996; Sakai et al., 1994a), NSP family members (Gotoh et al., 2000; Lu et al., 1999; Sakakibara and Hattori, 2000), Grb2 (Wang et al., 2000), Nephrocystin (Donaldson et al., 2000), PI3K (Li et al., 2000), ID2 (Law et al., 1999), CIZ (Nakamoto et al., 2000)

There are two other family members that share suggest that they have potentially distinct functions. considerable structure and sequence homology with First, the expression patterns of these three proteins Cas. Human enhancer of filamentation HEF1/CasL dier signi®cantly. Whereas Cas mRNA and protein (HEF1) was identi®ed in 1996 as a `lymphocyte-type' are expressed in most adult tissues, HEF1 mRNA Cas family member that promoted pseudohyphal levels are signi®cantly reduced in brain and liver, and growth in the budding yeast Saccharomyces cerevisiae HEF1 protein levels appear to be greatest in (Law et al., 1996; Minegishi et al., 1996). Embryonal lymphocytes, lung and breast epithelium (Law et al., Fyn-associated substrate (Efs)/Src-interacting protein 1996, 1998; Minegishi et al., 1996; Sakai et al., 1994a). (Sin) was identi®ed about the same time as a Fyn/Src- Expression of Efs/Sin is considerably more restricted, associated protein (Alexandropoulos and Baltimore, with the highest levels of mRNA being present in 1996; Ishino et al., 1995). Both HEF1 and Efs/Sin embryonic tissues (Ishino et al., 1995). Second, there is share a similar domain structure with Cas, with the some indication that the three family members may greatest sequence similarity present in the SH3 undergo distinct post-translational modi®cations and domains and the carboxy-terminal 200 amino acids exhibit unique localization patterns within the cell. All (Figure 2). One of the most notable dierences three molecules are predominantly cytoplasmic, but a between the functional sequence motifs of these fraction of Cas and HEF1 is found in focal adhesions proteins is found in the Src binding sequences. The of adherent cells (Harte et al., 1996, 2000; Law et al., bipartite binding site present in Cas and Efs/Sin 1996, 2000; Nakamoto et al., 1997; Petch et al., 1995; includes a proline-rich region that binds to the Src Polte and Hanks, 1995). HEF1 undergoes a speci®c SH3 domain and a pTyr-containing sequence that cleavage event during mitosis, culminating in the binds to the Src SH2 domain (Alexandropoulos and appearance of a biologically active amino-terminal Baltimore, 1996; Burnham et al., 1999, 2000; fragment that localizes to the mitotic spindle (Law Nakamoto et al., 1996). The SH3-binding sequence et al., 1998). Cas also undergoes post-translational is absent from HEF1 (Figure 2). It is unclear how this modi®cations during mitosis, characterized by a change aects Src binding to HEF1, but Cas dramatic loss of tyrosine phosphorylation and a molecules with proline-to-alanine substitutions in these concomitant increase in serine phosphorylation sequences exhibit reduced binding to Src (Burnham et (Yamakita et al., 1999). Third, there is evidence to al., 1999, 2000; Nakamoto et al., 1996). suggest that HEF1 and Efs/Sin are unable to While the three Cas family members share a genetically and/or functionally substitute for Cas common domain structure, several lines of evidence during embryonic development. Mouse embryos con-

Oncogene Functions of the adapter protein Cas AH Bouton et al 6450

Figure 2 Cas family members. The domain structures of Cas, HEF1 and Efs/Sin are shown. Domains include the SH3 domain (SH3), proline-rich region (PRO), substrate-binding YXXP domain (YXXP), serine-rich region (SER) and carboxy-terminus (C- terminus) The bipartite Src binding sequences present on Cas and Efs/Sin are indicated by single letter amino acid codes. Regions that are absent from HEF1 are indicted by hatched/dotted lines

taining a homozygous deletion of the cas gene do not Nojima et al., 1995; Polte and Hanks, 1997; Vuori survive embryogenesis, despite the fact that the genes and Ruoslahti, 1995). c-Src and other Src family encoding HEF1 and Efs/Sin are still present (Honda et kinases, as well as focal adhesion kinase (Fak) and al., 1998). Cas7/7 embryos obtained at embryonic day its close relative Pyk2 (also known as cell adhesion 11.5 ± 12.5 exhibit severe cardiovascular de®ciencies, kinase b; CAKb), have been implicated in integrin- which correlate with the appearance of defects in dependent phosphorylation of Cas (Hamasaki et al., myo®bril and Z-disk organization in cardiocytes 1996; Klinghoer et al., 1999; Schlaepfer et al., 1997; isolated from these animals. These studies indicate Tachibana et al., 1997; Ueki et al., 1998; Vuori et al., that the genes encoding HEF1 and Efs/Sin are not 1996). sucient to overcome the extensive structural abnorm- There is considerable evidence to suggest that Cas alities that occur as a consequence of the Cas plays a role in cytoskeletal regulation and cell de®ciency. adhesion. Perhaps the most compelling data come While Cas, HEF1 