EFFICACY OF AGONIST-STIMULATED MEK ACTIVATION DETERMINES THE SUSCEPTIBILITY OF MITOGEN-ACTIVATED PROTEIN (MAP) KINASE TO INHIBITION IN RAT AORTIC SMOOTH-MUSCLE CELLS

In rat aortic smooth muscle cells, platelet-derived growth factor (PDG F) stimulated a sustained activation of mitogen-activated protein kina se (MAP kinase) while the response to angiotensin II (AII) was transie nt. This was due to a relatively greater initial activation of MAP kin ase kinase (MEK) and a correspondingly greater residual MEK activity a t later time points. Pretreatment of cells with the novel MEK inhibito r PD 098059 reduced MEK activation at 5 min in response to each agonis t by a similar proportion (70%); however, at this time point MAP kinas e activation in response to PDGF was only marginally affected while th e response to AII was substantially reduced. PD 098059 did, however, r educe PDGF-stimulated MK activity after 30 min and this correlated wit h a loss in MAP kinase activity and DNA synthesis. Pretreatment with f orskolin also caused a similar pattern of inhibition of agonist-stimul ated MEK and MAP kinase activity. Only following protein kinase C down -regulation were both AII- and PDGF-stimulated MAP kinase activation s ubstantially reduced and this correlated with the virtual loss of both MEK and c-Raf-1 activity in response to both agents. The differential inhibition of MAP kinase activation by forskolin was not due to speci fic activation of A-Raf by PDGF; both PDGF and AII stimulated A-Raf ki nase and this activity was strongly inhibited by forskolin. These resu lts suggest that the efficacy of MEK activation determines the duratio n of MAP kinase activation and the susceptibility of MAP kinase activa tion to inhibition by different agents. The results also argue against the selective activation of A-Raf by PDGF as a mechanism to explain t he differences in the kinetics of MAP kinase activity stimulated by AI I and PDGF.