The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments.
Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.
Janosch
P
P
The Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, U.K. pjanosch@beatson.gla.ac.uk
Kieser
A
A
Eulitz
M
M
Lovric
J
J
Sauer
G
G
Reichert
M
M
Gounari
F
F
Büscher
D
D
Baccarini
M
M
Mischak
H
H
Kolch
W
W
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004789:Enzyme Activation; D007382:Intermediate Filaments / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D014746:Vimentin / Q000378:metabolism*
D010748:Phosphopeptides; D014746:Vimentin; C101571:vimentin kinase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf