Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src.
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.
Ruest
P J
PJ
Department of Cell Biology, Vanderbilt University School of Medicine, Nahville, Tennessee 37232, USA.
Shin
N Y
NY
Polte
T R
TR
Zhang
X
X
Hanks
S K
SK
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D050719:Crk-Associated Substrate Protein; D051416:Focal Adhesion Protein-Tyrosine Kinases; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
D050719:Crk-Associated Substrate Protein; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases