Regulation of Protein Phosphatase One During Cell Cycle

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2016 Regulation of Protein Phosphatase One During Cell Cycle

Nasa, Isha

Nasa, I. (2016). Regulation of Protein Phosphatase One During Cell Cycle (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/27589 http://hdl.handle.net/11023/3446 doctoral thesis

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Regulation of Protein Phosphatase One During Cell Cycle

Isha Nasa

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN BIOLOGICAL SCIENCES

CALGARY, ALBERTA

OCTOBER, 2016

Protein phosphatase 1 (PP1) is a highly conserved enzyme that controls the majority of serine/threonine (Ser/Thr) dephosphorylation reactions in eukaryotes. PP1 gains substrate specificity through binding to a large number (> 200) of regulatory proteins, which control PP1 localization, activity, and substrate interaction. PP1 recognizes the majority of these regulatory proteins via well-characterized RVxF binding motif generating hundreds of distinct PP1 holoenzymes. The main objective of this research was to uncover the regulatory mechanisms that govern the interaction of PP1 with its regulatory proteins during the cell cycle.

The progression of cell cycle is largely governed by reversible protein phosphorylation. I showed that a subset of the RVxF binding motifs, in which x is a phosphorylatable amino acid (RV[S/T]F), are phosphorylated specifically during mitosis and that this phosphorylation event abrogates the interaction of PP1 with the regulatory protein. This phosphorylation is primarily governed by mitotic protein kinase Aurora B and is crucial to maintain phosphorylation of PP1 substrates during mitosis. In addition, I showed that PP1 itself dephosphorylates RVp[S/T]F motifs during mitotic exit, which allows the phosphatase to re-associate with the regulatory proteins and dephosphorylate other mitotic substrates.

To gain further insight into the regulation of PP1 function in cell cycle, I characterized the novel cell cycle dependent interactome of PP1. Using quantitative mass spectrometry, I identified 113 novel RVxF containing potential PP1 binding partners including 17 mitosis-specific partners. Furthermore, using immunoblotting, I validated 9 of the PP1 interactions both in asynchronous and mitotic populations with proteins

ii involved in cell cycle regulation (Aurora B, Aurora A, TPX2, CDCA2 (RM), TACC3,

GCN2, DBC1, BRCA1 and RIF1). In addition, I demonstrated a novel interaction of PP1 with centrosomal protein, CEP192 via its ‘KHVTF’ motif.

The work presented here expands our understanding of the regulation of PP1 in the cell cycle, and also suggests a novel regulatory mechanism by which the coordinated activities of Aurora B kinase and PP1 drive mitotic progression, which is crucial to maintain the genomic stability.

iii Acknowledgements

My Ph.D. journey would not have been the same without the support and encouragement of all the amazing people in my life. First and foremost, I offer my sincere gratitude to my supervisor and mentor, Dr. Greg Moorhead, for the continuous support and guidance throughout my Ph.D. research. His patience, motivation and knowledge, steered me through this journey. He was an exceptional supervisor who always inspired me to grow as a scientist by giving me intellectual and scientific freedom, engaging me in new ideas, and encouraging me to be a better version of myself in every aspect of student life. I would also like to thank my committee members Dr. Aaron

Goodarzi and Dr. Carrie Shemanko, for fruitful discussions and insightful ideas which helped me carve my work into its present form. I truly appreciate their taking out time for all the committee meetings and discussions.

I have been fortunate to be able to collaborate and be trained by excellent mentors throughout my graduate school journey. My sincere thanks to our collaborator Dr.

Arminja Kettenbach, whose expertise and knowledge in the field of mass spectrometry were a great help for this project and my research. I owe my gratitude to Dr. Veerle de

Wever, Dr. Pauline Douglas and Dr. Susan Lees-Miller, for their experimental advice, technical training and resources for microscopy experiments. All the past and present members of the Moorhead lab, who have always been truly helpful and have given me fond memories of my graduate school deserve a special mention. I would like to thank

Anne-Marie Labandera, David Lloyd, Dr. Dylan Silver, Dr. Glen Uhrig, Sibapriya

Chaudhuri, Brooke Rackel, Nic Sieben, Ahmad Vahab and Ryan Toth for all lunch-time and coffee conversations. In particular, I would like to extend my appreciation to Anne-

iv Marie who has been a tremendous support, both in the lab and outside, and lifted up my spirit in days of frustration. I also owe thanks to all undergraduates and summer students who contributed to my research: Nic Sieben, Simon Hassan, Drishti Mannan, Brooke

Rackel and Guneet Khosa. I am also grateful to the Samuel research group for their participation and fruitful discussions in our joint lab meetings, Prenner and Turner research groups for allowing me to use their equipment and supplies when I needed them.

I would extend my gratitude to all the funding agencies who have kept me going in my research including Alberta Cancer Foundation, URGC Thesis Research Grant, URGC

International Graduate Travel Award, University of Calgary Faculty of Graduate Studies

Doctoral Scholarship, Chancellor’s Challenge Graduate Scholarship, Eyes High

International Graduate Scholarship, and EMBO Conference Travel Award.

Lastly, I would like to thank the most important people of my life, my family, who have supported me unconditionally throughout my journey. My Mom, Dad, Saurabh and

Sakshi, who made it possible for me to follow my dreams away from home. A special thanks to my fiancée Rahul who has been a constant support through all these years and has virtually lived through all the ups and downs of this journey with me. Living away from my home, my friends became my family in Calgary through all these years. I would like to thank Bhairavi Sohoni, Navneet Randhawa, Neha Dawar, Rosy Dabas, and Tushar

Sharma for their unyielding support and for always being there.

To my Family, for their endless love and support

vi Table of Contents

Abstract ...... ii

Acknowledgements ...... iv

Dedication ...... vi

Table of Contents ...... vii

List of Tables ...... xii

List of Figures and Illustrations ...... xiii

List of Symbols, Abbreviations and Nomenclature ...... xvi

Epigraph ...... xviii

List of Manuscripts from Ph.D. thesis ...... xix

Chapter 1. Introduction ...... 1

1.1 Reversible protein phosphorylation and the cell cycle ...... 1

1.2 Mitotic entry and exit as controlled by phosphorylation ...... 4

1.3 Protein phosphatases ...... 6

1.3.1 Classification and function ...... 6

1.3.2 Phosphatases as mitotic guards ...... 9

1.4 Protein phosphatase one and its regulation ...... 15

1.4.1 The catalytic subunit of protein phosphatase 1 (PP1) ...... 15

1.4.2 Mechanism of dephosphorylation by PP1 ...... 15

1.4.3 PP1 Regulatory proteins: Diversity and specificity ...... 17

1.5 Role of PP1 during mitosis ...... 20

1.5.1 Mitotic entry ...... 20

1.5.2 Centrosomal splitting and spindle formation ...... 21

vii 1.5.3 Spindle assembly checkpoint ...... 23

1.5.4 Mitotic exit ...... 26

1.6 Research Objectives ...... 29

Chapter 2. Reversible phosphorylation within PP1 docking RVxF motif

controls the function of PP1 during cell cycle ...... 31

2.1 Introduction ...... 31

2.2 Methods ...... 34

2.2.1 Cell culture and synchronization ...... 34

2.2.2 Antibodies and reagents ...... 35

2.2.3 Affinity purification of phospho-specific antibodies ...... 36

2.2.4 PP1 expression and purification ...... 36

2.2.5 PP1 binding assays ...... 37

2.2.6 Kinase inhibitor assays ...... 37

2.2.7 Phosphatase inhibitor assays ...... 38

2.2.8 Immunoprecipitation ...... 38

2.2.9 Mass spectrometry analysis ...... 39

2.2.10 Data analysis ...... 41

2.2.11 Immunofluorescence staining and microscopy...... 42

2.3 Results ...... 42

2.3.1 PP1 preferentially binds non-phosphorylated RV[S/T]F motifs in-vitro ...... 42

2.3.2 RV[S/T]F motifs are phosphorylated during mitosis ...... 45

2.3.3 Aurora B-dependent phosphorylation of the RV[S/T]F motifs ...... 50

viii 2.3.4 Proteomics analysis of Aurora B phosphorylation-dependent p-RV[S/T]F

proteins ...... 50

2.3.5 PP1 dephosphorylates the RV[S/T]F motifs at mitotic exit ...... 59

2.4 Discussion ...... 62

2.5 Conclusion ...... 67

Chapter 3. Identification of the Cell Cycle Specific Interactome of PP1 ...... 69

3.1 Introduction ...... 69

3.2 Materials and Methods ...... 71

3.2.1 Cell synchronization and mitotic shake-off ...... 71

3.2.2 GFP-TRAP using U2OS GFP-tagged PP1 cells ...... 71

3.2.3 Mass spectrometry sample preparation, analysis and iBAQ ...... 73

3.2.4 Data analysis ...... 73

3.2.5 Immunoblotting and validation ...... 74

3.3 Results ...... 75

3.3.1 Validation of GFP-TRAP pull-downs ...... 75

3.3.2 Proteomic analysis of GFP-TRAP eluates ...... 75

3.3.3 Validation of mass spectrometry results ...... 86

3.4 Discussion ...... 89

3.5 Conclusion ...... 93

Chapter 4. Validation and characterization of interaction between

centrosomal protein CEP192 and PP1...... 94

4.1 Introduction ...... 94

4.2 Materials and Methods ...... 98

ix 4.2.1 Conservation of potential PP1 binding motif among eukaryotes ...... 98

4.2.2 Molecular cloning and site directed mutagenesis of CEP192 ...... 99

4.2.3 Expression and purification of human CEP192 and its RARA mutant ...... 99

4.2.4 In-vitro PP1 binding assay ...... 100

4.2.5 GFP-TRAP with U2OS-GFP-PP1 and HeLa-GFP-CEP192 cells ...... 101

4.2.6 Generation and validation of phospho ‘KHVTF’ CEP192 antibody ...... 102

4.2.7 Kinase inhibitor assay ...... 102

4.2.8 Immunofluorescence and microscopy ...... 102

4.3 Results ...... 103

4.3.1 The potential PP1 binding ‘KHVTF’ motif in CEP192 ...... 103

4.3.2 CEP192 co-localizes with PP1 ...... 103

4.3.3 CEP192 interacts with PP1 ...... 105

4.3.4 ‘KHVTF’ motif in CEP192 is phosphorylated during mitosis ...... 105

4.3.5 PLK1 regulates the phosphorylation of the ‘KHVTF’ motif in CEP192 .... 108

4.4 Discussion ...... 108

4.5 Conclusion ...... 112

Chapter 5. Perspectives and Future Directions ...... 113

5.1 PP1 as the master controller of cell cycle ...... 113

5.1.1 Aurora B and PP1: the essential kinase-phosphatase equilibrium ...... 113

5.1.2 The novel cell cycle dependent PP1 interactome ...... 116

5.2 The therapeutic potential of PP1 ...... 117

References ...... 124

x Appendix A: Mass Spectrometry data from p-RV[S/T]F IP ...... 140

A.1. List of proteins specifically enriched in p-RV[S/T]F IP with their binding

behavior upon Aurora B inhibition and SAINT scores ...... 140

Appendix B: Mass Spectrometry data from PP1 GFP-TRAP...... 157

B.1. List of mitosis-specific or asynchronous-specific proteins enriched in PP1

immunoprecipitations from all the three isoforms...... 157

xi List of Tables

Table 1.1: Classification of protein phosphatases...... 7

Table 1.2: PP1 binding motifs in different regulatory proteins...... 19

Table 2.1: List of peptide sequences used in this study ...... 43

Table 2.2: List of proteins containing "RV[S/T]F" motifs specifically enriched in

the p-RV[S/T]F immunoprecipitation...... 55

Table 3.1: List of specific PP1-binding proteins (SAINT AvgP score  0.9)

containing one or more “RVxF” motifs...... 80

Table 5.1: List of known PP1 regulatory proteins with mutations in one or more

residues within the PP1 binding “RVxF” motif...... 120

xii List of Figures and Illustrations

Figure 1.1: Regulation of cell cycle by phosphorylation...... 3

Figure 1.2: Regulation of mitosis by coordinated activities of protein kinases and

protein phosphatases...... 11

Figure 1.3: PP2A-Shugoshin complex regulates cohesion during mitosis...... 14

Figure 1.4: Human PP1 isoforms are highly conserved among each other...... 16

Figure 1.5: Protein phosphatase 1 and its regulation...... 18

Figure 1.6: Mitotic roles of protein phosphatase 1...... 22

Figure 2.1: Consensus RV[S/T]F motif in PP1-interacting proteins...... 34

Figure 2.2: PP1 preferentially binds dephosphorylated RV[S/T]F motifs in-vitro. .. 44

Figure 2.3: RV[S/T]F motifs are phosphorylated during mitosis...... 46

Figure 2.4: The phosho-specificity of the p-RV[S/T]F antibody...... 48

Figure 2.5: Immunofluorescence also confirms that p-RV[S/T]F motifs are

phosphorylated during mitosis...... 49

Figure 2.6: Aurora B phosphorylates PP1 binding RV[S/T]F motifs during

mitosis...... 51

Figure 2.7: p-RV[S/T]F containing proteins during mitosis...... 53

xiii Figure 2.8: Phosphorylation within the RV[S/T]F motifs of RIF1 and CDCA2

(RM) is governed by Aurora B and regulates binding to PP1...... 58

Figure 2.9: PP1 dephosphorylates the RV[S/T]F motifs at mitotic exit...... 61

Figure 2.10: Proposed model of PP1 regulation during the cell cycle...... 63

Figure 3.1: Known protein-protein interaction network of PP1...... 70

Figure 3.2: Methodology for GFP-TRAP experiment...... 72

Figure 3.3: Validation of the GFP-TRAP pull-down...... 76

Figure 3.4: Clustering of PP1 binding proteins identified shows mitosis-specific

and asynchronous-specific partners...... 78

Figure 3.5: “RVxF” motifs from mitosis-specific and asynchronous-specific

clusters reveal subtle differences...... 83

Figure 3.6: Clustering of PP1 binding proteins identified shows isoform-specific

clustering in mitotic and asynchronous GFP-TRAP eluates...... 85

Figure 3.7: Validation of mass spectrometry results by immunoblotting...... 88

Figure 4.1: Regulation of the centrosome...... 96

Figure 4.2: Regulation of CEP192 by phosphorylation...... 98

Figure 4.3: Potential PP1 binding ‘KHVTF’ motif in CEP192...... 104

Figure 4.4: CEP192 interacts with PP1...... 106

xiv Figure 4.5: Validation of the phospho-KHVTF CEP192 antibody...... 107

Figure 4.6: Phosphorylation of the ‘KHVTF’ motif in CEP192...... 109

Figure 5.1: Interplay between CDK1, Aurora B and PP1 activities during cell

cycle...... 115

xv List of Symbols, Abbreviations and Nomenclature

Symbol Definition ATF3 Activating transcription factor 3 AP1 Activator protein one APC/C Anaphase promoting complex/cyclosome ARPP19 Cyclic adenosine monophosphate (cAMP)- regulated phosphoprotein of 19kDa ASPM Abnormal spindle microtubule assembly ATP Adenosime triphosphate BME Beta mercaptoethanol BRCA1 Breast cancer 1 BSA Bovine serum albumin c-myc Cellular-myelocytomatosis CaMKII Ca2+ /calmodulin-dependent protein kinase II CDC14 Cell division cycle 14 CDC25 Cell division cycle 25 CDCA2 Cell division cycle associated 2 CDK Cyclin dependent kinase CENP-E Centromere protein E CEP192 Centrosomal protein of 192kDa CHK1/2 Checkpoint kinase 1/2 CTD Carboxy-terminal domain DAPI 4',6-diamidino-2-phenylindole DBC1 Deleted in breast cancer 1 DMEM Dulbecco's Modified Eagle Medium DNA Deoxyribonucleic acid DSP Dual specificity phosphatase DTT Dithiothreitol EDTA Ethylene diamine tetraacetic acid ENSA Endosulfine alpha FACS Fluorescent activated cell sorting FBS Fetal bovine serum FoxK2 Forkhead Box K2 FoxM1 Forkhead Box M1 GCN2 General Control Nonderepressible 2 GFP Green fluorescent protein GO Gene ontology HCD Higher-energy collisional dissociation HIV Human immunodeficiency virus iBAQ Intensity-based absolute quantification KLH Keyhole limpet hemocyanin KNL1 Kinetochore Null 1 homolog LC Liquid chromatography MAPK Mitogen activated protein kinase

xvi Symbol Definition MLCP Myosin light chain phosphatase mTOR Mammalian target of rapamycin MYPT1 Myosin phosphatase targeting subunit 1 Nek NimA related kinase OA Okadaic acid ORC2 Origin recognition complex subunit 2 PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffer saline PCM Pericentriolar material PCR Polymerase chain reaction PKA Protein kinase A PKB Protein kinase B PLK1 Polo like kinase 1 PMSF Phenyl methyl sufonyl fluoride PP1 Protein phosphatase one PP2A Protein phosphatase 2A PPM Metallo protein phosphatase PPP Phospho protein phosphatase pRB Retinoblastoma protein PTEN Phosphatase and tension homolog PTP Protein tyrosine phosphatase RM Repoman (Recruits PP1 onto mitotic anaphase chromosome) RIF1 Rap1 interacting factor 1 homolog RRP1B Ribosomal RNA processing 1 homolog B SAC Spindle assembly checkpoint SAINT Significance analysis of interactome SDS Sodium dodecyl sulfate Ser (S) Serine TACC3 Transforming acidic coiled-coil containing protein 3 TCA Trichloro acetic acid Thr (T) Threonine TSC2 Tuberous sclerosis 2 Tyr (Y) Tyrosine UBR5 Ubiquitin Protein Ligase E3 Component N- Recognin 5

xvii Epigraph

Progress is made by trial and failure; the failures are generally a hundred times more numerous than the successes; yet they are usually left unchronicled. -William Ramsay

xviii List of Manuscripts from Ph.D. thesis

Chapter 2 I. Nasa, S. Rusin, A. Kettenbach and G. Moorhead (2016) Aurora B opposes PP1 function in mitosis through phosphorylation of the conserved RVxF binding motif in PP1 regulatory proteins. Science Signaling. Manuscript in revision.

