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Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src
P J Ruest1, N Y Shin, T R Polte
1Department of Cell Biology, Vanderbilt University School of Medicine, Nahville, Tennessee 37232, USA.
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.
Insights
Focal adhesion kinase (FAK) acts as a scaffold to recruit Src kinase for CAS (Crk-associated substrate) phosphorylation, rather than directly phosphorylating it. This clarifies CAS signaling in cell behavior and cancer progression.
Area of Science:
- Cell Biology
- Molecular Signaling
- Cancer Research
Background:
- Tyrosine phosphorylation of Crk-associated substrate (CAS) is crucial for integrin-mediated cell functions like motility and survival.
- Dysregulated CAS phosphorylation by oncoproteins (e.g., v-Crk, v-Src) is linked to cancer development and metastasis.
- The CAS substrate domain (SD) has multiple Tyr-X-X-Pro motifs, serving as docking sites for signaling proteins like c-Crk and Nck.
Purpose of the Study:
- To experimentally test proposed models for the mechanism of CAS-SD phosphorylation by tyrosine kinases Focal Adhesion Kinase (FAK) and Src.
- To elucidate the precise roles of FAK and Src in regulating CAS tyrosine phosphorylation.
Main Methods:
- In vitro kinase assays to assess the direct phosphorylation capacity of FAK and Src on CAS-SD.
- Expression studies in COS-7 cells to evaluate the combined effect of FAK and Src on CAS phosphorylation.
- Structure-functional studies of FAK and CAS to understand their interaction and regulatory mechanisms.
Main Results:
- FAK demonstrated minimal direct capacity to phosphorylate CAS-SD compared to Src.
- Co-expression of FAK and Src significantly increased CAS tyrosine phosphorylation levels in cells.
- FAK functions primarily as a scaffold, recruiting Src to phosphorylate CAS-SD, with a secondary FAK-independent mechanism involving direct Src binding to the CAS Src-binding domain (SBD).
Conclusions:
- FAK plays a critical scaffolding role in promoting Src-mediated CAS-SD phosphorylation.
- A FAK-independent pathway exists where Src directly phosphorylates CAS.
- The findings refute models suggesting direct FAK phosphorylation of CAS-SD or CAS-SBD.