MOLECULAR-CLONING, EXPRESSION, AND CHARACTERIZATION OF THE HUMAN MITOGEN-ACTIVATED PROTEIN-KINASE P44ERK1
MOLECULAR-CLONING, EXPRESSION, AND CHARACTERIZATION OF THE HUMAN MITOGEN-ACTIVATED PROTEIN-KINASE P44ERK1
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DOI：
10.1128/mcb.13.8.4679
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发表时间：
1993-08-01
影响因子：
5.3
通讯作者：
PELECH, SL
中科院分区：
文献类型：
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作者：
CHAREST, DL;MORDRET, G;PELECH, SL
p44 erk 1是参与细胞周期控制的酪氨酰磷酸化和促分裂原活化蛋白（MAP）激酶家族的成员。从人肝癌细胞系Hep G2文库中克隆了erk 1全长cDNA。erk 1 cDNA克隆与啮齿动物erk 1同源物的部分序列共享约96%的预测氨基酸同一性，并且erk 1基因通过杂交板分析被分配到人类16号染色体。人erk 1在大肠杆菌中表达为谷胱甘肽S-转移酶融合蛋白（GST-Erk 1），在体内基本上是酪氨酸磷酸化。它进行了进一步的自磷酸化在体外（高达0.01摩尔的P每摩尔）在监管Tyr-204网站和在额外的酪氨酸和丝氨酸残基。推测在调节Thr-202位点的苏氨酸自磷酸化，也检测到弱时，重组激酶在锰的存在下孵育，但不存在镁。在用凝血酶切割GST-Erk 1蛋白之前和之后，它表现出相对高水平的髓鞘碱性蛋白磷酸转移酶活性，其可以通过用蛋白酪氨酸磷酸酶CD 45处理激酶而降低8倍，但不能通过用蛋白丝氨酸/苏氨酸磷酸酶2A处理。蛋白酪氨酸激酶p56 lck在两个自磷酸化位点（包括Tyr-204）和一个新位点催化GST-Erk 1的磷酸化。进一步的5倍刺激的髓鞘碱性蛋白磷酸转移酶活性的GST-Erk 1的部分纯化的MAP激酶激酶从绵羊血小板的存在下实现。在这些情况下，主要是增强了GST-Erk 1的酪氨酸磷酸化。这种MAP激酶激酶也类似地磷酸化GST-Erk 1的催化受损版本，其中Lys-71通过定点突变转化为Ala。
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.