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Ml 48106-1346 USA 313/761-4700 800/521-0600 MITOSIS-SPECIFIC PHOSPHORYLATION OF MAP4 AND CHARACTERIZATION OF THE MPM-2 EPITOPE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yunhi Choi, B.S, M.S ★ ★ ★ ★ ★ The Ohio State University 1996 Dissertation Committee: Approved by Dale D.Vandre, Ph.D. John M.Robinson, Ph.D. Arthur R.Strauch, Ph.D. .dviser R.Tom Boyd, Ph.D. Ohio State Biochemistry Program UMI Number: 9620003 UMI Microform 9620003 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 TO MY PARENTS ii ACKNOWLEDGMENTS I wish to express my sincere appreciation to my adviser, Dr. Dale D.Vandre, for his guidance, support, and insight throughout my graduate study. I also thank him for his patience, understanding, and encouragement. I would like to thank to members of my dissertation committee, Drs. John M.Robinson, Arthur R.Strauch, and R.Tom Boyd for their time, helpful suggestions, and interest in my studies. Special thanks to my colleagues, Min Ding, Yang Feng, Christine Kondratick, and Colin Lowry, for their friendship, encouragement, sharing their ideas, and support. I extend my gratitude to my family, especially my parents and my mother-in-law for thier love and support. Finally, I wish to thank to my wonderful husband, Siyoung, and my precious son, Jaesuk, for their love and patience in allowing me to complete my studies. iii VITA September 26, 1965 --------- Born - Seoul, Korea 1988 ---- ------------------ B.S. Department of Biochemistry Yonsei University Seoul, Korea 1990 ------------------ M.S. Department of Biochemistry Yonsei University Seoul, Korea 1991-1995 ------------------ Graduate Research Associate Ohio State Biochemistry program, The Ohio State University, Columbus, Ohio PUBLICATIONS Choi, Y., Wills,V.L., and Vandre, D.D.(1993) Mitosis dependent phosphorylation of MAP4 and 125 kD MAP. Mol. Biol. Cell 4: 269a. [Abstract] Choi, Y. and Vandre, D.D.(1994) Interphase MAP4 becomes MPM-2 immunoreactive following phosphorylation by cdc2 kinase. M o l .Biol. Cell 5: 168a. [Abstract] Choi, Y. and Vandre, D.D.(1995) Phosphorylation of interphase MAP4 and formation of the mitosis-specific MPM-2 phosphoepitope in vitro. J . Biol. Chem. [manuscript submitted] FIELD OF STUDY Major field: Biochemistry v TABLE OF CONTENTS DEDICATION ............................................ ii ACKNOWLEDGEMENTS ..................................... iii VITA .................................................. iv LIST OF TABLES .......................................vii LIST OF FIGURES .....................................viii LIST OF ABBREVIATIONS ................................. x ABSTRACT .............................................. xi CHAPTERS I. INTRODUCTION ..................................... 1 II. MATERIALS AND METHOODS ......................... 19 III. RESULTS .........................................31 IV. DISSCUSSION ................................... 102 LIST OF REFERENCES .................................. 116 vi LIST OF TABLES TABLES PAGE 1. Purification of microtubules and MAP4 from HeLa cells .................................. 50 2. Potential phosphorylation sites are present on and conserved betweendifferent MAP4s ..........51 3. Fractionation of microtubule-associated kinases from mitotic HeLa cells .................. 52 vii LIST OF FIGURES FIGURES PAGE 1. Purification and immunoblot analysis of MAP4 and 125 kD MAP from interphase and mitotic HeLa cells ........................................ 55 2. Cell cycle dependent phosphorylation of HeLa MAP4 ......................................... 57 3. In vitro phosphorylation of MAP4 by exogenous kinases .............................. 59 4. Assay of endogenous kinase activity and analysis of proteins associated with mitotic HeLa microtubules ......................... 61 5. Phosphorylation of interphase HeLa MAP4 by endogenous kinases present in mitotic HeLa samples ...................................... 