B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts.
Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.
Pritchard
Catrin A
CA
Department of Biochemistry, University of Leicester, United Kingdom. cap8@le.ac.uk
Hayes
Louise
L
Wojnowski
Leszek
L
Zimmer
Andreas
A
Marais
Richard M
RM
Norman
Jim C
JC
D051339:Actin Depolymerizing Factors; D000199:Actins; D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D003599:Cytoskeleton / Q000502:physiology / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000648:ultrastructure*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D054461:Lim Kinases; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D022502:Stress Fibers / Q000502:physiology / Q000648:ultrastructure*; D014162:Transfection; D054460:rho-Associated Kinases
D051339:Actin Depolymerizing Factors; D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D011494:Protein Kinases; C482119:BRAF protein, human; C482120:Braf protein, mouse; C515892:LIMK1 protein, human; D054461:Lim Kinases; C515893:Limk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D054460:rho-Associated Kinases; D020928:Mitogen-Activated Protein Kinases