Chapter 3 I. Nasa, G. Khosa, S. Lyons, A. Kettenbach and G. Moorhead (2016) Proteomics analysis of PP1 interactome highlights cell cycle specific roles for PP1. Manuscript in- preparation.

Chapter 4 I. Nasa, G. Moorhead (2016) CEP192 interacts with PP1 to regulate phosphorylation of centrosomal proteins. Manuscript in-preparation.

Other notable contributions during Ph.D. thesis V. de Wever*, I. Nasa*, D. Chamousset*, D. Lloyd, M.Nimick, H. Xu, L. Trinkle- Mulcahy, G. Moorhead (2014) The human mitotic kinesin KIF18A binds protein phosphatase 1 (PP1) through a highly conserved docking motif. Biochem Biophys Res Commun. 453 (3): 432-7. * These authors contributed equally to this work.

M. Prévost, D. Chamousset, I. Nasa, E. Freele, J. Andersen, N. Morrice, G. Moorhead and L. Trinkle-Mulcahy (2013) Quantitative fragmentome mapping reveals novel, domain specific partners for the modular protein RepoMan .Mol Cell Proteomics. 12 (5): 1468-86.

V. De Wever, D.C. Lloyd, I. Nasa, M. Nimick, L. Trinkle-Mulcahy, R. Gourlay, N. Morrice and G. Moorhead (2012) Identification of mitotic protein phosphatase complexes in human cells –novel connections between Protein Phosphatase 1 and the nucleolar RNA helicase DDX21. PLoS one 7 (6): e39510.

xix CHAPTER 1. INTRODUCTION

1.1 Reversible protein phosphorylation and the cell cycle

Reversible protein phosphorylation is a prevalent regulatory post-translational modification that is catalyzed by protein kinases and protein phosphatases in the cell. In eukaryotes, protein phosphorylation typically takes place on serine, threonine and tyrosine residues with the percentage phosphorylation on each residue in the human proteome being 86.4%, 11.8% and 1.8% respectively (Olsen, Blagoev et al. 2006, Olsen,

Vermeulen et al. 2010). Recent mass spectrometry-based phosphoproteomics has revealed that more than 70% of the human proteome is phosphorylated in its lifetime with high occupancy during cellular events like mitosis (Sharma, D'Souza et al. 2014).

Genomic studies also show that about 2-4% of the protein encoding genes in most eukaryotes constitute protein kinase and protein phosphatase gene families (Kerk,

Templeton et al. 2008, Choudhary, Olsen et al. 2009). This highlights the importance of the switch-like mechanism of protein phosphorylation in controlling diverse signaling pathways in a eukaryotic cell.

The eukaryotic cell cycle is a highly regulated series of events including chromosome duplication, segregation, and packaging into separate daughter cells. The timely execution of these events is crucial for accurate cell cycle progression. Transitions between cell cycle phases, including the replication of genetic material in S-phase and the segregation of chromosomes during M-phase, are largely governed by reversible protein phosphorylation (Hunt 2002). The benchmark discoveries of cyclins and cyclin- dependent kinases (CDKs) by Tim Hunt and Paul M. Nurse, respectively, during the

1980s, were fundamental to the field of cell cycle regulation (Morgan 1995, Nurse, Masui et al. 1998, Hunt 2002, Nurse 2002). The CDKs are considered as the master regulators or the “engines” of the cell cycle, driving cell cycle progression by associating with their partner cyclins. This association of cyclins with different CDKs turns the engine ‘on’ and sets the stage for each cell cycle phase (Obaya and Sedivy 2002).

Most cells remain in the quiescent Go state unless stimulated by internal or external factors to enter the G1 phase of cell cycle. Upon mitogenic stimulation, which may be via RAS, MAPK, and mTOR signaling pathways, the transcription of early response genes is initiated (Figure 1.1) (Loyer, Cariou et al. 1996). This upregulation of gene transcription, including transcription of factors like AP1, ATF3 and c-Myc, also leads to the upregulation of the D-type cyclin gene (Mateyak, Obaya et al. 1999). Cyclin

D activates CDK4 or CDK6 by directly binding to them. The activated CDKs (CDK4 and

CDK6) promote G1 progression by the phosphorylation of retinoblastoma pocket proteins including pRb, p107, and p130. The activation of pRb de-represses the activity of E2F transcription factors, inducing the expression of G1-S target genes including cyclin A and cyclin E (Bartek, Bartkova et al. 1997). Activated CDK2/cyclin E complex drives the cell through the G1-S transition by also inducing histone synthesis and centrosome duplication at this point (Bertoli, Skotheim et al. 2013). Followed by this, CDK2/cyclin A initiates DNA replication at the S-phase entry and drives the progression of cell cycle through S-phase. At G2 phase of cell cycle, the sequential activation of transcription factors FoxM1 and FoxK2 by CDK1/cyclin A and CDK1/cyclin B complexes activates

Figure 1.1: Regulation of cell cycle by phosphorylation.

Stimulation by mitogenic signals upregulates the expression of cyclin D which binds with CDK4/6. This complex triggers the phosphorylation of the retinoblastoma pocket proteins which activate the transcription of E2F regulated genes. These genes include S- phase genes including cyclin E, which after binding to CDK2, transitions the cell from G1 to S phase. During the S-phase, CDK2/cyclin A complex controls chromosome duplication and enables the cell to enter next growth G2 phase where the cell prepares for mitosis. At G2/M transition, the activation of CDK1 forms an active CDK1/cyclin B complex which phosphorylates multiple proteins necessary for controlling mitotic processes of chromosome condensation and separation. Cyclin B is degraded by APC/CCDC20 complex at anaphase, which leads to decline in the CDK1 activity, eventually leading to cytokinesis and formation of two daughter cells.

the genes involved in mitotic entry (Laoukili, Alvarez et al. 2008, Marais, Ji et al. 2010,

Sadasivam, Duan et al. 2012). CDK1/cyclin B complex phosphorylates multiple proteins at mitotic entry through metaphase, which are crucial for cellular processes like nuclear envelope breakdown, chromatin condensation and spindle assembly (Section 1.2). At anaphase, the APC/CCDC20 degrades cyclin B which leads to the decline of CDK1 activity. This decrease in CDK1 activity is important for the cells to exit mitosis and split into two daughter cells.

1.2 Mitotic entry and exit, as controlled by phosphorylation

Mitosis is the process in which duplicated chromosomes are segregated and partitioned into two daughter cells. Mitosis is divided into sub-stages based on particular sets of events– prophase, metaphase, anaphase, and telophase. This process of mitotic cell division is controlled by a number of post-translational modifications, among which, protein phosphorylation is the most crucial. During mitosis, proteins are phosphorylated by the mitotic serine/threonine protein kinases of the cyclin-dependent kinase (CDK),

Polo, Aurora and Nek families (Nigg 2001, O'Farrell 2001, Barr, Sillje et al. 2004,

Ruchaud, Carmena et al. 2007, Lindqvist, Rodriguez-Bravo et al. 2009). As protein phosphorylation is dynamic in nature, the phosphorylated proteins are dephosphorylated after signaling their downstream effectors. This dephosphorylation in mitosis has been attributed to specific serine/threonine and tyrosine phosphatases (Trinkle-Mulcahy and

Lamond 2006, Chen, Archambault et al. 2007, Gharbi-Ayachi, Labbe et al. 2010,

Wlodarchak and Xing 2016). The maintenance of accurate stoichiometric balance in

phosphorylation that is controlled by the activities of protein kinase and protein phosphatase families is critical for proper progression of mitosis.

The CDK1-cyclin B complex is the master controller of mitosis regulating mitotic entry and progression. At the mitotic entry, CDK1 activity requires its binding to the partner cyclin B and phosphorylation of its T-loop residue Thr161. The activity of CDK1 is also regulated by inhibitory phosphorylation of the neighboring residues in the ATP- binding site of CDK1 (Thr14 and Tyr15), catalyzed by Wee1 and Myt1 protein kinases

(Gould and Nurse 1989, Lundgren, Walworth et al. 1991, Izumi and Maller 1993,

Mueller, Coleman et al. 1995). The phosphorylation of these residues keeps CDK1 inactive prior to the G2/M transition, when their dephosphorylation by the dual specificity phosphatase CDC25 fully activates CDK1 (Russell and Nurse 1986, Kumagai and

Dunphy 1992, Trunnell, Poon et al. 2011). Thus, an appropriate balance between the phosphatase (CDC25) and kinase (Wee1 and Myt1) activity regulates CDK1 during cell cycle. Once activated, the CDK1-cyclin B complex can phosphorylate its regulators:

Wee1, Myt1, and CDC25. This phosphorylation negatively regulates the activity of inhibitory kinases Wee1 and Myt1 while positively regulating its positive regulator

CDC25 phosphatase, exhibiting an elegant double negative and positive feedback mechanism for CDK1 activation. The establishment of mitotic entry requires the complete activation of CDK1, which further phosphorylates and activates its downstream kinases, Polo-like kinase 1 (PLK1), and Aurora kinases, to drive cell cycle progression.

Mitosis is thus characterized by a global increase in phosphorylation of multiple proteins at numerous sites which act as a switch to activate the downstream processes (Dephoure,

Zhou et al. 2008, Olsen, Vermeulen et al. 2010, Kettenbach, Schweppe et al. 2011). All

these processes allow the cells to equally and accurately segregate their chromosomes.

This segregation is followed by decline in the kinase activities of the mitotic kinases, mostly by proteolytic degradation of the activators or kinases themselves. CDK1 activity declines at the metaphase-anaphase transition when cyclin B is degraded by the APC/C complex. However, the small percentage of proteins degraded at the mitotic exit (about

2.5% in humans and 20% in yeast) (Min, Mayor et al. 2014) cannot account for the dephosphorylation of about 32,000 phosphorylation events that take place in mitosis

(Olsen, Vermeulen et al. 2010, Sharma, D'Souza et al. 2014). Therefore, along with the decline in kinase activity, the critical regulator of mitotic exit is also the activation of phosphatase activity towards the mitotic phospho-proteins. Many phosphatases have been implicated to play a role throughout the cell cycle and at the mitotic exit to maintain the phospho-status of cell cycle regulatory proteins. These phosphatases will be discussed in future sections.

1.3 Protein phosphatases

1.3.1 Classification and function

Protein phosphorylation is a dynamic post-translational switch that regulates the cellular response to internal and external cues. Protein phosphatases, enzymes which dephosphorylate specific amino acid residues, have been characterized into four classes based on their substrate preference and catalytic signature (Table 1.1). These classes include PTPs (protein tyrosine phosphatases), aspartate-based protein phosphatases,

PPMs (Mg2+/Mn2+-dependent protein phosphatases) and PPPs (phosphoprotein phosphatases).

Table 1.1: Classification of protein phosphatases.

Adapted from (Kerk, Templeton et al. 2008). Protein phosphatases as classified into four families: PPP (Phosphoprotein phosphatases), PPM (Mg2+/Mn2+-dependent protein phosphatases), PTP (Protein tyrosine phosphatases) and Asp-based catalysis phosphatases. The table shows the number of genes in humans which encode each type of phosphatase.

Protein phosphatase Subclass Human genes family PPP family Total 13 PP1 3 PP2A 2 PP2B/PP3 3 PP4 1 PP5 1 PP6 1 PP7 2 PPM (PP2C) family Total 20 PTP family Total 106 Class I PTPs (Classic) Total 37 Receptor 20 Non-receptor 17 Class I PTPs (DSPs) Total 67 MAPKP 11 Slingshots 3 PRL 3 Atypical DSP 19 CDC14 5 PTEN 7 Myotubularins 16 Other 3 Class II PTPs (CDC25) 3 Class III PTPs 1 (LMWPTP) Asp-based catalysis Total 13 FCP-like 8 Chronophins 1 EYA 4 Total phosphatases 158

PTP (Protein Tyrosine Phosphatase) family members contain a unique catalytic signature, CX5R, and have a varied substrate preference, which includes complex carbohydrates, mRNA, and phosphoinositides (Fischer, Charbonneau et al. 1991, Guan and Dixon 1991). There are 106 genes that encode for PTPs in the human genome (Tonks

2006). Based on primary sequence of catalytic domains, the PTPs are divided into four separate classes. Class I PTPs include “classical” PTPs, which dephosphorylate tyrosine residues, and “dual-specificity phosphatases”, which dephosphorylate both serine/threonine and tyrosine residues in protein substrates and non-protein substrates.

The DSP group includes phosphatases like CDC14 (cell division cycle 14), PTEN, slingshot, and myotubularins. The class II PTPs are structurally related to bacterial arsenate reductases and are represented by low molecular mass PTP (LMPTP) in humans

(Bottini, Bottini et al. 2002). Class III PTPs are tyrosine/threonine specific phosphatases and include the crucial cell cycle phosphatase CDC25. These enzymes are thought to have evolved from the bacterial rhodanese-like enzyme (Bordo and Bork 2002).

The aspartate-based family of phosphatases has the catalytic signature DXDXT/V, with an Asp-based catalytic mechanism of dephosphorylation. The founding member of this family FCP1 [TFIIF (transcription initiation factor IIF)-associating component of

CTD (C-terminal domain) phosphatase] dephosphorylates the CTD of the largest subunit of Pol II and very recently has been implicated in cell cycle regulation in a transcription independent manner (Visconti, Palazzo et al. 2012, Hegarat, Vesely et al. 2014, Della

Monica, Visconti et al. 2015, Visconti, Della Monica et al. 2015).

The PPP and the PPM families of protein phosphatases, both of which depend on metal ions for catalysis, are responsible for the majority of phospho-serine and threonine

dephosphorylation reactions in a eukaryotic cell. The PPM family members, including

PP2C and pyruvate dehydrogenase phosphatase, are characterized by 11 conserved motifs with nine highly conserved amino acids including four aspartate residues necessary for coordination of metal ions for catalysis (Cohen 1997, Andreeva and Kutuzov 2001). The

PPP family, defined by three signature motifs (GDXHG-, -GDXVDRG- and -GNHE-) within a 280 amino acid catalytic domain, includes the crucial cell cycle regulatory protein phosphatases PP1 and PP2A (Cohen 2002, Honkanen and Golden 2002, Wang,

Zhang et al. 2008). Most of the PPP family members work as multimeric holoenzyme complexes, formed by a specific phosphatase catalytic subunit and one or more associated regulatory/scaffolding subunits. PP1 associates with many large complexes but typically contacts a single binding partner or the regulatory subunit which is responsible for targeting the catalytic subunit to the substrate. PP2A works with a scaffolding A subunit and a regulatory B subunit (Cohen, Brewis et al. 1990, Yanagida, Kinoshita et al.

1992). These regulatory subunits provide diversity and substrate specificity to the phosphatases. More than 200 regulatory proteins have been identified for PP1 which are discussed in further detail in Section 1.4. The PP2A B subunits belong to four unrelated families (B/PR55/B55, B′/PR61/B56, B′′/PR72, and B′′′/the striatins, STRN), which are encoded by 15 different genes and translate into 26 different B subunits by alternative splicing.

1.3.2 Phosphatases as mitotic guards

The entry into mitosis and the maintenance of the mitotic state involves timely phosphorylation of multiple proteins by mitotic kinases including CDK1, Greatwall

kinase, NIMA-related kinases (Nek), Polo-like kinase 1 (PLK1), Aurora A, Aurora B and

Wee1-related kinases (Figure 1.2). To counteract the activity of these kinases, multiple phosphatase complexes work throughout mitosis to ensure accurate and timely mitotic progression and mitotic exit. The major mitotic phosphatases include PPP family members PP1, PP2A and PP6, PTP family members CDC14 and CDC25, and aspartate based phosphatase FCP1 (Figure 1.2).

The CDK1-cyclin B complexes in the cell are kept in an inactive state by inhibitory phosphorylations on Thr14 and Tyr15 by Wee1 and Myt1 protein kinases as described previously (Section 1.2) (Lundgren, Walworth et al. 1991, Mueller, Coleman et al. 1995).

The removal of these phosphorylations activates the CDK1 kinase, and this function has been attributed to dual specificity CDC25 phosphatase (Honda, Ohba et al. 1993).

Humans have three paralogs of CDC25: CDC25A, CDC25B, and CDC25C, where B and

C have more prominent roles in mitotic entry. Interestingly, the CDC25 phosphatase is directly regulated by a positive feedback loop with CDK1, which drives the activation of the kinase even further. CDK1 also activates other kinases like PLK1, Aurora A, and

Casein kinase 1, which indirectly activate the CDC25 phosphatase activity. Another mechanism of the regulation of CDC25 is through the recruitment of protein phosphatase

1 to dephosphorylate its Ser287 (inhibitory phosphorylation site for CDC25) and is explained in further detail in Section 1.4. The full activation of CDK1 also requires that the counteracting protein phosphatases be inactivated at mitotic entry. This phosphatase activity is essential during interphase to prevent premature entry into mitosis but needs to be reduced to allow the cell to enter mitosis and maintain the directionality of mitosis.