and Efs/Sin share structural and from studies using ®broblasts isolated from embryos sequence homology, the data described above suggest containing a targeted disruption of the cas gene. that they may have distinct functions that arise from Cas7/7 cells contain disorganized, short actin ®laments dierences in tissue distribution, subcellular localiza- relative to the normal actin stress ®ber organization tion, post-translational modi®cations and primary exhibited by analogous cells that have been engineered sequence divergence. The remainder of this review will to express ectopic Cas (Honda et al., 1998, 1999). focus predominantly on Cas, although other family However, Cas does not appear to be required for focal members will be discussed in those cases where unique adhesion formation, since both Cas7/7 and Cas+/+ cells functions are suggested. For additional information show normal levels of vinculin staining of focal about possible functions of Cas family members, adhesions (Honda et al., 1998). readers are directed to a recent review by O'Neill et During the process of cell migration, actin stress al. (2000). ®bers undergo dissolution and reformation. Conse- quently, it is not surprising that Cas and other focal adhesion proteins play a role in cell migration. Cas- Regulation of the actin cytoskeleton and cell migration de®cient ®broblasts show decreased haptotaxis toward FN, a decreased ability to migrate into the gap in a The process of cell migration is functionally linked to wound healing assay, and decreased basal and serum- cell-extracellular matrix (ECM) interactions. The ®rst induced invasion through a 3-dimensional collagen hint that Cas played a role in this process came from matrix (Cho and Klemke, 2000; Honda et al., 1999). studies demonstrating that Cas was present in sub- This reduced migratory phenotype is similar to that cellular structures called focal adhesions that form observed for FN-, Fak- and Src-de®cient ®broblasts molecular bridges between the ECM and the actin (George et al., 1993; Hamasaki et al., 1996; Ilic et al., cytoskeleton (Harte et al., 1996, 2000; Nakamoto et al., 1995; Klinghoer et al., 1999; Owen et al., 1999), 1997; Petch et al., 1995; Polte and Hanks, 1995; for suggesting that these molecules may function in a review of focal adhesion structure and function, see common pathway to promote migration. Critchley, 2000). Cas becomes phosphorylated in In many cases, migration can be correlated with response to integrin engagement by a wide variety of increased tyrosine phosphorylation of Cas. Src/Yes/ ECM components, including ®bronectin (FN), vitro- Fyn (SYF)-de®cient ®broblasts, which show decreased nectin, laminin and collagen (Harte et al., 1996; levels of adhesion-dependent Cas phosphorylation,

Oncogene Functions of the adapter protein Cas AH Bouton et al 6451 exhibit marked defects in cell migration (Klinghoer et no eect on myosin light chain phosphorylation and al., 1999). Fibroblasts that overexpress the protein cellular contraction in a 3-dimensional collagen matrix tyrosine phosphatase (PTPase) PTP-PEST also demon- (Cheresh et al., 1999). strate defects in cell migration that coincide with signi®cantly reduced levels of tyrosine phosphorylated Cas (Garton and Tonks, 1999). Finally, expression of Growth regulation the dual-speci®city phosphatase PTEN inhibits cell migration and invasion through a process that involves As discussed above, focal adhesion proteins can dephosphorylation of Fak and Cas (Tamura et al., regulate cell adhesion to the ECM and migration. 1999). When either Fak or Cas is co-expressed with However, these same proteins also play a role in cell PTEN, phosphorylation of these molecules increases cycle progression and proliferation by transmitting and PTEN-induced inhibition of migration and growth and survival signals from the ECM (for review, invasion is reversed. see Aplin and Juliano, 1999; Giancotti, 1997; Howe et The close functional relationship between Fak and al., 1998). A number of studies suggest that Cas, Cas likely contributes to the role of Cas in cell through interactions with its binding partners Fak, Src migration. Cas and Fak colocalize in focal adhesions and Crk, is involved in this process. (Harte et al., 1996, 2000; Polte and Hanks, 1995, 1997) The requirement for cells to adhere in order to and, as discussed above, Cas can be a substrate of Fak. progress through the cell cycle is ®rmly established for Cary et al. (1996, 1998) showed that overexpression of many cells (Aplin and Juliano, 1999; Giancotti, 1997; Fak in Chinese Hamster Ovary (CHO) cells enhances Howe et al., 1998). However, upon entry into mitosis, FN-mediated haptotaxis and that this eect can be cells detach from the ECM and remain detached until inhibited by co-expression of the Cas SH3 domain. cytokinesis is complete. This suggests that there must Under these conditions, it is thought that the Cas SH3 be mechanisms in place