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A study on the signaling pathway activating CAKbeta, second protein-tyrosine kinase of the FAK subfamily.

关于激活 CAKbeta（FAK 亚家族的第二种蛋白酪氨酸激酶）的信号通路的研究。

基本信息

批准号：
09670137
负责人：
SASAKI Terukatsu
金额：
$ 1.92万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

1. A cDNA for a CAKbeta/PYK2-binding protein (CBP-1) was cloned by screening a human brain cDNA library with the CAKbeta C-domain as a probe. CBP-1 was the human homologue of Hic-5. Our CBP-1 cDNA provided an evidence that Hic-5 has more than 16 additional amino acid residues at its N-terminal region in addition to the sequence previously described for mouse Hic-5. Hic-5 localizedatfocal adhesions. Hic-5 has 4 LIM domains and 5 LD motifs and is most closely related to paxillin. Hic-5 bound to the C-terminal half of the CAKbeta C-domain with its N-domain. CAKbeta was coimmunoprecipitated with Hic-5 from the WFB cell lysate. When WFB cells were stimulated to enhance the tyrosine-phosphorylation of CAKbeta, the tyrosine-phosphorylation of Hic-5 was also enhanced.2. In COS-7 cells, the tyrosine-phosphorylation of Hic-5 was enhanced by co-expression of CAKbeta and was further enhanced by stimulating the coexpressed cells with osmotic-stress. The tyrosine-60 residue of Hic-5 was the major site of the tyrosine-phosphorylation because the Y6OF mutant of Hic-5 was nottyrosine-phosphorylated. The CAKbeta mutant deleted at the Hic-5 binding site was defective in the tyrosine-phosphorylation of Hic-5. The tyrosine-phosphorylated Hic-5 was bound to the SH2 domain of Csk but not to the SH2 domains of Fyn, Src, and Crk.3. The wild type CAKbeta localized in cytoplasm. A single amino acidsubstituted mutant of CAKbeta designated "A mutant" was found to localize exclusively in nucleus. A translocation of wild type CAKbeta to nucleus was observed under certain culture conditions. A portion of Hic-5 was also translocated to nucleus when coexpressed with the A mutant of CAKbeta.

1. 以CAKbeta c结构域为探针，筛选人脑cDNA文库，克隆了CAKbeta/ pyk2结合蛋白（CBP-1） cDNA。CBP-1是Hic-5的人类同源物。我们的CBP-1 cDNA提供了证据，证明Hic-5在其n端区域除了先前描述的小鼠Hic-5序列外，还有超过16个额外的氨基酸残基。Hic-5定位病灶粘连。Hic-5具有4个LIM结构域和5个LD基序，与paxillin关系最为密切。Hic-5结合到cak - β c-结构域的c端和n -结构域。cakβ与WFB细胞裂解液中的Hic-5共免疫沉淀。当刺激WFB细胞增强cakβ的酪氨酸磷酸化时，Hic-5的酪氨酸磷酸化也增强。在COS-7细胞中，CAKbeta的共表达增强了Hic-5酪氨酸磷酸化，并通过渗透胁迫刺激共表达细胞进一步增强了Hic-5酪氨酸磷酸化。由于Hic-5的Y6OF突变体不被酪氨酸磷酸化，Hic-5的酪氨酸-60残基是酪氨酸磷酸化的主要位点。在Hic-5结合位点缺失的CAKbeta突变体在Hic-5的酪氨酸磷酸化中存在缺陷。酪氨酸磷酸化的Hic-5与Csk的SH2结构域结合，而不与Fyn、Src和Crk.3的SH2结构域结合。野生型CAKbeta定位于细胞质中。发现CAKbeta的单氨基酸取代突变体“A突变体”只定位于细胞核。在一定的培养条件下，观察到野生型CAKbeta向细胞核的易位。当与CAKbeta的A突变体共表达时，Hic-5的一部分也易位到细胞核中。