The Raf‐1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments

• Published in: The FASEB journal 2000-10, Vol.14 (13), p.2008-2021
• Main Authors: Janosch, Petra, Kieser, Arnd, Eulitz, Manfred, Lovric, Josip, Sauer, Guido, Reichert, Manuela, Gounari, Fotini, Büscher, Dirk, Baccarini, Manuela, Mischak, Harald, Kolch, Walter
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; casein ki‐nase 2; Cells, Cultured; cytoskeleton; Enzyme Activation; Intermediate Filaments - ultrastructure; Molecular Sequence Data; Peptide Mapping; Phosphopeptides - isolation & purification; Phosphorylation; Protein Binding; Protein-Serine-Threonine Kinases - metabolism; Proto-Oncogene Proteins c-raf - metabolism; Raf; Vimentin - metabolism
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.99-0883com

ABSTRACT 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 ki‐nases. 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 poly‐merization 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., Kieser, A., Eulitz, M., Lovric, J., Sauer, G., Reichert, M., Gounari, F., Büscher, D., Baccarini, M., Mischak, H., Kolch, W. The Raf‐1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments. FASEB J. 14, 2008–2021 (2000)