Jang Dong Man, Boxer Kayla, Ha Byung Hak, Tkacik Emre, Levitz Talya, Rawson Shaun, Metivier Rebecca J, Schmoker Anna, Jeon Hyesung, Eck Michael J
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.
Nat Commun. 2025 Sep 1;16(1):8150. doi: 10.1038/s41467-025-63227-2.
CRAF (RAF1) is one of three RAF-family kinases that initiate MAP kinase signaling in response to activated RAS and is essential for oncogenic signaling from mutant KRAS. Like BRAF, CRAF is regulated by 14-3-3 engagement and by intramolecular autoinhibitory interactions of its N-terminal regulatory region. Unlike BRAF, it is thought to require tyrosine phosphorylation in its N-terminal acidic (NtA) motif for full catalytic activation. Here we describe cryo-EM reconstructions of full-length CRAF in complex with MEK1 and a 14-3-3 dimer. These structures reveal a fully autoinhibited conformation analogous to that observed for BRAF and two "open monomer" states in which the inhibitory interactions of the CRD and 14-3-3 dimer are released or rearranged, but the kinase domain remains inactive. Structure-function studies of the NtA motif indicate that phosphorylation or acidic mutations in this segment increase catalytic activity by destabilizing the inactive conformation of the kinase domain. Collectively, these studies provide a structural foundation for understanding the shared and unique regulatory features of CRAF and will inform efforts to selectively block CRAF signaling in cancer.
CRAF（RAF1）是三种RAF家族激酶之一，可响应激活的RAS启动丝裂原活化蛋白激酶（MAP激酶）信号传导，并且对于来自突变型KRAS的致癌信号传导至关重要。与BRAF一样，CRAF受14-3-3结合以及其N端调节区域的分子内自抑制相互作用调控。与BRAF不同的是，人们认为它需要其N端酸性（NtA）基序中的酪氨酸磷酸化才能实现完全催化激活。在此，我们描述了全长CRAF与MEK1和一个14-3-3二聚体复合物的冷冻电镜重建结构。这些结构揭示了一种类似于BRAF中观察到的完全自抑制构象，以及两种“开放单体”状态——在这两种状态下，CRD和14-3-3二聚体的抑制性相互作用被释放或重新排列，但激酶结构域仍处于无活性状态。对NtA基序的结构-功能研究表明，该片段中的磷酸化或酸性突变通过破坏激酶结构域的无活性构象来增加催化活性。总的来说，这些研究为理解CRAF的共同和独特调节特征提供了结构基础，并将为在癌症中选择性阻断CRAF信号传导的研究提供信息。