during mitosis that promote domain eectively competes for endogenous Cas in disruption of cell adhesions and temporarily prevent binding to Fak, thus inhibiting functional interactions reattachment. During mitosis, Cas, Fak and the focal between Fak and full-length Cas. adhesion protein paxillin become phosphorylated on Cas also plays a role in migration through its serine (Ser) and threonine (Thr) residues, and are association with Crk. Integrin-dependent phosphoryla- concomitantly dephosphorylated on tyrosine residues tion of Cas often results in the establishment of Cas- (Yamakita et al., 1999). Furthermore, levels of Fak- Crk complexes (Cheresh et al., 1999; Garton and Cas and Fak-Src complexes are signi®cantly reduced Tonks, 1999; Klemke et al., 1998; Mielenz et al., 2001; during mitosis. Re-entry into the G1 phase of the cell Vuori et al., 1996). The role of these complexes in cell cycle is accompanied by a loss of Ser/Thr phosphor- migration was directly established in a study by ylation, a return to normal levels of Fak, Cas and Klemke et al. (1998) which showed that Cas over- paxillin tyrosine phosphorylation, a restoration of Fak- expression could enhance cell migration of COS and Cas and Fak-Src protein complexes, and reattachment FG-M pancreatic carcinoma cells plated on vitronectin. to the ECM. Expression of a substrate binding domain deletion of One downstream consequence of integrin-mediated Cas that is de®cient for Crk binding inhibited this cell adhesion is activation of the Jun NH2-terminal eect. By expressing putative downstream molecules, kinase (JNK), leading to phosphorylation of its target the authors concluded that this migration pathway c-jun and regulation of AP-1-dependent transcription. involved Cas, CrkII and Rac1. Cas-Crk signaling also JNK activation in response to cell adhesion may appears to be important for haptotaxis involving ECM therefore be an important factor in cell cycle components other than vitronectin. For example, cells progression (Oktay et al., 1999). Expression of expressing a mutant of the a7 integrin subunit exhibit dominant-negative Fak variants inhibits integrin-de- diminished Cas phosphorylation and Cas-Crk associa- pendent JNK activation, indicating that Fak plays an tion that coincides with decreased migration on the important role in this process. Expression of a Cas ECM component laminin (Mielenz et al., 2001). The mutant lacking the substrate-binding YXXP domain is Cas-Crk pathway has also been shown to be important also inhibitory for JNK activation and cell cycle for chemotaxis induced by insulin, epidermal growth progression, implicating Cas in these processes as well. factor (EGF) and serum (Cheresh et al., 1999; Cho and It has been proposed that protein complexes containing Klemke, 2000; Klemke et al., 1998). Fak, Src and Cas recruit the small adapter protein Crk In many cases, Cas-Crk complexes function to in order to promote JNK activation and cell prolifera- promote cytoskeletal rearrangements through activation. The requirement for Fak-Cas interactions in cell tion of Rac1. Molecules that may link Cas-Crk proliferation is supported by a second study, which interactions to Rac1 activation include C3G and showed that G1 progression and DNA synthesis DOCK180 (Arai et al., 1999; Kiyokawa et al., requires the direct association of Fak and Cas (Reiske 1998a,b; Matsuda et al., 1996; Tanaka et al., 1994). et al., 2000). In this case, expression of a Fak molecule Interestingly, Cas-dependent cell migration pathways de®cient for Cas binding was found to decrease 5- may not involve actin-myosin contraction, as indicated Bromo-2'deoxy-uridine (BrdU) incorporation by 50%. by the ®nding that a mutant form of Cas that blocks There is also evidence in support of a role for Src- insulin-induced membrane ruing and migration has Cas interactions in cell proliferation. Overexpression of

Oncogene Functions of the adapter protein Cas AH Bouton et al 6452 Cas in C3H10T1/2-5H cells that stably overexpress c- Cytoskeletal and adhesive changes that accompany Src (Wilson and Parsons, 1990), and the ensuing programmed cell death are often associated with accumulation of Src-Cas complexes, correlates with cleavage of a number of cytoskeletal regulatory an increase in BrdU incorporation under serum-free proteins, including Cas, Fak, paxillin and Src (Chan conditions and a marked enhancement of anchorage- et al., 1999; Kook et al., 2000b). Multiple pro- independent growth (Burnham et al., 2000). Because apoptotic stimuli have been shown to induce Cas Cas binding to Src serves to increase the enzymatic cleavage, including Eschericia coli lipopolysaccharide activity of Src, it has been proposed that the enhanced (LPS), nocodazole, etoposide, adenosine and UV ability of these cells to grow in a serum- and ECM- irradiation (Bannerman et al., 1998; Chan et al., independent fashion may stem from an overall increase 1999; Harrington et al., 