Phosphorylation of 338SSYY341 regulates specific interaction between Raf-1 and MEK1
Phosphorylation of 338SSYY341 regulates specific interaction between Raf-1 and MEK1
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DOI：
10.1074/jbc.m203953200
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发表时间：
2002-11-22
影响因子：
4.8
通讯作者：
Luo, ZJ
中科院分区：
文献类型：
--
作者：
Xiang, XQ;Zang, MW;Luo, ZJ
本研究表征Raf-1激酶结构域和MEK 1之间的相互作用，并检查其相互作用的大小是否与Raf磷酸化MEK 1的能力相关。在这里，我们表明Raf激酶活性所需的最小结构域从色氨酸342开始。Raf激酶结构域与MEK 1的最大结合及其激酶活性是在响应4 β-12-O-十四烷酰基佛波醇-13-乙酸酯（TPA）的区域（SSYY 341）-S-338磷酸化或Y340 Y341突变为天冬氨酸后实现的。相反，当该区域缺失或Ser（338）和Ser（339）突变为丙氨酸时，TPA刺激的MEK结合和激酶活性降低。我们还表明，完整的Raf ATP结合位点是必要的Raf-1和MEK 1之间的相互作用。此外，还鉴定了两个MEK结合位点;第一个位于氨基酸325和349之间，第二个位于氨基酸350和648之间的区域内。单独地，每个位点与MEK 1的结合是弱的，但在顺式情况下，它们产生更强的关联，这可以被TPA进一步刺激。最后，我们发现，色氨酸342，这是保守的Raf家族和其他蛋白激酶，是必不可少的丝氨酸（338）磷酸化的全长Raf和它的结合MEK 1。综上所述，我们的结果表明Raf上Ser（338）和Tyr（341）的磷酸化对这两个MEK结合位点的重构产生重要影响。因此，这两个位点协调形成高亲和力MEK结合表位，导致Raf激酶活性显著增加。
The present study characterizes the interaction between the Raf-1 kinase domain and MEK1 and examines whether the magnitude of their interaction correlates to the ability of Raf to phosphorylate MEK1. Here we show that the minimal domain required for the Raf kinase activity starts from tryptophan 342. Maximal binding of the Raf kinase domain to MEK1 and its kinase activity are achieved upon phosphorylation of the region (SSYY341)-S-338 in response to 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA), or mutation of Y340Y341 to aspartic acids. Conversely, the TPA-stimulated MEK binding and kinase activity are diminished when this region is deleted or Ser(338) and Ser(339) are mutated to alanines. We also show that the integrity of the Raf ATP-binding site is necessary for the interaction between Raf-1 and MEK1. Furthermore, two MEK-binding sites are identified; the first is localized between amino acids 325 and 349, and the second is within the region between amino acids 350 and 648. Separately, the binding of each site to MEK1 is weak, but in a cis context, they give rise to a much stronger association, which can be further stimulated by TPA. Finally, we find that tryptophan 342, which is conserved among the Raf family and other protein kinases, is essential for the Ser(338) phosphorylation of the full-length Raf and its binding to MEK1. Taken together, our results indicate that the phosphorylation of Ser(338) and Tyr(341) on Raf exerts an important effect on reconfiguring the two MEK-binding sites. As a result, these two sites coordinate to form a high affinity MEK-binding epitope, leading to a marked increase in Raf kinase activity.