Oncogene (2001) 20, 6448 ± 6458 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc 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.