2001; Kook et al., 2000a,b). in Src activity. Cas dephosphorylation, Cas cleavage and apoptosis are From these data, it appears that Cas can function to also induced by overexpression of the receptor-like regulate progression through the cell cycle, both by PTPase LAR (Weng et al., 1999). Several lines of transmitting survival signals from the ECM and by evidence indicate that caspases are responsible for Cas modulating Src kinase activity. The Cas family member cleavage following treatment with these apoptosis- HEF1 appears to have a dierent role in cell cycle inducing agents. First, caspase-3 can directly cleave progression. Unlike Cas, which is expressed at Cas in vitro and caspase inhibitors prevent cleavage of relatively equivalent levels throughout the cell cycle, Cas in vivo (Kook et al., 2000a). Second, Cas cleavage HEF1 expression levels are low in quiescent cells and is prevented when a consensus caspase cleavage site on increase following induction of cell growth by either Cas is mutated. The functional signi®cance of this serum or release from thymidine block (Law et al., modi®cation is suggested by the ®nding that etoposide- 1998). The levels of full-length HEF1 then undergo a dependent Cas cleavage correlates with a loss of focal dramatic decrease at the onset of mitosis. The adhesions, and that focal adhesions are maintained in appearance of a 55 kDa amino-terminal fragment of the presence of a Cas mutant that lacks the consensus HEF1 (p55) coincides with this decrease in the full- caspase cleavage site (Kook et al., 2000a). length molecule. Immuno¯uorescence studies showed Cleavage of Cas during apoptosis and the accom- that p55 is localized to the mitotic spindle from panying changes in focal adhesion architecture may prophase to late anaphase, and it localizes to the serve to prevent transmission of survival signals from midbody during cytokinesis. Interestingly, a yeast two- the ECM. Several studies support the idea that Cas is hybrid screen identi®ed Dim1p as a binding partner of an important transmitter of survival signals. FG-M HEF1 (Law et al., 1996). This protein is essential for pancreatic adenocarcinoma cells show increased survi- passage through the G2/M checkpoint in Sacchar- val as compared to the nonmetastatic FG cell line from omyces pombe. While further analysis is required to which they were selected (Cho and Klemke, 2000; fully understand the role of p55 HEF1 in cell cycle Klemke et al., 1998). FG-M cells contain an increased progression, it appears that this Cas family member number of Cas-Crk complexes and disruption of these has a unique function in cell proliferation, one that complexes promotes apoptosis. Cas-Crk complexes may involve transmission of pro-proliferative signals also promote invasion and survival in COS-7 cells, directly to the mitotic spindle. both of which are blocked by expression of a dominant-negative form of Rac1. These data indicate that the Cas-Crk-Rac1 pathway that was established Apoptosis for cell migration (Klemke et al., 1998) is also important for transmission of survival signals emanat- The ECM also provides an important survival signal to ing from the ECM. adherent cells (for review, see Frisch and Ruoslahti, The Cas family member HEF1 has a somewhat 1997; Howe et al., 1998). Thus the presence of Cas in dierent role in programmed cell death. Whereas focal adhesions and its potential role in integrin increased Cas expression can result in protection from signaling may contribute to cell survival and the apoptosis (Cho and Klemke, 2000; Klemke et al., 1998; prevention of apoptosis. The precise molecular me- Weng et al., 1999), overexpression of HEF1 in MCF-7 chanism by which Cas might mediate these eects is human breast cancer cells leads to increased apoptosis not yet clear, although evidence points toward a role (Law et al., 2000). Endogenous HEF1 is cleaved in for Fak-Cas, Src-Cas and/or Cas-Crk protein com- MCF-7 cells in a caspase-dependent manner upon plexes. For example, in rabbit synovial ®broblasts, pro- induction of apoptosis by TNF-a. The carboxy- survival signals emanating from the ECM proceed terminal 28 kDa cleavage product has been shown to through Fak to Cas and require the small GTPases be both necessary and sucient to promote pro- Ras and Rac1 as well as activation of JNK2 (Almeida grammed cell death (Law et al., 2000). These data et al., 2000). Phosphorylation of Cas and its ability to implicate a pro-apoptotic function within the extreme associate with Fak are essential steps in this survival carboxy-terminus of HEF1, which shares considerable pathway. Similarly, treatment of cells with reactive sequence homology with Cas. Interestingly, Cas oxygen species (ROS) induces JNK activation through cleavage during apoptosis also produces a 28-kDa a pathway that involves Src and Cas (Yoshizumi et al., carboxy-terminal fragment but there is no evidence 2000). that this fragment is capable of inducing apoptosis

Oncogene Functions of the adapter protein Cas AH Bouton et al 6453 (Law et al., 2000). Further research is needed to invasin-b1 integrin binding, the interaction between determine if Cas, and possibly Efs/Sin, can promote Adenovirus proteins and a7 integrins induces tyrosine apoptosis upon cleavage. phosphorylation of Cas (Li et al., 2000). Treatment of cells with PTK inhibitors both inhibits Cas phosphorylation and results in decreased viral internalization, Microbial pathogenesis indicating that PTK activity and perhaps speci®cally Cas phosphorylation are important events in the In addition to its function in signaling pathways that process of viral endocytosis. Although speci®c Cas- regulate cell adhesion, migration, proliferation and dependent mechanisms for Adenovirus uptake have not survival, the actin cytoskeleton also plays an important been identi®ed, infection has been shown to promote role in a myriad of host-pathogen interactions (for an association between Cas and the p85 subunit of review, see Frischknecht and Way, 2001). For example, phosphatidylinositol-3 kinase (PI3K) (Li et al., 2000). phagocytosis of Yersinia pseudotuberculosis is largely Overexpression of a Cas construct in which the initiated by a mechanism that involves many of the carboxy-terminal PI3K binding site is mutated inhibits same proteins that regulate cell migration. Y. pseudo- Adenovirus uptake, suggesting that the association of tuberculosis expresses a surface protein, termed invasin, PI3K and Cas may be critical for ecient Adenovirus that binds with high anity to b1 integrins present on endocytosis. the surface of host cells (Dersch and Isberg, 2000; In light of these studies, it is clear that internalization Isberg et al., 2000). As is the case for integrin of pathogens that bind to integrin receptors can be engagement by ECM components, invasin-b1 interac- initiated through PTK-dependent signaling pathways tions induce the activation of cellular PTKs and that promote rearrangements of the actin cytoskeleton downstream signaling cascades. This results in a coincident with uptake. The demonstration that en- dramatic rearrangement of the actin cytoskeleton that docytosis of intracellular microorganisms such as accompanies bacterial uptake and clearance. Patho- Adenovirus involves Cas, and that Cas is a target of genic Yersiniae have developed a mechanism to remain eectors designed to inhibit phagocytosis of extracel- extracellular by expressing virulence proteins that block lular pathogens such as Y. pseudotuberculosis, highlights these cellular signaling pathways and thereby inhibit its important function in these processes. Although its phagocytosis. A role for Cas in bacterial internalization precise role is not well understood, the ability of Cas to was ®rst suggested when two laboratories reported that either directly or indirectly modulate the function and Cas is a substrate of an antiphagocytic eector of Y. activities of molecules such as Crk, Rac1, PI3K and Src pseudotuberculosis, the PTPase YopH (Black and is likely to be critical for its function in microbial Bliska, 1997; Persson et al., 1997). The antiphagocytic pathogenesis as well as in migration. function of YopH is believed to be due to its ability to dephosphorylate focal adhesion proteins such as Cas, Fak and paxillin, thereby preventing cytoskeletal Cancer rearrangements and inhibiting signaling pathways that lead to bacterial uptake (Black and Bliska, 1997; Black Deregulation of the signaling pathways that control et al., 1998; Persson et al., 1997). cell adhesion, migration, survival and proliferation can Y. pseudotuberculosis strains that do not express result in oncogenic transformation and metastasis. YopH are eciently internalized by a wide range of Consequently, molecules such as Cas that participate mammalian cells (Andersson et al., 1996; Black and in multiple facets of these processes are likely to play a Bliska, 1997; Persson et al., 1997). The requirement for critical role in oncogenesis. In fact, Cas was ®rst Cas function in the process of Yersinia uptake was identi®ed as a molecule whose phosphorylation established through the expression of a variant of Cas correlated with transformation by the v-Src and v- that contains a deletion of the substrate-binding YXXP Crk oncoproteins (Matsuda et al., 1990; Reynolds et domain. Expression of this molecule was found to al., 1989). Studies using Cas-null ®broblasts provide inhibit the uptake of Yersinia strains that do not strong evidence that Cas is required for Src transfor- express YopH (Weidow et al., 2000). Further char- mation (Honda et al., 1998). In the absence of Cas acterization of the Cas-dependent pathway of Yersinia expression, mouse embryo ®broblasts were shown to be uptake placed Crk and Rac1 downstream of Cas and resistant to transformation by constitutively active Src. also implicated additional binding partners of Crk. The ability of activated