The actions of luteinizing hormone (LH) to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific cellular locations by A-kinase anchoring proteins (AKAPs). I previously showed that follicle-stimulating hormone (FSH) induces expression of the AKAP microtubule-associated protein (MAP) 2D and that MAP2D co-immunoprecipitates with PKA regulatory subunits in rat granulosa cells. Here I describe a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with LH receptor agonist hCG. This event is mimicked by treatment with forskolin or a cAMP analog and blocked by the PKA inhibitor myristoylated-PKI, indicating a role for PKA signaling in phospho-regulation of granulosa cell MAP2D. I show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase (PP) 2A inhibitor okadaic acid and demonstrate interactions between MAP2D and PP2A by co-immunoprecipitation and microcystin-agarose pulldown. I also show that MAP2D interacts with glycogen synthase kinase (GSK) 3β and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3β at Ser9 and PP2A B56δ subunit at Ser566 is observed after treatment with hCG, corresponding to LH receptor-mediated inhibition of GSK3β and activation of PP2A, respectively. MAP2D dephosphorylated at Thr256/Thr259 appears to redistribute into a vimentin-enriched cell fraction coincident with hCG-stimulated phosphorylation of vimentin on two PKA sites (Ser38 and Ser72). I show that MAP2D is localized to vimentin filaments and microtubules in granulosa cells and that LH receptor activation induces remodeling of the vimentin cytoskeleton. MAP2D binds directly to immobilized vimentin protein in overlay assays and this binding is diminished by GSK3β phosphorylation of MAP2D in vitro. These results are consistent with the hypothesis that LH-stimulated dephosphorylation of MAP2D may facilitate the progesterone biosynthesis that is obligatory for fertility by altering intermediate filament dynamics.

Last modified

• 10/03/2018

Creator

• Maxfield Patrick Flynn

DOI

• https://doi.org/10.21985/N2H743

Subject

• Integrated Graduate Program in the Life Sciences

Keyword

• cyloskeleton
• MAP2
• vimentin
• ovary
• granulosa cell
• luteinizing hormone

Date created

• 2008-08-11

Resource type

• Dissertation

Rights statement

• In Copyright