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Molecular and Cellular Biology, November 2001, p. 7641-7652, Vol. 21, No. 22
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.22.7641-7652.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Mechanisms of CAS Substrate Domain Tyrosine Phosphorylation by FAK and Src

Paul J. Ruest, Nah-Young Shin, Thomas R. Polte,dagger Xiaoe Zhang,Dagger and Steven K. Hanks*

Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received 9 May 2001/Returned for modification 25 June 2001/Accepted 23 August 2001

Tyrosine phosphorylation of CAS (Crk-associated substrate, p130Cas) 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.

* Corresponding author. Mailing address: Department of Cell Biology, Vanderbilt, University School of Medicine, Nashville, TN 37332. Phone: (615) 343-8502. Fax: (615) 343-4539. E-mail: hankss{at}ctrvax.vanderbilt.edu.

dagger Present address: Department of Surgical Research, Children's Hospital, Harvard Medical School, Boston, MA 02115.

Dagger Present address: Aventis Pharmaceuticals Inc., Bridgewater, NJ 08807.

Molecular and Cellular Biology, November 2001, p. 7641-7652, Vol. 21, No. 22
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.22.7641-7652.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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