Agents which elevate intracellular cyclic AMP (cAMP) have been shown to promote adipose conversion of preadipocyte cell lines. Modulation of cell growth by cAMP often correlates with either activation or inhibition of the MAP kinase pathway. In light of this, we have investigated the effect of cAMP on the activation of MAP kinases in 3T3F442A pre-adipocytes. The effects of raising intracellular cAMP by treatment of cells with either forskolin, CPTcAMP, which is a cell permeable analogue of cyclic AMP, or the cAMP phosphodiesterase inhibitors isobutylmethylxanthine and RO-201724, were first examined. All agents tested induced an approximate 2-fold increase in MAP kinase enzyme activity in phenyl Sepharose-purified cell extracts. Moreover, immunoblotting of cell extracts revealed that this correlated with the appearance of the hyper-phosphorylated forms of p42 and p44 MAP kinases. Activation of MAP kinase by cAMP was prevented by the structurally distinct inhibitors of cyclic AMP-dependent protein kinase (PKA), H-89 and (Rp)-8BrcAMPS. In addition, PD098059, the specific inhibitor of the MAP kinase kinase MEK1, also prevented activation of MAP kinase by cAMP. These results demonstrate that cAMP exerts a positive influence on the MAP kinase pathway in 3T3-F442A cells which is mediated by PKA. In common with some other cell types in which cAMP activates MAP kinase this effect parallels a potentiating effect on cellular differentiation.
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