Interaction of two proline-rich sequences of cell adhesion kinase beta with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf.
Cell adhesion kinase beta (CAKbeta) is a protein tyrosine kinase closely related to focal adhesion kinase (FAK) in structure. CAKbeta 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 CAKbeta to a panel of SH3 domains. Affinity precipitation from rat brain lysate revealed selective interactions of CAKbeta with glutathione S-transferase (GST)-fused SH3 domains of p130(Cas)(Cas)-related proteins and Graf. Mutational analysis indicated that the proline-rich sequences of CAKbeta 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 CAKbeta preferentially associated with the SH3 domain of Cas. The second proline-rich sequence of CAKbeta bound to the SH3 domain of Graf with higher specificity than the corresponding proline-rich sequence of FAK. Finally, we showed co-immunoprecipitation of CAKbeta with Graf from rat brain lysate. These results indicate that CAKbeta associates in vivo with Graf through its SH3 domain.
Ohba
T
T
Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan.
Ishino
M
M
Aoto
H
H
Sasaki
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D016384:Consensus Sequence; D050719:Crk-Associated Substrate Protein; D051418:Focal Adhesion Kinase 2; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase / Q000096:biosynthesis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D011392:Proline; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000737:chemistry / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D018909:src Homology Domains
C493843:Bcar1 protein, rat; D050719:Crk-Associated Substrate Protein; D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D050717:Retinoblastoma-Like Protein p130; D011392:Proline; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496054:Ptk2b protein, rat