Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.
The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.
Ramocki
M B
MB
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA.
Johnson
S E
SE
White
M A
MA
Ashendel
C L
CL
Konieczny
S F
SF
Taparowsky
E J
EJ
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D048369:MAP Kinase Kinase 1; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D018482:Muscle, Skeletal / Q000166:cytology*; D017570:MyoD Protein / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D015398:Signal Transduction; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins
D004791:Enzyme Inhibitors; D005419:Flavonoids; D017570:MyoD Protein; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one