BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling

Abstract

The nonreceptor protein-tyrosine kinase c-Src is frequently overexpressed and/or activated in a variety of cancers, including those of the breast. Several heterologous binding partners of c-Src have been shown to regulate its catalytic activity by relieving intramolecular autoinhibitory interactions. One such protein, p130Cas (Cas), is expressed at high levels in both breast cancer cell lines and breast tumors, providing a potential mechanism for c-Src activation in breast cancers. The Cas-binding protein BCAR3 (breast cancer antiestrogen resistance-3) is expressed at high levels in invasive breast cancer cell lines, and this molecule has previously been shown to coordinate with Cas to increase c-Src activity in COS-1 cells. In this study, we show for the first time using gain-and loss-of-function approaches that BCAR3 regulates c-Src activity in the endogenous setting of breast cancer cells. We further show that BCAR3 regulates the interaction between Cas and c-Src, both qualitatively as well as quantitatively. Finally, we present evidence that the coordinated activity of these proteins contributes to breast cancer cell adhesion signaling and spreading. Based on these data, we propose that the c-Src/Cas/BCAR3 signaling axis is a prominent regulator of c-Src activity, which in turn controls cell behaviors that lead to aggressive and invasive breast tumor phenotypes.

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

“…In ad-dition, the interaction of BCAR3 with Cas/Src is eliminated by a mutation in the BCAR3 GEF domain, while this mutant BCAR3 can still interact with phospho-Tyr789 PTP␣. All together, this evidence confirms BCAR3 as an activator of Src (38) and reveals BCAR3 as a molecular linker of PTP␣ and Cas.…”

“…A remarkably similar phenotype to Y789F PTP␣-expressing cells is reported for cells depleted in a protein called BCAR3 (also known as AND-34 in mice) (5), including impaired Src-Cas association, tyrosine phosphorylation of Cas, relocalization of Cas to the cell membrane, and cell migration (37,38). Thus, BCAR3 and phospho-Tyr789 PTP␣ may act within the same signaling pathway.…”

“…To confirm the role of BCAR3 in regulating Cas tyrosine phosphorylation and localization, we depleted ϳ90% of BCAR3 from MEFs using siRNA. As reported (38), the depletion of BCAR3 inhibited the integrin-induced tyrosine phosphorylation of Cas. It also inhibited PAK activation (Fig.…”

“…Importantly, BCAR3 can regulate cell spreading and movement. Its overexpression promotes breast cancer cell or fibroblast spreading on fibronectin, migration, and invasion, while depletion of BCAR3 inhibits these cell properties (32,37,38). These actions of BCAR3 are dependent upon its ability to bind to Cas, since BCAR3-enhanced migration is abolished or reduced when the BCAR3 GEF domain responsible for Cas binding is deleted or mutated (32,43).…”

“…5B), in accord with the latter being a downstream target of BCAR3-Cas signaling (6). BCAR3 is reported to regulate Src activity (32,38), and integrin-induced Src phosphorylation at its activating Tyr416 site was indeed abrogated in BCAR3-deficient ; ***, P ϭ 5 ϫ 10 Ϫ8 ), and n.s. indicates no significant difference (P ϭ 0.210).…”