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

Alison Yu; Duc Huy Nguyen; Thomas Joseph Nguyen; Zhihong Wang

doi:10.1016/j.jbc.2023.105188

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

J Biol Chem. 2023 Oct;299(10):105188. doi: 10.1016/j.jbc.2023.105188. Epub 2023 Aug 23.

Authors

Alison Yu¹, Duc Huy Nguyen¹, Thomas Joseph Nguyen¹, Zhihong Wang²

Affiliations

¹ Department of Chemistry & Biochemistry, Rowan University, Glassboro, New Jersey, USA.
² Department of Chemistry & Biochemistry, Rowan University, Glassboro, New Jersey, USA. Electronic address: wangz@rowan.edu.

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, BRAF^S732 (equivalent in CRAF^S624), 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 BRAF^pS729 (CRAF^pS621) and enhances RAF dimerization. We conducted mutational analysis of BRAF^S732A/E and CRAF^S624A/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 BRAF^S732 and CRAF^S624 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.

Keywords: 14-3-3; ATP-competitive inhibitor; RAF kinase; cancer; dimerization; kinase signaling; paradoxical activation; phosphorylation; regulatory mechanism; scaffolding protein.

Abstract

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