p130Cas is the founding member of the “Cas family” of proteins that, in mammals, also includes HEF-1/Cas-L/NEDD9, Efs/Sin, and HEPL (Tikhmyanova et al. 2010). One Cas family member is present in Drosophila, but there are none in yeast or C. elegans. All Cas family proteins have the following domain organization: an N-terminal SH3 domain, a central substrate domain (SD), and a C-terminal Cas-family homology (CCH) domain (Fig. 1). However, they appear to be functionally nonredundant due to differences in their tissue distribution and small nuances in primary structure. Of the vertebrate Cas family members, p130Cas is most widely expressed in embryonic and adult tissues. In adult tissues, highest p130Cas expression levels are in the brain, lung, intestine, kidney, and testis (Sakai et al. 1994).
Early studies implicated p130Cas as a signaling component of the focal adhesion protein complex that assembles at sites of integrin-mediated cell adhesion to the extracellular matrix (ECM). Focal adhesions are major cellular sites of tyrosine kinase signaling, in addition to their functions in establishing the transmembrane linkage between the ECM and actin cytoskeleton. Like its binding partner FAK, p130Cas localizes prominently to focal adhesions where it undergoes tyrosine phosphorylation in response to cellular adhesion and contractile force generation. Both the SH3 and CCH domains have important functions in targeting p130Cas to focal adhesions (Donato et al. 2010). FAK aids in the recruitment of p130Cas to focal adhesions via interactions made by the p130Cas SH3 domain (Donato et al. 2010). The CCH region of p130Cas is known to bind NSP family proteins (NSP1, NSP2/AND-34/BCAR3, NSP3/Chat/Shep-1) as well as a zyxin family member Ajuba, but none of these proteins are implicated in p130Cas focal adhesion targeting. They may, however, play a role in targeting p130Cas to plasma membrane ruffles and protrusions.
Regulation of p130Cas Tyrosine Phosphorylation
Major sites of p130Cas tyrosine phosphorylation reside in the SD, which is defined by 15 scattered Tyr-x-x-Pro (YxxP) motifs. From phospho-proteomics studies, all but one of the YxxP tyrosines have been detected as in vivo phosphorylation sites. Most notable are nine major sites within Tyr-Asp-x-Pro (YDxP) motifs, where tyrosine phosphorylation generates optimal binding sites for SH2 domains of Crk and Nck adaptor proteins (Shin et al. 2004). Crk and Nck proteins are the best-characterized downstream effectors in p130Cas-mediated signaling pathways.
Cellular stretching enhances tyrosine phosphorylation of the p130Cas SD in focal adhesions, and mechanical extension of the SD increases the accessibility of SD tyrosines for phosphorylation by Src (Sawada et al. 2006). These observations implicate p130Cas SD tyrosine phosphorylation in the process of mechanotransduction whereby cells sense a physical force and initiate a biochemical response. While the tertiary structure of the SD is undetermined, intramolecular interactions within the SD may maintain a conformational state wherein the YDxP tyrosine hydroxyl groups are hidden from Src until exposed by traction forces. The unique structural properties of the SD also appear to account for the retarded SDS-PAGE mobility of p130Cas, since deletion of this domain results in a protein that migrates according to the predicted molecular mass.
In addition to integrin-mediated cell adhesion, many studies have shown that p130Cas tyrosine phosphorylation occurs in response to ligand stimulation of various receptor tyrosine kinases and G-coupled protein receptors (see Tikhmyanova et al. 2010 for more details). In many of these cases, p130Cas phosphorylation may be a secondary response brought about by increased cellular contractility.
Tyrosine phosphorylation of the p130Cas SD is rapid and greatly enhanced upon inhibition of cellular tyrosine phosphatases, indicating a signaling transience subject to negative regulation by phosphatases. p130Cas has been characterized as a substrate of several tyrosine phosphatases including PTP1B, PTP-PEST, RPTP-α, and SHP-2. PTP1B and PTP-PEST bind the p130Cas SH3 domain.
