TY - JOUR
T1 - A novel phosphorylation site involved in dissociating RAF kinase from the scaffolding protein 14-3-3 and disrupting RAF dimerization
AU - Yu, Alison
AU - Nguyen, Duc Huy
AU - Nguyen, Thomas Joseph
AU - Wang, Zhihong
N1 - Publisher Copyright:
© 2023 The Authors
PY - 2023/10
Y1 - 2023/10
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85171740707&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85171740707&partnerID=8YFLogxK
U2 - 10.1016/j.jbc.2023.105188
DO - 10.1016/j.jbc.2023.105188
M3 - Article
C2 - 37625591
AN - SCOPUS:85171740707
SN - 0021-9258
VL - 299
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
M1 - 105188
ER -