Functional Expression and Growth Factor Activation of an Epitope-Tagged p 44 Mitogen-Activated Protein Kinase , p 44 mapk
Functional Expression and Growth Factor Activation of an Epitope-Tagged p 44 Mitogen-Activated Protein Kinase , p 44 mapk
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表位标记的 p 44 丝裂原激活蛋白激酶的功能表达和生长因子激活，p 44 mapk
DOI：
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发表时间：
--
期刊：
影响因子：
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通讯作者：
J. Pouysségur
中科院分区：
文献类型：
--
作者：
S. Meloche;G. Pagès;J. Pouysségur
丝裂原活化蛋白激酶（MAPKs）或细胞外信号调节激酶（ERKs）是表观Mr 42-44 kDa的丝氨酸/苏氨酸激酶，在许多细胞类型中被各种细胞外信号迅速激活。这种激活与它们在酪氨酸和苏氨酸残基上的磷酸化一致，这些共价修饰是酶充分活性所必需的。它们被认为在整合和传递跨膜信号以促进生长和分化中起关键作用。在这里，我们报道了仓鼠p44 MAP激酶（p44maPk）的克隆、序列和在成纤维细胞中的功能表达。从几乎全长cDNA的核苷酸序列中推断出的蛋白质与大鼠p44maPk （ERK1）同源性为98.6%。为了区分克隆cDNA与内源性p44maPk的表达，我们在cDNA的5'端融合了一个启动密码子，后跟一个流感血凝素9-残基肽表位（HAP）。嵌合激酶HAP/p44maPk在巨细胞病毒启动子转录调控下，在中国仓鼠肺成纤维细胞中以功能形式稳定表达。我们发现它的基础活性，通过底物髓鞘碱性蛋白的磷酸化来测量，被丝裂原a-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，我们报告说，在对a-凝血酶的反应中，这种激活是快速的（1分钟内6倍），双相的（第一个峰在5分钟，第二个更宽的峰在1-2小时），持续的（2小时）。4 h)，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明显保留了内源性形式的所有激素调节特征。该系统现在提供了研究结构-功能关系和确定该激酶在生长控制中的作用的可能性。
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 fulllength 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 a-thrombin, platelet-derived growth factor, and fetal calf serum. In addition, we report that in response to a-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 .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.
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胰岛素刺激的微管相关蛋白激酶在体内的酪氨酸和苏氨酸上被磷酸化。
DOI：
10.1073/pnas.85.11.3753
发表时间：
1988
影响因子：
11.1
作者：
Ray,LB;Sturgill,TW
通讯作者：
Sturgill,TW
p42/丝裂原激活蛋白激酶作为不同生长因子信号通路的汇聚靶标：使用百日咳毒素作为区分因素。
DOI：
10.1091/mbc.2.8.675
发表时间：
1991
期刊：
Cell regulation
影响因子：
--
作者：
L'Allemain,G;Pouyssegur,J;Weber,MJ
通讯作者：
Weber,MJ
DOI：
10.1089/dna.1983.2.329
发表时间：
1983-01-01
期刊：
DNA-A JOURNAL OF MOLECULAR & CELLULAR BIOLOGY
影响因子：
--
作者：
CATHALA, G;SAVOURET, JF;BAXTER, JD
通讯作者：
BAXTER, JD
影响因子：
56.9
作者：
BOULTON, TG;YANCOPOULOS, GD;COBB, MH
通讯作者：
COBB, MH
DOI：
10.1073/pnas.82.15.5131
发表时间：
1985-01-01
影响因子：
11.1
作者：
HOUGHTEN, RA
通讯作者：
HOUGHTEN, RA