Robbins D J, Zhen E, Owaki H, Vanderbilt C A, Ebert D, Geppert T D, Cobb M H
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas.
J Biol Chem. 1993 Mar 5;268(7):5097-106.
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.
细胞外信号调节蛋白激酶（ERK）1和2及其各自的突变体在带有六组氨酸标签的细菌中表达，并通过镍螯合层析进行纯化。野生型ERK2的基础活性约为2 nmol/分钟/毫克。在体外，分子内反应在酪氨酸磷酸化的主要生理位点发生了自催化磷酸化。兔肌肉ERK激活剂将ERK2激活了500 - 1000倍，直至其比活性（约2 μmol/分钟/毫克）接近从受刺激细胞中纯化得到的ERK1的比活性（Boulton, T.G., Gregory, J.S., and Cobb, M.H. (1991) Biochemistry 30, 278 - 286）。ERK1也能被ERK激活剂激活到相同程度。缺乏酪氨酸磷酸化主要位点的突变体自磷酸化速率大幅降低，并且不再被ERK激酶高度激活。缺乏苏氨酸磷酸化主要位点的突变体自磷酸化速率相同或有所增强，但其激酶活性取决于用于取代苏氨酸的残基。用谷氨酸取代能使激酶被ERK激活剂激活，而用丙氨酸取代则不能。因此，谷氨酸的羧基至少能提供一些由磷酸苏氨酸在活化的ERK中引入的特性。
