MEK-1 PHOSPHORYLATION BY MEK KINASE, RAF, AND MITOGEN-ACTIVATED PROTEIN-KINASE - ANALYSIS OF PHOSPHOPEPTIDES AND REGULATION OF ACTIVITY
MEK-1 PHOSPHORYLATION BY MEK KINASE, RAF, AND MITOGEN-ACTIVATED PROTEIN-KINASE - ANALYSIS OF PHOSPHOPEPTIDES AND REGULATION OF ACTIVITY
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DOI：
10.1091/mbc.5.2.193
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发表时间：
1994-02-01
影响因子：
3.3
通讯作者：
JOHNSON, GL
中科院分区：
文献类型：
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作者：
GARDNER, AM;VAILLANCOURT, RR;JOHNSON, GL
MEK-1 是一种苏氨酸和酪氨酸双重识别激酶，可磷酸化并激活丝裂原激活蛋白激酶 (MAPK)。 MEK-1 又被磷酸化激活。 Raf 和 MAPK/细胞外信号调节激酶激酶 (MEKK) 独立磷酸化和激活 MEK-1。重组 MEK-1 也能够自动激活。纯化的重组野生型 MEK-1 和突变激酶失活 MEK-1 用作 MEKK、Raf 和自磷酸化的底物。由 Raf、MEKK 或自磷酸化催化的 MEK-1 磷酸化导致 MEK-1 激酶活性的激活，通过突变激酶失活 MAPK 的磷酸化来测量。磷酸氨基酸分析和肽图分析在 MEK 激酶、Raf 或 MEK-1 自磷酸化磷酸化后鉴定出类似的 MEK-1 胰蛋白酶磷酸肽。 MEK-1 在与 Raf 和 MEKK 不同的位点被 MAPK 磷酸化。 MAPK 对 MEK-1 的磷酸化不会影响 MEK-1 激酶活性。用表皮生长因子刺激后，从 P-32 标记的细胞中免疫沉淀 MEK-1 中存在的几个磷酸化位点与体外磷酸化酶相同。 MEK-1 中 MAPK 磷酸化的主要位点是苏氨酸 292。苏氨酸 292 突变为丙氨酸消除了 90% 的 MAPK 催化的 MEK-1 磷酸化，但不影响 MEK-1 活性。结果表明，MEKK和Raf通过共同残基的磷酸化来调节MEK-1活性，因此，两个独立的蛋白激酶在MEK-1处汇聚来调节MAPK的活性。
MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in tum activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from P-32-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.