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

MAP激酶超家族成员在多种信号转导途径中发挥重要作用。我们报道了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类的cdna，编码一种新型蛋白激酶的完整开放阅读框，称为MOK。MOK包含蛋白丝氨酸/苏氨酸激酶共识基序，与MAP激酶超家族成员、MAK和MAK相关激酶（MRK）有一定的相似性。此外，与经典的MAP激酶一样，MOK在激活环域中具有一个Thr-Glu-Tyr （TEY）基序。MOK能够磷酸化几种已知的MAP激酶底物并进行自磷酸化。AEF的TEY基序突变可消除MOK的激酶活性，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远亲关系，因此可以归类为新成员。接下来，我们寻找与MOK特异性相关的细胞蛋白。几个细胞蛋白，包括一个主要的90 kda分子伴侣HSP90，被发现与MOK相关。用格尔达霉素（一种hsp90特异性抑制剂）处理细胞后，MOK蛋白水平迅速降低，这是由于蛋白酶体依赖途径增强了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的稳定性和MOK的溶解度。密切相关的蛋白激酶MAK和MRK也被发现与HSP90相关，而传统的MAP激酶（ERK、p38和SAPK/JNK）与HSP90无关。此外，我们还发现在MOK-HSP90免疫复合物中特异性检测到Cdc37、HSC70、HSP70和HSP60等分子伴侣。

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.