Figure 1.2: Regulation of mitosis by coordinated activities of protein kinases and protein phosphatases.

The activation of protein kinases and protein phosphatases is temporally and spatially controlled to regulate the transition into each mitotic phase. While different protein kinases are activated at different phases during mitosis, protein phosphatases gain their spatial control through different regulatory proteins as depicted. The upper panel shows the protein kinases activated at locations indicated in arrow heads while the lower panel shows protein phosphatase complexes activated during various mitotic phases at locations indicated in arrow heads.

Pioneering experiments to determine the phosphatase responsible for this function showed that this phosphatase activity is sensitive to okadaic acid (OA), a PPP serine/threonine phosphatase inhibitor (Vandre and Wills 1992, Zhao, Haccard et al.

2008, Castilho, Williams et al. 2009, Vigneron, Brioudes et al. 2009, Burgess, Vigneron et al. 2010). OA triggers mitotic entry even in the presence of CDK1 inhibitors suggesting the importance of this phosphatase activity in preventing premature mitotic entry (Skoufias, Indorato et al. 2007). A series of studies showed that this protein phosphatase is PP2A, and specifically PP2A with the B55 regulatory protein (Mochida,

Ikeo et al. 2009, Burgess, Vigneron et al. 2010, Lorca, Bernis et al. 2010, Mochida,

Maslen et al. 2010). The regulation of this phosphatase holoenzyme was attributed to the inhibitors ENSA and ARPP19 (Gharbi-Ayachi, Labbe et al. 2010, Mochida, Maslen et al.

2010). Both ENSA and ARPP19 are substrates for the protein kinase Greatwall, which is in turn activated by CDK1. Once phosphorylated by Greatwall at the mitotic entry, ENSA and ARPP19 can inhibit the activity of PP2A-B55. This switch is crucial to fully activate

CDK1 and trigger further mitotic events. Another mitotic phosphatase FCP1 regulates the activity of PP2A-B55 by directly dephosphorylating Greatwall kinase at the CDK1 phosphorylation sites (Visconti, Palazzo et al. 2012, Hegarat, Vesely et al. 2014, Della

Monica, Visconti et al. 2015). In this way, the phosphatase FCP1 can reduce the activity of Greatwall towards ENSA/ARPP19 and drive the mitotic progression.

As the cell enters mitosis, DNA condenses to form two sister chromatids linked by the proteinaceous multi-subunit Cohesin complex (Nasmyth and Haering 2009). During prophase most of the cohesin is removed from the sister chromatids by PLK1 and Aurora

B mediated phosphorylation (Kitajima, Hauf et al. 2005). However, the cohesin-mediated

linkage is protected at the centromeres, which is essential for the attachment of chromosomes to the mitotic spindle. In mammalian cells, this centromeric protection is attributed to the Shugoshin protein (Sgo1 and Sgo2) (Kitajima, Kawashima et al. 2004,

Salic, Waters et al. 2004, Kitajima, Hauf et al. 2005, McGuinness, Hirota et al. 2005).

Sgo1 and Sgo2 can bind PP2A-B56 holoenzyme complexes and recruit the phosphatase to centromeres (Xu, Cetin et al. 2009). This localized pool of the phosphatase counteracts the activity of PLK1 and Aurora B at centromeres by dephosphorylating centromere- specific substrates (Figure 1.3). This pathway is critical for chromosome attachment and separation during mitosis.

During mitosis, multiple proteins are phosphorylated with the majority of them being CDK1 substrates. These mitotic phospho-proteins need to be dephosphorylated for the cell to exit mitosis and come back to the interphase level of phosphorylation (Queralt and Uhlmann 2008). CDC14 has been recognized as the essential phosphatase required to dephosphorylate CDK1 substrates to achieve mitotic exit in budding yeast. The cdc14 yeast mutants arrest in a telophase-like mitotic state with a bilobed nucleus and elongated mitotic spindle (Stegmeier and Amon 2004). The vertebrate orthologs, CDC14A and

CDC14B, can rescue these mutants in yeast suggesting functional conservation

(Bremmer, Hall et al. 2012, Yellman and Roeder 2015). However, in higher eukaryotes,

CDC14 is dispensable for mitotic exit and its role as a CDK-counteracting phosphatase is taken over by okadaic acid-sensitive phosphatases as described above (Queralt and

Uhlmann 2008, Mocciaro and Schiebel 2010, Bouchoux and Uhlmann 2011).

Figure 1.3: PP2A-Shugoshin complex regulates cohesion during mitosis.

The cohesin complexes (shown in orange) are removed from the chromosomes during prophase by phosphorylation triggered by protein kinases Aurora B and PLK1. However, the centromeric pool of cohesin is protected at this point by the recruitment of the PP2A- Sgo1/2 holoenzyme, which keeps centromeric substrates of the kinases in dephosphorylated state. At the end of anaphase, this pool of cohesin is cleaved by the activated separase enzyme so that chromatids can segregate to the opposite spindle poles.

Studies in Drosophila show that the abnormal anaphase resolution (aar) mutations, which result in abnormal separation of chromosomes at anaphase, map to PP2A-B complex (Mayerjaekel, Ohkura et al. 1993). In addition, mutations in Drosophila PP1 also cause defective sister chromatid segregation and abnormal chromatin condensation

(Axton, Dombradi et al. 1990). PP1 and PP2A have both been implicated in normal

mitotic exit and are critical for cell division (Mochida, Ikeo et al. 2009, Wu, Guo et al.

2009, Schmitz, Held et al. 2010).

1.4 Protein Phosphatase One and its regulation

1.4.1 The catalytic subunit of protein phosphatase 1 (PP1)

Protein phosphatase 1 (PP1) was initially identified as the protein phosphatase responsible for conversion of phosphorylase a to phosphorylase b, and since then it has been shown to be critical for many cellular events including metabolism, cell division, apoptosis, cytoskeleton reorganization and protein translation. The catalytic subunit of

PP1 is encoded by multiple genes in most eukaryotes, the exception being

Saccharomyces cerevisiae which has only a single gene (Glc7). There are three mammalian isoforms of PP1: PP1, PP1 and PP1. PP1 exists in two forms: PP11 and

PP12, which arise through alternative splicing of the same gene (Bollen and Stalmans

1992, Cohen 2002). The human PP1 isoforms are 90% identical in their amino acid sequence with differences in the N- and the C-terminal residues which provides specificity to each isoform in cells (Figure 1.4).

1.4.2 Mechanism of dephosphorylation by PP1

PP1 is a member of the serine/threonine specific PPP family of phosphatases which utilize metal ions to catalyze the dephosphorylation reaction. The catalytic site of

PP1 contains two metal ions located at the intersection of three clefts: the hydrophobic, the acidic and the C-terminal groove (Shi 2009). The metal ions are coordinated by oxygen atoms of the oxyanion of a water molecule (Goldberg, Huang et al. 1995).

Figure 1.4: Human PP1 isoforms are highly conserved among each other.

The protein sequences for each of the PP1 isoforms were aligned using Clustal Omega. The fully conserved residues are indicated with an asterisk (*), amino acid side chains with highly similar properties are indicated with a colon (:) and amino acid side chains with weakly similar properties are indicated with a period (.).

The metal-coordinating aspartate, histidine, and arginine, as well as the phosphate (from phosphorylated residue)-interacting arginine and histidine residues, are conserved within the PPP family of phosphatases including PP1 (Barford, Das et al. 1998, Terrak, Kerff et al. 2004, Shi 2009). PP1 catalyzes the dephosphorylation in a single step with the metal-

activated water molecule or hydroxide ion (Figure 1.5A). Other catalytic site residues also enhance the catalytic activity of PP1. This includes arginine residues (Arg96 and

Arg221), which stabilize the transition state and increase the Kcat, and His125, which donates a proton to the leaving group oxygen atom of the P-O bond.

1.4.3 PP1 Regulatory proteins: Diversity and specificity

The catalytic subunit of PP1 works in conjunction with diverse regulatory subunits to form holoenzymes that function in various cellular pathways. More than 200 regulatory proteins have been identified for PP1, with more awaiting identification

(Bollen 2001, Cohen 2002, Hendrickx, Beullens et al. 2009, Bollen, Peti et al. 2010,

Heroes, Lesage et al. 2013). The large number of regulatory subunits not only provide diversity to PP1 but also confer specificity towards particular substrates and restrict subcellular locations. The binding of PP1 with its regulatory proteins is mediated by short docking motifs. About nine different docking motifs have been identified in PP1 interactors (Ceulemans, Stalmans et al. 2002, Meiselbach, Sticht et al. 2006, Roy and

Cyert 2009). Some of these motifs are specific to particular interactors, and some are present in multiple PP1 interacting proteins (Table 1.2).

More than 90% of PP1 regulatory subunits bind the phosphatase through a short degenerate motif called the RVxF motif, which conforms to the sequence [RK]x0-

1[VI]{P}[FW], where x can be any residue and {P} refers to any residue but proline

(Bollen 2001, Wakula, Beullens et al. 2003). An example of this is MYPT1 which uses its 35KVKF38 sequence to bind PP1 (Figure 1.5B). This peptide sequence from the

Figure 1.5: Protein phosphatase 1 and its regulation.

(A) The active site of PP1 contains two metal ions (Mn2+) coordinated by a water molecule. PP1 catalyzes the dephosphorylation with metal activated water molecule which can attack the phosphate bond in the substrate (shown in red). This is aided by the proton donating positive charged His125 in the active site of PP1. (B) The active site of PP1 (purple) contains two metal ions (red spheres) and is 20Å away from the RVxF binding site as shown in case of the regulatory protein MYPT1 (green). The inset shows the 35KVKF38 sequence of MYPT1 (green) in the RVxF binding pocket of PP1 (purple). The figure was made using Protein Workshop with PDB ID 1S7O.

Table 1.2. PP1 binding motifs in different regulatory proteins.

Nine types of PP1 binding motifs have been identified in different regulatory proteins. These motifs bind to the catalytic subunit of PP1 at different or same site. Although binding of these motifs does not change the catalytic activity of PP1, they bring the interacting protein close to the catalytic subunit thereby aiding in the interaction.

Motif Sequence/Type Examples from PP1 regulatory

proteins

RVxF [KR]-[KR]-[VI]--[FW] 90% of PP1 regualory proteins

including KNL1 (CASC5), RIF1,

CDCA2 (RM)

SILK [GS]-I-L-[RK] KNL1 (CASC5), RIF1

MyPhone R-x-x-Q-[VIL]-[KR]-x-[YW] MYPT1, MYPT2, MYPT3,

Myosin phosphatase N-terminal element MYR8

SpiDoC Spinophilin Docking site for the C- Spinophilin

terminal groove

IDoHA Inhibitor-2 Docking site for Hydrophobic Inhibitor-1

and Acidic grooves

BiSTriP Bipartite docking site of Sds22 interacting Sds22

with Triangular Region delineated by a4-6

helices of PP1

AnkCap Ankyrin repeat cap MYPT1

NiHiP NIPP1 Helix that interacts with PP1 NIPP1

regulatory proteins bind PP1 in a hydrophobic channel that is remote from the active site.

The binding of RVxF motif does not alter catalytic activity of the phosphatase but serves as the primary binding site for regulatory proteins thereby promoting the secondary binding of these proteins with the catalytic subunit.

It is important to note here that although the activity of the PP1 catalytic subunit is largely governed by its regulatory proteins, CDK1 can also regulate the activity of PP1 during mitosis. The activation of CDK1 at G2/M checkpoint leads to the phosphorylation of Thr320 at the C-terminus of PP1 (Dohadwala, da Cruz e Silva et al. 1994, Kwon, Lee et al. 1997). This phosphorylation by CDK1 has been shown to have an inhibitory effect on the catalytic activity of PP1 during mitosis and is crucial to keep this phosphatase activity in check during mitosis. In addition, the activity of PP1 is also blocked by the binding of Inhibitor-1 phosphorylated by PKA. However, upon the decline of CDK1 activity at anaphase, PP1 can autodephosphorylate the Thr320 and regain partial activity

(Wu, Guo et al. 2009). This partially active PP1 can then dephosphorylate the PKA phosphorylated residue in inhibitor-1 which leads to its dissociation from PP1 rendering

PP1 fully activated. This phosphatase can then actively dephosphorylate the mitotic phospho-proteins and drive mitotic exit.

1.5 Role of PP1 during mitosis

1.5.1 Mitotic entry

Entry into mitosis is largely governed by the activation of the master kinase

CDK1 through multiple positive and negative feedback signaling loops (Section 1.2)

(Morgan 1995). As described before, CDC25 phosphatase plays a crucial role in this

activation of CDK1 by dephosphorylating critical inhibitory phosphorylated residues on

CDK1. However, CDC25 itself is under strict regulation to control the precise timing of mitotic entry. The checkpoint suppression of CDC25 is maintained through the phosphorylation of Ser287, which is essential for the recruitment of 14-3-3 proteins

(Figure 1.6A) (Margolis, Perry et al. 2006). The phosphorylation of this critical residue has been attributed to multiple kinases including CHK1, CHK2, and protein kinase A

(PKA). It has been shown that PP1 is an essential regulator of this pathway as it dephosphorylates the Ser287 of CDC25, thereby derepressing the activity of CDC25 at

G2/M transition.

1.5.2 Centrosomal splitting and spindle formation

As cells enter mitosis, the two duplicated centrosomes are separated to form microtubule-organizing centers that eventually form the mitotic spindle. The separation of centrosomes at G2/M transition is regulated by protein phosphorylation. Nek2 serine/threonine protein kinase regulates this process by phosphorylating key substrates.

Nek2 forms a complex with mammalian STE2-like protein kinase 2 (MST2) and PP1 during interphase (Helps, Luo et al. 2000, Meraldi and Nigg 2001, Mi, Guo et al. 2007).

As cells enter mitosis, PLK1 phosphorylates MST2, which dissociates PP1 from the

Nek2-MST2-PP1 complex and allows Nek2 to autophosphorylate and activate itself. The active Nek2 can then phosphorylate the linker proteins C-Nap1 and Rootelin, which allows the centrosomes to move apart (Figure 1.6B) (Helps, Luo et al. 2000). Thus, PP1 plays a critical role in the process of centrosomal splitting essential for the formation of mitotic spindle.

Figure 1.6: Mitotic roles of protein phosphatase 1.

(A) CDK1 is kept inactive during the interphase by inhibitory phosphorylations on Thr14 and Tyr15. CDC25 phosphatase removes these phosphorylations to activate CDK1 at the

G2/M transition. The CDC25 phosphatase itself is regulated by Ser287 phosphorylation which keeps the phosphatase suppressed. PP1 dephosphorylates the Ser287, thereby activating CDC25 and allowing the activation of CDK1 for mitotic entry. (B) The formation of PP1-Nek2-MST2 complex keeps the Nek2 kinase inactive until the G2/M transition when PLK1-mediated phosphorylation of MST2 dissociates PP1 from the complex. Nek2 can then phosphorylate itself and its substrates C-Nap1 and Rootelin which initiate the separation of centrosomes before the cells enter mitosis. (C) Unattached kinetochores are detected by the spindle assembly continued…

1.5.3 Spindle assembly checkpoint

The bipolar orientation of sister chromatid kinetochores through the microtubules emanating from opposite poles is essential for error-free segregation of chromosomes.

The Spindle Assembly Checkpoint (SAC) is the surveillance mechanism of the cell that ensures correct bipolar attachment, and delays anaphase until this has been achieved.

However, after this bi-orientation is achieved, it is important to silence the checkpoint for the cell to progress into anaphase and exit mitosis. Reversible protein phosphorylation is a key regulatory mechanism in SAC activation and silencing. The protein kinase critical for this process has been identified to be Aurora B, which is localized in the inner centromeres during metaphase where it phosphorylates different members of SAC to activate arrest and eventually moves to central spindle in anaphase (Krenn and

Musacchio 2015).

Figure 1.6: continued…checkpoint proteins, preventing the cells to enter anaphase by phosphorylation of SAC proteins. Once the chromosomes achieve bipolar attachment, the SAC is silenced by dephosphorylation of SAC proteins mostly by the kinetochore recruitment of PP1 through various regulatory proteins including KNL1, CENP-E, and Sds22. PP1 antagonizes the phosphorylations by Aurora B kinase which resides at the centromeres and is pulled away from the kinetochores once the tension is established. (D) The inhibitory phosphorylation of PP1 at Thr320 by CDK1 kinase keeps it inactive during mitosis. At anaphase, cyclin B is degraded by APC/C mediated proteasomal degradation which leads to the decline of CDK1 activity. This leads to auto- dephosphorylation by PP1 at Thr320 and activates PP1, which can then dephosphorylate multiple mitotic phospho-proteins by forming complexes with CDCA2 (RM), PNUTS and AKAP149 among others.

Aurora B together with proteins Survivin, INCENP and Borealin form the chromosome passenger complex, which are important for its localization and activity. The binding of

INCENP around the Aurora B active site leads to the phosphorylation of TSS motif on

INCENP and autophosphorylation of the T-loop site of Aurora B, Thr232.

Aurora B regulates the incorrect microtubule-kinetochore attachments by sensing tension across the centromere. Lack of bipolar attachment of sister chromatids leads to the kinetochore recruitment of multiple checkpoint proteins like Mps1, Bub1, CENP-E,

Bub3, Mad1 and Mad2. All spindle checkpoints proteins recruited to unattached kinetochores contribute to the formation of the mitotic checkpoint complex and therefore to APC/C inhibition which delays the onset of anaphase until correct bi-orientation is achieved.

