Butch E R, Guan K L
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, 48109-0606, USA.
J Biol Chem. 1996 Feb 23;271(8):4230-5. doi: 10.1074/jbc.271.8.4230.
To discern MEK1 and MEK2 specificity for their substrate, extracellular signal-regulated kinase (ERK), site-directed mutagenesis was performed on the amino acid residues flanking the regulatory phosphorylation sites of ERK1. These ERK1 mutants were analyzed for the ability to act as a substrate for MEK1 and MEK2. Based on both phosphorylation and activation analyses, the mutants could be divided into four classes: 1) dramatically decreased phosphorylation and activation, 2) enhanced basal kinase activity, 3) preferentially enhanced phosphorylation of tyrosine and decreased phosphorylation of threonine, and 4) increased threonine phosphorylation with an increase in activation. In general, the residues proximal to the regulatory phosphorylation sites of ERK1 had greater influence on both phosphorylation and activation. This is consistent with the highly specific recognition of the ERK1 regulatory sites by MEK. Mutation of Arg-208 or Thr-207 to an alanine residue significantly altered the relative phosphorylation on Thr-202 and Tyr-204. The Arg-208 to alanine mutant increased the phosphorylation of Tyr-204 approximately 4-fold yet almost completely eliminated the phosphorylation on Thr-202. In contrast, mutation of Gly-199 to alanine resulted in an increased phosphorylation of Thr-202 relative to Tyr-204. This suggests that both Gly-199 and Arg-208 play important roles in determining the relative phosphorylation of Thr-202 and Tyr-204. Our results demonstrate that residues in the phosphorylation lip of ERK play an important role in the recognition and phosphorylation by MEK.
为了辨别丝裂原活化蛋白激酶激酶1（MEK1）和丝裂原活化蛋白激酶激酶2（MEK2）对其底物细胞外信号调节激酶（ERK）的特异性，对ERK1调节性磷酸化位点两侧的氨基酸残基进行了定点诱变。分析了这些ERK1突变体作为MEK1和MEK2底物的能力。基于磷酸化和活化分析，突变体可分为四类：1）磷酸化和活化显著降低；2）基础激酶活性增强；3）酪氨酸磷酸化优先增强而苏氨酸磷酸化降低；4）苏氨酸磷酸化增加且活化增强。一般来说，ERK1调节性磷酸化位点近端的残基对磷酸化和活化的影响更大。这与MEK对ERK1调节位点的高度特异性识别一致。将精氨酸-208（Arg-208）或苏氨酸-207（Thr-207）突变为丙氨酸残基会显著改变苏氨酸-202（Thr-202）和酪氨酸-204（Tyr-204）上的相对磷酸化。精氨酸-208突变为丙氨酸的突变体使酪氨酸-204的磷酸化增加了约4倍，但几乎完全消除了苏氨酸-202上的磷酸化。相反，甘氨酸-199（Gly-199）突变为丙氨酸导致苏氨酸-202相对于酪氨酸-204的磷酸化增加。这表明甘氨酸-199和精氨酸-208在决定苏氨酸-202和酪氨酸-204的相对磷酸化中都起重要作用。我们的结果表明，ERK磷酸化环中的残基在MEK的识别和磷酸化中起重要作用。
