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Studies on the function of Hic-5, a CAKβ-binding protein localized at focal adhesions

位于粘着斑的 CAKβ 结合蛋白 Hic-5 的功能研究

基本信息

批准号：
10670124
负责人：
SASAKI Hiroko
金额：
$ 1.98万
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：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670124/
关键词：
Hic-5 protein-tyrosine kinase tyrosine-phosphorylation nuclear translocation SH3 domain CAKβ PYK2 焦点接着 SH2ドメイン

项目摘要

Hic-5 is a CAKβ-binding protein localized at focal adhesions. We showed that overexpression of CAKβ or Fyn, but not FAK, enhanced the tyrosine-phosphorylation of coexpressed Hic-5. These phosphoorylations were further augmented by stimulating cells with osmotic stress. The Y60F mutant of Hic-5 was not phosphorylated, and Hic-5 phosphorylated on tyrosine 60 was bound specifically to the SH2 domain of Csk. Coexpression experiments revealed that the phosphorylation of Hic-5 by CAKβ required the kinase activation of CAKβ and binding of Hic-5 by CAKβ. Specific phosphorylation of Hic-5 by CAKβ and Fyn may activate a signaling pathway mediated by Hic-5.CAKβ/PYK2 is a protein-tyrosine kinase of the focal adhesion kinase (FAK) family. Whereas FAK predominantly localizes at focal adhesions, CAKβ localizes at the perinuclear region. Here we expressed in cultured cells two point mutants of CAKβ, P717A and P859A, which each lost one of the two PXXP motifs, the ligand sequenc3 for SH3 domains, found at the CAKβC-terminal region. We found a remarkable change in the subcellular distribution of the P859A mutant; that of the P717A mutant was the same as the wild type. The P859A mutant localized exclusively in the cell nucleus in all cell lines examined. Wild-type CAKβ also accumulated in the nucleus when cells were treated with an inhibitor of the nuclear export of proteins. These results indicate that CAKβ shuttles between the cytoplasm and the nucleus. On nuclear accumulation of P859A-CAKβ, a CAKβ-binding protein, Hic-5, also accumulated in the nucleus. P859A-CAKβ and co-expressed Hic-5 formed nuclear speckles, in which one other CAKβ-binding proteins, p130ィイD1CasィエD1, was also concentrated. These findings on nuclear translocation of CAKβ imply that CAKβmay regulate nuclear processes such as transcription, particularly because Hic-5 was recently shown to be a coactivator of nuclear receptors.

HIC-5是一种定位于局部粘连的β结合蛋白。我们的研究结果表明，高表达的钙结合蛋白β或Fyn，而不是粘着斑蛋白，促进了共表达的Hic-5的酪氨酸磷酸化。通过用渗透胁迫刺激细胞，这些磷酸化作用进一步增强。Hic-5的Y60F突变体没有被磷酸化，Hic-5在酪氨酸60上被磷酸化，并与CSK的SH2结构域特异结合。共表达实验表明，细胞角蛋白β对Hic-5的磷酸化需要激活细胞角蛋白β并与细胞角蛋白β结合。CAKβ和FYN对Hic-5的特异性磷酸化可能激活了Hic-5介导的信号通路。CAKβ/PYK2是粘着斑激酶家族中的一种蛋白酪氨酸激酶。FAK主要定位于局灶性粘连，而CAKβ定位于核周。在此，我们在培养细胞中表达了CAKβ、P717A和P859A的两个点突变体，每个突变体都丢失了两个PXXP基序中的一个，即在CAKβC-末端发现的SH3结构域的配体序列3。我们发现P859A突变体的亚细胞分布发生了显著的变化，P717A突变体的亚细胞分布与野生型相同。P859A突变体仅定位于所有细胞系的细胞核中。当用蛋白质核输出抑制剂处理细胞时，野生型CAKβ也在细胞核内积聚。这些结果表明，细胞角蛋白β在细胞质和细胞核之间穿梭。在P859A-CAKβ的核积累方面，CAKβ结合蛋白HIC-5也在核内积累。P859A-CAKβ和共表达的Hic-5形成核斑点，另一种CAKβ结合蛋白p130ィイD1CasィエD1也集中在其中。这些关于CAKβ核转位的研究结果表明，CAKβ可能调控转录等核过程，特别是因为最近发现Hic-5是核受体的辅助激活因子。