Related Experiment Video
Updated: Jun 25, 2026

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells
Published on: June 30, 2021
A-RAF kinase functions in ARF6 regulated endocytic membrane traffic
Elena Nekhoroshkova1, Stefan Albert, Matthias Becker
1Institut für Medizinische Strahlenkunde und Zellforschung, University of Würzburg, Würzburg, Germany.
Background:
RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation.
Methodology/Principal Findings:
Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment.
Conclusions/Significance:
A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.
More Related Videos
06:44Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells
Published on: March 1, 2024
07:49Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
Published on: July 17, 2019
Related Concept Videos
MAPK Signaling Cascades
Rab Cascades
Rab Proteins
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
Small GTPases - Ras and Rho
Three regulatory proteins control their activity:
Recycling Endosomes and Transcytosis
The recycling endosome is not a single organelle but an extensively tubulated network of recycling pathways. It functions in storing molecules or transporting them across...
The Ras Gene
Ras is a superfamily...