Extracellular Signal-regulated Kinase (ERK)-dependent Phosphorylation of Y-Box-binding Protein 1 (YB-1) Enhances Gene Expression in Granulosa Cells in Response to Follicle-stimulating Hormone (FSH)

Abstract

Within the ovarian follicle, immature oocytes are surrounded and supported by granulosa cells (GCs). Stimulation of GCs by FSH leads to their proliferation and differentiation, events that are necessary for fertility. FSH activates multiple signaling pathways to regulate genes necessary for follicular maturation. Herein, we investigated the role of Y-box-binding protein-1 (YB-1) within GCs. YB-1 is a nucleic acid binding protein that regulates transcription and translation. Our results show that FSH promotes an increase in the phosphorylation of YB-1 on Ser 102 within 15 min that is maintained at significantly increased levels until ϳ8 h post treatment. FSH-stimulated phosphorylation of YB-1(Ser 102 ) is prevented by pretreatment of GCs with the PKA-selective inhibitor PKA inhibitor (PKI), the MEK inhibitor PD98059, or the ribosomal S6 kinase-2 (RSK-2) inhibitor BI-D1870. Thus, phosphorylation of YB-1 on Ser 102 is PKA-, ERK-, and RSK-2-dependent. However, pretreatment of GCs with the protein phosphatase 1 (PP1) inhibitor tautomycin increased phosphorylation of YB-1(Ser 102 ) in the absence of FSH; FSH did not further increase YB-1(Ser 102 ) phosphorylation. This result suggests that the major effect of RSK-2 is to inhibit PP1 rather than to directly phosphorylate YB-1 on Ser 102 . YB-1 coimmunoprecipitated with PP1␤ catalytic subunit and RSK-2. Transduction of GCs with the dephospho-adenoviral-YB-1(S102A) mutant prevented the induction by FSH of Egfr, Cyp19a1, Inha, Lhcgr, Cyp11a1, Hsd17b1, and Pappa mRNAs and estradiol-17␤ production. Collectively, our results reveal that phosphorylation of YB-1 on Ser 102 via the ERK/ RSK-2 signaling pathway is necessary for FSH-mediated expression of target genes required for maturation of follicles to a preovulatory phenotype.In the female, fertility requires maturation of the ovarian follicle that contains the oocyte surrounded by granulosa cells (GCs) 2 and theca cells. Follicular maturation is a tightly regulated process that is initiated by FSH. FSH regulates at least 500 target genes within GCs whose expression drives development of the follicle, allowing it to respond to the surge of luteinizing hormone (LH) that promotes ovulation, oocyte maturation, and formation of the corpus luteum that serves to support the developing embryo after fertilization and implantation (for review, see Refs. 1 and 2). The mechanism by which FSH signals to regulate gene and protein expression in GCs has been extensively investigated. FSH binds to its G protein-coupled receptor (GPCR) expressed exclusively on GCs to activate adenylyl cyclase, raise intracellular cAMP levels, and activate PKA (3-6). PKA then either directly phosphorylates proteins that regulate transcription or indirectly activates signaling cascades whose targets regulate primarily transcription and translation. Direct PKA targets in GCs include cAMP-response element binding protein (CREB) (Ser 133 ) (7), histone H3 (Ser 10 ) (8), and ␤-catenin (Ser 552 and Ser 675 ) (9). Upon phosphorylation, these direct PKA target pro...

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

“…Collectively, these results indicate that FSH, via PKA, regulates at least two established RTK signaling cascades through regulation of phosphatases, seemingly hijacking these cascades. FSH, via PKA, also seems to enhance the transcriptional events by promoting the phosphorylation of at least one transcriptional factor via the inhibition of a phosphatase (5). Together, these results indicate that, at least in GCs, PKA either directly phosphorylates target proteins or enhances the phosphorylation of key proteins in RTK pathways by regulating the activity of protein phosphatases.…”

“…Immunoprecipitation-Immunoprecipitations were performed as described previously (5). Inputs represent ϳ2% and bound sample ϳ98% of total sample.…”

“…The ability of ERK to promote phosphorylation/activation of the transcriptional factor YB1 is based on a prior publication (5). The ability of PKA to sensitize IRS1 to the tyrosine kinase activity of the insulin-like growth factor 1 receptor (IGF 1 R) is also based on prior publications (30,58).…”

“…Although it is well recognized that the ERK signaling pathway plays a predominant role in the ovulatory response to luteinizing hormone in preovulatory GCs (4), the functional significance of the ERK signaling pathway in immature GCs is less understood. We recently showed that the ERK signaling pathway in immature GCs is required for the induction of at least a subset of FSH gene targets that define the mature preovulatory GC, including Inha (which encodes the ␣ subunit of the hormone inhibin), Lhcgr (which encodes the luteinizing hormone receptor), Egfr (which encodes the EGF receptor (EGFR) required for ovulation), and Cyp19a1 (which encodes the rate-limiting enzyme in estrogen biosynthesis) (5). ERK-dependent gene expression in immature GCs is mediated in part by phosphorylation of the transcriptional activator Y-box binding protein 1 (YB-1) on Ser 102 (5).…”

“…We recently showed that the ERK signaling pathway in immature GCs is required for the induction of at least a subset of FSH gene targets that define the mature preovulatory GC, including Inha (which encodes the ␣ subunit of the hormone inhibin), Lhcgr (which encodes the luteinizing hormone receptor), Egfr (which encodes the EGF receptor (EGFR) required for ovulation), and Cyp19a1 (which encodes the rate-limiting enzyme in estrogen biosynthesis) (5). ERK-dependent gene expression in immature GCs is mediated in part by phosphorylation of the transcriptional activator Y-box binding protein 1 (YB-1) on Ser 102 (5). Based on the relevance of the ERK signaling pathway to immature GC maturation, we sought to better understand the mechanism by which FSH activates ERK.…”