Ohba T, Ishino M, Aoto H, Sasaki T
Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan.
Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1249-54. doi: 10.1042/bj3301249.
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.
细胞黏附激酶β（CAKβ）是一种蛋白酪氨酸激酶，在结构上与粘着斑激酶（FAK）密切相关。CAKβ在其C末端区域包含两个富含脯氨酸的序列。由于已知FAK相应区域中存在的富含脯氨酸的序列通过与SH3结构域结合来介导蛋白质-蛋白质相互作用，我们研究了CAKβ与一组SH3结构域的结合情况。从大鼠脑裂解物中进行亲和沉淀，结果显示CAKβ与p130（Cas）（Cas）相关蛋白和Graf的谷胱甘肽S-转移酶（GST）融合的SH3结构域存在选择性相互作用。突变分析表明，CAKβ的富含脯氨酸的序列介导了这种相互作用。在斑点印迹分析中，与GST融合的两个富含脯氨酸的序列中的每一个都直接与这些SH3结构域结合。竞争性结合试验表明，CAKβ的第一个富含脯氨酸的序列优先与Cas的SH3结构域结合。CAKβ的第二个富含脯氨酸的序列与Graf的SH3结构域结合的特异性高于FAK相应的富含脯氨酸的序列。最后，我们展示了从大鼠脑裂解物中CAKβ与Graf的共免疫沉淀。这些结果表明，CAKβ在体内通过其SH3结构域与Graf结合。
