Regulation of CDK1 Activity during the G1/S Transition in S. cerevisiae through Specific Cyclin

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University of Massachusetts Medical School
eScholarship@UMMS
GSBS Dissertations and Theses Graduate School of Biomedical Sciences
10-21-2014
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
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Recommended Citation
Bhaduri, S. Regulation of CDK1 Activity during the G1/S Transition in S. cerevisiae through Specific Cyclin-Substrate Docking: A
Dissertation. (2014). University of Massachusetts Medical School.GSBS Dissertations and Theses.Paper 871. DOI: 10.13028/
M2130G. http://escholarship.umassmed.edu/gsbs_diss/871
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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.
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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
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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.
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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
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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 motif promotes Cln2-Cdc28 phosphorylation of Ste5
Figure 2.3. The Cln2 docking region in Ste5, and requirements for Cdk
phosphorylation and regulation
Figure 2.4. Cyclin-specific binding by docking motifs in Ste20 and Ste5
Figure 2.5. Ste20-Cln2 binding and conservation of the Cln2 docking site
Figure 2.6. Re-wiring a Cdk regulatory circuit with a Ste20Ste5PM chimera
Figure 2.7. Ste20Ste5PM chimeras: cyclin specificity, protein levels, and minimal
motif
Figure 2.8. Identification of additional candidate Cln2 docking sites
Figure 2.9. Sources of candidate Cln2 docking sites from other Cdk substrates
Figure 2.10. Docking sites drive cyclin-specific phosphorylation
Figure 3.1. Sequence constraints and evolutionary conservation of docking
function.
Figure 3.2. Leucine zippers, hydrophobic patch, and cyclin domains
Figure 3.3. Identification of a docking-defective Cln2 mutant
Figure 3.4. Site-directed mutations in Cln2