Src to induce morphological Perhaps not surprisingly, this pathway is similar to the transformation and anchorage-independent growth was Cas-dependent pathway that is involved in aspects of restored when wild type Cas was ectopically expressed cell migration (Klemke et al., 1998). in the Cas-null cells. Furthermore, Auvinen et al. Interestingly, studies investigating Adenovirus inter- (1995) demonstrated that expression of antisense Cas nalization showed that overexpression of a Cas mutant constructs could partially reverse cellular transforma- containing a similar deletion of the substrate-binding tion induced by ornithine decarboxylase, Ha-Ras and YXXP domain also inhibits Adenovirus entry into host v-Src, indicating that Cas plays a direct role in cellular cells (Li et al., 2000). Adenovirus internalization is transformation. initiated when surface penton proteins bind to host cell Although the precise mechanism by which Cas a7 integrins (Mielenz et al., 2001). As is the case for participates in Src-mediated transformation is not

Oncogene Functions of the adapter protein Cas AH Bouton et al 6454 known, the direct interaction between Cas and Src may not only regulate aspects of normal cell appears to be important for this process. In Rat 1- proliferation, but it may also promote unregulated LA29 cells, which express a temperature-sensitive v- growth and survival, which are hallmarks of cellular Src allele, Cas is tyrosine phosphorylated and transformation. associates with Src only at permissive temperatures It is therefore not surprising that there is evidence (Burnham et al., 1999). When the carboxy-terminus of suggesting Cas involvement in the initiation and/or Cas (Cas-CT) was ectopically expressed in these cells, progression of human cancers. Retroviral insertion the transformed phenotype was maintained despite the mutagenesis of the estrogen receptor-positive ZR-75-1 fact that v-Src-Cas complexes were replaced by v-Src- human breast cancer cell line resulted in the identi®ca- Cas-CT complexes. This suggests that Cas-CT, which tion of three loci that promote tamoxifen resistance contains the bipartite Src-binding sequence, can when they become upregulated (Brinkman et al., 2000). functionally substitute for full length endogenous The breast cancer anti-estrogen resistance 1 (BCAR1) Cas in this process. locus was subsequently identi®ed as the human Src-Cas complexes may function to promote homologue of Cas. When Cas was stably transfected transformation through the modulation of Src activity. into ZR-75-1 cells, it conferred the ability to proliferate In support of this model, a dramatic increase in Src in the presence of either tamoxifen or the anti-estrogen PTK activity is observed upon Src binding to either ICI 182,780. This coincided with Cas-dependent full length Cas or Cas-CT (Burnham et al., 2000). The alterations in cell morphology and cytoskeletal archi- biological signi®cance of this activation is suggested tecture. Moreover, high levels of Cas expression in by studies that examined the growth properties of breast tumors correlated with decreased patient stable cell lines that overexpress Src and either full survival and poor response to tamoxifen therapy (van length Cas or Cas-CT. These cells, which contain der Flier et al., 2000a). Although high Cas expression elevated levels of Src-Cas or Src-Cas-CT protein did not correlate with the acquisition of tamoxifen complexes, exhibited enhanced serum- and ancho- resistance, it was found to be a signi®cant risk factor rage-independent growth. for the roughly 40% of breast tumors that exhibit Numerous events occur downstream of Src catalytic intrinsic tamoxifen resistance (Foekens et al., 1994; van activation that may be impacted by Cas. For example, der Flier et al., 2000b). v-Src expression results in the transcriptional activa- The mechanism by which Cas promotes tamoxifen tion of a set of immediate early genes that are resistance in breast tumors has yet to be elucidated. An controlled by serum response elements (SREs) (Meijne attractive hypothesis, based on the in vitro data et al., 1997). Hakak and Martin showed that co- presented above, is that increased expression of both expression of Cas with v-Src enhanced transcription Cas and c-Src could result in increased levels of Src- from these sites. These investigators went on to Cas complexes, leading to elevated Src activity and demonstrate that Src-dependent SRE activation in- enhanced cell growth. In fact, 25 ± 30% of breast volved Cas binding to Src, the establishment of Grb2- tumors exhibit elevated c-Src mRNA and protein Shc and Grb2-Shp2 complexes, and activation of the levels, which correlate with increased Src activity in Ras/MEK/Erk signaling cascade (Hakak and Martin, the absence of activating mutations of Src (Biscardi et 1999). al., 1998; Koster et al., 1991; Verbeek et al., 1996). Like Cas, Efs/Sin can bind to, and activate, c-Src Many of the