The protein phosphatase involved in the silencing of spindle checkpoint is PP1, which is targeted to the kinetochores by several kinetochore-associated proteins (Lesage,

Qian et al. 2011). The functional disruption of the specific kinetochore-associated PP1 causes a metaphase arrest (Liu, Vleugel et al. 2010). Consistent with this, Pinsky et al., reported that, in budding yeast, overexpression of PP1 prevents checkpoint activation in response to both tension and attachment defects, suggesting an important role of PP1 in silencing the spindle checkpoint (Pinsky, Nelson et al. 2009). KNL1 (also called CASC5), a kinetochore resident protein, recruits PP1γ to the kinetochore where PP1 can oppose the phosphorylation by checkpoint kinase Aurora B. KNL1 binds PP1 through a conserved

RVSF motif and mutations in this motif lead to persistent activation of the spindle assembly checkpoint (Liu, Vleugel et al. 2010, Rosenberg, Cross et al. 2011). The level of PP1 recruited at the kinetochores is also critical for checkpoint silencing, as mutants

that display excess amount of PP1 at kinetochores are lethal (Rosenberg, Cross et al.

2011). Additionally, recruitment of PP1 is dependent on kinetochore tension. Once bi- orientation of sister kinetochores is achieved, the pulling forces generated by microtubules emanating from opposing poles create tension thereby stretching kinetochores. This pulling of kinetochores spatially separates Aurora B, which is localized to centromeres, from its kinetochore substrates and promotes the recruitment of

PP1 at kinetochores (Figure 1.6C). The spatial separation decreases Aurora B-dependent phosphorylation and increases the dephosphorylation of SAC proteins by PP1 at the kinetochores, thereby silencing the checkpoint and signaling cells to progress into anaphase.

The targeting of PP1 to the kinetochore has also been attributed to plus-end directed motor protein Klp5/6 (kinesin 8) by Meadows et al. (2011), where they show in fission yeast that the recruitment of PP1 by both KNL1 and Klp5/6 is essential for checkpoint silencing (Meadows, Shepperd et al. 2011). The vertebrate orthologs of Kpl5/6

(KIF18A) and kinesin-7 (CENP-E) have also been shown to bind PP1 suggesting a similar mechanism in higher vertebrates (Kim, Holland et al. 2010, De Wever, Nasa et al.

2014, Hafner, Mayr et al. 2014). The motor protein CENP-E has a RVxF docking motif for PP1 with threonine at the x position. Phosphorylation of this site by Aurora B at the kinetochores disrupts its association with PP1, decreasing its affinity towards the microtubules. However, spatial separation of Aurora B after bi-orientation of sister chromatids and dephosphorylation of threonine in the RVxF motif helps CENP-E to recruit PP1 to kinetochores where it can dephosphorylate its target proteins (Figure

1.6C). This adds to another regulatory mechanism by which PP1 can be recruited to the kinetochores and silence the spindle checkpoint.

Another protein that targets PP1 to the kinetochores is Sds22, which does not have a RVxF type docking motif but forms a trimeric complex with PP1 and RVxF containing

Inhibitor-3 (Posch, Khoudoli et al. 2010). The localization of Sds22 and PP1 to the kinetochores is mutually dependent on each other, suggesting a prior interaction between the two proteins for kinetochore recruitment. Evidence from Posch et al. (2010), suggests that at the onset of anaphase, Sds22 regulates the activity of Aurora B at kinetochores and maintains the interactions between the kinetochores and microtubules

(Figure 1.6C) (Posch, Khoudoli et al. 2010). Another study used an RNAi based screening and phosphorylation biosensor methodology to confirm that the PP1-Sds22 complex counteracts Aurora B-dependent phosphorylation at kinetochores

(Wurzenberger, Held et al. 2012). These studies validate the importance of PP1 at kinetochores to maintain the fidelity of chromosome segregation and drive the cell towards exit from mitosis.

1.5.4 Mitotic exit

CDCA2 (RM)/PP1 Complex

Several studies have suggested that PP1 is important for the cells to exit mitosis. PP1 mutants in Drosophila show abnormal sister chromatid segregation and excessive chromosome condensation. CDCA2 (RM) was identified as a regulatory protein of PP1, which targets PP1 to chromatin at the onset of anaphase (Trinkle-Mulcahy, Andersen et al. 2006). This localization of CDCA2 and PP1 is inhibited by CDK1 phosphorylation of

CDCA2 during early mitosis and depends on the inactivation of CDK1 after metaphase to anaphase transition (Figure 1.6D). In fact, CDCA2 is a major player in the phosphatase relay where PP2A-B56 recruited by active CDK1 in pro-metaphase dephosphorylates

CDCA2 Ser 893 which is crucial for the recruitment of PP1 during anaphase (Qian,

Beullens et al. 2015). Once active at anaphase, the CDCA2/PP1 complex dephosphorylates the CDCA2 Ser893, H3 Thr3, Ser10 and Ser28 and other mitotic phospho-proteins (Qian, Lesage et al. 2011, Qian, Beullens et al. 2013). Phosphorylated

H3 Thr3 is the established docking site for Aurora B, the master kinase of spindle checkpoint, on mitotic chromosomes and the dephosphorylation of this site might contribute to re-localization of Aurora B and the chromosome passenger complex to spindle mid-zone after spindle checkpoint. Interestingly, dephosphorylation of H3 Ser10 regulates the binding of heterochromatin protein 1 (HP1) which directs heterochromatin formation (Vagnarelli, Hudson et al. 2006). The CDCA2 and PP1 complex is also shown to be responsible for regulating the phospho-switch which mediates the re-establishment of heterochromatin in post-mitotic cells (Vagnarelli, Ribeiro et al. 2011).

During late anaphase, a pool of CDCA2/PP1 complex localizes to the periphery of chromosomes, where it contributes to the recruitment of importin β, a critical player in the re-assembly of nuclear envelope. Further experimental insight is required to determine the chromosomal substrates of CDCA2/PP1 and elucidate the mechanism of its action in reforming nuclei (Vagnarelli, Ribeiro et al. 2011).

PNUTS/PP1 Complex

As mentioned above, the post-mitotic decondensation of the chromatin is crucial for the cells to return to the basal state of functional interphase cells. PNUTS (PP1 nuclear targeting subunit) is a ubiquitously expressed regulatory subunit of PP1 that targets PP1 to the nucleus (Allen, Kwon et al. 1998, Rebelo, Santos et al. 2015). Interestingly,

PNUTS is exclusively nuclear in interphase and co-localizes with chromatin during telophase. PNUTS is targeted to the reforming nucleus in telophase after the assembly of nuclear membranes (Landsverk, Kirkhus et al. 2005). PNUTS has been shown to enhance the in-vitro decondensation of mitotic chromosomes in a PP1-dependent manner

(Landsverk, Kirkhus et al. 2005). Lee et al. (2010) identified a novel multi-subunit PP1 complex, PTW/PP1 complex, that consists of catalytic subunit of PP1, Wdr82 a regulatory component of the SET1 methyl-transferase complex and Tox4, along with

PNUTS (Lee, You et al. 2010). Mutation in the PP1 binding motif of PNUTS causes defects in the process of chromosome decondensation at late telophase suggesting a potential role of PP1-PNUTS in targeting the methyl-transferase complex to chromatin, thereby, regulating the chromatin structure at mitotic exit. Interestingly, PNUTS itself degrades at the mitotic exit and the disruption of this process delays the exit of cells from mitosis suggesting that this complex is also an important regulator of mitotic exit (Fisher,

Wang et al. 2014).

AKAP149/PP1 Complex

The nuclear envelope (NE) consists of two concentric membranes, nuclear pores and the nuclear lamina, a meshwork of intermediate filaments called A- and B-type

lamins. Disassembly of the NE in mitosis correlates with the phosphorylation of the proteins of nuclear membranes and nuclear lamina. The dephosphorylation of these proteins is crucial for the re-assembly of NE at mitotic exit. A-kinase anchoring protein

149 (AKAP149) was identified as a component of the ER-NE system that also interacts with A- and B-type lamins. AKAP149 has been shown to target PP1 to the nuclear envelope, where it enhances the dephosphorylation of B-type lamins to promote the reformation of nuclear envelope at mitotic exit (Steen, Martins et al. 2000, Steen,

Beullens et al. 2003, Kuntziger, Rogne et al. 2006).

1.6 Research Objectives

PP1 has been shown to regulate the cell division cycle in multiple ways by forming complexes with diverse regulatory subunits. Although the motif involved in binding of

PP1 regulatory proteins with PP1 catalytic subunit has been identified and studied, the binding mechanism in the context of cell cycle is not known. The main objective of this

Ph.D. thesis is to determine how PP1 is regulated through the cell cycle and how this regulation drives the cell cycle.

I hypothesize that the docking of PP1 to its regulatory proteins is controlled in a cell cycle-dependent manner and is critical to drive cell cycle progression.

I will address this hypothesis with three specific aims:

1. To decipher the mechanism by which reversible phosphorylation within the RVxF

motif controls the docking of PP1 with its regulatory proteins in mitosis.

The first section of this thesis addresses this objective. Using biochemical assays and quantitative mass spectrometric analysis, I will demonstrate the effect of phosphorylation within the PP1 binding RVxF motif in controlling PP1 docking with its regulatory proteins. I will also show how this phosphorylation is dependent on the cell cycle. Lastly,

I address the possibility of a novel interplay between the kinase activity that can phosphorylate these RVxF motifs within multiple PP1 binding proteins and the counteracting phosphatase activity of PP1 which might be crucial for cell cycle progression.

2. To identify and characterize cell cycle specific interactome of PP1

The interaction of PP1 with its binding partners during the cell cycle is not only regulated by localization (spatial regulation), but is also dependent on the specific cell cycle stage

(temporal regulation). Hence, the interactome of PP1 is very dynamic throughout the cell cycle. Next, I analyze the interactome of PP1 in non-synchronized cells vs synchronized mitotic cells to determine cell cycle specific binding tendencies of PP1 with potential novel regulatory proteins.

3. To understand the regulation of centrosomal cycle by PP1: Novel interaction with

CEP192

Finally, I have validated a novel centrosomal interactor of PP1, centrosomal protein of

192 kDa (CEP192), which is crucial for the centrosomal maturation and splitting during cell cycle.

CHAPTER 2. REVERSIBLE PHOSPHORYLATION WITHIN PP1 DOCKING

RVxF MOTIF CONTROLS THE FUNCTION OF PP1 DURING CELL CYCLE

2.1 Introduction

Mitosis involves equal segregation of the genome into two daughter cells, regulated by reversible protein phosphorylation. The opposing activities of protein kinases and protein phosphatases are responsible for maintaining the phosphorylation stoichiometry of mitotic regulatory proteins. Imbalance in either of these activities can lead to mitotic defects and genomic instability (Kastan and Bartek 2004).

Progression into mitosis is regulated by the activation of cyclin-dependent kinase 1

(CDK1) at the G2/M transition which in turn activates other mitotic kinases (Malumbres

2014). Phosphoproteomic profiling has shown a dramatic increase in phosphorylation of a subset of proteins during mitosis (Dephoure, Zhou et al. 2008, Olsen, Vermeulen et al.

2010, Kettenbach, Schweppe et al. 2011). However, to achieve the lower interphase level of protein phosphorylation it is necessary that the mitotic phospho-proteins be dephosphorylated in a timely manner as the cell transitions up to and through the mitotic exit (McCloy, Parker et al. 2015). In vertebrates, most of the dephosphorylation reactions during mitotic exit have been attributed to the phosphoprotein phosphatase (PPP) family of serine/threonine phosphatases, in particular, PP1 and PP2A (Barr, Elliott et al. 2011,

Rusin, Schlosser et al. 2015) and CDK1 has been reported to inversely regulate the activity of these major mitotic protein phosphatases (Qian, Winkler et al. 2013). CDK1 phosphorylation of PP1 at Thr320 has been shown to inhibit its phosphatase activity during mitosis. However, after the decline of CDK1 activity at anaphase, PP1 can auto-

dephosphorylate Thr320 and regain full activity to dephosphorylate multiple mitotic phospho-proteins thereby driving mitotic exit (Wu, Guo et al. 2009). PP1 is responsible for one-third of all the dephosphorylation reactions in a eukaryotic cell (Moorhead,

Trinkle-Mulcahy et al. 2007). The binding of the PP1 catalytic subunits with its regulatory proteins is governed by docking motifs (Hurley, Yang et al. 2007, Moorhead,

Trinkle-Mulcahy et al. 2008). Nearly 90% of PP1 binding proteins dock PP1 through a short degenerate motif called the RVxF motif described as [RK]-X(0,1)-[VI]-{P}-[FW], where X is any residue and {P} any residue but proline (Egloff, Johnson et al. 1997,

Wakula, Beullens et al. 2003, Ceulemans and Bollen 2004, Meiselbach, Sticht et al.

2006). The RVxF containing regulatory proteins bind the catalytic PP1 in a hydrophobic channel located 20Å away from the active site (Egloff, Johnson et al. 1997, Terrak, Kerff et al. 2004).

PP1 has been previously identified to antagonize the activity of mitotic protein kinase

Aurora B, the catalytic component of the chromosomal passenger complex (Francisco,

Wang et al. 1994). The Aurora B-PP1 axis has been reported as the key mechanism responsible for establishing chromosome bi-orientation and regulate the spindle assembly checkpoint (SAC) (Emanuele, Lan et al. 2008, Meadows 2013). During early mitosis,

Aurora B localizes to centromeres and chromosomes but relocalizes to the spindle midzone during mitotic exit. Incorrect microtubule-kinetochore attachments or the lack of tension at kinetochores leads to phosphorylation of outer kinetochore proteins by Aurora

B and activation of the spindle assembly checkpoint (Lampson and Cheeseman 2011).

Once the correct bipolar attachment has been established, PP1 dephosphorylates the

Aurora B substrates at the kinetochores to silence the SAC and drive mitotic exit (Lesage,

Qian et al. 2011, Meadows, Shepperd et al. 2011). This is a well-established mechanism that is essential for the faithful segregation of chromosomes at anaphase. PP1 is enriched on chromosomes and centrosomes during mitosis (Trinkle-Mulcahy, Andrews et al. 2003,

Trinkle-Mulcahy, Chusainow et al. 2007) and multiple PP1 targeting subunits have been identified at kinetochores, mitotic spindle, centromeres, spindle midzone and the nuclear envelope (Steen, Beullens et al. 2003, Trinkle-Mulcahy, Andersen et al. 2006, Rosenberg,

Cross et al. 2011, De Wever, Nasa et al. 2014, Rodrigues, Lekomtsev et al. 2015). One of the key candidate substrates regulated by the Aurora B-PP1 axis during the SAC is the kinetochore protein KNL1/CASC5 which recruits PP1 to the kinetochores via its RVSF motif. Phosphorylation within the RVSF motif of KNL1/CASC5 by Aurora B kinase dissociates PP1 from the regulatory protein (Liu, Vleugel et al. 2010, Rosenberg, Cross et al. 2011).

Characterization of the RVxF motif from known PP1 interactors shows an over- representation of serine and threonine at the ‘x’ position as depicted by the WebLogo

(Crooks, Hon et al. 2004), where serine occurs in ~21% of known PP1 binding partners and threonine in ~18% (Figure 2.1) (Hendrickx, Beullens et al. 2009). Previous studies with specific regulatory proteins GM, KNL1/CASC5, and CENP-E have shown that phosphorylation within the RV[S/T]F motif regulates the association of PP1 with these partners (Dent, Campbell et al. 1990, Kim, Holland et al. 2010, Liu, Vleugel et al. 2010).

In this section, phosphorylation within the RV[S/T]F motif in many potential regulatory proteins of PP1 was evaluated and characterized using quantitative mass spectrometry. It can be speculated that this phosphorylation is an important widespread regulatory mechanism that dissociates the phosphatase (PP1) from its regulatory proteins during

Figure 2.1: Consensus RV[S/T]F motif in PP1 interacting proteins.

Graphical representation of the frequency of amino acids in the RVxF motif in validated PP1 binding proteins. Motif sequences were taken from (Hendrickx, Beullens et al. 2009) and image created using WebLogo (Crooks, Hon et al. 2004) .

mitosis, thereby affecting its targeting to substrates and maintaining the phosphorylation of a multitude of PP1 substrates during mitosis.

2.2 Methods

2.2.1 Cell culture and synchronization

Mycoplasma free HeLa cells were obtained from ATCC and cultured in DMEM, supplemented with 10% (v/v) FBS and 100 U ml-1 penicillin-streptomycin. For cell cycle analysis, cells were arrested with 2 mM thymidine for 17 h followed by a 7 h release.

Following release, cells were blocked in prometaphase with 40 ng ml-1 nocodazole. After

9 h arrest, the cells were released into fresh media and harvested at different time points within a 24 h cycle. A part of the sample was fixed in ethanol and stained with propidium iodide for FACS analysis performed at the Flow Cytometry Facility at the University of

Calgary.

