The focal adhesion localization of p130Cas: dynamics, targeting mechanism, and signaling.

Abstract

Focal adhesions (FAs) are sites at the interface between the cell and the ECM, linking integrin receptors and the actin cytoskeleton. In addition to serving as a structural platform, these sites are also robust sites of tyrosine phosphorylation and integrin signaling. When cells become adherent to the ECM, p130Cas (Crk-associated substrate) becomes tyrosine phosphorylated. Since p130Cas is primarily phosphorylated at tyrosines when it is localized to FAs, the localization of p130Cas to these sites appears critical to its ability to promote cell motility. The observation that with the exception of the SH3 domain, the C-terminus of p130Cas is the most highly conserved area of the protein, suggests an important role for this domain. Further observations that this domain has some sequence similarity to the FAK FAT domain is suggestive of this domain having an FA targeting function. The research in this dissertation aims to answer the following questions: 1) What contributions do the conserved N- and C-terminal domains make in the targeting of p130Cas to FAs and 2) What are the dynamics of p130Cas localization to FAs? In order to do so, fluorescently-tagged mutants of p130Cas were used to map the domain requirements for its FA localization. The localization of p130Cas to these sites was dependent on both the SH3 and CCH domains. The interaction of the SH3 domain with FAK was implicated as the major interaction mediating the localization of p130Cas through this domain. The SH3 and CCH domains were furthermore shown to be required for efficient p130Cas tyrosine phosphorylation to occur and the loss of tyrosine phosphorylation in deletion mutants was correlated with their inability to promote efficient cellular migration during wound-healing. Studies of the fluorescently-tagged p130Cas in live cells revealed that p130Cas localizes to FAs throughout their lifetime and exists in FAs with a high mobile fraction. Additionally, preliminary data suggested alternate sites of subcellular localization for p130Cas including filopodia and cell-cell contacts.