MAK(雄性生殖细胞相关蛋白激酶)和MRK/ICK(MAK相关激酶/肠细胞激酶)是酿酒酵母中Ime2p的人类同源物,而裂殖酵母中Mde3和Pit1的人类同源物。与人细胞周期蛋白依赖性激酶2(CDK2)和细胞外信号调节激酶2(ERK2)相似。MAK和MRK需要在未鉴定的人苏氨酸激酶和酪氨酸自磷酸化催化的TDY基序中双重磷酸化。在这里,我们确定人CDK相关激酶CCRK(细胞周期相关激酶)是MRK的激活T157激酶,而活性CDK7/细胞周期蛋白H/MAT1复合物磷酸化CDK2而不是MRK。蛋白磷酸酶5(PP5)与复合物中的MRK相互作用,并在体外和原位在T157处脱磷酸化MRK。因此,CCRK和PP5是T157磷酸化的阴阳调节因子。为了确定底物共识,我们筛选了具有活性MRK的组合肽库。MRK优先磷酸化R-P-X-S/T-P位点,在位置-3(P-3)对精氨酸的偏好比在P-2和P1对脯氨酸更严格。使用共识,我们确定了人镰刀菌MRK的推定磷酸化位点(RPLT(1080)S),与MRK相互作用的抗凋亡蛋白。通过定点诱变和质谱法测定,MRK在T1080的体外磷酸化Scythe,支持共识,并建议镰刀菌作为MRK的生理底物。
MAK (male germ cell-associated protein kinase) and MRK/ICK (MAK-related kinase/intestinal cell kinase) are human homologs of Ime2p in Saccharomyces cerevisiae and of Mde3 and Pit1 in Schizosaccharomyces pombe and are similar to human cyclin-dependent kinase 2 (CDK2) and extracellular signal-regulated kinase 2 (ERK2). MAK and MRK require dual phosphorylation in a TDY motif catalyzed by an unidentified human threonine kinase and tyrosine autophosphorylation. Herein, we establish that human CDK-related kinase CCRK (cell cycle-related kinase) is an activating T157 kinase for MRK, whereas active CDK7/cyclin H/MAT1 complexes phosphorylate CDK2 but not MRK. Protein phosphatase 5 (PP5) interacts with MRK in a complex and dephosphorylates MRK at T157 in vitro and in situ. Thus, CCRK and PP5 are yin-yang regulators of T157 phosphorylation. To determine a substrate
consensus, we screened a combinatorial peptide library with active MRK. MRK preferentially phosphorylates R-P-X-S/T-P sites, with the preference for arginine at position -3 (P-3) being more stringent than for prolines at P-2 and P+1. Using the
consensus, we identified a putative phosphorylation site (RPLT(1080)S) for MRK in human Scythe, an antiapoptotic protein that interacts with MRK. MRK phosphorylates Scythe at T1080 in vitro as determined by site-directed mutagenesis and mass spectrometry, supporting the
consensus and suggesting Scythe as a physiological substrate for MRK.