A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK
A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK
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DOI：
10.1038/nature09860
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发表时间：
2011-04-21
期刊：
影响因子：
64.8
通讯作者：
Barford, David
中科院分区：
文献类型：
--
作者：
Brennan, Damian F.;Dar, Arvin C.;Barford, David
在多细胞动物中，Ras - Raf - MEK（丝裂原活化蛋白激酶激酶） - ERK（细胞外信号调节激酶）信号通路传递细胞外刺激，引发细胞功能和基因表达的变化。通过致癌突变导致该通路的异常激活在很大比例的人类癌症中起作用。Ras激酶抑制因子（KSR）（1 - 3）作为一种必需的支架蛋白发挥作用，协调Raf - MEK - ERK复合物的组装（4,5）。在此，我们综合结构和生化研究来理解KSR如何促进MEK的刺激性Raf磷酸化（参考文献6,7）。我们从人KSR2激酶结构域（KSR2（KD））与兔MEK1复合物的晶体结构中表明，KSR2（KD）和MEK1之间的相互作用是由它们各自的激活片段和C叶αG螺旋介导的。与BRAF（参考文献8,9）类似，KSR2通过一个涉及Arg 718的侧向界面自我结合，Arg 718是在基因筛选中被鉴定为Ras信号抑制因子的一个残基（1 - 3）。ATP结合到KSR2（KD）催化位点，并且我们通过体外实验和化学遗传学证明了KSR2对MEK1的激酶活性。在KSR2（KD） - MEK1复合物中，两种激酶的激活片段相互约束，并且KSR2呈现一种无活性构象。BRAF变构刺激KSR2的激酶活性，这依赖于侧向KSR2 - BRAF异二聚体的形成。此外，KSR2 - BRAF异二聚化通过KSR2介导的从BRAF传递的信号，使MEK的激活片段释放以便磷酸化，从而导致BRAF诱导的MEK磷酸化增加。我们提出，KSR与顺式的调节性Raf分子相互作用，诱导MEK的构象转变，促进MEK被反式的一个单独的催化性Raf分子磷酸化。
In metazoans, the Ras-Raf-MEK (mitogen-activated protein-kinase kinase)-ERK (extracellular signal-regulated kinase) signalling pathway relays extracellular stimuli to elicit changes in cellular function and gene expression. Aberrant activation of this pathway through oncogenic mutations is responsible for a large proportion of human cancer. Kinase suppressor of Ras (KSR)(1-3) functions as an essential scaffolding protein to coordinate the assembly of Raf-MEK-ERK complexes(4,5). Here we integrate structural and biochemical studies to understand how KSR promotes stimulatory Raf phosphorylation of MEK (refs 6, 7). We show, from the crystal structure of the kinase domain of human KSR2 (KSR2(KD)) in complex with rabbit MEK1, that interactions between KSR2(KD) and MEK1 are mediated by their respective activation segments and C-lobe alpha G helices. Analogous to BRAF (refs 8, 9), KSR2 self-associates through a side-to-side interface involving Arg 718, a residue identified in a genetic screen as a suppressor of Ras signalling(1-3). ATP is bound to the KSR2(KD) catalytic site, and we demonstrate KSR2 kinase activity towards MEK1 by in vitro assays and chemical genetics. In the KSR2(KD)-MEK1 complex, the activation segments of both kinases are mutually constrained, and KSR2 adopts an inactive conformation. BRAF allosterically stimulates the kinase activity of KSR2, which is dependent on formation of a side-to-side KSR2-BRAF heterodimer. Furthermore, KSR2-BRAF heterodimerization results in an increase of BRAF-induced MEK phosphorylation via the KSR2-mediated relay of a signal from BRAF to release the activation segment of MEK for phosphorylation. We propose that KSR interacts with a regulatory Raf molecule in cis to induce a conformational switch of MEK, facilitating MEK's phosphorylation by a separate catalytic Raf molecule in trans.