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

我们最近鉴定并克隆了MAP激酶超家族的一个新成员MOK。为了解决其调节机制，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HSP 90被发现与MOK。用热休克蛋白90特异性抑制剂格尔德霉素处理细胞，迅速降低MOK的蛋白水平，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，与热休克蛋白90的关联可能会调节细胞内蛋白质的稳定性和溶解性的MOK。MOK的一系列缺失突变体的实验表明，该区域包含的蛋白激酶催化亚结构域I-IV是所需的HSP 90结合。此外，我们发现其他分子伴侣包括Cdc 37，HSC 70和HSP 70在MOK-HSP 90免疫复合物中特异性检测。的 ...更多信息 这些结果共同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的作用。我们还研究了分子伴侣与其他密切相关的激酶，包括ERK，p38，JNK/SAPK，MAK，MRK，HIPK 1，HIPK 2，Dyrk 1A和Dyrk 2的关联。其中只有MAK和MRK与HSP 90和Cdc 37相关，表明HSP 90-Cdc 37分子伴侣识别并稳定具有相似催化结构域的特定蛋白激酶亚群。以MOK全长cDNA为探针，通过筛选小鼠基因组文库，获得MOK基因组DNA片段。MOK基因编码区位于14 q32的一个80 kbp的区域内，有12个外显子。所获得的14 kbp的基因组片段含有MOK的外显子2-4。进行限制性内切酶图谱分析，并构建具有TK/neo选择标记的靶向载体。我们目前正在尝试通过同源重组技术将靶向载体导入ES细胞Less

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