P130Cas Src-Binding and Substrate Domains Have Distinct Roles in Sustaining Focal Adhesion Disassembly and Promoting Cell Migration

Abstract

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas −/− mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.

Targeting Cas Family Proteins as a Novel Treatment for Breast Cancer

“…Utilizing p130 Cas SH3 domain deletion mutants, studies indicated that this domain and its interaction with FAK are necessary for phosphorylation and the localization of p130 Cas to focal adhesions (FAs) . More recent studies, further explored the temporal and spatial involvement of p130 Cas in FA dynamics and showed the influence of p130 Cas in FA turnover and controlling the migratory response (Donato et al 2010;Meenderink et al 2010). The p130 Cas SH3 domain was shown to be necessary for tyrosine phosphorylation of the SD and the promotion of cell migration (Donato et al 2010), in conditional FAK-deficient mammary tumor cells a reduction in the phosphorylation of Y249 within the p130 Cas SD was observed (Provenzano et al 2008).…”

“…More recent studies, further explored the temporal and spatial involvement of p130 Cas in FA dynamics and showed the influence of p130 Cas in FA turnover and controlling the migratory response (Donato et al 2010;Meenderink et al 2010). The p130 Cas SH3 domain was shown to be necessary for tyrosine phosphorylation of the SD and the promotion of cell migration (Donato et al 2010), in conditional FAK-deficient mammary tumor cells a reduction in the phosphorylation of Y249 within the p130 Cas SD was observed (Provenzano et al 2008). These studies uncovered the significance of the Cas SH3 domain in the spatial regulation of Cas protein function and in particular its role for the phosphorylation of the SD of the Cas proteins (as described in the next section).…”

The focal adhesion targeting domain of p130Cas confers a mechanosensing function

“…The Cas-family proteins NEDD9 (also known as HEF1 and Cas-L) and p130Cas (also known as BCAR1) are examples of signalling proteins that are recruited to focal adhesions, where they regulate cell migration signals (Cary et al, 1998;Klemke et al, 1998;Fashena et al, 2002;Natarajan et al, 2006;Meenderink et al, 2010;Kong et al, 2011;Baquiran et al, 2013;Bradbury et al, 2014). Both proteins contain a C-terminal focal adhesion targeting (FAT) domain (Arold et al, 2002;Mace et al, 2011), characterised by extensive primary sequence and tertiary structural similarity.…”

Displacement of p130Cas from focal adhesions links actomyosin contraction to cell migration

“…Focal adhesions also provide platforms where extracellular cues are converted into intracellular signals (Schwartz, 2010;Zaidel-Bar et al, 2007). Integrinextracellular-matrix (ECM) binding initiates the tyrosine phosphorylation of focal adhesion proteins such as paxillin, FAK (also known as PTK2) and p130Cas (Crk-associated substrate, hereafter referred to as Cas; also known as BCAR1), which then promotes actin remodeling, actomyosin contraction and focal adhesion turnover (Kawauchi et al, 2012;Meenderink et al, 2010;Parsons, 2003).…”

“…Individual focal adhesions in fibroblasts have a typical lifespan of ,30 min (Delorme-Walker et al, 2011;Kuo et al, 2011;Meenderink et al, 2010). Both the assembly and disassembly of focal adhesions are reportedly modulated by tyrosine phosphorylation of several focal adhesion proteins including FAK, paxillin and Cas (Geiger et al, 2001).…”