Luteinizing Hormone Receptor Activation in Ovarian Granulosa Cells Promotes Protein Kinase A-Dependent Dephosphorylation of Microtubule-Associated Protein 2D

Abstract

The actions of 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 locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pull-down. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3beta at Ser9 and the PP2A B56delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes

“…Studies of rat granulosa cells in culture have shown that LH activation of G s leads to the PKA-dependent activation of PPP2 and that PPP2 is associated with the MAP2D A-kinase anchoring protein (Flynn et al, 2008). One possible scenario, although not the only one, is that NPR2 could also be associated with MAP2D, such that it might be dephosphorylated by PPP2 in response to LH-G s -PKA signaling.…”

Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells

“…We initially sought to determine whether MAP2D, the majority of which is highly phosphorylated in naive granulosa cells (Flynn et al, 2008), colocalized with the microtubule, microfilament and/or intermediate cytoskeleton in granulosa cells. Untreated fixed granulosa cells appear flattened and fibroblastic with long bundles of phalloidin-stained F-actin (Karlsson et al, 2010).…”

“…MAP2D is the only MAP2 isoform detected in rat granulosa cells (Salvador et al, 2004). The majority of MAP2D in preovulatory granulosa cells is phosphorylated, based on its migration on SDS-PAGE gels at 80 kDa rather than at 70 kDa with dephosphorylated MAP2D (Flynn et al, 2008). Addition to primary granulosa cells of an ovulatory concentration of luteinizing hormone, mimicked by the luteinizing hormone chorionic gonadotropin receptor agonist human choriogonadotropin (hCG), promotes rapid dephosphorylation of MAP2D on Thr256 and Thr259 in a PKA-dependent manner (Flynn et al, 2008).…”

“…The majority of MAP2D in preovulatory granulosa cells is phosphorylated, based on its migration on SDS-PAGE gels at 80 kDa rather than at 70 kDa with dephosphorylated MAP2D (Flynn et al, 2008). Addition to primary granulosa cells of an ovulatory concentration of luteinizing hormone, mimicked by the luteinizing hormone chorionic gonadotropin receptor agonist human choriogonadotropin (hCG), promotes rapid dephosphorylation of MAP2D on Thr256 and Thr259 in a PKA-dependent manner (Flynn et al, 2008). Surprisingly, hCG does not promote phosphorylation of the Ser residue within the KXGS motif of the MTBDs (Flynn et al, 2008), although these Ser residues can be phosphorylated by PKA in vitro using recombinant MAP2D (Flynn et al, 2008) and are recognized PKA targets in other cells (Ozer and Halpain, 2000).…”

Pharmacological Interference With Protein-protein Interactions of Akinase Anchoring Proteins as a Strategy for the Treatment of Disease

“…In addition, AKAPs bind GPCRs [11], ACs [12,13], exchange proteins directly activated by cAMP (Epac) [14], phosphodiesterases (PDEs) [15,16], protein kinases (e.g. protein kinases C, D and N) [17], protein phosphatases such as PP1 [18] and PP2B (calcineurin) [19,20] and ion channels and pumps (e.g. L--type Ca 2+ channels, Na + --Ca 2+…”