Charest D L, Mordret G, Harder K W, Jirik F, Pelech S L
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Mol Cell Biol. 1993 Aug;13(8):4679-90. doi: 10.1128/mcb.13.8.4679-4690.1993.
p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.
p44erk1是酪氨酰磷酸化且有丝分裂原激活的蛋白（MAP）激酶家族的成员，参与细胞周期调控。从人肝癌细胞系（Hep G2）文库中分离出全长erk1 cDNA。erk1 cDNA克隆与啮齿动物erk1同源物的部分序列预测氨基酸同一性约为96%，通过杂交板分析将erk1基因定位于人类16号染色体。在大肠杆菌中表达为人谷胱甘肽S - 转移酶融合（GST - Erk1）蛋白的人erk1在体内酪氨酸上大量磷酸化。它在体外于调节性Tyr - 204位点以及其他酪氨酸和丝氨酸残基处进一步自磷酸化（每摩尔高达0.01摩尔磷）。当重组激酶在锰存在下孵育时，也微弱检测到苏氨酸自磷酸化，推测发生在调节性Thr - 202位点，但在镁存在下未检测到。用凝血酶切割GST - Erk1蛋白前后，它表现出相对较高水平的髓鞘碱性蛋白磷酸转移酶活性，用蛋白酪氨酸磷酸酶CD45处理激酶可使其降低8倍，但用蛋白丝氨酸/苏氨酸磷酸酶2A处理则不能。蛋白酪氨酸激酶p56lck在两个自磷酸化位点（包括Tyr - 204）以及一个新位点催化GST - Erk1的磷酸化。在存在来自绵羊血小板的部分纯化的MAP激酶激酶的情况下，GST - Erk1的髓鞘碱性蛋白磷酸转移酶活性进一步提高了5倍。在这些情况下，主要是GST - Erk1的酪氨酸磷酸化增强。这种MAP激酶激酶同样磷酸化了通过定点诱变将Lys - 71转化为Ala的催化受损版本的GST - Erk1。
