Haystead C M, Wu J, Gregory P, Sturgill T W, Haystead T A
Department of Pharmacology, University of Virginia, Charlottesville 22908.
FEBS Lett. 1993 Feb 8;317(1-2):12-6. doi: 10.1016/0014-5793(93)81481-e.
Mitogen-activated protein (MAP) kinases p42mapk and p44mapk are activated by dual tyrosine and threonine phosphorylation in vivo. Both MAPKs are phosphorylated and activated in vitro by an activator recently identified as a protein-tyrosine/threonine kinase. We have isolated a putative cDNA for a MAP kinase kinase (MAPKK) and determined its structure [Proc. Natl. Acad. Sci. USA, in press]. The protein encoded by this cDNA shares sequence homology with two yeast protein kinases byr1 and STE7. We now report that stimulation with serum of COS cells expressing this shares sequence homology with two yeast protein kinases byr1 and STE7. We now report that stimulation with serum of COS cells expressing this protein amplifies MAPK activator activity markedly. The increased activity co-migrates during chromatography with the expressed 45 kDa protein, recognized by an anti-STE7/byr1 antibody, and is abrogated by treatment with phosphatase 2A. Thus, this cDNA encodes a functional MAPKK. The anti-STE7/byr1 antibody also recognized a 46 kDa COS cell protein that was resolved from the expressed MAPKK by anion-exchange chromatography. This immunoreactive protein co-eluted with endogenous MAPKK activity, suggesting identification of the immunoreactive band as monkey MAPKK.
丝裂原活化蛋白（MAP）激酶p42mapk和p44mapk在体内通过酪氨酸和苏氨酸双磷酸化而被激活。这两种MAP激酶在体外都能被一种最近鉴定为蛋白酪氨酸/苏氨酸激酶的激活剂磷酸化并激活。我们分离出了一种假定的MAP激酶激酶（MAPKK）的cDNA并确定了其结构[《美国国家科学院院刊》，即将发表]。该cDNA编码的蛋白质与两种酵母蛋白激酶byr1和STE7具有序列同源性。我们现在报告，用血清刺激表达这种与两种酵母蛋白激酶byr1和STE7具有序列同源性的蛋白质的COS细胞，能显著增强MAPK激活剂活性。增加的活性在色谱分析中与表达的45 kDa蛋白质共迁移，该蛋白质能被抗STE7/byr1抗体识别，并且用磷酸酶2A处理后活性被消除。因此，该cDNA编码一种功能性的MAPKK。抗STE7/byr1抗体还识别出一种46 kDa的COS细胞蛋白质，该蛋白质通过阴离子交换色谱与表达的MAPKK分离。这种免疫反应性蛋白质与内源性MAPKK活性共洗脱，表明将该免疫反应条带鉴定为猴MAPKK。
