AKT-independent phosphorylation of TSC2 and activation of mTOR and ribosomal protein S6 kinase signaling by prostaglandin F2α

Prostaglandin F2 alpha (PGF2 alpha) is an important mediator of corpus luteum (CL) regression, although the cellular signaling events that mediate this process have not been clearly identified. It is established that PGF2 alpha binds to a G-protein-coupled receptor (GPCR) to stimulate protein kinase C (PKC) and Raf-MEK-Erk signaling in luteal cells. The present experiments were performed to determine whether PGF2 alpha stimulates the mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase 1 (S6K1) signaling pathway in steroidogenic luteal cells. We demonstrate that PGF2 alpha treatment results in a time- and concentration-dependent stimulation of the phosphorylation and activation of S6K1. The stimulation of S6K1 in response to PGF2 alpha treatment was abolished by the mTOR inhibitor rapamycin. Treatment with PGF2 alpha did not increase AKT phosphorylation but increased the phosphorylation of Erk and the tumor suppressor protein tuberous sclerosis complex 2 (TSC2), an upstream regulator of mTOR. The effects of PGF2 alpha were mimicked by the PKC activator PMA and inhibited by U0126, a MEK1 inhibitor. The activation of mTOR/S6K1 and putative down stream processes involving the translational apparatus (i.e. 4EBP1 phosphorylation, release of 4EBP1 binding in m(7)G cap binding assays, and the phosphorylation and synthesis of S6) were completely sensitive to treatment with rapamycin, implicating mTOR in the actions of PGF2 alpha. Taken together, our data suggest that GPCR activation in response to PGF2 alpha stimulates the mTOR pathway which increases the translational machinery in luteal cells. The translation of proteins under the control of mTOR may have implications for luteal development and regression and offer new strategies for therapeutic intervention in PGF2 alpha-target tissues.