2.2.2 Antibodies and reagents

Commercial antibodies and their fold-dilution used in this study: anti-PP1

(SC7482, Santa Cruz, 1:500), anti-RIF1 (A300-569A, Bethyl Antibodies, 1:500), anti-

SFI1 (13550-1-AP, Proteintech Group Inc., 1:250), anti-BRCA1 (9010S, Cell Signaling,

1:1,000), anti-CDCA2 (RM) (ab45129, Abcam, 1:1,000), anti-Cyclin B (SC594, Santa

Cruz, 1:500), anti-tubulin (T9026, Sigma-Aldrich, 1:1,000), H3S10ph (06-570, Millipore

(Upstate), 1:1,1000), anti-Aurora B (ab45145, Abcam, 1:5,000). HRP-conjugated goat anti-Mouse and goat anti-Rabbit were obtained from Pierce and Thermo Fisher Scientific and were used at 1:5,000. All the peptides used in this study, unmodified or phosphorylated within RV[S/T]F motif (ZAP: GKKRVRWADLE, ZAPRARA:

GKKRARAADLE, RIF1: KRRVSFADK, CEP192: SEKHVTFENHK, SFI1:

SRKVTFEGPK, CDCA2 (RM): KRKRVTFGED, TSC2: QLHRSVSWADSAK,

RRP1B: SSKKVTFGLN, MPP10: KESLKRVTFAL, ORC2: KTPQKSVSFSLK,

BRCA1: QSPKVTFECEQK, Ki67: KRRRVSFGGH) were synthesized with 98% purity at GL Biochem (Shanghai) Ltd., China. For the generation of phospho-specific antibodies, the RVp[S/T]F peptides derived from the proteins were coupled to keyhole limpet haemocyanin (KLH) (Imject Maleimide-activated KLH kit, Pierce) and bovine serum albumin (BSA) before injecting them into rabbits. In-house generated antibodies were affinity purified on phospho-peptide columns (Section 2.2.3) and used in western blots at the following concentrations: p-RIF1 (2 g ml-1), p-CEP192 (2 g ml-1), p-SFI1

(1 g ml-1), p-CDCA2 (RM) (5 g ml-1), p-BRCA1 (5 g ml-1). NaF (25 mM) and 5 g ml-1 of the respective dephospho-peptides were added to all the phospho-epitope specific antibody solutions.

2.2.3 Affinity purification of phospho-specific antibodies

The serum from the rabbit was diluted (1:10) with 10 mM Tris, pH 7.5, and loaded onto the phospho-peptide coupled activated CH-Sepharose beads (GE Healthcare) for 4 h at 4°C. The column was washed with at least 100 column volumes of 10 mM Tris, pH 7.5 with 500 mM NaCl, followed by 100 column volumes of 10 mM Tris, pH 7.5.

The antibody was eluted from the column using 100 mM glycine, pH 2, in a tube containing 1 volume of 1 M Tris, pH 8, to neutralize the pH of eluate. The antibody was dialyzed against PBS overnight and concentrated the next day using 10 kDa cut-off

Amicon Centrifugal Filter Units (EMD Millipore). The total protein was estimated using

Bradford reagent with BSA as standard and tested for purity and specificity using western blots and dot blots.

2.2.4 PP1 expression and purification

The three isoforms of human PP1 (//) were transformed in BL-21 DE3 E.Coli cells and proteins purified as described in (Moorhead, MacKintosh et al. 1994). Briefly, cells expressing pCW-PP1 construct were grown to an O.D.600 of 0.4 and induced with

0.3 mM isopropyl-thio--D-galactopyranoside at 28°C for 16 h. The cells were then lysed by sonication (6-8 cycles of 30 sec pulse each) in resuspension buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 1 mM EDTA, 2 mM MnCl2, 5% (v/v) glycerol, 0.1% (v/v) BME and 0.1 mM PMSF). The homogenate was centrifuged for 30 mins at 35,000 rpm, and the supernatant was loaded onto microcystin-Sepharose column for 1.5 h. The column was washed with wash buffer (50 mM Tris, pH 7.5, 0.1 mM EGTA, 5% (v/v) glycerol, 1 mM

MnCl2, 0.1% (v/v) BME) with 0.5 M NaCl, followed by no salt wash before eluting with

elution buffer (wash buffer + 3 M NaSCN). The eluate was dialyzed overnight against

PBS with 0.2 M KCl and 0.1% (v/v) BME, concentrated and stored at -80°C for further use.

2.2.5 PP1 binding assays

For the peptide pull-downs, the RV[S/T]F containing peptides (either unmodified or phosphorylated within RV[S/T]F motif) derived from different proteins were coupled to activated CH-Sepharose 4B beads (GE Healthcare) using coupling buffer (0.1 M

NaHCO3 + 500 mM NaCl, pH 8.0) for 4 h at room temperature. Peptide coupled beads were incubated with HeLa whole cell extracts for 3 h at 4°C. The beads were washed with wash buffer (25 mM Tris, pH 7.5, 500 mM NaCl and 0.5% (v/v) NP-40) and the bound proteins were eluted and analyzed for the presence of PP1 by immunoblotting.

For PP1 overlays, recombinant PP1 was expressed in bacteria and affinity purified as described in Section 2.2.4. RV[S/T]F containing peptides were coupled to

BSA using glutaraldehyde (G5882, Sigma-Aldrich) as the coupling agent. These peptides were spotted onto nitrocellulose membrane in different amounts and overlaid with a mixture equal amounts of recombinant human PP1(//) (1μg ml-1). Phosphatase inhibitors (25 mM NaF and 0.5 μM microcystin-LR) were included at all steps. PP1 binding was analyzed by immunoblotting.

2.2.6 Kinase inhibitor assays

HeLa cells were treated with 100 ng ml-1 nocodazole for 15 h for inducing a prometaphase arrest. The respective inhibitors (Aurora A inhibitor I (S1451,

Selleckchem, 100 nM), Hesperadin (S1529, Selleckchem, 100 nM), BI2536 (S1109,

Selleckchem, 100 nM) and Roscovitine (557360, Millipore (Calbiochem), 50 M) were added for 1 h before the nocodazole was washed off the cells. The cells were released into media with inhibitors for 30 mins or 90 mins after nocodazole wash-off. For hesperadin and roscovitine-treated cells, MG132 (10 M, 474790, Millipore

(Calbiochem)) was added along with the protein kinase inhibitors to prevent mitotic exit.

2.2.7 Phosphatase inhibitor assays

HeLa cells were treated with the phosphatase inhibitors: calyculin A (C5552,

Sigma Aldrich), okadaic acid (350-003, Enzo Lifesciences) and fostriecin for 2 h before lysis or fixing for DAPI staining. Lysates were analyzed for changes in phosphorylation of the RV[S/T]F motifs by immunoblotting. DAPI stained cells were visualized with a

Leica DMIRE2 microscope equipped with a digital charge-coupled device (CCD) camera

(Hamamatsu, Photonics, K.K.)

2.2.8 Immunoprecipitation

Nocodazole-treated (100 ng ml-1) HeLa mitotic cells with or without 1 h hesperadin treatment were collected by mitotic shake-off and released for 30 mins in the presence or absence of hesperadin. The cells were lysed using nucleosome preparation buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1.5 mM MgCl2, 0.34 M sucrose, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100 with 100 U ml-1 micrococcal nuclease and 100 U ml-1 TurboNuclease). After 10 min incubation at 37C, an equal volume of high salt buffer (600 mM NaCl with 2% (v/v) Triton X-100) was added 38

followed by sonication and clearing of extract at 10,000 rpm for 10 mins. All the buffers were supplemented with protease inhibitors (EDTA-free protease inhibitor cocktail tablets, S8830, Sigma Aldrich) and phosphatase inhibitors (25 mM NaF, 0.5 M microcystin-LR). Prior to the IP, lysate was pre-cleared with CL-4B Sepharose beads in the ratio of 1:10 v/v of the lysate.

For the IP, the p-RV[S/T]F IgG and the control IgG were coupled to protein A-

Sepharose beads using dimethyl pimelimidate before incubation with the cell extracts.

The beads were washed 3 times with high salt wash buffer (400 mM NaCl with 0.5%

(v/v) NP-40 in PBS) followed by 2 washes with low salt wash buffer (150 mM NaCl with

0.2% (v/v) NP-40 in PBS). The beads were washed two times with PBS before release with the elution buffer (1% (w/v) SDS, 15% (v/v) glycerol, 50 mM Tris-HCl, pH 8.7 and

150 mM NaCl).

2.2.9 Mass spectrometry analysis

Immunoprecipitations were precipitated using 10% (v/v) v/v TCA and sent to Dr.

Arminja Kettenbach, Geisel School of Medicine at Dartmouth, for mass spectrometry analysis. The samples were digested in solution with trypsin in 50 mM ammonium bicarbonate. Reactions were quenched by the addition of 50% (v/v) acetonitrile/5% (v/v) formic acid and dried. Peptides were analyzed on a Q-Exactive Plus mass spectrometer

(Thermo Scientific) equipped with an Easy-nLC 1000 (Thermo Scientific) as previously reported (Rusin, Schlosser et al. 2015). Peptides were resuspended in 5% (v/v) methanol/1% (v/v) formic acid and loaded on to a trap column (1 cm length, 100 μm inner diameter, ReproSil, C18 AQ 5 μm 120 Å pore (Dr. Maisch, Ammerbuch, Germany))

vented to waste via a micro-tee and eluted across a fritless analytical resolving column

(35 cm length, 100 μm inner diameter, ReproSil, C18 AQ 3 μm 120 Å pore) pulled in- house (Sutter P-2000, Sutter Instruments, San Francisco, CA) with a 60 minute gradient of 5-30% LC-MS buffer B (LC-MS buffer A: 0.0625% formic acid, 3% ACN; LC-MS buffer B: 0.0625% formic acid, 95% ACN). The Q-Exactive Plus was set to perform an

Orbitrap MS1 scan (R=70K; AGC target = 3e6) from 350 – 1500 Thomson, followed by

HCD MS2 spectra on the 10 most abundant precursor ions detected by Orbitrap scanning

(R=17.5K; AGC target = 1e5; max ion time = 75 ms) before repeating the cycle.

Precursor ions were isolated for HCD by quadrupole isolation at width = 0.8 Thomson and HCD fragmentation at 26 normalized collision energy. Charge state 2, 3 and 4 ions were selected for MS2. Precursor ions were added to a dynamic exclusion list +/- 20 ppm for 20 seconds. The resulting data files were searched using Comet (release version

2014.01) in high resolution mode (Eng, Jahan et al. 2013) against a target-decoy

(reversed) (Elias and Gygi 2007) version of the human proteome sequence database

(UniProt; downloaded 2/2013, 40482 entries of forward and reverse protein sequences) with a precursor mass tolerance of +/- 1 Da and a fragment ion mass tolerance of 0.02

Da, and requiring fully tryptic peptides with up to three miscleavages.

Carbamidomethylcysteine was enabled as a fixed modification and oxidized methionine as variable modification. The resulting peptide spectral matches were filtered to < 1% false discovery rate based on reverse-hit counting (Elias and Gygi 2010). Peptide quantification was performed using MassChroQ (Valot, Langella et al. 2011). Protein quantification was performed by iBAQ (Schwanhausser, Busse et al. 2011).

2.2.10 Data analysis

Total peptide counts from p-RV[S/T]F and control immunoprecipitations from mitotically-arrested HeLa cells were input into SAINT (Choi, Larsen et al. 2011) using the CRAPome interface (Mellacheruvu, Wright et al. 2013). For a protein to be considered specific to the p-RV[S/T]F immunoprecipitations, we required the interaction to have an AvgP score in the SAINT analysis of 0.9 or above based on in at least two of the five replicates. Changes in p-RV[S/T]F binding upon Aurora B kinase inhibitor addition were determined by Student’s T-Test. Proteins not quantified in immunoprecipitations upon Aurora kinase B inhibitor addition were called “lost”; proteins quantified in four or five out of five replicates of control immunoprecipitations and only one out of five replicates of immunoprecipitations upon Aurora kinase inhibitor addition (p-value: nd) were called “lost”; proteins quantified in two or three out of five replicates of control immunoprecipitations and only one out of five replicates of immunoprecipitations upon Aurora kinase inhibitor addition (p-value: nd) with a decrease of 2-fold ore more were called “lost”; proteins quantified in immunoprecipitations in the presence or absence of Aurora kinase inhibitor with a p-value < 0.05 and a fold-change decrease of 2-fold or more were called “reduced”; protein quantified in immunoprecipitations in the presence or absence of Aurora B inhibitor with a p-value >

0.05 or p-value: nd were called “change not significant”. Protein-protein interactions between p-RV[S/T]F specific proteins were determined using the STRING database and analyzed in Cytoscape (Shannon, Markiel et al. 2003, Saito, Smoot et al. 2012). Edges represent protein-protein interactions based on the STRING database. GO annotations were performed in Cytoscape using BiNGO to test for ontology enrichment. To

determine the significance of enrichment of terms a Bonferroni corrected p-value cutoff of 0.05 was used.

2.2.11 Immunofluorescence staining and microscopy

For immunofluorescence studies, HeLa cells were grown on poly-lysine coated coverslips, fixed with 3.7% (v/v) formaldehyde, permeabilized with 0.5% (v/v) Triton X-

100 and blocked in 1% (v/v) BSA/PBS. The coverslips were then incubated with primary antibodies for 2 h followed by Alexa Fluor 488-conjugated goat anti-rabbit and Alexa

Fluor 594-conjugated goat anti-mouse secondary antibodies (Molecular Probes, Thermo

Fisher Scientific) for 1 h. Nuclei were counterstained with DAPI (Sigma-Aldrich, 1g ml-

1) and images acquired using a Leica DMIRE2 microscope as described in Section 2.2.7.

2.3 Results

2.3.1 PP1 preferentially binds non-phosphorylated RV[S/T]F motifs in-vitro

To determine if the phosphorylation status of the RV[S/T]F motifs plays a role in the association of PP1 with its regulatory proteins, the docking of PP1 with unmodified or phosphorylated versions of RV[S/T]F peptides (Table 2.1) from different known and potential regulatory proteins of PP1 was examined. PP1 binding to these peptides was tested using two independent biochemical experiments- overlays using recombinant human PP1 (Figure 2.2A) and in-vitro peptide pull-downs (Figure 2.2B). In PP1 overlays, RVRW peptide from the protein ZAP was used as a positive control (Ulke-

Lemee, Trinkle-Mulcahy et al. 2007), while its mutated RARA version, which does not bind PP1 (Ulke-Lemee, Trinkle-Mulcahy et al. 2007), was used as negative control.

Table 2.1: List of peptide sequences used in this study

NAME ACCESSION DESCRIPTION PEPTIDE PEPTIDE SEQUENCE: SEQUENCE: UNMODIFIED MODIFIED RIF1_HUMAN Q5UIP0 Telomere-associated KRRVSFADK KRRVpSFADK protein RIF1

CE192_HUMAN Q8TEP8 Centrosomal protein of SEKHVTFENHK SEKHVpTFENHK 192 kDa SFI1_HUMAN A8K8P3 Protein SFI1 homolog SRKVTFEGPK SRKVpTFEGPK

CDCA2_HUMAN Q69YH5 Cell division cycle- KRKRVTFGED KRKRVpTFGED associated protein 2 (Repoman, RM) TSC2_HUMAN P49815 Tuberin QLHRSVSWADSAK QLHRSVpSWADSAK

RRP1B_HUMAN Q14684 Ribosomal RNA SSKKVTFGLN SSKKVpTFGLN processing protein 1 homolog B MPP10_HUMAN O00566 U3 small nucleolar KESLKRVTFAL KESLKRVpTFAL ribonucleoprotein protein MPP10 BRCA1_HUMAN P38398 Breast cancer type 1 QSPKVTFECEQK QSPKVpTFECEQK susceptibility protein KI67_HUMAN P46013 Antigen Ki-67 KRRRVSFGGH KRRRVpSFGGH

YLPM1_HUMAN P49750 YLP motif-containing GKKRVRWADLE protein 1 (ZAP) YLPM1_HUMAN P49750 YLP motif-containing GKKRARAADLE protein 1 (ZAP)

Figure 2.2: PP1 preferentially binds dephosphorylated RV[S/T]F motifs in-vitro.

(A) Phospho or dephospho versions of different RV[S/T]F containing peptides were spotted on nitrocellulose membranes at indicated amounts. The membranes were overlaid with a mixture of bacterially expressed human PP1 (⍺/β/γ) 1 µg ml-1 and probed with anti-PP1. A peptide derived from PP1 binding protein ZAP (RVRW) is the positive control and the mutated version of this peptide (RARA) is the negative control in the experiment. (B) Activated CH-Sepharose beads were coupled to the phospho or dephospho versions of the indicated peptides and incubated with clarified HeLa whole cell extracts. After washing, proteins bound to the beads were eluted with SDS sample buffer, run on SDS-PAGE and analyzed for PP1 association by immunoblotting. The 35 kDa mass marker is indicated. Peptides derived from the indicated proteins and sequences are shown in Table 2.1. The data represents one of the three independent experiments for both (A) and (B).

A clear preference of PP1 binding to the unmodified versions of all peptides used in both experiments (Figure 2A, B) was noted suggesting loss of PP1 binding upon phosphorylation within RV[S/T]F motifs is a general phenomenon consistent with previous observations (Kim, Holland et al. 2010, Liu, Vleugel et al. 2010).

2.3.2 RV[S/T]F motifs are phosphorylated during mitosis

Next, I explored if the phosphorylation of RV[S/T]F motifs is a cell cycle dependent event. Phospho-specific antibodies against the RV[S/T]F motifs from known and potential PP1 binding proteins were validated using dot blots (Figure 2.3A) and were used to monitor the phosphorylation status of the RV[S/T]F motifs in different proteins during cell cycle. Increased phosphorylation at RV[S/T]F motifs was observed in the mitotic extracts after the release from thymidine-nocodazole arrest as shown with p-RIF1, p-CEP192, p-SFI1, p-BRCA1 and p-CDCA2 (RM) antibodies (Figure 2.3B). Cell cycle synchronization was confirmed by FACS analysis (Figure 2.3C) and immunoblotting with the mitotic marker cyclin B (Figure 2.3B).

