A novel phosphorylation site involved in dissociating RAF kinase from the scaffolding protein 14-3-3 and disrupting RAF dimerization.

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-23 DOI:10.1016/j.jbc.2023.105188
Alison Yu, Duc Nguyen, Thomas Joseph Nguyen, Zhihong Wang
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Abstract

Rapidly accelerated fibrosarcoma (ARAF, BRAF, CRAF) kinase is central to the MAPK pathway (RAS-RAF-MEK-ERK). Inactive RAF kinase is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane via RAS-GTP, leading to the relief of autoinhibition, phosphorylation of key regulatory sites, and dimerization of RAF protomers. Although it is well known that active and inactive BRAF have differential phosphorylation sites that play a crucial role in regulating BRAF, key details are still missing. In this study, we report the characterization of a novel phosphorylation site, BRAFS732 (equivalent in CRAFS624), located in proximity to the C-terminus binding motif for the 14-3-3 scaffolding protein. At the C terminus, 14-3-3 binds to BRAFpS729 (CRAFpS621) and enhances RAF dimerization. We conducted mutational analysis of BRAFS732A/E and CRAFS624A/E and revealed that the phosphomimetic S→E mutant decreases 14-3-3 association and RAF dimerization. In normal cell signaling, dimerized RAF phosphorylates MEK1/2, which is observed in the phospho-deficient S→A mutant. Our results suggest that phosphorylation and dephosphorylation of this site fine-tune the association of 14-3-3 and RAF dimerization, ultimately impacting MEK phosphorylation. We further characterized the BRAF homodimer and BRAF:CRAF heterodimer and identified a correlation between phosphorylation of this site with drug sensitivity. Our work reveals a novel negative regulatory role for phosphorylation of BRAFS732 and CRAFS624 in decreasing 14-3-3 association, dimerization, and MEK phosphorylation. These findings provide insight into the regulation of the MAPK pathway and may have implications for cancers driven by mutations in the pathway.

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一种新的磷酸化位点,参与从支架蛋白14-3-3中解离RAF激酶并破坏RAF二聚化。
快速加速纤维肉瘤(ARAF、BRAF、CRAF)激酶是MAPK通路(RAS-RAF-MEK-ERK)的核心。非活性RAF激酶被认为是单体的、自动抑制的和胞质的,而活化的RAF通过RAS-GTP募集到膜上,导致自动抑制的缓解、关键调控位点的磷酸化和RAF原聚体的二聚化。尽管众所周知,活性和非活性BRAF具有差异磷酸化位点,在调节BRAF中起着至关重要的作用,但关键细节仍然缺失。在本研究中,我们报道了一个新的磷酸化位点BRAFS732(相当于CRAFS624)的特征,该位点位于14-3-3支架蛋白的C末端结合基序附近。在C末端,14-3-3与BRAFpS729(CRAFpS621)结合并增强RAF二聚化。我们对BRAFS732A/E和CRAFS624A/E进行了突变分析,发现→E突变体降低14-3-3缔合和RAF二聚化。在正常细胞信号传导中,二聚化的RAF磷酸化MEK1/2,这在磷酸缺乏的S中观察到→突变体。我们的结果表明,该位点的磷酸化和去磷酸化微调了14-3-3和RAF二聚化的结合,最终影响MEK磷酸化。我们进一步鉴定了BRAF同源二聚体和BRAF:CRAF异二聚体,并确定了该位点的磷酸化与药物敏感性之间的相关性。我们的工作揭示了BRAFS732和CRAFS624磷酸化在减少14-3-3结合、二聚化和MEK磷酸化中的新的负调控作用。这些发现提供了对MAPK途径调控的深入了解,并可能对由该途径突变驱动的癌症具有启示。
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