University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2014-10-21 Regulation of CDK1 Activity during the G1/S Transition in S. cerevisiae through Specific Cyclin-Substrate Docking: A Dissertation Samyabrata Bhaduri University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Biochemistry Commons, Cell Biology Commons, and the Molecular Biology Commons Repository Citation Bhaduri S. (2014). Regulation of CDK1 Activity during the G1/S Transition in S. cerevisiae through Specific Cyclin-Substrate Docking: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/ M2130G. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/871 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. REGULATION OF CDK1 ACTIVITY DURING THE G1/S TRANSITION IN S. cerevisiae THROUGH SPECIFIC CYCLIN-SUBSTRATE DOCKING A Dissertation Presented By Samyabrata Bhaduri Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY October 21, 2014 Interdisciplinary Graduate Program iii REGULATION OF CDK1 ACTIVITY DURING THE G1/S TRANSITION IN S. cerevisiae THROUGH SPECIFIC CYCLIN-SUBSTRATE DOCKING A Dissertation Presented By Samyabrata Bhaduri The signatures of the Dissertation Committee signify completion and approval as to style and content of the Dissertation _____________________________________________ Peter Pryciak, Ph.D., Thesis Advisor _____________________________________________ Jennifer Benanti, Ph.D., Member of Committee _____________________________________________ Amy Gladfelter, Ph.D., Member of Committee _____________________________________________ Dannel McCollum, Ph.D., Member of Committee _____________________________________________ Eduardo Torres, Ph.D., Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee _____________________________________________ Nicholas Rhind, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school _______________________________________________ Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Interdisciplinary Graduate Program October 21, 2014 iv Acknowledgements I am deeply grateful to my parents who have always encouraged me to choose for myself and take risks. Everyday of my life, I try to imbibe their great values and use that in decision-making. I want to sincerely thank Peter for being an excellent mentor to me over so many years and for constantly helping me develop analytical and presentation skills. I will continue to learn from him for years to come. These last seven years would have been stale and lackluster, had it not been for the constant companionship of my beautiful wife, Seemin, who always knows how to make it right, aptly demonstrated by her latest creation, Zoya – our five month old daughter. I cannot even begin to describe how much more fun life has become after Zoya took over, so a big “thank you” to her. I want to specially thank Matt, my lab-mate, for not only helping me with countless experiments and writing, but also for being a patient friend and a source of American political history, religious guidance, cultural sensitivity, etc. My sincere thanks goes out to my other lab-mates over the years, Patty, Rachel and Brian for helping with experiments while carrying on lighthearted banter. I also thank the members of my TRAC and DEC Committees for their constant guidance over the years and Dr. Gladfelter for kindly agreeing to be my external examiner. A special thanks goes out to Param, Shubham and Sebastian from Dan’s lab for a variety of scientific and non-scientific help and discussions. I would also like to thank my extended family back home in India for a lot of things and the large Indian diaspora here that makes us feel at home away from home in the USA. v Abstract Several cell cycle events require specific forms of the cyclin-CDK complexes. It has been known for some time that cyclins not only contribute by activating the CDK but also by choosing substrates and/or specifying the location of the CDK holoenzyme. There are several examples of B-type cyclins identifying certain peptide motifs in their specific substrates through a conserved region in their structure. Such interactions were not known for the G1 class of cyclins, which are instrumental in helping the cell decide whether or not to commit to a new cell cycle, a function that is non-redundant with B-type cylins in budding yeast. In this dissertation, I have presented evidence that some G1 cyclins in budding yeast, Cln1/2, specifically