Critical Roles for p130Cas in Development
As a major signaling component in focal adhesions, it is not surprising that p130Cas is critical for many aspects of development. Heart and brain development are two well-documented examples. Cas−/− mice die at embryonic day 12.5 due to severe cardiac abnormalities (Honda et al. 1998). In the myocardium of cas−/− embryos, myofibrils and Z-disks show extensive disorganization. In cultured cardiac myocytes, p130Cas is prominently localized in the Z-disks where, via its interaction with FAK, it functions to regulate sarcomeric organization as well as the program of gene expression associated with hypertrophy (Kovacic-Milivojević et al. 2001).
Cas−/− mouse embryos exhibit an overall growth retardation, and have decreased brain size. p130Cas is expressed to particularly high levels in the developing cerebellum, and p130Cas tyrosine phosphorylation is enriched in the growth cones of extending neurites (Huang et al. 2006). Impairment of p130Cas expression or SD tyrosine phosphorylation in cultured cerebellar granule cells results in defects in growth cone elongation (Huang et al. 2006). The Drosophila p130Cas homolog was similarly shown to function in integrin-dependent neurite outgrowth and axon guidance during development (Huang et al. 2007), indicating conserved neuronal functions of Cas family proteins.
The above studies indicate a general role for p130Cas in mediating actin cytoskeletal rearrangement in various settings. Cas−/− mouse embryo fibroblasts (MEFs) also have defects in actin organization and cell motility (Honda et al. 1998), and these cells have been instrumental to understanding p130Cas-activated signaling events impacting the actin cytoskeleton.
p130Cas Signaling Functions Leading to Enhanced Cell Motility
The requirements for p130Cas functional domains in promoting cell motility have been evaluated through expression of wild-type versus mutational variants in cas−/− MEFs (Shin et al. 2004; Donato et al. 2010; Meenderink et al. 2010). These studies demonstrated that p130Cas functions to enhance cell motility, while also showing requirements for all major p130Cas functional domains (SH3, SD, SBD, and CCH). Mutation or deletion of any of the domains has a negative impact on tyrosine phosphorylation of the SD, which is a key signaling function of p130Cas in cell motility. The small adaptor proteins Crk and Nck, which bind to tyrosine phosphorylated motifs in the SD, have been implicated in p130Cas-motility responses. These adaptors consist essentially of SH2 domains (with high binding affinity for phosphorylated YDxP motifs in the SD) and SH3 domains for mediating interactions with downstream effector proteins. Most notably, p130Cas coupling to Crk adaptors (Crk-II and Crk-L) has been implicated in promoting plasma membrane protrusion by activating Rac1 GTPase via a Crk SH3 domain interaction with the guanine nucleotide exchange factor complex DOCK180/ELMO (reviewed in Chodniewicz and Klemke 2004). Activated Rac1 can promote membrane protrusion by activating the Arp2/3 complex to stimulate branched actin polymerization (Fig. 2). The p130Cas-Crk-Rac pathway has been implicated in other actin-mediated cellular processes, including the phagocytosis of the pathogenic bacteria Yersinia (reviewed in Tikhmyanova et al. 2010). Nck adaptors (Nck1 and Nck2) can also promote Arp2/3 complex activation via the interaction of their SH3 domains with other proteins including N-WASP and Pak1. The interaction of p130Cas with Nck adaptors has been linked to cytoskeletal reorganization and chemotaxis stimulated by platelet-derived growth factor (PDGF) (Rivera et al. 2006). It is not clear, however, if p130Cas/Nck coupling in response to PDGF stimulation occurs solely in dorsal membrane ruffles or if this coupling also has a focal adhesion signaling component.
In addition to its role in promoting plasma membrane protrusion via coupling to Crk and/or Nck, p130Cas has another role in cell motility to sustain the disassembly of mature focal adhesions (Meenderink et al. 2010). SD tyrosine phosphorylation was implicated in the focal adhesion disassembly process, while the SBD was also found to have a role in this process distinct from its ability to promote SD phosphorylation (Meenderink et al. 2010). These observations emphasize two distinct signaling functions of p130Cas: (1) SD tyrosine phosphorylation to recruit SH2-containing effectors and (2) SBD-mediated activation of Src (and possibly other signaling proteins that bind to this site) to act on substrates other than p130Cas.
