Regulation of Protein Phosphatase 1, Pp1 2, in Testis/Spermatozoa by Ppp1r11, Ppp1r7 and Ppp1r2
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REGULATION OF PROTEIN PHOSPHATASE 1, PP1γ2, IN TESTIS/SPERMATOZOA BY PPP1R11, PPP1R7 AND PPP1R2 A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Lina Cheng May, 2008 Dissertation written by Lina Cheng B.S., M.D., Shanxi Medical University, 1998 M.S., Shanghai Medical University, 2001 Ph.D., Kent State University, 2008 Approved by ______________________________,Dr. S. Vijayaraghavan (Chair, Doctoral Dissertation Committee) ______________________________,Dr. Douglas Kline ______________________________,Dr. Jennifer L. Marcinkiewicz ______________________________,Dr. Bansidhar Datta ______________________________,Dr. Roger B. Gregory Accepted by ______________________________, Dr.Robert V. Dorman (Director, School of Biomedical Sciences) ______________________________, Dr.John Starlvey (Dean, College of Arts and Sciences) ii TABLE OF CONTENTS LIST OF FIGURES………………………………………………………………………iv ACKNOWLEDGEMENTS………………………………………………………….…..vii AIMS………..............................................................................................................…..viii INTRODUCTION.......................................................................................………….…...1 MATERIALS AND METHODS………...........................................................................28 RESULTS..........................................................................................................................37 DISCUSSION……………..............................................................................................107 REFERENCES……………………………………………………………………........127 iii LIST OF FIGURES Fig. 1 The + and t homologs of the mouse t complex region ………………….………..25 Fig. 2 TCTEX5 antibody recognizes an I3-unrelated protein in testis/sperm…………...38 Fig. 3. Validation of the I3 antibody raised against the two peptides at N-terminus of I3 protein……………………………………………………………………………………39 Fig. 4. Northern blot analysis of Tctex5 mRNA expression in multiple mouse tissues…41 Fig. 5. I3 is a heat-stable protein present in nearly equal abundance in mouse sperm, testis and a wide variety of somatic tissues…………………………………………………….42 Fig.6. GST-I3 binds Sds22 and PP1γ2 from testis or sperm extracts in vitro……….......44 Fig. 7. Anti-PP1γ2 can co-precipitate Sds22 and I3, and anti-I3 can co-precipitate PP1γ2 and Sds22 from sperm (A) or testis (B).............................................................................46 Fig. 8. I3, Sds22 and PP1γ2 co-elute during chromatographic purification of testis extracts...............................................................................................................................50 Fig. 9. I3, Sds22, and PP1γ2 from co-eluting column fractions are reciprocally co- immunoprecipitated...........................................................................................................54 Fig. 10. I3, Sds22, and PP1γ2 from co-eluting column fractions co-migrate by native PAGE...............................................................................................................................,.55 Fig. 11. A protein molecular weight calibration curve for Superdex 200 column...........56 Fig.12. Microsequencing of Coomassie-blue stained protein band containing Sds22/ PP1/I3.................................................................................................................................58 iv Fig 13. Evidence that PP1γ2, Sds22, and I3 are bound to each other in a male germ cell complex that is larger than a trimer...................................................................................62 Fig. 14. Actin co-precipitates with PP1γ2, Sds22, and/or I3 from testis...........................63 Fig. 15. Macromolecular complexes containing I3 and Sds22 are not PP1γ2 isoform- specific...............................................................................................................................66 Fig. 16. Actin is also complexed with PP1β or PP1γ1 bound with I3 and Sds22.............68 Fig. 17. PP1γ2 is inactive in Superose 6 fractions containing PP1γ2, Sds22, I3, and actin....................................................................................................................................69 Fig. 18. Increasing steady state levels of I3, PP1γ2, and Sds22 in the testis parallel the temporal progression of spermiogenesis............................................................................71 Fig. 19. Cellular localization of PP1γ2, I3 and Sds22 in wild-type mouse testis sections...............................................................................................................................72 Fig. 20. Both the steady state level