PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT in ovarian granulosa cells

Abstract

Controlled maturation of ovarian follicles is necessary for fertility. Follicles are restrained at an immature stage until stimulated by FSH secreted by pituitary gonadotropes. FSH acts on granulosa cells within the immature follicle to inhibit apoptosis, promote proliferation, stimulate production of steroid and protein hormones, and induce ligand receptors and signaling intermediates. The phosphoinositide 3-kinase (PI3K)/AKT (protein kinase B) pathway is a pivotal signaling corridor necessary for transducing the FSH signal. We report that protein kinase A (PKA) mediates the actions of FSH by signaling through multiple targets to activate PI3K/AKT. PKA uses a route that promotes phosphorylation of insulin receptor substrate-1 (IRS-1) on Tyr 989, a canonical binding site for the 85-kDa regulatory subunit of PI3K that allosterically activates the catalytic subunit. PI3K activation leads to activation of AKT through phosphorylation of AKT on Thr 308 and Ser 473. The adaptor growth factor receptor bound protein 2-associated binding protein 2 (GAB2) is present in a preformed complex with PI3K heterodimer and IRS-1, it is an A-kinase anchoring protein that binds the type I regulatory subunit of PKA, and it is phosphorylated by PKA on Ser 159. Overexpression of GAB2 enhances FSHstimulated AKT phosphorylation. GAB2, thus, seems to coordinate signals from the FSH-stimulated rise in cAMP that leads to activation of PI3K/AKT. The ability of PKA to commandeer IRS-1 and GAB2, adaptors that normally integrate receptor/nonreceptor tyrosine kinase signaling into PI3K/AKT, reveals a previously unrecognized route for PKA to activate a pathway that promotes proliferation, inhibits apoptosis, enhances translation, and initiates differentiation of granulosa cells. F ertility in females requires controlled maturation of the oocyte and supporting theca and granulosa cells (GCs) that comprise the ovarian follicle. Follicles are restrained at the preantral stage until they are stimulated by FSH synthesized and secreted from pituitary gonadotropes. FSH directs GCs to proliferate and produce steroid hormones, such as estrogen and progesterone, protein hormones, including inhibin, and growth factors, such as VEGF. These hormones and growth factors not only regulate oocyte maturation and support the growth and differentiation of follicles but also regulate uterine receptivity and provide feedback to the hypothalamus and pituitary (reviewed in ref. 1). In response to FSH, follicles develop to a mature preovulatory stage competent to receive the surge of luteinizing hormone (LH) that promotes ovulation and terminal differentiation of GCs and theca cells to luteal cells.FSH signals through its surface G protein-coupled receptor (GPCR) localized to GCs (2). A crucial pathway by which FSH signals is the PI3K pathway that leads to the phosphorylation and activation of the nodal kinase AKT (protein kinase B). Studies using dominant negative and constitutively active AKT showed that the AKT pathway is necessary but not sufficient for activation of...

Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation

“…3D and 5A). 3 As IGF-1 concentration exceeds endogenous levels and becomes sufficient to stimulate IRS1/PI3K/AKT activation, FSH via PKA continues to enhance IRS1 Y*MXM phosphorylation with parallel activation of PI3K/ AKT (see Fig. 4).…”

“…Following reverse transduction of GCs with PKA-CQR or control EGFP, 3 GC suspension was seeded on calf serum-coated plates in serum-free M199 medium containing 30 ng/ml testosterone. Plated GCs were then treated without or with FSH, as indicated, and collected for Western blotting analysis.…”

“…However, the mechanisms by which GPCRs signal to activate PI3K are much less understood compared with classical activation by receptor tyrosine kinases (1). We have used rat ovarian granulosa cells (GCs) as a model to elucidate the mechanism by which the GPCR agonist folliclestimulating hormone (FSH) activates PI3K, based on prior evidence that FSH activates PI3K in a protein kinase A (PKA)-dependent manner (3).…”

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

“…As casein kinase 2␣ is a constitutively active kinase (71), it does not appear to be a PKA-regulated candidate to inhibit MKP3(DUPS6) in GCs. Although FSH activates PI3K in a PKA-dependent manner (30,58), phosphorylation of YB-1(Ser 102 ) downstream of ERK and RSK-2 is not inhibited by the PI3K inhibitor wortmannin (5). These results thus indicate that PKA activation of the PI3K pathway in GCs is not the mechanism by which PKA inactivates MKP3(DUPS6).…”

“…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).…”