Luteinizing hormone receptor activation in ovarian granulosa cells promotes protein kinase A-dependent dephosphorylation of microtubule-associated protein 2D.
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.
Flynn
Maxfield P
MP
Department of Cell and Molecular Biology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA.
Maizels
Evelyn T
ET
Karlsson
Amelia B
AB
McAvoy
Thomas
T
Ahn
Jung-Hyuck
JH
Nairn
Angus C
AC
Hunzicker-Dunn
Mary
M
D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005260:Female; D006107:Granulosa Cells / Q000166:cytology*; D006801:Humans; D007986:Luteinizing Hormone / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D010053:Ovary / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011974:Receptors, LH / Q000378:metabolism*; D015398:Signal Transduction
D008869:Microtubule-Associated Proteins; D011974:Receptors, LH; D007986:Luteinizing Hormone; D017868:Cyclic AMP-Dependent Protein Kinases