Regulation of Raf by Akt controls growth and differentiation in vascular smooth muscle cells.
The stimulation of platelet-derived growth factor (PDGF) receptors shifts vascular smooth muscle (VSM) cells toward a more proliferative phenotype. Thrombin activates the same signaling cascades in VSM cells, namely the Ras/Raf/MEK/ERK and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways. Nonetheless, thrombin was not mitogenic, but rather increased the expression of the smooth muscle-specific myosin heavy chain (SM-MHC) indicative of an in vitro re-differentiation of VSM cells. A more detailed analysis of the temporal pattern and relative signal intensities revealed marked differences. The strong and biphasic phosphorylation of ERK1/2 in response to thrombin correlated with its ability to increase the activity of the SM-MHC promoter whereas Akt was only partially and transiently phosphorylated. By contrast, PDGF, a potent mitogen in VSM cells, induced a short-lived ERK1/2 phosphorylation but a complete and sustained phosphorylation of Akt. The phosphorylated form of Akt physically interacted with Raf. Moreover, Akt phosphorylated Raf at Ser(259), resulting in a reduced Raf kinase activity and a termination of MEK and ERK1/2 phosphorylation. Disruption of the PI 3-kinase signaling prevented the PDGF-induced Akt and Raf-Ser(259) phosphorylation. Under these conditions, PDGF elicited a more sustained MEK and ERK phosphorylation and increased SM-MHC promoter activity. Consistently, in cells that express dominant negative Akt, PDGF increased SM-MHC promoter activity. Furthermore, expression of constitutively active Akt blocked the thrombin-stimulated SM-MHC promoter activity. Thus, we present evidence that the balance and cross-regulation between the PI 3-kinase/Akt and Ras/Raf/MEK signaling cascades determine the temporal pattern of ERK1/2 phosphorylation and may thereby guide the phenotypic modulation of vascular smooth muscle cells.
Reusch
H P
HP
Institut für Klinische Pharmakologie und Toxikologie, Freie Universität Berlin, Garystr. 5, 14195 Berlin, Germany. reusch@medizin.fu-berlin.de
Zimmermann
S
S
Schaefer
M
M
Paul
M
M
Moelling
K
K
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D015151:Immunoblotting; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D010641:Phenotype; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D002867:Chromones; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D009025:Morpholines; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012694:Serine; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin