Abstract
Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS–vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS–vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS–vinculin interaction in mechanosensing and focal adhesion dynamics.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-013-1450-x) contains supplementary material, which is available to authorized users.
Highlights
Crk-associated substrate (CAS) is an important substrate of Src
We have previously shown that phosphorylation on tyrosine 12 within the SRC homology 3 (SH3) domain of CAS suppresses FAK binding and deactivates CAS-mediated signaling [27, 28]
Vinculin binding was only present in CAS–SH3 domain constructs with WT tyrosine at position 12 or with a non-phosphorylatable mutation Y12F (Tyr 12 changed to phenylalanine)
Summary
Crk-associated substrate (CAS) is an important substrate of Src. CAS is dominantly localized in focal adhesions of adherent cells and plays a central role in the integrinmediated control of cell behavior [1]. Src family kinases seem to phosphorylate numerous, if not all, of the CAS SD YxxP tyrosines either by binding directly to the CAS SBD, or by indirect association with CAS via a FAK bridge [6, 7]. CAS SD tyrosine phosphorylation, in untransformed cells, takes place at the integrin-mediated adhesion sites [8], and has been shown to be associated with integrin signaling pathways that regulate cell movement and survival [9,10,11,12]
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