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 FSH-stimulated 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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

FSH-stimulated phosphorylation of AKT on Thr308 and Ser473 is mediated by PKA. (A) GCs were treated for indicated times without (time 0) or with 50 ng/mL FSH. After heat denaturation in SDS sample buffer and SDS/PAGE, a blot of total cell extracts was sequentially probed with indicated antibodies. Separate panels reflect results with the same samples run on different gels (hence, distinct loading controls). Results are representative of three independent experiments. The rest of the conditions are described in Materials and Methods. (B) GCs were treated for 15 min with vehicle (DMEM; lane 1), FSH (lane 2), 0.25% ethanol/DMEM (lane 3), or 10 μM forskolin in 0.25% ethanol/DMEM (lane 4). The rest of the conditions are described in A. Results are representative of four independent experiments. (C) GCs were treated without or with FSH or with indicated concentrations of 8-CPT-cAMP or 8-CPT-2-O-Me-cAMP for 1 h. Dotted lines between lanes represent cropped images. Results are representative of four independent experiments. (D) GCs were pretreated with vehicle (water) or 50 μM Myr-PKI for 1 h followed by treatment without or with FSH for 1 h. FSH-stimulated phosphorylations of AKT(Ser473), IRS-1(Tyr989), and GAB2(Ser159) were inhibited by Myr-PKI [93.8 ± 2.4% (n = 4), 88.0 ± 10.8% (n = 2), and 95.3 ± 2.2% (n = 2), respectively]. (E) GCs were transduced overnight with Ad-GFP (5.8 × 103 OPU/cell) or PKI (1.8 × 104 OPU/cell) that expresses the full-length PKI protein. Cells were then treated 15 min without or with FSH. FSH-stimulated phosphorylations of AKT(Ser473), GAB2(Tyr452), and AKT (Thr308) were inhibited by PKI [97.0 ± 2.3% (n = 5), 96.9 ± 3.2% (n = 4), and 100 ± 0% (n = 4), respectively]. The rest of the conditions are described in A.

Fig. 2.

The PI3K inhibitor wortmannin blocks FSH-stimulated phosphorylations of AKT(Ser308) and (Thr473). GCs were pretreated with vehicle (DMSO) or 100 nM wortmannin for 1 h and then treated without or with FSH for 15 min. FSH-stimulated phosphorylations of AKT(Ser473), CREB(Ser133), and AKT(Thr308) were inhibited [98.1 ± 2.2% (n = 4), 5.2 ± 2.9% (n = 3), and 96.6 ± 2.5% (n = 3), respectively] by wortmannin. The rest of the conditions are described in Fig. 1A.

Fig. 3.

FSH regulates the phosphorylation of YXXM binding motifs for p85 R-subunit on both GAB2 and IRS-1. (A) GCs were treated without or with FSH or 50 ng/mL IGF1 for 1 h. Blot of total cell extracts was probed with indicated antibodies. Molecular weight markers are shown only for binding motif and total GAB2, and the latter shows a characteristic upshift that is, in part, attributable to phosphorylation on Ser159. Results are representative of two independent experiments. Equivalent results for vehicle and FSH treatments were seen in three additional experiments. Full p-YXXM blot is show in Fig. S3. (B) GCs were transduced overnight with Ad-LacZ (1.6 × 105 OPU/cell) or WT GAB2 (5.4 × 105 OPU/cell). Cells were then treated for 15 min without or with FSH and probed for indicated antibodies. The rest of conditions are described in Fig. 1A. Results are representative of three independent experiments. (C) GCs were treated with vehicle, FSH, or IGF1 for 10 min or 1 h. Cells were scraped into lysis buffer and sonicated, and insoluble particulate was removed by centrifugation. Soluble extracts were incubated with anti–IRS-1 agarose-conjugated antibody. Immunoprecipitated (bound) and input fractions were probed with indicated antibodies. Results are representative of three independent experiments. (D) GCs were treated without or with FSH for 1 h. Soluble extracts were subjected to immunoprecipitation with anti–IRS-1 agarose-conjugated antibody, which was described in B. Input and unbound (flow-through) fractions were probed with indicated antibodies. Results are representative of two independent experiments.

Fig. 4.

