Prostaglandin F2α stimulates the Raf/MEK1/mitogen-activated protein kinase signaling cascade in bovine luteal cells

Upon binding to its G protein-coupled transmembrane receptors, the actions of PGF(2 alpha) on the corpus luteum are initiated by the phospholipase C/diacylglycerol-inositol 1,4,5-trisphosphate (InsP(3))/Ca2+-protein kinase C (PKC) pathway. However, little is known about the downstream intracellular signaling events that can lead to transcriptional activation in response to PGF(2 alpha). The present study was conducted to examine the involvement of the mitogen-activated protein kinase (MAPK) signaling cascade in the corpus luteum. Three isoforms of the Raf family of oncoprotein kinases (A-Raf, B-Raf, and Raf-1 or c-Raf) were detected in bovine luteal cells. Raf-1 and B-Raf, but not A-Raf, were activated by PGF(2 alpha) (1 mu M) and the pharmacological PKC activator phorbol myristate acetate (PMA, 20 nM). Kinetic analysis revealed that PGF(2 alpha) rapidly and transiently activated Raf-1. In vitro protein kinase assays demonstrated that activation of Raf-1 and B-Raf resulted in the phosphorylation and activation of MAPK kinase (MEK1), which subsequently phosphorylated p42(mapk). As de termined by hyperphosphorylation, tyrosine phosphorylation, and enzymatic activity, p42(mapk) and p44(mapk) were rapidly and transiently activated by both PGF(2 alpha) (1 mu M) and PMA (20 nM). Additionally, both PGF(2 alpha) (1 mu M) and PMA (20 nM) stimulated phosphorylation of Raf-1, MEK1, and p42(mapk) in P-32-labeled cells. Our data demonstrate that PGF(2 alpha) activates the Raf/MEK1/p42/44(mapk) signaling cascade in bovine luteal cells and that the actions of PGF(2 alpha) are mimicked by the PKC activator PMA. Activation of the Raf/MEK1/MAPK signaling cascade by PGF(2 alpha) in luteal cells provides a mechanism to transduce signals initiated by PGF(2 alpha) receptors on the cell surface into the nucleus. Activation of the Raf/MEK1/MAPK signaling cascade may be associated with transcriptional activation of luteal genes possessing activator protein-1-binding sites.