Prostaglandin E2, the most abundant prostaglandin produced by tumor cells and by the tumor microenvironment, plays a critical role in regulation of tumor cell functions by activating signaling pathways that ultimately lead to increased tumor growth, proliferation, invasion, and survival. We previously demonstrated a growth regulatory role of PGE2 in the established glioblastoma multiforme cell line U87-MG. Unexpectedly, stimulation of U87-MG cells by PGE2 resulted in inhibition of the ERK 1/2 pathway. The present study was, therefore, undertaken to characterize PGE2-induced inhibition of ERK 1/2 and elucidate the underlying signaling mechanisms. We demonstrate that ERK 1/2 phosphorylation was transiently inhibited by PGE2 in a time-dependent manner. Dephosphorylation of ERK1/2 was detected at 5 min exposure to PGE2, reached maximum levels between 20-180 min and was sustained for 6 h. Levels of phosphorylated ERK 1/2 increased thereafter and at 24-48 h were higher than those found in unstimulated cells. Raf-1 phosphorylation was also transiently inhibited by PGE2. Desphosphorylation of Raf-1 at Ser338 was detected at 10 min exposure to PGE2 and followed thereafter kinetics identical to those of ERK 1/2. Receptor tyrosine kinase dependent- (EGF) and independent (TPA)- phosphorylation of ERK and Raf-1 were also attenuated by PGE2. PGE2-induced dephosphorylation of ERK1/2 and Raf-1 was not unique to U87-MG cells, as it was detected in other glioma cell lines, including T98G, U118-MG, but not in primary astrocytes or brain endothelial cells. Stimulation of U87-MG cells with other prostaglandins, including prostaglandin D2, had no effect on Raf-1 and ERK dephosphorylation. Pretreatment of U87-MG with okadaic acid (OA), an inhibitor of several Ser/Thr phosphatases, including PP2A, PP2B, PP1 and PP5, rescued Raf-1 and ERK dephosphorylation detected at 20 min exposure to PGE2, while an inactive analogue of OA had no effect. In contrast, fostriecin, a selective inhibitor of PP2A and PP1, failed to rescue PGE2-induced ERK 1/2 and Raf-1 dephosphorylation. Sodium orthovanadate, a tyrosine phosphatase inhibitor, had no effect on PGE2-induced ERK 1/2 dephosphorylation. RNAi-mediated silencing of PP5, but not PP2A, and forced expression of the TPR domain of PP5, which acts as a dominant negative PP5 construct, partially rescued PGE2-induced Raf-1 and ERK-dephosphorylation. Taken together, these results demonstrate that PGE2 transiently dephosphorylates Raf-1 and ERK 1/2 via an okadaic acid-sensitive pathway involving activation of PP5 and suggest the potential involvement or cooperation of additional Ser/Thr phosphatases. We speculate that transient inhibition of Raf-1 and ERK 1/2 activation by PGE2 could represent a mechanism by which glioma cells escape apoptosis caused by sustained activation of the ERK 1/2 pathway. (Supported by NIH Grant RO3 NS076765 to M.T.R). Citation Format: Maria Teresa Rizzo, Wilmer Mata-Castro, Yoo Seung Ko, Aaron Cohen-Gadol. Prostaglandin E2 activates an okadaic acid-sensitive Ser/Thr phosphatase that leads to transient inhibition of the ERK 1/2 pathway in glioblastoma multiforme cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4193. doi:10.1158/1538-7445.AM2014-4193