To monitor changes in phosphorylation of the RV[S/T]F motifs present in numerous PP1 binding partners, the in-house generated p-RV[S/T]F antibody developed against the phospho-peptide sequence (KRRVpSFAD) was used. The characterization of this antibody confirmed its specificity for many RV[S/T]F containing proteins including known PP1-interacting proteins RIF1, Ki67 and KNL1/CASC5 (Rosenberg, Cross et al.

2011, Takagi, Nishiyama et al. 2014, Sreesankar, Bharathi et al. 2015).

Figure 2.3: RV[S/T]F motifs are phosphorylated during mitosis.

(A) Different amounts of phospho and dephospho versions of the indicated peptides were spotted on nitrocellulose membranes and probed with the in-house generated phospho- epitope specific antibodies for the respective proteins. Peptide sequences are shown in Table 2.1. (B) HeLa cells were synchronized using thymidine-nocodazole (T/N) block and released in fresh media after that. Samples were collected at the indicated time- points, blotted for cyclin B, and analyzed for phosphorylation within the RV[S/T]F motifs of the indicated proteins using in-house generated phospho-epitope specific antibodies. The peptide sequences are shown in Table 2.1. Samples were collected three times with the same synchronization technique, and the data represents one of the three independent experiments. (C) Cell cycle synchronization was confirmed by FACS analysis.

The phospho-specificity of the antibody was validated using different versions of the RV[S/T]F peptides (Figure 2.4A). This shows that the antibody raised against

KRRVpSFAD is phospho-specific and recognizes to some degree related ‘RV[S/T]F’ motifs. Cell cycle analysis using this antibody also showed a marked increase in phosphorylation within the RV[S/T]F motifs in different proteins during mitosis (Figure

2.4B). This increase in phosphorylation was validated in single cells by immunofluorescence staining using the phospho-RV[S/T]F antibody or specific phospho-

RVxF epitope antibodies (p-CEP192, p-SFI1, p-BRCA1) (Figure 2.5A, 2.5B). Compared to an interphase cell, metaphase cells show a dramatic increase in phosphorylation of the

RV[S/T]F motifs diffused through the cytoplasm and enriched at spindle poles. This phosphorylation continues at spindle poles and the central spindle in anaphase and the midbody and telophasic bridge during cytokinesis (Figure 2.5A). This increased phosphorylation during mitotic phase of the cell cycle compared to interphase is consistent with our immunoblotting results.

The phosphorylation of PP1 Thr 320 by CDK1 during mitotic entry inactivates the enzyme until anaphase during which PP1 can autodephosphorylate Thr320 and fully activate itself (You and Bird 1995, Helps, Luo et al. 2000, Wu, Guo et al. 2009). This dephosphorylation happens within 2 h of nocodazole-release corresponding with the dephosphorylation of the RV[S/T]F motifs (Figure 2.4B). This suggests that along with the regulation of the catalytic subunit (PP1), there is as an additional targeting subunit based regulatory mechanism that controls PP1 activity during mitotic exit.

Figure 2.4: The phospho-specificity of the p-RV[S/T]F antibody.

(A) Different versions of the RV[S/T]F containing peptides derived from the indicated proteins were spotted on nitrocellulose membrane and probed with the phospho- RV[S/T]F antibody to identify peptides and peptide variants recognized by the antibody.

Phosphorylated S and T are indicated in red. (B) The phospho-epitope-specific antibody generated against the peptide RRVpSFADK is able to recognize a number of proteins in the HeLa cell lysates. Synchronized HeLa cell lysates were probed with this phospho- RV[S/T]F antibody to monitor changes in this motif phosphorylation during cell cycle. Phosphorylation of PP1 Thr320 (p-Thr320) was also assessed at these time points. Equal loading was confirmed by tubulin blotting and mass markers are shown in kDa.

Figure 2.5: Immunofluorescence also confirms that p-RV[S/T]F motifs are phosphorylated during mitosis.

(A) Immunofluorescence staining of HeLa cells with DAPI, anti-tubulin and with p- RV[S/T]F antibody (Anti-RRVpSFADK, hereafter referred to as p-RV[S/T]F antibody) at different phases of the cell cycle, and the images merged. >50 cells were imaged for each condition in four independent experiments. Blue indicates DAPI, red indicates tubulin and green indicates p-RV[S/T]F antibody. Scale bar, 20µm. (B) HeLa cells were grown on coverslips, fixed and stained for the respective antibodies to monitor the phosphorylation status of the RV[S/T]F motifs within these proteins. Blue indicates DAPI, red indicates tubulin and green indicates the test antibody as indicated. Images representative of > 50 cells imaged in each of the three independent experiments.

2.3.3 Aurora B-dependent phosphorylation of the RV[S/T]F motifs

The next aim was to investigate the protein kinase(s) responsible for phosphorylation of the RV[S/T]F motifs during mitosis. To test this, protein kinase inhibition assays were performed. Since this phosphorylation is a mitotic event, different protein kinases which are activated at mitotic entry and play a role in driving the cell cycle were inhibited (Kettenbach, Schweppe et al. 2011, McCloy, Parker et al. 2015).

Among these, only the inhibition of Aurora B kinase showed a marked decrease in the phosphorylation of RV[S/T]F motifs (Figure 2.6A). Furthermore, a time-dependent reduction in the phosphorylation of these RV[S/T]F containing proteins using the Aurora

B inhibitor in cells released from nocodazole prometaphase arrest was seen (Figure

2.6B). These findings were also validated by p-RV[S/T]F immunofluorescence of Aurora

B kinase inhibitor-treated cells (Figure 2.6C). The inhibition of Aurora B activity was confirmed by the specific lack of phosphorylation of its substrate histone H3 ser10 (Giet and Glover 2001).

2.3.4 Proteomic analysis of Aurora B phosphorylation-dependent p-RV[S/T]F proteins

To explore these observations further, immunoprecipitations with the p-RV[S/T]F antibody and control IgG were performed. This showed a marked enrichment of proteins in the p-RV[S/T]F compared to control IgG immunoprecipitations in mitotic cells

(Figure 2.7A). To determine the identity of the p-RV[S/T]F antibody bound proteins, p-

RV[S/T]F and control IgG immunoprecipitations from mitotically-arrested

Figure 2.6: Aurora B phosphorylates PP1 binding RV[S/T]F motifs during mitosis.

(A) HeLa cells were arrested in prometaphase with nocodazole for 15 h, treated with the respective protein kinase inhibitors (Aur Ai: Aurora A inhibitor I (100 nM), Aur Bi: Hesperadin (100 nM + MG132 at 10 µM), PLK1i: BI2536 (100 nM) and CDK1i: Roscovitine (50 µM + MG132 at 10 µM) for an additional 1 h and released into media with inhibitors in the absence of nocodazole for indicated times (0.5 or 1.5 h). The cells were harvested, and the lysates run on SDS-PAGE and immunoblotted with indicated antibodies. Samples were collected in four independent replicates. (B) HeLa cells arrested in mitosis were treated with either DMSO or Aur Bi for up to 2 h and phosphorylation of the RV[S/T]F motifs was examined by immunoblot analysis. continued…

HeLa cells were performed and analyzed by LC-MS/MS (Appendix A). The degree of confidence for p-RV[S/T]F specific binding was assessed by SAINT (Choi, Larsen et al.

2011, Mellacheruvu, Wright et al. 2013) (Appendix A). Using this approach, 421 specific p-RV[S/T]F bound proteins were identified (Appendix A).

Next, we investigated these proteins for their binding behavior upon Aurora B inhibition. Comparison of p-RV[S/T]F immunoprecipitations in the presence and absence of Aurora B inhibitor by label-free intensity-based absolute quantification (iBAQ)

(Schwanhausser, Busse et al. 2011) (Appendix A) revealed that 227 (~54%) of the 421 specifically-bound proteins were either lost or significantly reduced (p-value < 0.05) upon Aurora B inhibition (Appendix A).

To determine the functional profile of the proteins enriched in the p-RV[S/T]F IPs, known protein-protein interactions among the 421 proteins were extracted from the

STRING database (Jensen, Kuhn et al. 2009, Szklarczyk, Franceschini et al. 2015). p-

RV[S/T]F-enriched proteins constituted a highly connected network between each other, with an average of 9.33 first-degree neighbors per node and only 44 isolated nodes

(Figure 2.7B). Gene ontology (GO) analysis showed an enrichment of proteins involved in mitosis, spindle organization and assembly, and chromosome segregation.

Furthermore, many of the proteins are localized to cellular structures such as the spindle,

Figure 2.6: continued… Mass markers are shown in kDa. (C) HeLa cells were either treated with the Aur Bi for 2 h or left untreated, fixed and stained with DAPI, anti-tubulin, and either p-H3S10 or phospho-RV[S/T]F antibody and the images merged. > 15 cells were imaged for three independent replicates. Blue indicates DAPI, red indicates tubulin and green indicates the test antibody as indicated. Scale bar, 20 µm.

Figure 2.7: p-RV[S/T]F containing proteins during mitosis.

(A) Immunoprecipitation was performed using equal amounts of the phospho-RV[S/T]F antibody (anti-RRVpSFADK) or control IgG with mitotic HeLa cell lysates. An equal amount of each IP was run on SDS-PAGE and stained with colloidal Commassie blue. Mass markers are shown in kDa. These samples were analyzed by mass spectrometry for the identification of RV[S/T]F containing proteins. The samples for the IP were collected in three different replicates, and the gel is representative from one experiment. (B) STRING protein-protein interaction network analysis for specifically p-RV[S/T]F bound proteins. Node color depicts Aurora B kinase inhibitor sensitivity. Grey - change not significant, dark green - significantly reduced upon inhibitor addition, light green - lost upon inhibitor addition.

kinetochore, chromosome, and midbody. This reiterates the importance of the interplay between the antagonistic activities of PP1 and Aurora B in regulating these processes

(Emanuele, Lan et al. 2008, Liu, Vleugel et al. 2010, Meadows 2013).

37 proteins containing the RV[S/T]F motif and phosphorylated within this motif during mitosis were identified, out of which 65% were either lost or significantly reduced in p-RV[S/T]F binding upon Aurora B inhibitor treatment (Table 2.2). The protein

KNL1/CASC5, a PP1 binding protein whose association with PP1 has already been shown to be regulated by the phosphorylation via Aurora B within its RVTF motif

(Rosenberg, Cross et al. 2011), was identified in this study along with other novel cell cycle regulatory proteins including RIF1, CDCA2 (RM), UBR5, ASPM, SEH1 and

ELYS. The results from the mass spectrometry analysis were further validated with reciprocal immunoprecipitations using antibodies against two known PP1 binding proteins: RIF1 and CDCA2 (RM) in the presence and absence of various mitotic kinase inhibitors (Figure 2.8A). Immunoblot analysis of RIF1 and CDCA2 (RM) immunoprecipitations with phospho-RVSF or -RVTF epitope-specific antibodies against the respective proteins confirmed the role of Aurora B in the phosphorylation of these motifs in RIF1 and CDCA2 (RM). The phosphorylation of RIF1 in mitosis negatively regulates binding with PP1. PP1-bound microcystin-Sepharose beads were used to pull- down proteins from asynchronous, mitotic or Aurora B inhibited extracts and the elution was analyzed for the presence of RIF1. Dephosphorylated RIF1 from asynchronous HeLa extracts shows an increased binding to PP1-bound microcystin-Sepharose beads (Figure

2.8B) compared to phosphorylated RIF1 from mitotic extracts. This association increases upon the inhibition of Aurora B kinase activity.

Table 2.2 List of proteins containing "RV[S/T]F" motifs specifically enriched in the p-RV[S/T]F immunoprecipitation.

Name Accession Description RVxF RVxF sequence Binding after position Aurora B inhibition ACACB_HUMAN O00763 Acetyl-CoA carboxylase 2 OS=Homo 1460-1463 DYGLRRITFLIAQEK lost sapiens GN=ACACB PE=1 SV=3 ADDG_HUMAN Q9UEY8 Gamma-adducin OS=Homo sapiens 465-468 TSPRTKITWMKAEDS change not GN=ADD3 PE=1 SV=1 significant AHNK2_HUMAN Q8IVF2 Protein AHNAK2 OS=Homo sapiens 4708-4711 KVPKVSFSSTKT change not GN=AHNAK2 PE=1 SV=2 significant AN13A_HUMAN Q8IZ07 Ankyrin repeat domain-containing 422-425 HVLNARITFGNVNGC lost protein 13A OS=Homo sapiens GN=ANKRD13A PE=1 SV=3 ARHGH_HUMAN Q96PE2 Rho guanine nucleotide exchange 374-377 AFRVAKVSFPSYLAS reduced factor 17 OS=Homo sapiens GN=ARHGEF17 PE=1 SV=1 ASPM_HUMAN Q8IZT6 Abnormal spindle-like microcephaly- 268-271 SANVSKVSFNEKAV change not associated protein OS=Homo sapiens significant GN=ASPM PE=1 SV=2 BRCA1_HUMAN P38398 Breast cancer type 1 susceptibility 898-901 QSPKVTFECEQK lost protein OS=Homo sapiens GN=BRCA1 PE=1 SV=2 CASC5_HUMAN Q8NG31 Protein CASC5 OS=Homo sapiens 58-61 KNSRRVS FADTI lost GN=CASC5 PE=1 SV=3 CDCA2_HUMAN Q69YH5 Cell division cycle-associated protein 2 392-395 MRKRKRVTFGEDLSP reduced OS=Homo sapiens GN=CDCA2 PE=1 SV=2 CLCN7_HUMAN P51798 H(+)/Cl(-) exchange transporter 7 res7-10 ANVSKKVSWSGRDRD reduced OS=Homo sapiens GN=CLCN7 PE=1 SV=2 DVL2_HUMAN O14641 Segment polarity protein dishevelled 494-497 RHTVNKITFSEQCYY change not homolog DVL-2 OS=Homo sapiens significant GN=DVL2 PE=1 SV=1 DYHC1_HUMAN Q14204 Cytoplasmic dynein 1 heavy chain 1 3695-3698 PDLCSRVTFVNFTVT reduced OS=Homo sapiens GN=DYNC1H1 PE=1 SV=5 ELYS_HUMAN Q8WYP5 Protein ELYS OS=Homo sapiens 1230-1233 RLKETRISFVEEDVH reduced GN=AHCTF1 PE=1 SV=3 GLCI1_HUMAN Q86VQ1 Glucocorticoid-induced transcript 1 500-503 QLSSRVSFTSLS lost protein OS=Homo sapiens GN=GLCCI1 PE=1 SV=1

NAME Accession Description RVxF RVxF sequence Binding position K1109_HUMAN Q2LD37 Uncharacterized protein KIAA1109 3657-3660 ASPGPRVTFNIQDTF change not OS=Homo sapiens GN=KIAA1109 significant PE=1 SV=2 KI67_HUMAN P46013 Antigen KI-67 OS=Homo sapiens 505-508 KRRRVSFGGHL reduced GN=MKI67 PE=1 SV=2 KIF5A_HUMAN Q12840 Kinesin heavy chain isoform 5A 529-532 HSQKQKISFLENNLE change not OS=Homo sapiens GN=KIF5A PE=1 significant SV=2 KIF5C_HUMAN O60282 Kinesin heavy chain isoform 5C 533-536 AAQKQKISFLENNLE change not OS=Homo sapiens GN=KIF5C PE=1 significant SV=1 KINH_HUMAN P33176 Kinesin-1 heavy chain OS=Homo 831-834 QKQKISFLENN change not sapiens GN=KIF5B PE=1 SV=1 significant MPRI_HUMAN P11717 Cation-independent mannose-6- 1185-1188 QRFSTRITFECAQIS reduced phosphate receptor OS=Homo sapiens GN=IGF2R PE=1 SV=3 NUMB_HUMAN P49757 Protein numb homolog OS=Homo 113-116 QTIEKVSFCA change not sapiens GN=NUMB PE=1 SV=2 significant PLPL6_HUMAN Q8IY17 Neuropathy target esterase OS=Homo 316-319 MVRLQRVTFLALHNY lost sapiens GN=PNPLA6 PE=1 SVR=2 RFIP1_HUMAN Q6WKZ4 Rab11 family-interacting protein 1 808-811 KKTKKRVSFSEQLFT reduced OS=Homo sapiens GN=RAB11FIP1 PE=1 SV=2 RIF1_HUMAN Q5UIP0 Telomere-associated protein RIF1 2203-2206 KVRRVSFADPI reduced OS=Homo sapiens GN=RIF1 PE=1 SV=2 RRP1B_HUMAN Q14684 Ribosomal RNA processing protein 1 683-686 PSSSKKVTFGLNRNM lost homolog B OS=Homo sapiens GN=RRP1B PE=1 SV=3 S4A7_HUMAN Q9Y6M7 Sodium bicarbonate cotransporter 3 1196-1199 KPVSVKISFEDEPRK reduced OS=Homo sapiens GN=SLC4A7 PE=1 SV=2 SCAPE_HUMAN Q9BY12 S phase cyclin A-associated protein in 1013-1016 VLFSNKITFLMDL change not the endoplasmic reticulum OS=Homo significant sapiens GN=SCAPER PE=1 SV=1 SEH1_HUMAN Q96EE3 Nucleoporin SEH1 OS=Homo sapiens 283-286 NSQVWRVSWNITGTV lost GN=SEH1L PE=1 SV=3 SPE39_HUMAN Q9H9C1 Spermatogenesis-defective protein 39 56-59 DDDLERVSWSGEPVG lost homolog OS=Homo sapiens GN=VIPAS39 PE=1 SV=1 TBCD4_HUMAN O60343 TBC1 domain family member 4 769-772 SVTPRRISWRQRIFL reduced OS=Homo sapiens GN=TBC1D4 PE=1 SV=2

NAME Accession Description RVxF RVxF sequence Binding position TF3C1_HUMAN Q12789 General transcription factor 3C 1262-1265 RMTRLRVTWSMQEDG change not polypeptide 1 OS=Homo sapiens significant GN=GTF3C1 PE=1 SV=4 TF3C3_HUMAN Q9Y5Q9 General transcription factor 3C 15-18 DYLEGKISFEEFERR reduced polypeptide 3 OS=Homo sapiens GN=GTF3C3 PE=1 SV=1 UBR5_HUMAN O95071 E3 ubiquitin-protein ligase UBR5 2278-2281 AVHRVKVTFKDEPG reduced OS=Homo sapiens GN=UBR5 PE=1 SV=2 VPRBP_HUMAN Q9Y4B6 Protein VPRBP OS=Homo sapiens 842-845 SPLIGRISFIRERPS reduced GN=VPRBP PE=1 SV=3 ZBED1_HUMAN O96006 Zinc finger BED domain-containing 350-353 MLVSNRVSWWGSTLA change not protein 1 OS=Homo sapiens significant GN=ZBED1 PE=1 SV=1 ZFHX3_HUMAN Q15911 Zinc finger homeobox protein 3 1195-1198 PATSKRISFPGSSES lost OS=Homo sapiens GN=ZFHX3 PE=1 SV=2 ZZEF1_HUMAN O43149 Zinc finger ZZ-type and EF-hand 1162-1165 DKWPKKVTFKAGPRL change not domain-containing protein 1 significant OS=Homo sapiens GN=ZZEF1 PE=1 SV=6

Figure 2.8: Phosphorylation within the RV[S/T]F motifs of RIF1 and CDCA2 (RM) is governed by Aurora B and regulates binding to PP1.

