Interaction of two proline-rich sequences of cell adhesion kinase β with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf

摘要

pCell adhesion kinase β (CAKβ) is a protein tyrosine kinase closely related to focal adhesion kinase (FAK) in structure. CAKβ contains two proline-rich sequences within its C-terminal region. Since proline-rich sequences present in the corresponding region of FAK are known to mediate protein-protein interactions by binding to SH3 domains, we investigated binding of CAKβ to a panel of SH3 domains. Affinity precipitation from rat brain lysate revealed selective interactions of CAKβ with glutathione S-transferase (GST)-fused SH3 domains of p130supiCas/i/sup(Cas)-related proteins and Graf. Mutational analysis indicated that the proline-rich sequences of CAKβ mediate this interaction. Each of the two proline-rich sequences fused to GST bound directly to these SH3 domains in dot blot analysis. A competitive binding assay revealed that the first proline-rich sequence of CAKβ preferentially associated with the SH3 domain of Cas. The second proline-rich sequence of CAKβ bound to the SH3 domain of Graf with higher specificity than the corresponding proline-rich sequence of FAK. Finally, we showed co-immunoprecipitation of CAKβ with Graf from rat brain lysate. These results indicate that CAKβ associates iin iv/iiiv/io/i with Graf through its SH3 domain./p