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Activation of the epidermal growth factor (EGF) receptor (EGFR) at the cell surface initiates signaling through the RAS-RAF-MAPK/ERK1/2 pathway and receptor endocytosis. Whether this signaling continues from endosomes remains unclear, because RAS is predominantly located on the plasma membrane, and the localization of endogenous RAF kinases, downstream effectors of RAS, is not defined. To examine RAF localization, we labeled endogenous RAF1 with mVenus using gene editing. From 10 to 15% of RAF1-mVenus (<2000 molecules/cell), which was initially entirely cytosolic, transiently translocated to the plasma membrane after EGF stimulation. Following an early burst of translocation, the membrane-associated RAF1-mVenus was undetectable by microscopy or subcellular fractionation, and this pool was estimated to be <200 molecules per cell. In contrast, persistent EGF-dependent translocation of RAF1-mVenus to the plasma membrane was driven by the RAF inhibitor sorafenib, which increases the affinity of Ras-GTP:RAF1 interactions. RAF1-mVenus was not found in EGFR-containing endosomes under any conditions. Computational modeling of RAF1 dynamics revealed that RAF1 membrane abundance is controlled most prominently by association and dissociation rates from RAS-GTP and by RAS-GTP concentration. The model further suggested that the relatively protracted activation of the RAF-MEK1/2-ERK1/2 module, in comparison with RAF1 membrane localization, may involve multiple rounds of cytosolic RAF1 rebinding to active RAS at the membrane.
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This content will become publicly available on December 1, 2026
Ras/Raf dimerization model for activation of Raf kinase
Not AvailableOur previously proposed Ras dimerization model is consistent with recent details observed by NMR in that Raf activation is centered on the Ras/Raf dimer, distinct from one in which Ras activates Raf as a monomer with the Raf cysteine rich domain inserted in the membrane. We review mechanistic understanding of Raf activation within nanoclusters of Ras on the membrane, with a shift to dimers upon binding Raf. This sets the stage for a signaling platform composed of Ras/Raf and Galectin dimers that facilitates the release of Raf autoinhibition and folding of the Raf intrinsically disordered region between the Ras-binding domains and the kinase bound to 14-3-3 and MEK. This platform could provide synchronized units for signal amplification and is consistent with a Ras stationary phase observed in cells.
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- Award ID(s):
- 2121426
- PAR ID:
- 10654681
- Publisher / Repository:
- ELSEVIER
- Date Published:
- Journal Name:
- Current Opinion in Structural Biology
- Volume:
- 95
- Issue:
- C
- ISSN:
- 0959-440X
- Page Range / eLocation ID:
- 103150
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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