breast cancer cell lines that exhibit (Alexandropoulos and Baltimore, 1996; Xing et al., increased c-Src activity also express high levels of Cas 2000). Using transcription from the SRE as a read-out and Src-Cas complexes (data not shown). The potential for c-Src activation, the Src-Efs/Sin protein complex contribution of these complexes to the growth, was shown to activate the small GTPase Rap1 progression and perhaps tamoxifen resistance of breast through a pathway that involves the adapter protein tumors remains to be determined. Crk and its association with the Rap1 exchange factor The involvement of Cas and its family members in C3G (Xing et al., 2000). GTP-bound Rap1, in turn, other human cancers remains an important question to mediates activation of the SRE through the ERK address. Recent evidence suggests that growth and pathway. invasion of malignant melanoma cells can be accom- Taken together, these data support a model in panied by increases in Cas tyrosine phosphorylation which the association of Cas or its family members (Eisenmann et al., 1999; Schraw and Richmond, 1995). with c-Src activates PTK activity, leading to increased Cas and its family members may also play a role in the tyrosine phosphorylation of Src substrates, activation development or progression of certain leukemias. Cells of downstream signaling pathways, and cell prolifera- expressing p190BCR/ABL, the causative agent of Philadel- tion. When spatially and temporally controlled, this phia-positive acute lymphoblastic leukemia (ALL) and process may contribute to normal growth regulation. chronic myeloid leukemia (CML), contain high levels However, when modulation of Src activity by Cas of tyrosine phosphorylated Cas and HEF1 coincident becomes deregulated as a consequence of increased with the presence of Cas/HEF1-Crk protein complexes protein expression or prolonged signaling of the Src- (Dejong et al., 1997; Salgia et al., 1996). Further Cas complex, this pathway may result in enhanced studies are needed to de®ne how the Cas family of growth and survival in the absence of growth factors proteins functions in the initiation and/or progression and/or the ECM. In this way, the Src-Cas complex of human cancer.

Oncogene Functions of the adapter protein Cas AH Bouton et al 6455 Cas phosphorylation and its binding partners: functional integrin-dependent Cas phosphorylation (Hamasaki et implications al., 1996; Vuori et al., 1996). In certain cases, Fak has also been shown to phosphorylate Cas in vivo (Bruce- While adhesion-dependent tyrosine phosphorylation of Staskal and Bouton, 2001; Schlaepfer et al., 1997). Cas has been extensively discussed above, activation of Because the induction of Fak phosphorylation and a wide range of other cellular receptors also results in catalytic activity often accompanies Cas phosphoryla- Cas phosphorylation (see Table 1). These include tion, Fak may play a central role in many cases of receptor PTKs, G-protein coupled receptors (GPCRs) agonist-induced tyrosine phosphorylation of Cas (for and hormone receptors (representative references example, see Bruce-Staskal and Bouton, 2001; Naka- include Casamassima and Rozengurt, 1997, 1998; mura et al., 1998; Zachary et al., 1993). Ferris et al., 1999; Larsson et al., 1999; Murakami et Tyrosine phosphorylation of Cas serves two known al., 1999; Needham and Rozengurt, 1998; Ojaniemi functions. First, it coincides with a relocalization of and Vuori, 1997; Ribon and Saltiel, 1996; Spencer et Cas from cytoplasmic to membrane-associated cell al., 2000; Takahashi et al., 1998; Zhu et al., 1998). The fractions (Sakai et al., 1994a). Second, it promotes pathways leading from these dierent receptors to Cas interactions with SH2-containing proteins. One phos- phosphorylation have both common and unique phorylation-dependent interaction that is observed features; this complexity is undoubtedly an important following receptor activation involves Cas and Src. feature of the biological function of Cas. The nature of The possible functions of Src-Cas interactions have both the ligand and activated receptor is likely to been explored in great detail above. The second such determine the speci®c signaling pathways that lead to interaction, which has perhaps been most frequently PTK activation and Cas phosphorylation. This can implicated in Cas function, involves Cas and Crk. The explain why PI3K, for example, appears to be required SH2 domain of Crk has the potential to bind to for Cas phosphorylation in response to platelet-derived multiple pTyr residues present within the substrate- growth factor (PDGF) treatment but not in response binding YXXP domain of Cas. As described above, to activation of GPCRs (Casamassima and Rozengurt, Cas-Crk signaling is critical for cell migration and 1997). internalization of the bacterial pathogen Y. pseudo- To date, four non-receptor PTKs have been shown tuberculosis (Cho and Klemke, 2000; Klemke et al., to