Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro.
Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro.
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DOI：
10.1016/s0021-9258(18)53507-9
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发表时间：
1993-03
期刊：
影响因子：
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通讯作者：
D. Robbins;E. Zhen;H. Owaki;C. Vanderbilt;D. Ebert;T. Geppert;M. Cobb
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文献类型：
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作者：
D. Robbins;E. Zhen;H. Owaki;C. Vanderbilt;D. Ebert;T. Geppert;M. Cobb
细胞外信号调节蛋白激酶（ERK）1和2和突变体的每一个在细菌中表达的六聚组氨酸标签，并使用镍螯合层析纯化。野生型ERK 2的基础活性约为2 nmol/min/mg。自催化磷酸化发生在体外酪氨酸磷酸化的主要生理位点上的分子内反应。兔肌肉ERK激活剂激活ERK 2 500-1000倍，达到接近从刺激细胞纯化的ERK 1的比活性（约2 μ mol/min/mg）（Boulton，T.G.，格雷戈里，J.S.，和Cobb，M.H.（1991）Biochemistry 30，278-286）。ERK 1也可以被ERK激活剂激活到相同的程度。缺乏酪氨酸磷酸化主要位点的突变体以大大降低的速率自磷酸化，并且不再被ERK激酶高度激活。缺乏苏氨酸磷酸化的主要位点的突变体以相同或增强的速率自磷酸化，但这些突变体的激酶活性取决于用于取代苏氨酸的残基。谷氨酸的替代使得激酶能够被ERK激活剂激活，而丙氨酸的替代则不能。因此，谷氨酸的羧基可以提供至少一些由磷酸苏氨酸在活化的ERK中引入的特征。
Extracellular signal-regulated protein kinases (ERK) 1 and 2 and mutants of each were expressed in bacteria with a hexahistidine tag and purified using nickel-chelate chromatography. Basal activity of wild type ERK2 was approximately 2 nmol/min/mg. Self-catalyzed phosphorylation occurred in vitro on the major physiological site of tyrosine phosphorylation in an intramolecular reaction. Rabbit muscle ERK activator activated ERK2 500-1000-fold up to a specific activity (approximately 2 mumol/min/mg) approximating that of ERK1 purified from stimulated cells (Boulton, T.G., Gregory, J.S., and Cobb, M.H. (1991) Biochemistry 30, 278-286). ERK1 could also be activated by the ERK activator to the same extent. Mutants lacking the major site of tyrosine phosphorylation were autophosphorylated at a greatly reduced rate and were no longer highly activated by the ERK kinase. Mutants lacking the major site of threonine phosphorylation were autophosphorylated at the same or an enhanced rate, but the kinase activity of these mutants depended on the residue used to replace the threonine. Replacement by glutamate rendered the kinase capable of being activated by ERK activator, while replacement by alanine did not. Thus, the carboxyl group of glutamate can provide at least some of the features introduced by phosphothreonine in activated ERKs.