Matsuya M, Sasaki H, Aoto H, Mitaka T, Nagura K, Ohba T, Ishino M, Takahashi S, Suzuki R, Sasaki T
Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan.
J Biol Chem. 1998 Jan 9;273(2):1003-14. doi: 10.1074/jbc.273.2.1003.
Cell adhesion kinase beta (CAKbeta/PYK2) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily. We identified a cDNA that encodes a CAKbeta-binding protein. This cDNA clone encodes the human homologue of Hic-5, the cDNA of which was cloned in 1994 as transforming growth factor beta1- and hydrogen peroxide-inducible mRNA. We found that Hic-5 exclusively localized at focal adhesions in a rat fibroblast line, WFB. This localization of Hic-5 was confirmed in WFB cells expressing Myc-tagged Hic-5. The amino acid sequence of Hic-5 is highly similar to that of paxillin in the four LD motifs as well as in the four contiguous LIM domains. The Hic-5 N-terminal domain directly associated in vitro with the extreme C-terminal region (residue 801 to the end) of CAKbeta. CAKbeta was coimmunoprecipitated with Hic-5 from the WFB cell lysate. The coimmunoprecipitation of CAKbeta with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAKbeta. Coimmunoprecipitation of Hic-5 with CAKbeta, which was shown in COS-7 cells doubly transfected with cDNA constructs of CAKbeta and Myc-tagged Hic-5, was lost when the CAKbeta amino acid residues 741-903 were deleted. Hic-5 was tyrosine-phosphorylated in Src-transformed 3Y1 cells and in cells treated with pervanadate. Hic-5 associated with CAKbeta was selectively tyrosine-phosphorylated in WFB cells exposed to hypertonic osmotic stress. These results indicate that Hic-5 is a paxillin-related component of focal adhesions and binds to CAKbeta, implying possible involvement of Hic-5 in the downstream signaling of CAKbeta.
细胞黏附激酶β（CAKβ/PYK2）是黏着斑激酶亚家族的第二个蛋白酪氨酸激酶。我们鉴定出一个编码CAKβ结合蛋白的cDNA。该cDNA克隆编码Hic-5的人类同源物，其cDNA于1994年作为转化生长因子β1和过氧化氢诱导型mRNA被克隆。我们发现Hic-5仅定位于大鼠成纤维细胞系WFB的黏着斑。在表达Myc标签的Hic-5的WFB细胞中证实了Hic-5的这种定位。Hic-5的氨基酸序列在四个LD基序以及四个连续的LIM结构域中与桩蛋白高度相似。Hic-5的N末端结构域在体外直接与CAKβ的极端C末端区域（第801位残基至末端）结合。CAKβ与来自WFB细胞裂解物的Hic-5共免疫沉淀。添加CAKβ的极端C末端区域可显著抑制CAKβ与Hic-5的共免疫沉淀。当缺失CAKβ的741 - 903位氨基酸残基时，在同时转染CAKβ和Myc标签的Hic-5 cDNA构建体的COS-7细胞中显示的Hic-5与CAKβ的共免疫沉淀消失。在Src转化的3Y1细胞和经过氧钒酸盐处理的细胞中，Hic-5发生酪氨酸磷酸化。在暴露于高渗应激的WFB细胞中，与CAKβ结合的Hic-5被选择性地酪氨酸磷酸化。这些结果表明，Hic-5是黏着斑中与桩蛋白相关的成分，并与CAKβ结合，这意味着Hic-5可能参与CAKβ的下游信号传导。
