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.
Highlights
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]
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
Expression of p130Cas Variants Tagged with Fluorophore Venus-YFP—To study p130Cas localization to FAs, mouse p130Cas variants tagged at their C-terminal ends with the fluorophore Venus-YFP were stably expressed in CasϪ/Ϫ mouse embryo fibroblasts (MEFs)
Summary
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]. 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.
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