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Title page for ETD etd-06302010-180728

Type of Document Dissertation
Author Donato, Dominique Maria
URN etd-06302010-180728
Title The focal adhesion localization of p130Cas: dynamics, targeting mechanism, and signaling.
Degree PhD
Department Cell and Developmental Biology
Advisory Committee
Advisor Name Title
Stephen Hann, Ph.D. Committee Chair
Bruce Carter, Ph.D. Committee Member
Irina Kaverina, Ph.D. Committee Member
Roy Zent, MD, Ph.D. Committee Member
Steven Hanks, Ph.D. Committee Member
  • cell motility
  • FAK
  • tyrosine phosphorylation
  • paxillin
  • focal adhesion
  • p130Cas
Date of Defense 2010-05-17
Availability unrestricted
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.

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