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Reconstitution and characterization of BRAF in complex with 14-3-3 and KRAS4B on nanodiscs.
- Source :
-
Protein science : a publication of the Protein Society [Protein Sci] 2024 Jun; Vol. 33 (6), pp. e5016. - Publication Year :
- 2024
-
Abstract
- RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.<br /> (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)
- Subjects :
- Humans
Lipid Bilayers chemistry
Lipid Bilayers metabolism
Protein Binding
Protein Multimerization
14-3-3 Proteins metabolism
14-3-3 Proteins chemistry
14-3-3 Proteins genetics
Nanostructures chemistry
Proto-Oncogene Proteins B-raf chemistry
Proto-Oncogene Proteins B-raf metabolism
Proto-Oncogene Proteins B-raf genetics
Proto-Oncogene Proteins p21(ras) chemistry
Proto-Oncogene Proteins p21(ras) metabolism
Proto-Oncogene Proteins p21(ras) genetics
Cell-Free System
Subjects
Details
- Language :
- English
- ISSN :
- 1469-896X
- Volume :
- 33
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Protein science : a publication of the Protein Society
- Publication Type :
- Academic Journal
- Accession number :
- 38747381
- Full Text :
- https://doi.org/10.1002/pro.5016