Meloche S, Pagès G, Pouysségur J
Centre de Biochimie-CNRS, Université de Nice, France.
Mol Biol Cell. 1992 Jan;3(1):63-71. doi: 10.1091/mbc.3.1.63.
Mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated kinases (ERKs) are serine/threonine kinases of apparent Mr 42-44 kDa that are rapidly activated by a variety of extracellular signals in many cell types. This activation coincides with their phosphorylation on tyrosine and threonine residues, and these covalent modifications are required for full activity of the enzymes. They are thought to play a pivotal role in integrating and transmitting transmembrane signals for growth and differentiation. Here, we report the cloning, sequence, and functional expression in fibroblasts of the hamster p44 MAP kinase (p44mapk). The protein deduced from the nucleotide sequence of an almost full-length cDNA is 98.6% homologous to the rat p44mapk (ERK1). To distinguish the expression of the cloned cDNA from the endogenous p44mapk, we fused to the 5' end of the cDNA an initiating codon followed by an influenza hemagglutinin 9-residue peptide epitope (HAP). The chimeric kinase HAP/p44mapk, under transcriptional control of the cytomegalovirus promoter, was stably expressed in Chinese hamster lung fibroblasts in a functional form. We show that its basal activity, measured by phosphorylation of the substrate myelin basic protein, is activated severalfold (up to 25) by the mitogens alpha-thrombin, platelet-derived growth factor, and fetal calf serum. In addition, we report that in response to alpha-thrombin, this activation is rapid (6-fold in 1 min), biphasic (first peak at 5 min, second broader peak at 1-2 h), persistent (for greater than or equal to 4 h), and parallel to an increased phosphorylation on tyrosine.We conclude that the constructed and stably expressed chimera, HAP/p44mapk, has retained apparently all the hormonal regulation features of the endogenous form. This system now offers the possibility to study structure-function relationships and to determine the role of this kinase in growth control.
丝裂原活化蛋白激酶（MAPKs）或细胞外信号调节激酶（ERKs）是表观分子量为42 - 44 kDa的丝氨酸/苏氨酸激酶，在许多细胞类型中可被多种细胞外信号迅速激活。这种激活与它们酪氨酸和苏氨酸残基的磷酸化同时发生，并且这些共价修饰是酶的完全活性所必需的。它们被认为在整合和传递生长与分化的跨膜信号中起关键作用。在此，我们报告仓鼠p44 MAP激酶（p44mapk）在成纤维细胞中的克隆、序列及功能表达。从一个几乎全长的cDNA核苷酸序列推导的蛋白质与大鼠p44mapk（ERK1）有98.6%的同源性。为了区分克隆的cDNA与内源性p44mapk的表达，我们在cDNA的5'端融合了一个起始密码子，随后是一个流感血凝素9残基肽表位（HAP）。嵌合激酶HAP/p44mapk在巨细胞病毒启动子的转录控制下，以功能形式稳定表达于中国仓鼠肺成纤维细胞中。我们表明，通过底物髓鞘碱性蛋白的磷酸化测量，其基础活性被丝裂原α - 凝血酶、血小板衍生生长因子和胎牛血清激活数倍（高达25倍）。此外，我们报告，响应α - 凝血酶时，这种激活迅速（1分钟内增加6倍）、呈双相性（第一个峰值在5分钟，第二个更宽的峰值在1 - 2小时）、持续（大于或等于4小时），并且与酪氨酸磷酸化增加平行。我们得出结论，构建并稳定表达的嵌合体HAP/p44mapk显然保留了内源性形式的所有激素调节特征。该系统现在为研究结构 - 功能关系以及确定这种激酶在生长控制中的作用提供了可能性。
