Follicle-Stimulating Hormone (FSH)-dependent Regulation of Extracellular Regulated Kinase (ERK) Phosphorylation by the Mitogen-activated Protein (MAP) Kinase Phosphatase MKP3

Abstract

Within the ovarian follicle, granulosa cells (GCs) surround and support immature oocytes. FSH promotes the differentiation and proliferation of GCs and is essential for fertility. We recently reported that ERK activation is necessary for FSH to induce key genes that define the preovulatory GC. This research focused on the phosphoregulation by FSH of ERK within GCs. FSH-stimulated ERK phosphorylation on Thr(202)/Tyr(204) was PKA-dependent, but MEK(Ser(217)/Ser(221)) phosphorylation was not regulated; rather, MEK was already active. However, treatment of GCs with the EGF receptor inhibitor AG1478, a dominant-negative RAS, an Src homology 2 domain-containing Tyr phosphatase inhibitor (NSC 87877), or the MEK inhibitor PD98059 blocked FSH-dependent ERK(Thr(202)/Tyr(204)) phosphorylation, demonstrating the requirement for upstream pathway components. We hypothesized that FSH via PKA enhances ERK phosphorylation by inhibiting the activity of a protein phosphatase that constitutively dephosphorylates ERK in the absence of FSH, allowing MEK-phosphorylated ERK to accumulate in the presence of FSH because of inactivation of the phosphatase. GCs treated with different phosphatase inhibitors permitted elimination of both Ser/Thr and Tyr phosphatases and implicated dual specificity phosphatases (DUSPs) in the dephosphorylation of ERK. Treatment with MAP kinase phosphatase (MKP3, DUSP6) inhibitors increased ERK(Thr(202)/Tyr(204)) phosphorylation in the absence of FSH to levels comparable with ERK phosphorylated in the presence of FSH. ERK co-immunoprecipitated with Myc-FLAG-tagged MKP3(DUSP6). GCs treated with MKP3(DUSP6) inhibitors blocked and PKA inhibitors enhanced dephosphorylation of recombinant ERK2-GST in an in vitro phosphatase assay. Together, these results suggest that FSH-stimulated ERK activation in GCs requires the PKA-dependent inactivation of MKP3(DUSP6).