Protein kinase A directly phosphorylates GSK3β, and regulates β-catenin via phosphorylation in granulosa cells

Estradiol serves an important role in female fertility and FSH drives estradiol production. Beta-catenin is a transcriptional co-factor that is required for FSH-induced estradiol production. Beta-catenin is activated via phosphorylation at Ser552 and Ser675 by protein kinase A (PKA) dependent event. Wingless-type mouse mammary tumor virus (WNT) also regulates the β-catenin pathway. Glycogen Synthase Kinase-3 β (GSK3β) is a component of the β-catenin degradation complex; canonical WNT signaling pathway phosphorylates protein kinase B (AKT) and inhibits GSK3β resulting in β-catenin accumulation in the cytoplasm. Work in our laboratory demonstrated that WNT downregulates steroidogenesis. Furthermore, AKT is required for β-catenin accumulation and FSH-induced estradiol production, suggesting convergence of the WNT and FSH pathways. We hypothesize that WNT inhibition of FSH signaling occurs through modulation of phosphorylation patterns on β-catenin. The objective of these experiments was to evaluate the phosphorylation pattern of β-catenin in response to PKA, AKT, and WNT signaling pathways. Granulosa cells (KGN cell line) were cultured and treated with vehicle control, phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (LY) (30 μ M; 30 min), Forskolin (FSK) (10 μ M; 1.5 h), WNT (50 ng/mL, 30 min) and the combination of these treatments. Western blot was used to detect total and phosphorylated β-catenin and phosphorylated GSK3β. Protein abundances were analyzed using densitometry software and densitometry values for treatments were analyzed using the GLM procedure of SAS. When significant model interactions were detected, means were separated using PDIFF. Treatment of FSK combined with WNT and LY enhanced GSK3β phosphorylation compared to control (P < 0.05). Similarly, FSK alone or in combination with WNT and LY enhanced (P < 0.05) phosphorylation of β-catenin at Ser675 and Ser552, but WNT did not alter β-catenin phosphorylation after FSK stimulation (P > 0.10). However, when the AKT pathway was blocked with LY, FSK, and WNT treatment increased phosphorylation of GSK3β and subsequently β-catenin at Ser552 suggesting that PKA can directly phosphorylate GSK3β. Results from these experiments demonstrate that WNT does not alter FSH-stimulated phosphorylation patterns of β-catenin indicating that WNT inhibits FSH signaling via other unknown mechanisms.