identify substrates by interacting with a leucine-proline rich sequence different from the ones used by B-type cyclins. These “LP” type docking motifs determine cyclin specificity, promote phosphorylation of suboptimal CDK sites and multi-site phosphorylation of substrates both in vivo and in vitro. Subsequently, we have discovered the substrate-binding region in Cln2 and further showed that this region is highly conserved amongst a variety of fungal G1 cyclins from budding yeasts to molds and mushrooms, thus suggesting a conserved function across fungal evolution. Interestingly, this region is close to but not same as the one implicated in B-type cyclins to binding substrates. We discovered that the main effect of obliterating this interaction is to delay cell cycle entry in budding yeast, such that cells begin DNA replication and budding only at vi a larger than normal cell size, possibly resulting from incomplete multi-site phosphorylation of several key substrates. The docking-deficient Cln2 was also defective in promoting polarized bud morphogenesis. Quite interestingly, we found that a CDK inhibitor, Far1, could regulate the Cln2-CDK1 activity partly by inhibiting the Cln2-substrate interaction, thus demonstrating that docking interactions can be targets of regulation. Finally, by studying many fungal cyclins exogenously expressed in budding yeast, we discovered that some have the ability to make the CDK hyper-potent, which suggests that these cyclins confer special properties to the CDK. My work provides mechanistic clues for cyclin- specific events during the cell cycle, demonstrates the usefulness of synthetic strategies in problem solving and also possibly resolves long-standing uncertainties regarding functions of some cell cycle proteins. vii Table of Contents Title Page……………………………………………………………………………ii Signature Page……………………………………………………………………..iii Acknowledgements………………………………………………………………...iv Abstract………………………………………………………………………………v List of Tables………………………………………………………………………...ix List of Figures………………………………………………………………………..x Preface………………………………………………………………………………xiii CHAPTER I: Introduction…………………………………………………………...1 CHAPTER II: Cyclin-specific docking motifs promote phosphorylation of yeast signaling proteins by G1/S Cdk complexes………………………………………37 Abstract……………………………………………………………………………...38 Introduction…………………………………………………………………………40 Results……………………………………………………………………………….43 Discussions…………………………………………………………………………81 Materials and Methods……………………………………………………………85 CHAPTER III: A conserved docking interface in the cyclin Cln2 controls multi-site substrate phosphorylation and ensures timely entry into the cell cycle………..93 Abstract………………………………………………………………………………94 viii Introduction…………………………………………………………………………95 Results……………………………………………………………………………….98 Discussions………………………………………………………………………..136 Materials and Methods…………………………………………………………...145 CHAPTER IV: Regulation of cyclin-substrate docking by a G1 arrest signaling pathway and the Cdk inhibitor Far1………………………………………………153 Abstract……………………………………………………………………………..154 Background and Results………………………………………………………...155 Discussions………………………………………………………………………...183 Materials and Methods……………………………………………………………188 CHAPTER V: Conservation of LP recognition in fungal G1 cyclins and unusual hyper-potency in some cyclins…………………………………………………….196 Introduction…………………………………………………………………………197 Results and Discussions…………………………………………………………201 Materials and Methods……………………………………………………………220 CHAPTER VI: Concluding remarks……………………………………………..223 CHAPTER VII: APPENDIX………………………………………………………...232 References…………………………………………………………………………..239 ix List of Tables Table 2.1. Yeast strains used in Chapter II Table 2.2. Plasmids used in Chapter II Table 3.1. Yeast strains used in Chapter III Table 3.2. Plasmids used in Chapter III Table 3.3 Oligonucleotide primers used for RT-qPCR analysis in Chapter III Table 4.1. Yeast strains used in Chapter IV Table 4.2. Plasmids used in Chapter IV Table 5.1. Cyclin gene deletion and their phenotypes Table 5.2. Yeast strains used in Chapter V Table 5.3. Plasmids used in Chapter V x List of Figures Figure 1.1. CDKs and their role in cell-cycle progression (Reproduced from Bardin and Amon, 2001) Figure 1.2. Positive feedback loops operating at G1/S transition Figure 2.1. Cyclin-specific phosphorylation of Ste5 and Ste20 regulatory domains Figure 2.2. A distal docking