GAB2, IRS-1, and PI3K are present in GCs in a preformed complex. (A) GCs were treated without or with FSH for 15 min, scraped into lysis buffer, and sonicated, and particulate was removed by centrifugation. After taking corresponding input aliquots, soluble extracts were precleared and incubated with control IgG or anti-GAB2 antibody overnight in the presence of protein A/G PLUS agarose. Blots of input (3% of total sample) and proteins bound to washed agarose beads (immunoprecipitated; 97% of total sample) were probed with indicated antibodies. Results are representative of three independent experiments. Depletion of GAB2 in unbound fractions (>90%) from a separate experiment is shown in Fig. S4A. (B) GCs were treated without or with FSH for 15 min; soluble precleared extracts were subjected to immunoprecipitation with IgG or anti–p85-PI3K agarose-conjugated antibodies. Results are representative of three independent experiments. In Lower, unbound fractions from a separate experiment were probed with indicated antibodies. (C) GCs were treated without or with FSH for 1 h or 30 min (Lower); soluble precleared extracts were subjected to immunoprecipitation with IgG or anti–IRS-1 agarose–conjugated antibodies. Results are representative of two independent experiments. Depletion of IRS-1 in unbound fractions (∼80%) is shown in Fig. S4B. (D) GCs were treated without or with FSH for 15 min; soluble precleared extracts were subjected to immunoprecipitation with anti–IRS-1 agarose–conjugated antibodies. Results are representative of 10 independent experiments.

Fig. 5.

GAB2 is an RI AKAP. (A) GCs were treated without or with FSH for 30 min; soluble precleared lysates were subjected to immunoprecipitation with IgG or anti-RI antibody. Results are representative of two independent experiments. (B) GCs were treated without or with FSH for 30 min; soluble precleared lysates were subjected to immunoprecipitation with anti-GAB2 antibody. Proteins separated by PAGE were transferred to PVDF membrane. The top portion of the membrane was incubated with purified RIα, which was described in Materials and Methods, and then, it was subjected to RI Western blotting; the lower portion of the membrane was subjected directly to RI Western blotting. The arrow marks the migration position for GAB2. Results for the RI far Western assay are representative of seven independent experiments; results of the RI Western assay are representative of five independent experiments. (C) GCs were pretreated for 2 h with 50 μM sHt31 or sHt31-P or 100 μM RIAD or RSCR, and then, they were treated without or with FSH for 10 min. The rest of the conditions are described in Fig. 1A. Results are representative of two and three independent experiments, respectively.

Fig. 6.

GAB2 is phosphorylated on Ser159 by PKA. (A) GCs were treated without or with FSH for 30 min; soluble precleared lysates were subjected to immunoprecipitation with IgG or antiphospho-PKA substrate (p-PKAsub) antibody. Results are representative of two independent experiments. (B) Soluble precleared lysates from untreated GCs were subjected to immunoprecipitation with anti-GAB2 antibody. Washed immunoprecipitates were then subjected to an in vitro phosphorylation assay in the absence or presence of the catalytic subunit of PKA (PKAC), which was described in Materials and Methods. Results are representative of three independent experiments. (C) Phosphorylations of GAB2(Ser159) and AKT(Ser473) (Fig. 1A) are plotted as a percent of the maximal signal (means ± SEMs; n = 3 for 1-, 5-, and 30-min points; n = 2 for 15-min point; n = 1 for 10-min point). (D) GCs were treated without or with FSH for 30 min; soluble precleared lysates were subjected to immunoprecipitation with IgG or anti-p-GAB2(Ser159) antibody. Two independent experiments are shown. Results are representative of four independent experiments. (E) GCs were treated without or with FSH for 30 min; soluble precleared lysates were subjected to immunoprecipitation with IgG or anti-p85 R-subunit antibodies. Results are representative of three independent experiments.

Fig. 7.

Model of the signaling pathway by which FSH promotes AKT activation in GCs. FSH binding to and activation of the FSH GPCR leads to activation of the stimulatory G protein (Gs), activation of adenylyl cyclase (AC), generation of cAMP, and activation of the type I PKA holoenzyme (RI2C2). PKA directly (unbroken line) or indirectly (broken line) promotes dephosphorylation (slash-P) of GAB2(Tyr452) and phosphorylation (P) of GAB2(Ser159) and IRS-1(Tyr989). Phosphorylation of IRS-1 on Tyr989 provides the binding site for the p85 R-subunit of PI3K to allosterically activate the p110 catalytic subunit, resulting in phosphorylation-dependent activation of AKT(Thr308)(Ser473) that promotes GC proliferation and differentiation.

Fig. P1.

Model of the signaling pathway by which FSH promotes AKT activation in GCs. FSH binding to and activation of the FSH G protein-coupled receptor leads to activation of the stimulatory G protein (Gs), activation of adenylyl cyclase (AC), generation of cAMP, and activation of type I PKA (RI2C2). PKA directly (unbroken line) or indirectly (broken line) promotes dephosphorylation (slashed P) of GAB2(Y452) and phosphorylation (P) of GAB2(Ser159) and IRS-1(Tyr989). Phosphorylation of IRS-1 on Tyr989 provides the binding site for the p85 R-subunit of PI3K to allosterically activate the p110 catalytic subunit, resulting in phosphorylation-dependent activation of AKT(Thr308)(Ser473) that promotes GC proliferation and differentiation.