Ras- and Raf-induced down-modulation of non-muscle tropomyosin are MEK-independent.
Transformation is accompanied by the down-regulation of the high molecular weight isoforms of non-muscle tropomyosin. Several lines of evidence suggest that tropomyosin down-regulation may be essential for ras-induced tumorigenicity. It is unclear which of the many signaling pathways downstream of Ras are involved in tropomyosin down-regulation. Here we demonstrate that Raf activation induces tropomyosin down-regulation comparable to that induced by Ras. Expression of the effector-domain mutant Ras-G12V,Y40C, which is unable to bind Raf, induced only modest down-modulation of tropomyosin. Treatment with the MEK-specific inhibitor PD98059 had little effect on tropomyosin levels in ras- or raf-transformed cells. In contrast, a mutant form of MEK-1, MEK-1-S218A,S222A, restored tropomyosin levels in ras-transformed NIH3T3 cells almost to the levels observed in non-transformed cells. MEK-1-S218A,S222A does not inhibit MEK phosphorylation and is a poor inhibitor of ERK phosphorylation. These data suggest that this mutant form of MEK-1 interferes with a yet uncharacterized pathway controlled by Raf. We conclude that the ras-induced down-modulation of tropomyosin is predominantly Raf-mediated, but MEK-independent, and that a novel pathway exists downstream of Raf which may play an important role in regulation of the cytoskeleton.
Janssen
R A
RA
Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Massachusetts, Boston 02115, USA.
Veenstra
K G
KG
Jonasch
P
P
Jonasch
E
E
Mier
J W
JW
D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002461:Cell Line, Transformed; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D011905:Genes, ras; D048369:MAP Kinase Kinase 1; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D027384:Oncogene Proteins v-raf; D009857:Oncogenes; D010766:Phosphorylation; D017354:Point Mutation; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection; D014335:Tropomyosin / Q000096:biosynthesis / Q000235:genetics*
D004791:Enzyme Inhibitors; D005419:Flavonoids; D020033:Protein Isoforms; D011994:Recombinant Proteins; D015682:Retroviridae Proteins, Oncogenic; D014335:Tropomyosin; D011505:Protein-Tyrosine Kinases; D027384:Oncogene Proteins v-raf; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one