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

Amy H. Bouton,Michael S. Guerrero,Randy S. Schrecengost,Natasha R. Schuh

doi:10.1074/jbc.m109.046631

Amy H. Bouton, Michael S. Guerrero + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.046631

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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, p130(Cas) (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.

Highlights

BCAR3 is a member of the novel Src homology 2 (SH2)-containing protein family [3] and was first identified in a screen for genes whose overexpression conferred resistance to antiestrogens [4]
c-Src-binding protein p130Cas (Cas)/c-Src Interactions Are Required for BCAR3-dependent Enhancement of Cas-mediated c-Src Kinase Activity—To assess the molecular requirements for c-Src activation by Cas and BCAR3, COS-1 cells were transfected with plasmids encoding c-Src, Myc-tagged Cas, and/or FLAG-tagged BCAR3
Similar results were obtained using paxillin as an exogenous substrate (Fig. 1B, top panel, lanes 2 and 5), demonstrating that the enhancement in c-Src substrate phosphorylation observed in the presence of BCAR3 and Cas was not restricted to cortactin

Summary

Introduction

BCAR3 ( known as AND-34 and NSP2) is a member of the novel Src homology 2 (SH2)-containing protein family [3] and was first identified in a screen for genes whose overexpression conferred resistance to antiestrogens [4]. We show for the first time that BCAR3 regulates c-Src activity and adhesion-dependent Cas phosphorylation in breast cancer cells. Cells overexpressing Cas exhibited increased activation of c-Src, as measured by the increased phosphorylation of cortactin, compared with cells expressing only vector (Fig. 1A, top panel, lanes 1 and 2).

Results

Conclusion