Localization dynamics of endogenous fluorescently labeled RAF1 in EGF-stimulated cells

Area of Science:

• Cell biology
• Molecular signaling
• Signal transduction pathways

Background:

• Epidermal growth factor (EGF) receptor (EGFR) activation triggers intracellular signaling cascades, including the RAS-RAF-MAPK/ERK1/2 pathway.
• The precise localization and dynamics of RAF kinases, key downstream effectors of RAS, during EGFR signaling, particularly from endosomes, remain poorly understood.

Purpose of the Study:

• To investigate the subcellular localization and dynamics of endogenous RAF1 during EGF-stimulated signaling.
• To determine if RAF1 participates in signaling originating from endosomes.
• To elucidate the factors controlling RAF1 membrane association.

Main Methods:

• Gene editing to label endogenous RAF1 with mVenus.
• Microscopy and subcellular fractionation to track RAF1-mVenus localization.
• EGF stimulation and treatment with the RAF inhibitor sorafenib.
• Computational modeling of RAF1 dynamics.

Main Results:

• Following EGF stimulation, a small fraction (10-15%) of cytosolic RAF1-mVenus transiently translocated to the plasma membrane.
• The membrane-associated RAF1 pool was transient and rapidly diminished (<200 molecules/cell).
• RAF1-mVenus was not detected in EGFR-containing endosomes under any experimental conditions.
• Sorafenib promoted persistent EGF-dependent RAF1-mVenus translocation by enhancing Ras-GTP:RAF1 interactions.
• Computational modeling indicated RAF1 membrane abundance is primarily regulated by RAS-GTP binding kinetics and concentration.

Conclusions:

• RAF1's plasma membrane association is transient after EGF stimulation and does not occur within EGFR-containing endosomes.
• The concentration and binding dynamics with RAS-GTP are critical determinants of RAF1 membrane localization.
• Prolonged RAF-MEK1/2-ERK1/2 pathway activation may involve repeated cycles of RAF1 rebinding to RAS-GTP at the plasma membrane.