(A) Protein kinase inhibited extracts were collected by treating 15 h nocodazole-arrested HeLa cells with the respective protein kinase inhibitors (Aur Ai: Aurora A inhibitor I (100 nM), Aur Bi: Hesperadin (100 nM + MG132 at 10 µM), and PLK1i: BI2536 (100 nM)) for an additional 1 h and releasing into media with inhibitors in the absence of nocodazole for 0.5 h. RIF1 or CDCA2 (RM) were immunoprecipitated from asynchronous, mitotic or indicated protein kinase inhibited extracts. The respective eluates were probed with pan-RIF1 or CDCA2 (RM) antibodies and phospho-specific antibodies against the RVSF motif in RIF1 or RVTF motif in CDCA2 (RM). (B) PP1 ⍺/β/γ (10 µg) was pre-incubated with microcystin-Sepharose beads to generate PP1- bound resin. This PP1-bound resin was used to test in-vitro binding with proteins from asynchronous, mitotic or Aurora B inhibited HeLa extracts. The bound proteins were eluted and tested for the presence of RIF1 and numbers refer to band quantitation using ImageJ. The data represents results from three independent experiments.

2.3.5 PP1 dephosphorylates the RV[S/T]F motifs at mitotic exit

The RV[S/T]F motifs are dephosphorylated at the end of mitosis as shown by the cell cycle immunoblotting (Figure 2.4B). To determine which phosphatase regulates this process, we inhibited the protein phosphatases that have been implicated in the regulation of cell cycle (Wu, Guo et al. 2009, Wurzenberger and Gerlich 2011, Grallert, Boke et al.

2015, Wlodarchak and Xing 2016). The serine/threonine phosphatase inhibitor okadaic acid was able to prevent the dephosphorylation of these motifs but only when used at 500 nM (Figure 2.9A). At this concentration, okadaic acid inhibits the PPP family phosphatases PP1, PP5 and PP2A-type phosphatases (PP2A, PP4 and PP6). Low concentration of okadaic acid (10-100 nM) which is sufficient to inhibit PP2A-type phosphatases, does not prevent the dephosphorylation of the RV[S/T]F motifs suggesting this to be a PP1-dependent event. Treatment with additional PPP family inhibitors, calyculin A and fostriecin, again suggested that this event is dependent on PP1 (Figure

2.9A). PP1 substrate H3 Thr3 was used as a readout for the inhibition of PP1 in the assay

(Qian, Lesage et al. 2011). The treatment of cells with phosphatase inhibitors has been shown to cause a “pseudomitotic” state. The increased phosphorylation of RV[S/T]F motifs in these cells as confirmed by immunofluorescence is not due to the arrest of cells in mitosis-like state implicated by the differences in condensation of the chromosomes in control and inhibitor treated cells but are an effect of the inhibition of the phosphatase activity (Figure 2.9B). The dephosphorylation of the RV[S/T]F motifs was also confirmed with an in-vitro assay using recombinant PP1 with mitotic cell extracts. The addition of 1 M PP1 completely dephosphorylates the RV[S/T]F motifs in cell extracts from nocodazole-arrested mitotic cells (Figure 2.9C).

Figure 2.9: PP1 dephosphorylates the RV[S/T]F motifs at mitotic exit.

(A) HeLa cells were treated with the indicated concentration of protein phosphatase inhibitor for 2 h, lysed, run on SDS-PAGE and immunoblotted with the indicated antibodies. (B) Control HeLa cells or cells treated with 500 nM okadaic acid were stained with Hoechst 33342 to look for condensation of chromatin. (C) HeLa cells were synchronized in mitosis with nocodazole (16 h) and harvested by mitotic shake-off. The mitotic extracts were incubated with or without 1 µM PP1 (⍺/β/γ) for the indicated times, boiled in SDS-cocktail, run on SDS-PAGE and immunoblotted with the indicated antibodies. Mass markers are shown in kDa. The data is a representation from three independent replicates.

2.4 Discussion

The specificity of PP1 towards its substrates is controlled by regulatory proteins which utilize the docking “RVxF” motif for interaction with the phosphatase. The data presented here suggests a novel mechanism of regulation of protein kinase and protein phosphatase activities in cell cycle (Figure 2.10). Aurora B-dependent phosphorylation of multiple RVxF motifs during mitosis abrogates the binding of PP1 with its regulatory proteins keeping its phosphatase activity in check. The dephosphorylation of these RVxF motifs at mitotic exit by PP1 allows the phosphatase to re-associate with the regulatory proteins and thus dephosphorylate multiple mitotic phospho-proteins.

The sequence characterization of RVxF motif from known PP1 binding protein shows an over-representation of either positively charged Arg/Lys residues or phosphorylatable Ser/Thr residues at the ‘x’ position (Figure 2.1). The positive charge of the Arg/Lys residue aids in the tight binding of regulatory proteins with the hydrophobic pocket on the surface of PP1; for example, in the case of GADD34 ‘KVRF’ docking motif (Choy, Yusoff et al. 2015). However, the phosphorylation at the Ser/Thr residue at the ‘x’ position might be a universal mechanism for regulating the reversible association of the phosphatase with its regulatory proteins under the control of specific protein kinases during specific cellular events. The phosphorylation within or in close proximity to the PP1 docking site has been shown to block the binding of PP1 in various known

Figure 2.10: Proposed model of PP1 regulation during the cell cycle.

PP1 binds its regulatory proteins through RV[S/T]F motifs during interphase. Upon the activation of Aurora B during mitosis, these regulatory proteins are phosphorylated within the RV[S/T]F motifs. This phosphorylation leads to the dissociation of PP1 from regulatory proteins thereby interfering with the targeting of the phosphatase to its substrates. At the end of mitosis, these motifs are dephosphorylated by PP1 itself to generate the docking site for its association with the regulatory proteins, which can then target PP1 to phospho-substrates for their dephosphorylation at mitotic exit.

regulatory proteins including GM, KNL1/CASC5 and CENP-E (Egloff, Johnson et al.

1997, Kim, Holland et al. 2010, Meadows, Shepperd et al. 2011, Rosenberg, Cross et al.

2011) and we have confirmed and extended that observation here (Figure 2.2). Results here, and any previously documented phosphorylation in the RV[S/T]F sequences indicates that the motif is exposed on the protein in vivo, further supporting the potential to recruit PP1. The in-house generated phospho-epitope specific RV[S/T]F antibody shows that in many PP1 interactors this phosphorylation is a cell cycle dependent event occurring specifically during mitosis (Figure 2.3). The p-RV[S/T]F antibody is an important tool that can be used as a marker for mitotic cells, much like the MPM2 antibody (Davis, Tsao et al. 1983).

Among the possible mitotic protein kinases, the RV[S/T]F motif fits perfectly with the Aurora kinase consensus sequence R-X-S/T-Φ, where X is any amino acid and Φ is a hydrophobic amino acid. Indeed, the data presented here shows that Aurora B phosphorylates the serine or threonine within the docking motif of PP1 in multiple known and potential regulatory proteins of PP1 (Figure 2.6, Table 2.2). This is consistent with previous studies where PP1 has been reported to antagonize the activity of protein kinase

Aurora B to establish proper chromosome bi-orientation and silence the spindle assembly checkpoint through its association with multiple regulatory proteins at centromeres and kinetochores (Emanuele, Lan et al. 2008, Lesage, Qian et al. 2011, Wurzenberger and

Gerlich 2011). However, not all [RK][VI][ST][FW] motifs are phosphorylated by Aurora

B and other basophilic kinases like Aurora A, protein kinase A (PKA), protein kinase B

(PKB), CaMKII, CHK2 and others could play a role in the phosphorylation of these motifs in other cellular events or subcellular compartments where PP1 is involved.

Undoubtedly, the RV[S/T]F motif has evolved to recruit PP1, but the motif also evolved to coordinate with the function of select protein kinases under specific cellular conditions.

Using quantitative phospho-proteomics and the p-RV[S/T]F antibody, novel phosphorylation sites for Aurora B protein kinase within the RV[S/T]F motif of 6 known and 23 potential PP1 binding proteins were identified. This includes RIF1, Ki67, CDCA2

(RM) and KNL1/CASC5, known PP1 binding proteins that are phosphorylated by Aurora

B within the RV[S/T]F motif during mitosis. In addition, the mass spectrometry data also revealed phosphorylation within these motifs in novel potential PP1 binding cell cycle regulatory proteins like E3 ubiquitin ligase UBR5/EDD, ASPM, nucleoporin SEH1 and protein ELYS.

UBR5/EDD is an E3 HECT-ubiquitin ligase that has been recently reported to regulate spindle assembly checkpoint by a forming complex with SAC proteins BUBR1 and BUB3 (Jiang, He et al. 2015, Scialpi, Mellis et al. 2015) (Table 2.2).

Phosphorylation of the “KVTF” sequence in UBR5 by Aurora B might represent a novel mechanism for PP1 dissociation to keep the SAC activated. My data suggests that upon the inhibition of Aurora B, there is not only a quantitative decrease in UBR5 enrichment in the p-RV[S/T]F IP but also similar decrease in enrichment of BUBR1 and BUB3 suggesting that the interaction between UBR5 and SAC proteins may be dependent on phosphorylation by Aurora B. Once this motif is dephosphorylated, PP1 can be recruited by UBR5/EDD to contribute to silencing the SAC. ASPM is a mitotic spindle-associated protein and is known to be involved in spindle microtubule organization and cytokinesis

(Higgins, Midgley et al. 2010). Similar to Aurora B, ASPM localizes at the minus ends of the central spindle microtubules during anaphase and at the mid-body during telophase

and cytokinesis. Phosphorylation within the “KVSF” motif of ASPM during mitosis has been reported in previous large scale mass spectrometry studies (Nousiainen, Sillje et al.

2006, Kettenbach, Schweppe et al. 2011). The data here confirms this phosphorylation during mitosis and this phosphorylation was identified to be primarily governed by

Aurora B kinase. This again suggests that this might be a potential recruiting platform and regulator for PP1 action during the SAC. Further experiments need to be done to test if these SAC regulators UBR5 and ASPM directly interact with PP1 through their respective “RVxF” motifs.

Nucleoporin SEH1 is required for the recruitment of Nup107-160 to kinetochores during mitosis and plays a critical role in establishing correct kinetochore-microtubule attachments (Zuccolo, Alves et al. 2007). The protein ELYS has been reported to recruit the Nup107-160 subcomplex to the kinetochores (Rasala, Orjalo et al. 2006). My data suggests that both these proteins are phosphorylated within their RV[S/T]F motifs during mitosis and that this phosphorylation is lost or significantly reduced upon Aurora B kinase inhibition (Table 2.2). Very recently, MEL28, the C.elegans homolog of ELYS was shown to interact with PP1 during anaphase to regulate the nuclear reformation at mitotic exit (Hattersley, Cheerambathur et al. 2016). Phospho-mimetic and phospho-null mutants of ELYS can help decipher the role of phosphorylation of the RVxF motif in regulating its association with PP1. Given the role of Aurora B and PP1 during cytokinesis, these potential novel PP1 binding partners might be crucial for the recruitment of PP1 and regulation of Aurora B-PP1 axis during cytokinesis and nuclear envelope re-assembly.

In budding yeast, RIF1 has been shown to bind PP1, and this interaction has been implicated in the regulation of replication origin firing (Dave, Cooley et al. 2014,

Mattarocci, Shyian et al. 2014, Sreesankar, Bharathi et al. 2015). Here, this interaction was confirmed in human cells and Aurora B-dependent phosphorylation of the “RVSF” motif of RIF1 during mitosis was identified (Figure 2.8A). In addition, it is also shown that the phosphorylation within RVSF of RIF1 reduces PP1 binding, at least in-vitro

(Figure 2.8B).

2.5 Conclusion

Collectively the data presented here suggests a widespread targeting-subunit based mechanism for the regulation of PP1 function during mitosis that coordinates with previously established PP1 regulation by CDK1 (Wu, Guo et al. 2009). The activity of

PP1 catalytic subunit during mitosis is regulated by the CDK1-dependent phosphorylation of the C-terminus of PP1 (Thr320). At this time, the RV[S/T]F motif containing proteins are also phosphorylated by Aurora B to inhibit the binding of the phosphatase and thereby, inhibiting the phosphatase activity. This acts as targeting subunit based mechanism to control PP1 activity and function during mitosis. As CDK1 activity declines at anaphase, PP1 can autodephosphorylate Thr320 to regain its activity above a minimum threshold. I propose that this, in turn, is sufficient to dephosphorylate multiple RV[S/T]F motifs. It is after this dephosphorylation event that PP1 can be recruited to regulatory proteins, which can be substrates themselves, or target the catalytic subunit to multiple other mitotic phospho-substrates, contributing to dephosphorylation that drives mitotic exit. This balance between the counteracting

activities of protein kinases and protein phosphatases is absolutely essential to maintain the genomic integrity and cell proliferation.

CHAPTER 3. IDENTIFICATION OF THE CELL CYCLE SPECIFIC

INTERACTOME OF PP1

3.1 Introduction

Protein phosphorylation, controlled by protein kinases and protein phosphatases, regulates cellular behaviour towards internal and external changes in cell environment.

Mammalian genomes encode for about 400 protein serine/threonine kinases and in contrast only about 40 protein serine/threonine phosphatases (Virshup and Shenolikar

2009). This discrepancy in the ratio of the number of protein kinases to protein phosphatases can be attributed to the formation of diverse stable phosphatase complexes with regulatory subunits which provide specificity to the protein phosphatase catalytic subunits, especially in the case of PPP family members, PP1 and PP2A. PP1 has the most diverse interactome among the PPP family of protein phosphatases with about 200 interacting partners identified so far (Bollen 2001, Cohen 2002, Bollen, Peti et al. 2010)

(Figure 3.1).

The interaction of PP1 with different regulatory proteins allows the phosphatase to regulate multiple different processes. Among the various cellular processes regulated by

PP1, cell cycle regulation accounts for the majority of PP1 interactors (Bollen 2001)

(Figure 3.1). Given its crucial role in regulating the cell cycle, it is critical to study and identify the specific interactome of PP1 at different stages of cell cycle. In-depth knowledge about PP1 interacting partners can help us understand the regulation of

Figure 3.1: Known protein-protein interaction network of PP1.

All the known protein-protein interactions for the three human PP1 isoforms (,  and ) were curated from Reactome database and merged in Cytoscape (Shannon, Markiel et al. 2003, Saito, Smoot et al. 2012). Nodes indicate individual proteins and edges indicate protein-protein interactions. The gene ontology analysis was performed using BiNGO in Cytoscape. Proteins which belong to ‘cell cycle regulation’ category of biological processes are highlighted in yellow. Human PP1 isoforms are depicted as PPP1CA, PPP1CB, and PPP1CC.

binding behaviour of PP1 during the cell cycle, particularly during mitosis and provide insight on specific roles for PP1 during this complex event. In this section, I characterize novel binding partners of PP1 in interphase (asynchronous cells) and mitosis using biochemical techniques and quantitative LC-MS/MS.

