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Identification and functional analysis of a novel type of MAP kinase superfamily.

新型 MAP 激酶超家族的鉴定和功能分析。

基本信息

批准号：
11680696
负责人：
MIYATA Yoshihiko
金额：
$ 2.24万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Members of the MAP kinase superfamily play important roles in a variety of signal transduction pathways. We report molecular cloning and characterization of a novel member of the MAP kinase superfamily. We isolated mouse and human cDNAs that encode complete open reading frames of a novel protein kinase, termed MOK.MOK contains the protein serine/threonine kinase consensus motifs and shows modest similarity to members of the MAP kinase superfamily and MAK and MAK-related kinase (MRK). In addition, MOK possesses a Thr-Glu-Tyr (TEY) motif in the activation loop domain, like classical MAP kinases. MOK is able to phosphorylate several known MAP kinase substrates and to undergo autophosphorylation. A mutation in the TEY motif to AEF abolished the kinase activity of MOK, and treatment of cells with a phosphatase inhibitor okadaic acid enhanced the kinase activity of MOK.Phorbol ester TPA was found to stimulate the kinase activity of MOK.These results indicate that MOK is distantly related to members of known subfamilies of the MAP kinase superfamily and therefore can be classified as a novel member.Next, we searched for cellular proteins that specifically associate with MOK.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.Closely related protein kinases MAK and MRK were also found to associate with HSP90 while conventional MAP kinases (ERK, p38, and SAPK/JNK) were not associated with HSP90. In addition, we found that other molecular chaperones including Cdc37, HSC70, HSP70, and HSP60, were detected specifically in the MOK-HSP90 immunocomplexes.

MAP激酶超家族成员在多种信号转导途径中发挥重要作用。我们报告的MAP激酶超家族的一个新成员的分子克隆和表征。我们分离了小鼠和人的cDNA，它们编码一种新的蛋白激酶的完整开放阅读框，称为MOK。MOK含有蛋白丝氨酸/苏氨酸激酶共有基序，并与MAP激酶超家族和MAK及MAK相关激酶（MRK）的成员显示出适度的相似性。此外，MOK在激活环结构域中具有Thr-Glu-Tyr（TEY）基序，类似于经典的MAP激酶。MOK能够磷酸化几种已知的MAP激酶底物并进行自磷酸化。TEY基序突变为AEF可使MOK的激酶活性消失，而用磷酸酶抑制剂冈田酸处理细胞可增强MOK的激酶活性。发现佛波酯TPA可刺激MOK的激酶活性。这些结果表明，MOK与MAP激酶超家族的已知亚家族成员有较远的亲缘关系，因此可以归类为一个新成员。我们寻找与MOK特异性相关的细胞蛋白。发现了几种与MOK相关的细胞蛋白，包括主要的90-kDa分子伴侣HSP 90。用格尔德霉素（一种HSP 90特异性抑制剂）处理细胞，迅速降低MOK的蛋白水平，而这种降低归因于通过蛋白酶体依赖性途径增强MOK的降解。我们的数据表明，与HSP 90的关联可以调节MOK的细胞内蛋白质的稳定性和溶解性，与HSP 90密切相关的蛋白激酶MAK和MRK也被发现与HSP 90相关，而传统的MAP激酶（ERK，p38和SAPK/JNK）与HSP 90无关。此外，我们发现其他分子伴侣包括Cdc 37，HSC 70，HSP 70和HSP 60，在MOK-HSP 90免疫复合物中特异性检测。