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A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling
Kathrin Kirsch1, Margaret Kensinger, Hidesaburo Hanafusa
1Laboratory of Molecular Oncology, The Rockefeller University, NY, NY 10021, USA. kirschk@bu.edu
Background:
The adaptor protein p130Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals.
Results:
We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk.
Conclusion:
Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.
Insights
Tyrosine phosphorylation of the p130Cas (Cas) substrate domain alone does not drive cellular signaling. A phosphorylated Cas substrate domain acts as a dominant-negative mutant, blocking Cas-mediated events by competing for binding partners.
Area of Science:
- Cellular signaling
- Molecular biology
- Protein phosphorylation
Background:
- p130Cas (Cas) is an adaptor protein crucial for cell adhesion, migration, and transformation.
- Cas features a substrate domain with multiple tyrosine residues serving as docking sites for SH2/PTB domain proteins.
- Cas interacts with focal adhesions and is phosphorylated by FAK and Src, with effector molecules like c-crk and v-crk influencing its function.
Purpose of the Study:
- To investigate if the tyrosine-phosphorylated Cas substrate domain alone can initiate signaling.
- To construct a chimeric Cas molecule for independent phosphorylation, bypassing upstream signals.
Main Methods:
- Construction of a chimeric Cas molecule.
- Analysis of the chimeric molecule's effect on Cas-mediated signaling and v-crk transformation.
- Assessment of binding competition with endogenous and exogenous c-crk and v-crk.
Main Results:
- A tyrosine-phosphorylated Cas substrate domain functions as a dominant-negative mutant.
- This mutant blocks Cas-mediated signaling, including JNK activation and v-crk transformation.
- The blocking mechanism involves competition for binding partners, such as c-crk and v-crk.
Conclusions:
- Mere tyrosine phosphorylation of the Cas substrate domain is insufficient for its functional signaling.
- The study proposes a novel method for studying phosphorylation-dependent adaptor proteins.