Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 293, Issue 37, Pages 14276-14284Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.004597
Keywords
cancer; cancer therapy; cell signaling; drug resistance; cancer biology; 14-3-3; AMP-activated protein kinase; paradoxical activation; RAF inhibitor; RAF kinase
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Funding
- National Cancer Centre Singapore Research Fund (NCCRF) Startup Grant NCCRF-SUG-JH
- National Cancer Centre Singapore Research Fund (NCCRF) Bridging Grant [NCCRF-YR2016-JUL-BG1]
- National Cancer Centre Singapore Research Fund (NCCRF) Pilot/Exploratory Project [NCCRF-YR2017-JULPG3]
- Duke-NUS Khoo Bridge Funding Award [Duke-NUS-KBrFA/2017/0003]
- SingHealth Foundation Startup Grant [SHF/FG692S/2016]
- Asia Fund for Cancer Research Grant [AFCR2017/2019-JH]
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The dimerization-driven paradoxical activation of RAF proto-oncogene Ser/Thr kinase (RAF) is the predominant cause of drug resistance and toxicity in cancer therapies with RAF inhibitors. The scaffold protein 14-3-3, which binds to the RAF C terminus, is essential for RAF activation under physiological conditions, but the molecular basis is unclear. Here we investigated whether and how 14-3-3 regulates the dimerization-driven transactivation of the RAF isoform CRAF by RAF inhibitors and affects drug resistance and toxicity by virtue of the dominant role of CRAF in these processes. We demonstrated that 14-3-3 enhances the dimerization-driven transactivation of CRAF by stabilizing CRAF dimers. Further, we identified AMP-activated protein kinase (AMPK) and CRAF itself as two putative kinases that redundantly phosphorylate CRAF's C terminus and thereby control its association with 14-3-3. Next, we determined whether the combinatory inhibition of AMPK and CRAF could overcome the paradoxical effect of RAF inhibitors. We found that the AMPK inhibitor (AMPKi) not only blocked the RAF inhibitor-driven paradoxical activation of ERK signaling and cellular overgrowth in Ras-mutated cancer cells by blocking phosphorylation of Ser-621 in CRAF but also reduced the formation of drug-resistant clones of BRAF(V600E)-mutated cancer cells. Last, we investigated whether 14-3-3 binding to the C terminus of CRAF is required for CRAF catalytic activity and observed that it was dispensable in vivo. Altogether, our study unravels the molecular mechanism by which 14-3-3 regulates dimerization-driven RAF activation and identified AMPKi as a potential agent to counteract drug resistance and adverse effects of RAF inhibitors in cancer therapies.
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