Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2012 Regulation of the Transcription Factor YY1 by Phosphorylation Ari Kassardjian Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES REGULATION OF THE TRANSCRIPTION FACTOR YY1 BY PHOSPHORYLATION By ARI KASSARDJIAN A Dissertation submitted to the Program of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Summer Semester, 2012 Ari Kassardjian defended this dissertation on June 21, 2012. The members of the supervisory committee were: Myra M. Hurt Professor Directing Dissertation Akash Gunjan University Representative Lloyd Epstein Committee Member Cathy Levenson Committee Member Hank Bass Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements. ii ACKNOWLEDGMENTS I feel very lucky and privileged that I had the opportunity to work in the laboratory of Dr. Myra Hurt; I am forever grateful for the support and guidance that I have received from my advisor. I am honored to have worked under the supervision of a great mentor and I cannot thank her enough! I would like to thank the members of my committee: Dr. Akash Gunjan, Dr. Lloyd Epstein, Dr. Hank Bass, and Dr. Cathy Levenson. Without their valuable feedback and accessibility, this work would not have been achieved. This work would have not been possible without Beth Alexander, our lab director. She taught me many techniques and was always there to help me with all my work. I would also like to thank Dr. Raed Rizkallah, an amazing researcher who led by example. He was always present and available to help answer my questions and guide me in the right direction. I like to thank Dr. Sarah Riman for being a great friend and colleague in the lab. Beth, Raed and Sarah had major, direct and indirect, contributions to this study and I will never forget our experiences together. Also, I would like to thank all the faculty members, staff, colleagues and friends in the Department of Biomedical Sciences and the Molecular Biophysics Graduate program at Florida State University. Lastly, I would like to thank my parents, Chahe and Rosine, as well as my brother, Araz for their unwavering love, unending support and encouragement. iii TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………..v LIST OF FIGURES……………………………………………………………………...vi ABSTRACT…………………………………………………………………………….viii CHAPTER 1 INTRODUCTION ........................................................................... 1 DISCOVERY AND CHARACTERIZATION OF YY1 ......................................... 2 YY1 AND CELL CYCLE REGULATION ........................................................... 7 YY1 REGULATION ........................................................................................... 9 POSTTRANSLATIONAL REGULATION OF YY1 ........................................... 11 KINASES THAT PHOSPHORYLATE YY1 ...................................................... 24 GOALS OF THE PROJECT ............................................................................ 28 CHAPTER 2 AURORA B KINASE PHOSPHORYLATES THE TRANSCRIPTION FACTOR YY1 AT G2/M TRANSITION OF THE CELL CYCLE AND MODULATES ITS TRANSCRIPTIONAL ACTIVITY ................................................................. 29 INTRODUCTION .................................................................................................. 30 MATERIALS AND METHODS ................................................................................. 31 RESULTS .......................................................................................................... 39 DISCUSSION ..................................................................................................... 59 CHAPTER 3 IDENTIFICATION OF TYROSINE KINASES THAT PHOSPHORYLATE YY1 IN VITRO ................................................................................................... 65 INTRODUCTION .................................................................................................. 65 MATERIAL AND METHODS .................................................................................. 66 RESULTS .......................................................................................................... 72 DISCUSSION ..................................................................................................... 87 CHAPTER 4 CONCLUSIONS .......................................................................... 92 APPENDIX ......................................................................................................... 95 REFERENCES ................................................................................................... 97 BIOGRAPHICAL SKETCH .............................................................................. 114 iv LIST OF TABLES Table I. Known in vivo Posttranslational modifications of YY1. ..................... 16 Table II. In vivo phosphorylation sites of YY1 ......................................... ……22 v LIST OF FIGURES Figure 1. Diagram of the different functional domains of human YY1 and the location of each phosphorylated amino acid residue. ................................................ 4 Figure 2. Structure of the YY1 cocrystal structure. ...................................................... 7 Figure 3. Protein phosphorylation and protein dephosphorylation regulate the activities of transcription factors through multiple mechanisms. ................. 17 Figure 4. Phosphorylation of YY1 in nocodazole blocked extracts is detected by antiphospho-S184 antibody.. ...................................................................... 42 Figure 5. Serine 184 phosphorylation on YY1 peaks at the G2/M stage of the cell cycle. .......................................................................................................... 45 Figure 6. Aurora B phosphorylates YY1 at serine 184 in vitro. .................................. 47 Figure 7. PKA and ROCK1 phosphorylate YY1 at serine 184 in vitro. ....................... 49 Figure 8. Aurora B phosphorylates YY1 at serine 184 in vivo. ................................... 51 Figure 9. Cell cycle analysis and cellular localization of YY1 phospho-mutants in HEK293 cells.. ............................................................................................ 53 Figure 10. Phosphorylation of YY1 in its regulatory domain modulates transcriptional activity......................................................................................................... 55 Figure 11. YY1 S180,184D phospho-mutant exhibits an increased DNA binding affinity in vitro.. ............................................................................................ 56 Figure 12. The histone acetyltransferase p300 acetylates full length YY1 wild type in vitro. ............................................................................................................ 58 Figure 13. Schematic model of the regulation of YY1 by Aurora B at G2/M.. ............... 64 Figure 14. YY1 is phosphorylated on tyrosine residue(s) in vitro and in vivo.. ............. 73 Figure 15. YY1 is phosphorylated on tyrosine residues during mitosis.. ...................... 75 Figure 16. Structural and functional domains of YY1 showing the location of the six tyrosine residues in YY1.. ........................................................................... 77 Figure 17. Phosphorylation of Y251 and Y254 by mitotic extracts. .............................. 79 Figure 18. Tyrosine kinases included in the kinase profiling screen. ............................. 81 vi Figure 19. Multiple tyrosine kinases phosphorylate YY1 in vitro .................................. 82 Figure 20. Dot blot assay of non-phosphorylated and tyrosine phosphorylated synthetic peptides of YY1 probed with anti-pY251 or anti-pY254. ............................. 83 Figure 21. FAK and SRC phosphorylate YY1 at Y251 and Y254 in vitro ..................... 84 Figure 22. Protein expression and DNA binding activity of EGFP-YY1 Y251F and Y254F in HeLa extracts. ............................................................................. 85 Figure 23. Examples of zinc finger containing proteins with conserved tyrosine (Y) or phenylalanine (F) residues at the linker region ........................................... 86 Figure 24. Protein expression and DNA binding activity of EGFP-YY1 Y383F in HeLa extracts.. ..................................................................................................... 87 vii ABSTRACT Reversible protein phosphorylation plays an integral role in the regulation of eukaryotic cellular signaling, especially for transcription factors. Yin Yang 1 (YY1) is a ubiquitously expressed and highly conserved multifunctional transcription factor that is involved in a variety of cellular processes. Many YY1-regulated genes have crucial roles in cell proliferation, differentiation, apoptosis, and cell cycle regulation. Numerous mechanisms have been shown to regulate the function of YY1, such as DNA binding affinity, subcellular localization, and posttranslational modification including phosphorylation. We have previously identified Polo-like kinase 1(Plk1) and Casein kinase 2α (CK2 α) as the tfirs two kinases proven to phosphorylate YY1. In this study, we