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

1.以CAKβC结构域为探针，通过筛选人脑cDNA文库，克隆了CAKβ/PYK2结合蛋白(CBP-1)的cDNA。CBP-1是Hic-5的人类同源物。我们的CBP-1基因证明Hic-5的N-末端除了先前描述的小鼠Hic-5的序列外，还有超过16个额外的氨基酸残基。HIC-5定位于DATFocus粘连。HIC-5有4个LIM结构域和5个LD基序，与paxlin的亲缘关系最近。HIC-5通过其N-结构域与CAKβC-结构域的C-末端半部分结合。CAKβ与WFB细胞裂解液中的Hic-5免疫共沉淀。结论：1.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，CAKβ的共表达增强了Hic-5的酪氨酸磷酸化，渗透压刺激共表达的细胞进一步增强了Hic-5的酪氨酸磷酸化。Hic-5的Y60OF突变体没有酪氨酸磷酸化，因此Hic-5的酪氨酸-60残基是酪氨酸磷酸化的主要部位。在Hic-5结合位点缺失的CAKβ突变体在Hic-5的酪氨酸磷酸化过程中存在缺陷。酪氨酸磷酸化的Hic-5与CSK的SH2结构域结合，但不与Fyn、Src和Crk的SH2结构域结合。野生型CAKβ定位于细胞质。CAKβ的一个氨基酸取代突变体被命名为“A突变体”，该突变体仅定位于细胞核。在一定的培养条件下，观察到野生型CAKβ向细胞核的移位。当Hic-5与CAKβ的A突变体共表达时，部分Hic-5也被移位到细胞核。

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.