Dynamics and Mechanism of p130Cas Localization to Focal Adhesions

Abstract

The docking protein p130Cas is a major Src substrate involved in integrin signaling and mechanotransduction. Tyrosine phosphorylation of p130Cas in focal adhesions (FAs) has been linked to enhanced cell migration, invasion, proliferation, and survival. However, the mechanism of p130Cas targeting to FAs is uncertain, and dynamic aspects of its localization have not been explored. Using live cell microscopy, we show that fluorophore-tagged p130Cas is a component of FAs throughout the FA assembly and disassembly stages, although it resides transiently in FAs with a high mobile fraction. Deletion of either the N-terminal Src homology 3 (SH3) domain or the Cas-family C-terminal homology (CCH) domain significantly impaired p130Cas FA localization, and deletion of both domains resulted in full exclusion. Focal adhesion kinase was implicated in the FA targeting function of the p130Cas SH3 domain. Consistent with their roles in FA targeting, both the SH3 and CCH domains were found necessary for p130Cas to fully undergo tyrosine phosphorylation and promote cell migration. By revealing the capacity of p130Cas to function in FAs throughout their lifetime, clarifying FA targeting mechanism, and demonstrating the functional importance of the highly conserved CCH domain, our results advance the understanding of an important aspect of integrin signaling.p130Cas (Crk-associated substrate) is a Src substrate that functions in integrin signaling to promote cell motility, invasion, proliferation, and survival (1, 2). p130Cas was first recognized as a prominent tyrosine-phosphorylated protein in cells transformed by v-crk (3) and v-src (4). The observation that p130Cas interacts directly with the Src homology 2 (SH2) 2 domain of the v-Crk protein (5, 6) contributed to the recognition of SH2 domains as phosphotyrosine-binding modules in signal transduction. The primary structure of p130Cas (7) indicated a function as a docking/scaffolding protein, lacking domains indicative of intrinsic enzymatic activity but having various domains and motifs for mediating interactions with other proteins.p130Cas was independently identified in a screen for proteins that interact with focal adhesion kinase (FAK) (8), a tyrosine kinase named for its prominent localization to sites of integrin-mediated cell adhesion. A Src homology 3 (SH3) domain at the N terminus of p130Cas mediates the FAK interaction. Like FAK, p130Cas localizes to focal adhesions (FAs) and undergoes tyrosine phosphorylation in response to adhesion (9 -11). Thus, p130Cas is a signaling component of the FA protein complex ("adhesome") that assembles to bring about cellular responses to integrin engagement. A primary role for p130Cas in integrin signaling is consistent with the phenotype of p130Cas-deficient mice, which die during embryonic development due to defects associated with a disorganized actin cytoskeleton (12). Despite the direct interaction with FAK, tyrosine phosphorylation of p130Cas is attributed to Src-family kinases (13-15). However, FAK can act as a scaffold to recru...

Dynamic Phosphorylation of Tyrosine 665 in Pseudopodium-enriched Atypical Kinase 1 (PEAK1) Is Essential for the Regulation of Cell Migration and Focal Adhesion Turnover

“…For example, Src-mediated phosphorylation triggers paxillin-promoted disassembly (32,36,37), which allows release of the ECM as new FAs form at the leading edge (11). We found that PEAK1 enters FAs after both paxillin (supplemental Table S1) and ␣-actinin (data not shown) and persists in adhesions after paxillin (supplemental Movie 4) is gone, suggesting that PEAK1 functions more in the later stages of FA development and turnover than in FA initiation (11,38). These findings are consistent with PEAK1 increasing FA length (17) and promoting longer FA lifetimes (Figs.…”

Protein Tyrosine Phosphatase α Phosphotyrosyl-789 Binds BCAR3 To Position Cas for Activation at Integrin-Mediated Focal Adhesions

“…9) where integrin activation induces Src-mediated phosphorylation of PTP␣ at Tyr789, resulting in formation and localization of a PTP␣-BCAR3-Cas complex at nascent adhesion sites. Since BCAR3 interacts with the C-terminal region of Cas, specifically with the helix-containing Cas family C-terminal homology do- main (CCH) region of Cas required for its optimal focal adhesion localization (13,17), we propose that this mechanism underlies the C-terminal-dependent focal adhesion localization of Cas. Consistent with this, BCAR3 has been suggested to regulate migration by promoting Cas localization to the membrane or leading edge (37).…”

“…However, virtually nothing is known of BCAR3-SH2 specificity, and besides PTP␣, no other target phosphotyrosyl proteins have been found. Two domains of Cas mediate its targeting to adhesion complexes, and both domains are required for optimal focal adhesion recruitment and tyrosine phosphorylation of Cas (13,18,28). The N-terminal SH3 domain of Cas associates with FAK to recruit Cas to focal adhesions.…”

Downregulation of microRNA-362-3p and microRNA-329 promotes tumor progression in human breast cancer

“…3,4 Tight regulation of p130Cas function via proteolytic cleavage or reversible phosphorylation of tyrosine residues is necessary for the maintenance of cell motility, survival, and apoptosis in various cell types. [5][6][7][8] Although high expression of p130Cas in primary breast tumors is linked to activation of cell proliferation and cancer progression, the detailed mechanisms governing p130Cas expression are not fully understood.…”