NORTHWESTERN UNIVERSITY Luteinizing Hormone Receptor Signaling Regulates MAP2D Phosphorylation in Preovulatory Granulosa Cells A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS for the degree DOCTOR OF PHILOSOPHY Field of Cell and Molecular Biology Integrated Graduate Program in Life Sciences By Maxfield Patrick Flynn CHICAGO, ILLINOIS December 2008 2 © Copyright by Maxfield P. Flynn 2007 All Rights Reserved 3 ABSTRACT Luteinizing Hormone Receptor Signaling Regulates MAP2D Phosphorylation in Preovulatory Granulosa Cells Maxfield P. Flynn 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 4 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. 5 ACKNOWLEDGEMENTS I would like to express my profound gratitude to a number of people who supported me during this work. First, I thank my mentor, Dr. Mary Hunzicker-Dunn, who has provided me invaluable support, guidance, scientific insight, and enthusiasm through all of my time in her lab. I am equally grateful to Dr. Evelyn T. Maizels, who has become a mentor for me in life as well as science, providing encouragement and advice in even the most difficult circumstances. I thank the members of our lab, past and present, Dr. Youngkyu Park, Dr. Carl Peters, Zach Feiger, and, in particular, Dr. Hena Alam, who taught me much by example with her wisdom and perseverance, and Amelia Karlsson, who has been a knowledgeable colleague and a dear friend. I would also like to thank my committee members, Dr. Lester Binder, Dr. Adriana Ferreira, and Dr. Kelly Mayo, for their mentorship, guidance, and advice. Additionally, I thank Dr. Daniel Carr and Sarah Fiedler, who performed the recombinant MAP2D solid phase overlay assays and provided advice on AKAP signaling, Dr. Robert Goldman and Dr. Ying-hao Chou, who provided advice and reagents for vimentin research, Dr. Angus Nairn, Dr. Jung-Hyuck Ahn, and Dr. Thomas McAvoy, who contributed PP2A B56δ antibodies and provided advice on PP2A signaling, and Dr. Teng-Leong Chew at the Northwestern Cell Imaging Facility for microscopy training and assistance. I would like to acknowledge the Endocrinology Training Grant and the Medical Scientist Training Program at Northwestern University for financial support. Last, I would like to acknowledge that this work relies heavily on the sacrifice of many research animals and hope that each one of us will continue to respect and advocate for the humane treatment of the animals that make much of our work possible. 6 LIST OF ABBREVIATIONS AKAP, A-kinase anchoring protein C, PKA catalytic subunit CaMK, Ca2+/calmodulin-dependent protein kinase CDK, cyclin-dependent kinase DMSO, dimethylsulfoxide EGF, epidermal growth factor Epac, Exchange protein activated by cAMP ERK, extracellular signal-regulated kinase FSH, follicle-stimulating hormone GSK, glycogen synthase kinase hCG, human chorionic gonadotropin IDPN, β,β’-iminodipropionitrile LH, luteinizing hormone LHRH, luteinizing hormone releasing hormone MAP, microtubule-associated protein MAPK, mitogen-activated protein kinase MEK, MAPK kinase MTBD, microtubule-binding domain MLC, myosin light chain Myr-PKI, myristoylated PKA inhibitor 7 P-450 SCC, cytochrome P-450 side chain cleaveage enzyme PI, phosphatidylinositol PKA, cAMP-dependent protein kinase PMSF, phenylmethylsulfonyl fluoride PMSG, pregnant mare serum gonadotropin PO, preovulatory PP, protein-phosphatase R, PKA regulatory subunit RIA, radioimmunoassay STI, soybean trypsin inhibitor StAR, steroidogenic acute regulator protein 8-CPT-cAMP, 8-(4-chlorophenylthio) cAMP 8-pCPT-2’-O-Me-cAMP, 8-(4-chlorophenylthio)-2’-O-methyl cAMP 8 DEDICATION For Kira And for my parents 9 TABLE OF CONTENTS CHAPTER I: INTRODUCTION ................................................................................................................................. 13 HYPOTHALAMIC-PITUITARY-OVARIAN AXIS ............................................................................................................... 13 GONADOTROPINS AND FOLLICLE MATURATION .......................................................................................................... 16 LH RECEPTOR SIGNALING............................................................................................................................................. 18 cAMP/PKA Signaling............................................................................................................................................... 18 Other Signaling Pathways ....................................................................................................................................... 21 GRANULOSA CELL CYTOSKELETON.............................................................................................................................. 23 Microtubules ............................................................................................................................................................. 23 Actin Microfilaments................................................................................................................................................ 24 Intermediate Filaments ............................................................................................................................................ 24 MAP2 FAMILY PROTEINS.............................................................................................................................................. 25 MAP2 and Cytoskeletal Interactions....................................................................................................................... 27 MAP2 as a Signaling Scaffold ................................................................................................................................. 28 MAP2 PHOSPHORYLATION ........................................................................................................................................... 29 CHAPTER II: LUTEINIZING HORMONE RECEPTOR ACTIVATION IN OVARIAN GRANULOSA CELLS PROMOTES PROTEIN KINASE A-DEPENDENT DEPHOSPHORYLATION OF MAP2D .......... 31 SUMMARY....................................................................................................................................................................... 31 INTRODUCTION............................................................................................................................................................... 32 RESULTS ......................................................................................................................................................................... 34 DISCUSSION .................................................................................................................................................................... 58 CHAPTER III: MAP2D PHOSPHORYLATION REGULATES BINDING TO VIMENTIN INTERMEDIATE FILAMENTS .................................................................................................................................. 65 SUMMARY....................................................................................................................................................................... 65 10 INTRODUCTION............................................................................................................................................................... 66 RESULTS ........................................................................................................................................................................