Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)

Anthony Makkinje,Pierre Vanden Borre,Richard I Near,Prayag S Patel,Adam Lerner

doi:10.1074/jbc.m112.389981

Anthony Makkinje, Pierre Vanden Borre + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m112.389981

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Abstract

The focal adhesion adapter protein p130(cas) regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130(cas). AND-34/BCAR3, one of three NSP family members, binds the p130(cas) carboxyl terminus, adjacent to a bipartite p130(cas) Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130(cas). Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130(cas) complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130(cas) to bind the Src SH3 domain through an RPLPSPP motif in the p130(cas) SBD. Although our prior work identified phosphorylation of the serine within the p130(cas) RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130(cas). The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130(cas) complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130(cas) substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130(cas). Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130(cas) and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130(cas) complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130(cas) SBD.

Highlights

BCAR3 binds to p130cas, a known substrate of Src
Is BCAR3-p130cas Complex-dependent—In a study examining the association of the murine homolog of human BCAR3, AND-34, with the p130cas family member HEF1, we found that mutation of HEF1 L751D or AND-34 R743A blocked association of these two molecules (Fig. 3C, left) [29]
We have demonstrated that expression of BCAR3 augments Src binding to the focal adhesion adapter protein p130cas, at least in part through augmentation of Src SH3 domain binding to a previously identified carboxyl-terminal p130cas RPLPSPP motif

Summary

Background

BCAR3 binds to p130cas, a known substrate of Src. Results: BCAR3 augments binding of the Src SH3 domain to p130cas as well as p130cas tyrosine phosphorylation in a BCAR3p130cas complex-dependent manner. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD. BCAR3 Regulates Src SH3 Domain Binding to p130cas by another group in COS-7 cells reported that mutation of the RPLPSPP motif to RAAASPP failed to alter the ability of transfected FAK and n-Src to induce p130cas tyrosine phosphorylation. In these studies, p130cas substrate domain phosphorylation was instead shown to require Src binding to phosphorylated FAK Tyr-397 [10]. We report that BCAR3 expression augments both Src SH3 domain binding to p130cas and tyrosine phosphorylation of the p130cas substrate domain in a BCAR3-p130cas complex-dependent manner

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DISCUSSION