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Analysis of physiological role of protein kinase MOK, a novel member of MAP kinase superfamily

MAP激酶超家族新成员蛋白激酶MOK的生理作用分析

基本信息

批准号：
13680781
负责人：
MIYATA Yoshihiko
金额：
$ 2.3万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680781/
关键词：
MOK MAPK Kinase Molecular Chaperone HSP90 Dyrk HIPK Knockout Mouse CK2 ゲノム構造 ES細胞

项目摘要

We have recently identified and cloned a novel member of MAP kinase superfamily protein, MOK. To address its regulatory mechanisms, we searched for cellular proteins that specifically associate with MOK by co-immunoprecipitation experiments. Several cellular proteins including a major 90-kDa molecular chaperone HSP90 were found associated with MOK. Treatment of cells with geldanamycin, an HSP90-specific inhibitor, rapidly decreased the protein level of MOK, and the decrease was attributed to enhanced degradation of MOK through proteasome-dependent pathways. Our data suggest that the association with HSP90 may regulate intracellular protein stability and solubility of MOK. Experiments with a series of deletion mutants of MOK indicated that the region encompassing the protein kinase catalytic subdomain I IV is required for HSP90 binding. In addition, we found that other molecular chaperones including Cdc37, HSC70, and HSP70 were detected specifically in the MOK-HSP90 immunocomplexes. The … More se results taken together suggest a role of a specific set of molecular chaperones in the stability of signal transducing protein kinases. We also examined the association of molecular chaperones with other closely related kinases including ERK, p38, JNK/SAPK, MAK, MRK, HIPK1, HIPK2, Dyrk1A, and Dyrk2. Only MAK and MRK among them were associated with HSP90 and Cdc37, suggesting that HSP90-Cdc37 molecular chaperones recognize and stabilize a specific subset of protein kinases with similar catalytic domainsTo analyze physiological role of MOK, we have started making a MOK-knockout mouse strain. A fragment of MOK genomic DNA was isolated by screening mouse genomic library with MOK full-length cDNA as a probe. The coding region of MOK with 12 exons locates within an 80kbp region in 14q32. The obtained 14kbp genomic fragment contains exon 2-4 of MOK. Restriction enzyme mapping was performed and a targeting vector with TK/neo selection markers was constructed. We are currently trying to introduce the targeting vector into ES cells by using homologous recombination technique Less

我们最近鉴定并克隆了一个新的MAP蛋白超家族成员MOK。为了探讨其调控机制，我们通过免疫共沉淀实验寻找与MOK特异性相关的细胞蛋白。几种细胞蛋白，包括一个主要的90 kDa分子伴侣HSP90，被发现与MOK相关。用HSP90特异性抑制剂格尔达那霉素处理细胞后，MOK的蛋白水平迅速下降，这种下降归因于通过蛋白酶体依赖的途径促进MOK的降解。我们的数据表明，与HSP90的关联可能调节细胞内蛋白质的稳定性和MOK的溶解性。用MOK的一系列缺失突变体进行的实验表明，HSP90结合所需的区域包含蛋白激酶催化亚域I和IV。此外，我们还发现在MOK-HSP90免疫复合体中特异性地检测到其他分子伴侣分子，包括CDC37、HSC70和HSP70。The…更多的Se结果综合起来表明，一组特定的分子伴侣在信号转导蛋白激酶的稳定性中发挥了作用。我们还研究了分子伴侣与其他密切相关的激酶的关系，包括ERK、p38、JNK/SAPK、MAK、MRK、HIPK1、HIPK2、Dyrk1A和Dyrk2。其中只有MAK和MRK与HSP90和CDC37相关，这表明HSP90-CDC37分子伴侣可以识别和稳定具有相似催化结构域的一组特定的蛋白激酶。为了分析MOK的生理作用，我们开始制作Mok基因敲除的小鼠品系。以MOK全长cDNA为探针，筛选小鼠基因组文库，获得MOK基因组DNA片段。MOK的编码区由12个外显子组成，位于14q32的80kbp范围内。获得的14kbp基因组片段含有MOK基因的外显子2-4。进行限制性内切酶图谱分析，构建带有Tk/neo选择标记的靶向载体。我们目前正在尝试通过使用同源重组技术将靶向载体引入ES细胞