Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling
Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling
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DOI：
10.1073/pnas.1318188110
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发表时间：
2013-11-12
影响因子：
11.1
通讯作者：
Chu, Steven
中科院分区：
文献类型：
--
作者：
Nan, Xiaolin;Collisson, Eric A.;Chu, Steven
RAF丝氨酸/苏氨酸激酶通过MAPK通路调节细胞生长，并且在人类癌症中是小分子RAF抑制剂（RAFis）的作用靶点。现在很明显，蛋白质多聚体在RAF激活以及肿瘤对RAFis的反应中起重要作用。然而，由于缺乏对其进行可视化的技术，这些多聚体的确切化学计量和细胞定位仍不清楚。在本研究中，我们证明光激活定位显微镜（PALM）与定量空间分析相结合，提供了足够的分辨率以直接可视化细胞中的蛋白质多聚体。定量PALM成像显示，在静息条件下，CRAF主要以细胞质单体形式存在，但在有活性RAS存在时，在细胞膜上会形成二聚体以及三聚体和四聚体。相比之下，缺失自身抑制结构域的N末端截短的CRAF（CatC）在细胞质中形成组成型二聚体，偶尔形成四聚体，而具有破坏的CRAF - CRAF二聚体界面的CatC突变体则不会。最后，通过与RAS CAAX基序融合人为地将CRAF强制定位到膜上会诱导多聚体形成，但只有当二聚体界面完整时才会激活RAF/MAPK。总之，这些定量结果直接证实了RAF二聚体以及可能的高阶多聚体的存在及其在细胞信号传导中的参与，并表明RAF多聚体的形成可由多种机制导致，并且是RAF激活的关键但非充分步骤。
The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF-CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.