Identification of Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities and for MEK binding.
Identification of Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities and for MEK binding.
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DOI：
10.1091/mbc.e05-02-0090
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发表时间：
2005-08
影响因子：
3.3
通讯作者：
Jun Zhu;V. Balan;Agnieszka Bronisz;Karina Balan;Hengrui Sun;Deborah T Leicht;Zhijun Luo;J. Qin;J. Avruch;G. Tzivion
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文献类型：
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作者：
Jun Zhu;V. Balan;Agnieszka Bronisz;Karina Balan;Hengrui Sun;Deborah T Leicht;Zhijun Luo;J. Qin;J. Avruch;G. Tzivion
Ras-Raf-MAPK级联信号通路是一条重要的生长信号通路，其失控激活导致细胞转化。虽然沿级联信号传递的一般特征已经被合理地定义，但Raf激活的机制仍然不完全清楚。在这里，我们表明，Raf-1去磷酸化，主要是在表皮生长因子(EGF)诱导的部位，取消了Raf-1激酶的活性。利用质谱学方法，我们在体内鉴定了5个新的Raf-1磷酸化位点，其中S471位于Raf-1激酶域的VIB亚区。突变分析表明，Raf-1 S471对Raf-1的活性及其与丝裂原激活的蛋白激酶(MEK)的相互作用至关重要。同样，相应的B-Raf位点S578的突变导致了一个失活的激酶，这表明激活Raf-1和B-Raf需要相同的Raf-1和B-Raf磷酸化。重要的是，自然发生的与癌症相关的B-Raf激活突变V599E抑制了S578A突变，这表明在该区域引入带电残基消除了激活磷酸化的需要。我们的结果证明了特定的EGF诱导的Raf-1磷酸化位点在Raf-1激活中起重要作用，确定Raf-1 S471是一个新的对Raf-1和B-Raf激酶活性至关重要的磷酸化位点，并指出V599E突变可能通过模拟S578位点的磷酸化来激活B-Raf。
The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.