Regulation of Raf by Akt controls growth and differentiation in vascular smooth muscle cells

The stimulation of platelet-derived growth factor (PDGF) receptors shifts v ascular smooth muscle (VSM) cells toward a more proliferative phenotype. Th rombin activates the same signaling cascades in VSM cells, namely the Ras/R af/MEK/ERK and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways . Nonetheless, thrombin was not mitogenic, but rather increased the express ion 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 t he temporal pattern and relative signal intensities revealed marked differe nces. The strong and biphasic phosphorylation of ERK1/2 in response to thro mbin correlated with its ability to increase the activity of the SM-MHC: pr omoter whereas Akt was only partially and transiently phosphorylated. By co ntrast, PDGF, a potent mitogen in VSM cells, induced a short-lived ERK1/2 p hosphorylation but a complete and sustained phosphorylation of Akt. The pho sphorylated form of Akt physically interacted with Raf. Moreover, Akt phosp horylated 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-kina se signaling prevented the PDGF-induced Akt and Raf-Ser(259) phosphorylatio n. Under these conditions, PDGF elicited a more sustained MEK and ERK phosp horylation and increased SM-MHC promoter activity. Consistently, in cells t hat 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 bal ance and cross-regulation between the PI 3-kinase/Akt and Ras/Raf/MEK signa ling cascades determine the temporal pattern of ERK1/2 phosphorylation and may thereby guide the phenotypic modulation of vascular smooth muscle cells .