63 6. Endogenous kinase activities of mitotic HeLa samples under different buffer conditions ....... ...65 7. Effects of MAP4 substrate concentration on the HeLa microtubule-associated kinase activity ........ 67 8. Time-course of interphase HeLa MAP4 phosphorylation by p34cdc2 kinase .................. 69 9. Phosphorylation of interphase HeLa MAP4 by mitotic HeLa microtubule-associated kinase activities ........................................ 71 10. Phosphorylation of interphase HeLa MAP4 by kinase activities present in mitotic HeLa microtubule salt-extracted supernatant or salt-extracted pellet ............................. 73 viii FIGURES PAGE 11. Phosphorylation of interphase HeLa MAP4 by mitotic HeLa microtubule-depleted supernatant ..75 12. The effect of phosphatase inhibitors on the phosphorylation of interphase HeLa MAP4 by mitotic microtubule-depleted supernatant ..........77 13. Fractionation of mitotic HeLa microtubule-depleted supernatant kinase activities by ammonium sulfate ................... 79 14. Analysis of distribution of human Plk 1 in mitotic HeLa cells ............................ 81 15. Immunoprecipitation of Plk 1 from mitotic HeLa cell ......................................... 83 16. In vitro kinase assay of Plk 1 immunoprecipitate ................................. 85 17. Time-course of phosphorylation of interphase HeLa MAP4 by Plk 1 immunoprecipitate .............. 87 18. Immunoprecipitation of Plk 1 and MAP4 indicate they are not associated ........................... 89 19. Peptide mapping of mitotic HeLa MAP4 by NCS cleavage ................................... 91 20. Peptide mapping of mitotic HeLa MAP4 by trypsin digestion .............................. 93 21. Peptide mapping of mitotic HeLa MAP4 by chymotrypsin digestion ......................... 95 22. Peptide mapping of mitotic HeLa MAP4 by Staphylococcus aureus V8 protease .............. 97 23. Microtubule binding assay of MAP4 peptide fragments generated by V8 protease digestion .......99 24. Peptide mapping of mitotic HeLa MAP4 by endoproteinase Arg-C .......................... 101 ix LIST OF ABBREVIATIONS AEBSF 4-(2-Aminoethyl)-benzenesulfonyl fluoride APMSF Amidinophelylmethylsulfonyl fluoride CAK Cyclin dependent kinase activating kinase CDK Cyclin dependent kinase ERK Extracellular regulated kinase kD kiloDaltons MAP Microtubule-associated protein MAPK Mitogen-activated protein kinase MPF Maturation promoting factor MPM-2 Mitotic protein monoclonal antibody-2 NCS N-chlorosuccinimide PBS Phosphate buffered saline PKC Protein kinase C Plk 1 Polo-like kinase 1 PMSF Phenylmethylsulfonyl fluoride PVDF Polyvinylidine difluoride SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis TBS Tris buffered saline x ABSTRACT Microtubule-associated proteins (MAPs) bind to microtubules and regulate their assembly in vitro. Phosphorylation of MAPs appears to regulate their affinity for microtubules, and changes in the phosphorylation state of MAPs may provide a mechanism for regulating microtubule dynamics in vivo. In particular, MAP phosphorylation may play an important role in microtubule rearrangements associated with the transition from interphase to mitosis. MAP4 is the major MAP found in non-neuronal tissues, and HeLa cell MAP4 is demonstrated to be phosphorylated in a cell cycle dependent fashion. Mitotic MAP4 is also reactive with the mitotic phosphoepitope specific monoclonal antibody MPM-2. It is shown here that interphase and mitotic HeLa cell MAP4 are both substrates for MAP kinase, p34cdc2 kinase, and polo-like kinase (plk 1) but they are not substrates for casein kinase-2 in vitro. Further, extensive in vitro phosphorylation of interphase HeLa MAP4 by p34cdc2 kinase converts it into an MPM-2 reactive form. Endogenous mitotic HeLa cell xi microtubule-associated kinase activity has been identified that will also convert interphase HeLa MAP4 into an MPM-2 reactive protein. In comparison to the p34cdc2 kinase, the endogenous