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Cloning and functional analusis of cell-cycle regulatory genes by complementation cloning.

通过互补克隆进行细胞周期调控基因的克隆和功能分析。

基本信息

批准号：
05454647
负责人：
NOJIMA Hiroshi
金额：
$ 4.22万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

By west-western hybridization using an anti-cyclin G antibody, we cloned a cDNA encoding a novel association partner of cyclin G.The cDNA encodes a protein harboring a predicted N-terminal Ser/Thr protein kinase catalytic domain with a long C-terminal extension.Hence we named it GAK (cyclin G-associated kinase) . The C-terminal extension shares homology with tensin and auxilin, and contains a leucine zipper. By coimmunoprecipitation and western blotting, we detected and association between GAK and cyclin G in NRK cell extracts, and in extracts of COS cells expressing myc-tagged cyclin G.GAK was also found to coprecipitate with CDK5 and actin, and CDK5 is shown to be associated with cyclin G in NRK extracts. A direct interaction between GAK and cyclin G was detected using immobilized GST-GK and recombinant GST-free cyclin G for BIAcore analysis. Recombinant and immunoprecipitated GAK were found to have kinase activity towards histone H1, but this activity was not enhanced (or decreased) by the presence of cyclin G.In contrast to brain specific expression of auxilin, GAK was found to be ubiquitously expressed. In HeLa cells synchronized from S phase by the thymidine double block, RNA and protein levels of GAK and cyclin G oscillated, peaking at G1 phase, and they showed a similar pattern of oscillation. These results suggest that GAK is somehow involved in cell cycle regulation, forming a complex with cyclin G.

利用抗细胞周期蛋白G抗体的东西杂交技术，我们克隆了一个编码细胞周期蛋白G新结合伴侣的cDNA，该cDNA编码一个具有预测的n端丝氨酸/苏氨酸蛋白激酶催化结构域的蛋白，该蛋白具有长c端延伸。因此我们将其命名为GAK （cyclin G-associated kinase）。c端延伸与张力素和辅助素具有同源性，并包含亮氨酸拉链。通过共免疫沉淀和western blotting，我们在NRK细胞提取物中检测到GAK和cyclin G之间的关联，在表达myc标记的cyclin G的COS细胞提取物中，GAK还与CDK5和actin共沉淀，并且在NRK提取物中CDK5被证明与cyclin G相关。利用固定化GST-GK和重组GST-free cyclin G检测GAK和cyclin G之间的直接相互作用，并进行BIAcore分析。重组和免疫沉淀GAK被发现对组蛋白H1具有激酶活性，但这种活性不因细胞周期蛋白g的存在而增强（或降低）。与auxilin的脑特异性表达相反，GAK被发现普遍表达。在由胸腺嘧啶双阻断同步的S期HeLa细胞中，GAK和cyclin G的RNA和蛋白质水平振荡，在G1期达到峰值，它们表现出类似的振荡模式。这些结果表明GAK以某种方式参与细胞周期调节，与细胞周期蛋白G形成复合物。