and molecular weight of I3 are diminished in the PP1γ-null testis, but its level and molecular weight increase in the PP1γ-null testis producing low levels of PP1γ2 protein via transgene expression......................................76 Fig. 21. Difference of microcystin pulldown PP1γ2 and its binding proteins from sperm and testis.............................................................................................................................78 Fig. 22. Amino acid sequence difference between t/t-I3 and wt-I3...................................80 Fig. 23. Validation of t/t-I3 antibody.................................................................................82 Fig. 24. Comparison of the inhibitions of His-PP1γ2 by His-wt-I3 and His-t/t-I3............85 v Fig. 25. Comparison of the binding of GST-wt-I3 and GST-t/t-I3 with PP1γ2 and Sds22 by GST pulldown assay.....................................................................................................88 Fig. 26. Anti-PP1γ2 can co-precipitate t/t-I3 and Sds22, and anti-t/t-I3 can co-precipitate PP1γ2 and Sds22 from t/t-testis protein extracts...............................................................89 Fig. 27. t/t-I3, Sds22 and PP1γ2 co-elute during chromatographic purification of t/t-testis extracts...............................................................................................................................91 Fig. 28. t/t-I3, Sds22, and PP1γ2 from co-eluting column fractions co-migrate by native PAGE.................................................................................................................................92 Fig. 29. In vitro phosphorylation study of GST-wt-I3 and GST-t/t-I3 using sperm extracts as a source of kinase..........................................................................................................94 Fig. 30. Characterization of I2 antibody and verification of the existence of inhibitor I2 in testis...................................................................................................................................96 Fig. 31. Immunoprecipitation with I2 antibody from sperm or testis................................98 Fig. 32. Northern blots of mouse tissue mRNAs.............................................................101 Fig. 33. PCR results of multiple tissue cDNAs. .............................................................104 Fig. 34. Western blot of recombinant I2(17) protein.......................................................105 Fig. 35. Our affinity purified I2 antibody recognizes a 55kDa protein on western blots..................................................................................................................................106 Table 1 Microsequencing Result......................................................................................59 vi ACKNOWLEDGMENTS I will express my deepest gratitude to my advisor, Dr. Srinivasan Vijayaraghavan, for his constant support, guidance, encouragement and kindness. I would like to thank him for training and teaching me how to be a scientist. He helped me to achieve my goals during my research. He is an excellent professor, a fully devoted scientist and also a great friend in life. I thank our collaberator, Dr. Steven Pilder, and my committee members, Dr. Douglas W. Kline, Dr. Jennifer L. Marcinkiewicz, and Dr. Bansidhar Datta. All your help and kindness are greatly appreciated. Without your help, I could not have come this far. I thank Kimbery Myers for her great help in the experiments and discussion, and Dr. Michael Model for his help in the confocal microscopy. I thank the following experts for their various help in my research, Dr.Edgar F. da Cruz e Silva, Dr.David Brautigan, Dr. Mathieu Bollen, and Dr.Angus Nairn. I thank all my friends for their kindness and their support. Thank them for listening to me, encouraging me, and all the laughs and tears shared with me: Hongmei Zhang, Kamil Gierszal, Qunying Zhu, Zhiping Jiang, Viorel Sandu, JJ Voelker, and Karen Bonfiglio. Finally I deeply thank my parents, my brothers and sister-in-laws, my lovely niece and nephew, and all my other relatives. They strongly support me from every aspects of my life. For my every achievement, I must share with them. vii AIMS The phosphatase PP1γ2 is the only detectable PP1 isoform in mammalian spermatozoa. Its activity is inversely related to sperm motility suggesting that PP1γ2 has a fundamental, isoform-specific role(s) in mammalian sperm function. However, the biochemical nature of its essential role(s) in either sperm development or subsequent function during the fertilization process remains unclear. A number of PP1-interacting proteins in somatic cells have been detected via either biochemical or bioinformatic approaches, and considerable