Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes
Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes
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DOI：
10.1038/s41586-019-1660-y
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发表时间：
2019-11-21
期刊：
影响因子：
64.8
通讯作者：
Eck, Michael J.
中科院分区：
文献类型：
--
作者：
Park, Eunyoung;Rawson, Shaun;Eck, Michael J.
RAF家族激酶是ras激活的开关，通过MAP激酶级联启动信号传导，控制细胞增殖、分化和存活（1-3）。RAF活性受到严格调控，不适当的激活是癌症的常见原因（4-6）；然而，目前对RAF监管的结构基础了解甚少。在这里，我们使用冷冻电子显微镜来确定与MEK1和14-3-3二聚体配合物的全长BRAF的自抑制和活性结构。重建结果显示，在14-3-3二聚体形成的摇篮中抑制了一个无活性的BRAF- mek1复合物，该复合物结合了BRAF激酶结构域两侧磷酸化的S365和S729位点。BRAF富含半胱氨酸的结构域占据稳定该组装的中心位置，但邻近的ras结合结构域秩序不佳且处于外围。14-3-3摇篮通过隔离膜结合的富含半胱氨酸的结构域和阻断BRAF激酶结构域的二聚化来维持自身抑制。在活性状态下，这些抑制相互作用被释放，单个14-3-3二聚体重排以桥接两个BRAF的c端pS729结合位点，从而驱动一个活性的背对背BRAF二聚体的形成。我们的结构快照为理解正常的RAF调控及其在癌症和发育综合征中的突变破坏提供了基础。
RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival(1-3). RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer(4-6); however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.