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PMID: 11604500 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src.

Molecular and cellular biology ·Vol. 21 ·No. 22 ·2001-11-00 ·Pages 7641-52

Ruest PJ, Shin NY, Polte TR, Zhang X, Hanks SK

Abstract

Tyrosine phosphorylation of CAS (Crk-associated substrate, p130(Cas)) has been implicated as a key signaling step in integrin control of normal cellular behaviors, including motility, proliferation, and survival. Aberrant CAS tyrosine phosphorylation may contribute to cell transformation by certain oncoproteins, including v-Crk and v-Src, and to tumor growth and metastasis. The CAS substrate domain (SD) contains 15 Tyr-X-X-Pro motifs, which are thought to represent the major tyrosine phosphorylation sites and to function by recruiting downstream signaling effectors, including c-Crk and Nck. CAS makes multiple interactions, direct and indirect, with the tyrosine kinases Src and focal adhesion kinase (FAK), and as a result of this complexity, several plausible models have been proposed for the mechanism of CAS-SD phosphorylation. The objective of this study was to provide experimental tests of these models in order to determine the most likely mechanism(s) of CAS-SD tyrosine phosphorylation by FAK and Src. In vitro kinase assays indicated that FAK has a very poor capacity to phosphorylate CAS-SD, relative to Src. However, FAK expression along with Src was found to be important for achieving high levels of CAS tyrosine phosphorylation in COS-7 cells, as well as recovery of CAS-associated Src activity toward the SD. Structure-functional studies for both FAK and CAS further indicated that FAK plays a major role in regulating CAS-SD phosphorylation by acting as a docking or scaffolding protein to recruit Src to phosphorylate CAS, while a secondary FAK-independent mechanism involves Src directly bound to the CAS Src-binding domain (SBD). Our results do not support models in which FAK either phosphorylates CAS-SD directly or phosphorylates CAS-SBD to promote Src binding to this site.

MeSH Terms
Amino Acid Motifs Animals Binding Sites COS Cells Chlorocebus aethiops Crk-Associated Substrate Protein Focal Adhesion Protein-Tyrosine Kinases Phosphoproteins/metabolism Phosphorylation Protein-Tyrosine Kinases/metabolism Proteins Retinoblastoma-Like Protein p130 Tyrosine/metabolism src Homology Domains src-Family Kinases/metabolism
Chemicals
Crk-Associated Substrate Protein Phosphoproteins Proteins Retinoblastoma-Like Protein p130 Tyrosine Protein-Tyrosine Kinases Focal Adhesion Protein-Tyrosine Kinases src-Family Kinases
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Ruest P J
Department of Cell Biology, Vanderbilt University School of Medicine, Nahville, Tennessee 37232, USA.
Shin N Y
Polte T R
Zhang X
Hanks S K
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.21.22.7641-7652.2001
Published
2001-11-00
Pages
7641-52
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC99935
Subset
IM
Grants
NIGMS NIH HHS · R01 GM049882 · United States
NCI NIH HHS · T32 CA078136 · United States
NIGMS NIH HHS · R01-GM49882 · United States
NCI NIH HHS · T32-CA78136 · United States
External Links
PubMed source DOI: 10.1128/MCB.21.22.7641-7652.2001 PMC Full Text View journal details
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