p130Cas and Cancer
While p130Cas is essential to normal developmental processes, elevated p130Cas expression and/or SD tyrosine phosphorylation is seen in various forms of cancer and is likely to contribute to the malignant progression of these diseases (reviewed in Defillipi et al. 2006; Tikhmyanova et al. 2010). A role for p130Cas in invasive and metastasic cell behavior was demonstrated in studies of Src-transformed fibroblasts, where the formation of invasive structures called podosomes, activation of matrix-degrading metalloproteases, and experimental metastasis were all shown to be greatly reduced in the absence of p130Cas SD tyrosine phosphorylation (Brabek et al. 2005). The p130Cas-Crk-Rac1 pathway is implicated in both plasma membrane protrusion and cell survival during invasion (reviewed in Chodniewicz and Klemke 2004).
Much attention has been directed to the role of p130Cas in breast cancer. In breast cancer patients, high p130Cas levels are associated with a poor response to tamoxifen therapy, early disease recurrence, and lower long-term survival (for details, see Dorssers et al. 2001; Tikhmyanova et al. 2010). In mouse transgenic studies, p130Cas overexpression in mammary gland epithelia was associated with hyperplasia and, in combination with overexpression of the HER2/Neu oncogene, with shorter tumor latency (Defillipi et al. 2006). Interestingly, p130Cas was independently identified as the product of the human gene BCAR1 that confers resistance to antiestrogen drugs (e.g., tamoxifen) in breast cancer cells (Dorssers et al. 2001). Tamoxifen is a common and effective treatment for estrogen-receptor positive (ER+) breast cancer, but patients frequently develop resistance to this treatment. The discovery that p130Cas confers tamoxifen resistance provided new mechanistic insight into antiestrogen resistance. Tamoxifen resistance conferred by p130Cas does not appear to be regulated by activation of ER target genes, but rather has been linked to Src-driven cell proliferation and survival pathways. These pathways appear to be mediated either by p130Cas-Src complexes formed with the ER where MAPK and cell cycle signaling has been implicated (reviewed in Defillipi et al. 2006; Tikhmyanova et al. 2010), or through an ER-independent pathway involving the epidermal growth factor receptor and Stat5b (Riggins et al. 2006). Other studies revealed a role for adhesion-dependent p130Cas signaling in promoting activation of the PI3K-Akt cell survival pathway in response to ER antagonism by antiestrogens (Cowell et al. 2006). Thus, p130Cas may confer antiestrogen resistance through multiple mechanisms.
p130Cas has also been implicated in the more aggressive ER-negative breast cancers. p130Cas SD tyrosine phosphorylation is commonly elevated in ER-negative breast cancer cell lines and linked to enhanced migration, invasion, and survival of ER-negative breast cancer cells (Cunningham-Edmondson and Hanks 2009).
p130Cas, first recognized as a prominent tyrosine-phosphorylated protein in cells transformed by retroviral oncogenes v-crk and v-src, is a widely expressed docking/scaffolding protein that functions as a major Src substrate in integrin-mediated signaling and mechanotransduction. p130Cas was independently identified as a FAK-interacting protein and as the product of the BCAR1 gene that confers antiestrogen resistance in breast cancer cells. Src is recruited to phosphorylate p130Cas by directly binding to the SBD and indirectly by binding to FAK. Src phosphorylates multiple YDxP motifs in the p130Cas SD to create high-affinity binding sites for Crk and Nck adaptor proteins that act as downstream effectors in p130Cas-mediated signaling pathways. p130Cas SD couplings to Crk and Nck are implicated in promoting leading edge actin polymerization and plasma membrane protrusion in cell motility and invasion processes. SBD-mediated binding and activation of Src is a distinct signaling function of p130Cas that may be involved in maintaining focal adhesion disassembly during cell migration. A general role for p130Cas in mediating actin and adhesion dynamics is further indicated by developmental studies showing that p130Cas deficiency causes functional defects in sarcomere organization of cardiac myocytes and neuronal outgrowth and axon guidance in the central nervous system. p130Cas expression and/or SD tyrosine phosphorylation is commonly elevated in various forms of cancer where it likely contributes to the malignant disease progression. In conferring antiestrogen resistance in breast cancer cells; p130Cas SD signaling has been linked to the activation of Src-driven cell proliferation and survival pathways.
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