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

我们利用抗细胞周期蛋白G的抗体，通过Western-Western杂交，克隆了一个新的细胞周期蛋白G结合蛋白的编码区，该基因编码的蛋白含有一个N-末端的Ser/Thr蛋白激酶催化结构域和一个较长的C-末端延伸，因此我们将其命名为GAK(细胞周期蛋白G相关蛋白)。C-末端延伸部分与张力蛋白和生长素同源，并含有亮氨酸拉链。通过免疫共沉淀和Western blotting，我们检测到在NRK细胞提取物和表达myc标记的Cyclin G的COS细胞提取物中，GAK与CDK5和肌动蛋白共沉淀，CDK5和Cyclin G在NRK提取物中存在关联。用固定化GST-GK和不含GST的重组细胞周期蛋白G进行Biacore分析，检测到GAK和细胞周期蛋白G之间的直接相互作用。重组和免疫沉淀的GAK对组蛋白H1具有激酶活性，但这种活性不会因细胞周期蛋白G的存在而增强(或降低)。与脑组织特异表达的生长素相反，GAK普遍表达。在经胸腺嘧啶核苷双阻断与S期同步的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，并显示出类似的振荡模式。这些结果表明，GAK在某种程度上参与了细胞周期的调节，与细胞周期蛋白G形成了复合体。

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.