phosphorylate Cas in vitro: Src, Fak, Pyk2 and Abl 1998; Weidow et al., 2000). In these cases, Cas appears (Astier et al., 1997a; Mayer et al., 1995; Sakai et al., to function as a regulator of Crk, allowing it to interact 1994a; Tachibana et al., 1997). As mentioned above, with downstream eectors that ultimately promote Src is considered to be predominantly responsible for activation of the small GTP-binding protein Racl. In this regard, Cas may be instrumental in localizing Crk and its associated proteins to regions of the cell where activation of Racl may be particularly important, such Table 1 Inducers of Cas phosphorylation as membrane rues at the leading edge of the cell Receptor/Ligand (Klemke et al., 1998). One of the downstream eects of Cas-Crk-Racl signaling appears to be JNK activation rPTK ligands EGF (Dol® et al., 1998). Cas may also play a role in FGF regulating Crk binding to other signaling molecules. IGF-1 For example, Crk has been found to shuttle from Cas- NGF Crk complexes to Crk-Gab, Crk-Cbl or Crk-insulin PDGF GDNF receptor substrate 1 (IRS-1) complexes in response to certain signals (Khwaja et al., 1996; Lamorte et al., Adhesion receptors 2000; Sorokin and Reed, 1998). By controlling the b3 integrin nature and dynamics of Crk-dependent downstream b integrin 1 signaling pathways, Cas can eectively impact a wide a7 integrin ICAM-1, 2, 3 range of cellular responses. With the exception of Src and Crk, the function of GPCR agonists interactions between Cas and the large array of LPA possible binding partners remains largely undeter- Bombesin Vasopressin mined. However, recent data have shed some light on Endothelin possible functions of a third Cas complex, which SPC contains members of the Nsp family of proteins (Nsp1; Thrombin Nsp2/BCAR3/AND-34 (AND-34); and Nsp3/Chat/ Angiotensin II SHEP1 (Chat) (Cai et al., 1999; Dodelet et al., 1999; Bradykinin Sphingosine 1-phosphate Gotoh et al., 2000; Lu et al., 1999; Sakakibara and CCKA Hattori, 2000; van Agthoven et al., 1998). These proteins share a common structural organization, Other receptor ligands characterized by an amino-terminal SH2 domain, a Growth hormone Urokinase central proline-rich domain and a carboxy-terminal guanine nucleotide exchange factor (GEF) domain that

Oncogene Functions of the adapter protein Cas AH Bouton et al 6456 has been shown in at least one instance to have activity by overexpressed Cas and that inhibition required Cas- for Ra1A, Rap1A and R-Ras (Gotoh et al., 2000). AND-34 association. Both Cas (BCAR1) and AND-34 (BCAR3) were Although functions for the majority of Cas protein isolated from the same genetic screen that was designed complexes still remain to be determined, it is clear that to isolate genes that conferred upon cells the ability to early predictions about Cas serving as a scaolding grow in the presence of tamoxifen, strongly suggesting protein have stood the test of time. The ability to that they function in a common growth-regulatory undergo rapid changes in phosphorylation, subcellular pathway (Brinkman et al., 2000; van Agthoven et al., localization, and association with heterologous proteins 1998). All three Nsp family members have been shown provides Cas with the means to both spatially and to bind to Cas (Cai et al., 1999; Gotoh et al., 2000; Lu temporally regulate the function of its numerous et al., 1999; Sakakibara and Hattori, 2000). In most binding partners. It remains to be determined how cases, this interaction appears to involve sequences this molecule serves as a point of convergence for so within the carboxy-terminal GEF domain of the Nsp many distinct signaling inputs and how it ultimately proteins and carboxy-terminal sequences of Cas that contributes to the generation of speci®c cellular are distinct from the Src binding sites. Although Cas- responses. Nsp interactions appear to be largely constitutive, Nsp phosphorylation is induced by a variety of growth factors (Cai et al., 1999; Lu et al., 1999; Sakakibara Acknowledgments and Hattori, 2000). Following EGF treatment, both The authors would like to apologize in advance for any Cas and Chat relocalize to membrane rues, suggest- omissions or oversights in this review. We would like to ing that they may function coordinately at these sites thank past and present members of the lab and colleagues within the Department of Microbiology for their insight (Sakakibara and Hattori, 2000). Additional indications and helpful discussions. We would like to gratefully that Cas and Nsp functions may be linked comes from acknowledge support of our work by the National Science studies that focused on the GEF activity of AND-34 Foundation (MCB-9723820 and MCB-0078022) and the (Gotoh et al., 2000). These investigators demonstrated Thomas F Jeress and Kate Miller Jeress Memorial Trust that the RalA GEF activity of AND-34 was inhibited (J-421 and J556).

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Oncogene