Wan Paul T C, Garnett Mathew J, Roe S Mark, Lee Sharlene, Niculescu-Duvaz Dan, Good Valerie M, Jones C Michael, Marshall Christopher J, Springer Caroline J, Barford David, Marais Richard
Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK.
Cell. 2004 Mar 19;116(6):855-67. doi: 10.1016/s0092-8674(04)00215-6.
Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.
已鉴定出30多种与人类癌症相关的B-RAF基因突变，其中大多数位于激酶结构域内。在此我们表明，在所分析的22种B-RAF突变体中，有18种在体内具有升高的激酶活性并向ERK发出信号。令人惊讶的是，三种突变体在体外对MEK的激酶活性降低，但通过在体内激活C-RAF，在细胞中向ERK发出信号。野生型和致癌性V599E B-RAF激酶结构域与RAF抑制剂BAY43-9006复合的结构表明，激活片段通过与P环结合而保持在无活性构象中。大多数突变聚集在这两个区域表明，这种相互作用的破坏将B-RAF转化为其活性构象。高活性突变体通过直接磷酸化MEK向ERK发出信号，而活性受损的突变体通过激活内源性C-RAF来刺激MEK，可能是通过变构或转磷酸化机制。