3.2 Materials and Methods

3.2.1 Cell synchronization and mitotic shake-off

U2OS cells expressing either GFP-only or one of the three isoforms of GFP- tagged PP1 (GFP-Control, GFP-PP1, GFP-PP1 and GFP-PP1) were a generous gift from Dr. Laura Trinkle-Mulcahy, University of Ottawa (Trinkle-Mulcahy, Chusainow et al. 2007). These cells were cultured in DMEM with 10% (v/v) FBS and 400 g mL-1

G418 antibiotic (Gold Biotechnology). Cells were synchronized in mitosis with 100 ng mL-1 nocodazole for 16 h and collected by mitotic shake-off. The mitotic cells or asynchronous cells were washed with PBS and lysed in the lysis buffer (10 mM Tris, pH

7.5, 150 mM NaCl, 0.5 mM EDTA and 0.5% (v/v) NP-40). The lysate was sonicated (5 x

30 s pulses) and clarified at 14,000 rpm for 10 mins at 4C. Protein concentration was determined using Bradford reagent with BSA as standard. The cell lysates were processed according to the methodology in Figure 3.2 and described in further sections.

3.2.2 GFP-TRAP using U2OS GFP-tagged PP1 cells

Mitotic and asynchronous cell lysates were incubated with pre-equilibrated GFP-

TRAP_A beads (Chromotek) for 2 h at 4 C. The beads were washed with wash buffer

Figure 3.2: Methodology for GFP-TRAP experiment.

Asynchronous or mitotic GFP-tagged PP1 expressing cells were lysed (Lysis) followed by enrichment of GFP-bound proteins using GFP-TRAP_A beads (Chromotek) (Enrichment). The specifically bound proteins were eluted after washing and were either digested with trypsin (Digest) and analyzed by LC-MS/MS, or run on SDS-PAGE.

(10 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM EDTA) with 0.5% (v/v) NP-40 followed by

1X wash with wash buffer without NP-40. Bound proteins were eluted with 2 X SDS-

PAGE cocktail for SDS-PAGE analysis or mass spectrometry elution buffer (1% (w/v)

SDS, 15% (v/v) glycerol, 50 mM Tris, pH 8.7 and 150 mM NaCl) for LC-MS/MS analysis.

3.2.3 Mass spectrometry sample preparation, analysis, and iBAQ

For the preparation of mass spectrometry samples, the eluted proteins were reduced with 5 mM DTT. The samples were alkylated using 15 mM iodoacetamide and precipitated with 20% (v/v) TCA before sending them to Dr. Arminja Kettenbach, Giesel

School of Medicine at Dartmouth, for mass spectrometry analysis. The samples were digested in solution with trypsin in 50 mM ammonium bicarbonate, and mass spectrometry analysis was performed as described in Section 2.2.9.

3.2.4 Data analysis

Total peptide counts from PP1α, PP1β, PP1γ and control immunoprecipitations from asynchronous or mitotically-arrested U2OS cells were input into SAINT (Choi,

Larsen et al. 2011) using the CRAPome interface (Mellacheruvu, Wright et al. 2013). For a protein to be considered specific to the PP1 immunoprecipitations, we required the interaction to have an AvgP score in the SAINT analysis of 0.9 or above in at least two of the four replicates. Known PP1 interactors were identified based on a previously published report (Heroes, Lesage et al. 2013). Average iBAQ area of the respective protein in the PP1 immunoprecipitation was based on all peptides as well as unique

matches only. Average iBAQ areas for unique matches were normalized based on the

PP1α, PP1β, and PP1γ abundance respectively and compared on a per protein basis between immunoprecipitations performed on asynchronous and mitotically-arrested cells.

For hierarchical clustering, average iBAQ areas for unique matches were imported into

Perseus (Tyanova, Temu et al. 2016), log2 transformed, and clustered based on protein abundance using average linkage and Euclidean distance similarity metric.

3.2.5 Immunoblotting and validation

Eluates from GFP-TRAP were run on SDS-PAGE, transferred onto nitrocellulose membrane for 200V-hr and probed with the appropriate antibodies. Antibodies and their fold dilutions used in this study: anti-GFP (11814460001, Roche Diagnostics, 1:1,000), anti-PP1 (SC7482, Santa Cruz, 1:500), anti-RIF1 (A300-569A, Bethyl Antibodies,

1:500), anti-BRCA1 (9010S, Cell Signaling, 1:1,000), anti-CDCA2 (RM) (ab45129,

Abcam, 1:1,000), anti-Aurora B (ab45145, Abcam, 1:5,000), anti-TACC3 (8069, Cell

Signaling Technology, 1:1,000), anti-GCN2 (ab134053, Abcam, 1:1,000), anti-TPX2

(NB500-179, Novus Biologicals, 1:2,000), anti-Aurora A (MCA2249, Biorad, 1:1,000), anti-DBC1(A300-432A, Bethyl Antibodies, 1:1,000). HRP-conjugated goat anti-mouse and goat anti-rabbit were obtained from Pierce and Thermo Fisher Scientific and were used at 1:5,000.

The immunoblot bands for all antibodies used were quantified using ImageJ and were normalized to the respective control immunoprecipitation bands and log2 transformed to calculate the fold-change in both mitotic and asynchronous samples. The total fold change from all the three eluates (GFP-PP1++) was then normalized to the

total GFP-PP1++ in the eluates to account for changes in PP1 expression between mitotic and asynchronous samples. To compare between the PP1 isoforms, the fold- change against the respective controls was normalized to the specific GFP-PP1 isoform abundance in the GFP-TRAP eluates.

3.3 Results

3.3.1 Validation of GFP-TRAP pull-downs

The eluates from GFP-TRAP pull downs from mitotic or asynchronous cells expressing either GFP or one of the three GFP-tagged PP1 isoforms (GFP-PP1, GFP-

PP1 and GFP-PP1) were blotted for GFP expression to confirm pull-down efficiency.

Immunoblotting with GFP antibody confirmed the presence of GFP or GFP-tagged PP1 in all cell lines and GFP-TRAP eluates (Figure 3.3).

3.3.2 Proteomic analysis of GFP-TRAP eluates

To determine the identity of proteins in GFP-TRAP pull-downs, the eluates from both the mitotic and asynchronous cell lysates were analyzed by LC-MS/MS. The degree of confidence for PP1 specific binding was assessed by SAINT (Choi, Larsen et al. 2011,

Mellacheruvu, Wright et al. 2013). 173 specific interactors with an AvgP score of 0.9 or above were identified in asynchronous immunoprecipitations and 78 specific interactors with an AvgP score of 0.9 or above were identified in mitotic immunoprecipitations using this strategy (Appendix B). Average iBAQ areas from the unique matches of each of the proteins were used to compare the asynchronous and mitotic immunoprecipitations.

Specific PP1 binding proteins were clustered based on the average iBAQ areas into three 75

Figure 3.3: Validation of the GFP-TRAP pull-down.

Input asynchronous or mitotic cell lysates from four different cell lines (GFP-control, GFP-PP1, GFP-PP1 and GFP-PP1) and the eluates from the GFP-TRAP pull-down were run on SDS-PAGE and immunoblotted for the presence of GFP.

groups: mitosis-specific binding proteins, asynchronous-specific binding proteins, and proteins binding in both mitotic and asynchronous groups. 46 proteins clustered into the mitosis-specific group, 129 in the asynchronous-specific group while 60 proteins were present in both mitotic and asynchronous immunoprecipitations (Figure 3.4). The protein sequences of the SAINT-filtered proteins were scanned for the presence of most common

PP1 binding “RVxF” motif ([RK]-[VI]-x-[FW] or [RK]-x-[VI]-x-[FW]). A total of 113

Figure 3.4: Clustering of PP1 binding proteins identified shows mitosis-specific and asynchronous-specific partners.

Significantly enriched proteins (SAINT AvgP score  0.9) in any of the three isoforms of PP1 were clustered into mitosis-specific and asynchronous-specific groups with some proteins present in both groups. Red in the heat map indicates the presence of the protein and black indicates the absence of the protein.

proteins from all the clusters had one or more “RVxF” motif suggesting the potential to directly bind PP1 (Table 3.1).

To determine the changes in the binding motifs of cell cycle-dependent protein interactors of PP1, motifs from proteins unique to each mitotic and asynchronous cluster were used to create a WebLogo (Crooks, Hon et al. 2004) (Figure 3.5). The logo revealed the prevalence of a basic arginine or lysine residue upstream which can aid in

PP1 docking. Interestingly, the proteins which associate with PP1 only during mitosis do not show any over-representation of phosphorylatable serine or threonine residues at the

‘x’ position (Figure 3.5A) while the proteins binding PP1 throughout the interphase show the presence of phosphorylatable serine or threonine residues at this position

(Figure 3.5B). This would suggest a loss of association of PP1 with RV[S/T]F containing proteins during mitosis as discussed in Chapter 2.

Clustering analysis of the proteomics data also showed isoform specificity in the interactions of PP1 with its binding partners. All the data taken together when clustered according to the average iBAQ areas could be divided into multiple clusters with some clusters specific to a PP1 isoform and some clusters with overlapping proteins between the isoforms (Figure 3.6). Moreover, all proteins present in the asynchronous-specific group clustered together irrespective of the PP1 isoform. Similarly, proteins from the mitotic-specific group clustered together regardless of the PP1 isoform.

Table 3.1: List of specific PP1-binding proteins (SAINT AvgP score  0.9) containing one or more “RVxF” motifs.

NAME ACCESSION MOTIF BINDING “RVXF” SEQUENCES OCCURRENCES [RK]- SPECIFICITY [VI]-X-[FW] OR [RK]- X-[VI]-X-[FW]

SC24C_HUMAN P53992 2 mitosis AIRVGFVTY, VQKVVGFDAV

TPX2_HUMAN Q9ULW0 2 mitosis EYKEVNFTSE

AURKA_HUMAN O14965 2 mitosis ALKVLFKAQ PALLD_HUMAN Q8WX93 2 mitosis TPRVRWFCE, KPKIYWFKDG CRIP2_HUMAN P52943 2 mitosis CDKTVYFAEK, CSKKVYFAEK PRDX6_HUMAN P30041 2 mitosis VGRIRFHDF, TARVVFVFGP IF2B_HUMAN P20042 1 mitosis KKKNVKFPDE GLRX1_HUMAN P35754 1 mitosis PGKVVVFIKP UB2L3_HUMAN P68036 1 mitosis PPKITFKTK GSHR_HUMAN P00390 1 mitosis PKKVMWNTA

TCPG_HUMAN P49368 1 mitosis AVKMVQFEEN CYTB_HUMAN P04080 1 mitosis VFKAVSFKSQ MAP4_HUMAN P27816 1 mitosis LVKDVRWPTE FUBP2_HUMAN Q92945 1 mitosis GVRIQFKQD CASC5_HUMAN Q8NG31 5 both SRRVSFADT, MKKEVNFSVD, KIDKTIVFSED, MDKTVVFVDNH, NDKTIVFSEN YLPM1_HUMAN P49750 3 both QHRVGFQYQ KKRVRWADL, RKRAIGFVVGQ

MYO1D_HUMAN O94832 3 both AMKVIGFKPE, LIRIVLFLQKV, YGKHVKWPSPP MCM7_HUMAN P33993 3 both GSRFIKFQEM, GGRSVRFSEA, NASRTRITFV NEB2_HUMAN Q96SB3 3 both SRKIHFSTA, KGRVRFMIG, MAP1B_HUMAN P46821 2 both IIRVLFPGN, GERSVNFSLTP K1967_HUMAN Q8N163 2 both PLKQIKFLLG, PKERISFEVMVL RRP1B_HUMAN Q14684 2 both SKKVTFGLN, GPSRVAFDPEQK TPRN_HUMAN Q4KMQ1 2 both ADRAIRWQRP, KKMKISFNDK NEB1_HUMAN Q9ULJ8 2 both ANRKIKFSSA, NEKVRWELE PP1RA_HUMAN Q96QC0 2 both KRKSVTWPEE, EEKVPWVCPR RIF1_HUMAN Q5UIP0 2 both IKKIAFIAW, KVRRVSFADPI

NAME ACCESSION MOTIF OCCURRENCE BINDING SEQUENCE

TACC3_HUMAN Q9Y6A5 1 both AMKVTFQTP ASPP1_HUMAN Q96KQ4 1 both GLRVRFNPLAA SON_HUMAN P18583 1 both SEPKPIFFNLN WBP11_HUMAN Q9Y2W2 1 both YGRKVGFALD SFPQ_HUMAN P23246 1 both QLRVRFATH CHCH6_HUMAN Q9BRQ6 1 both GRRVSFGVD KI67_HUMAN P46013 1 both RRRVSFGGHL AURKB_HUMAN Q96GD4 1 both ALKVLFKSQ CDCA2_HUMAN Q69YH5 1 both RKRVTFGED PPR37_HUMAN O75864 1 both AKRVTFPSD STAU1_HUMAN O95793 1 both GRKVTFFEP ZFY16_HUMAN Q7Z3T8 1 both QKRVWFADG TCPB_HUMAN P78371 1 both EDKLIHFSGV HCFC1_HUMAN P51610 1 both RWKRVVGWSGPV HUWE1_HUMAN Q7Z6Z7 1 both LLKVIKFLGDE ZN185_HUMAN O15231 1 both TTKGILFVKEY SC16A_HUMAN O15027 1 both KNKSIVWDEK PP1RB_HUMAN O60927 1 both EKKVEWTSD ASPP2_HUMAN Q13625 1 both GMRVKFNPL PP1R8_HUMAN Q12972 1 both NSRVTFSED PPR18_HUMAN Q6NYC8 1 both QLKISFSET PP16A_HUMAN Q96I34 1 both LLKQVLFPPS MYPT2_HUMAN O60237 1 both SPRVRFEDG PP12C_HUMAN Q9BZL4 1 both RARTVRFERA MYPT1_HUMAN O14974 1 both KTKVKFDDGA AHNK_HUMAN Q09666 4 async SHGKIKFPTM, APKIGFSGPK EAKIKFPKFS, SGKVTFPKMKI PGK2_HUMAN P07205 3 async LGKDVLFLKDC NGVRITFPVDF, QARLIVWNGP STRAP_HUMAN Q9Y3F4 2 async YGRSIAFHSA KPKIGFPETTE PGK1_HUMAN P00558 2 async LGKDVLFLKD, RAKQIVWNG CD2B2_HUMAN O95400 2 async KRKVTFQGV CSRN2_HUMAN Q9H175 2 async RRKNVRFDQVT TKT_HUMAN P29401 2 async NTKGICFIRTS CPSM_HUMAN P31327 2 async RAKEIGFSDK FSCN1_HUMAN Q16658 2 async VDRDVPWGVD, SGKVAFRDCE PRS7_HUMAN P35998 2 async SVERDIRFEL

NAME ACCESSION MOTIF OCCURRENCE BINDING SEQUENCE

HS105_HUMAN Q92598 2 async KPRIVVFVDM, TPVKVRFQEA GLYM_HUMAN P34897 2 async EDRINFAVF RL8_HUMAN P62917 2 async PLAKVVFRDP CTND1_HUMAN O60716 2 async ALKNISFGRDQ, KPRHIEWESV ABCE1_HUMAN P61221 2 async PQSKIAWISE, ADRVIVFDG ACTN4_HUMAN O43707 2 async IRRTIPWLED, HIRVGWEQL CLCN7_HUMAN P51798 2 async SKKVSWSGR, TWRIFFASMI NOC2L_HUMAN Q9Y3T9 2 async SSKPINFSVIL, RQRVSFGVSEE PCIF1_HUMAN Q9H4Z3 2 async LSRIKFREE, DSRKVVKWNV S12A2_HUMAN P55011 2 async RFRVNFVDPA ZFYV9_HUMAN O95405 2 async EQRRVWFADGI, NGKVIRWTEV SAHH2_HUMAN O43865 1 async PKKQIQFADDM LMTK2_HUMAN Q8IWU2 1 async EKKAVTFFDDV SCRIB_HUMAN Q14160 1 async SVKGVSFDQA K6PL_HUMAN P17858 1 async KKKAVAFSPV NOP2_HUMAN P46087 1 async SKKVAFLRQN RL19_HUMAN P84098 1 async PEKVTWMRR IF4G1_HUMAN Q04637 1 async TSSRIRFMLQ IMA2_HUMAN P52292 1 async IPKFVSFLGR FKBP4_HUMAN Q02790 1 async YKKIVSWLEY LYPA2_HUMAN O95372 1 async PARVQFKTY

TRIP6_HUMAN Q15654 1 async HPRVNFCPLP IF4B_HUMAN P23588 1 async VSKPVSWADE TARA_HUMAN Q9H2D6 1 async TNKDIPWASF IDHC_HUMAN O75874 1 async EMTRIIWELIK TSP1_HUMAN P07996 1 async HDPRHIGWKDF TPIS_HUMAN P60174 1 async ITEKVVFEQTKV PDIA1_HUMAN P07237 1 async FKGKILFIFIDS FA83H_HUMAN Q6ZRV2 1 async EEFRILFAQSE PRDX5_HUMAN P30044 1 async GKKGVLFGVPG AK1C3_HUMAN P42330 1 async NGKVIFDIVDL UBP5_HUMAN P45974 1 async LERAVDWIFSH S10AA_HUMAN P60903 1 async DGKVGFQSFF COPG1_HUMAN Q9Y678 1 async PSKYIRFIYNR FOXK1_HUMAN P85037 1 async AIKIQFTSLY RL27_HUMAN P61353 1 async EAKVKFEER CAN2_HUMAN P17655 1 async KTRGIEWKR CHCH3_HUMAN Q9NX63 1 async TRRVTFEADE MPP10_HUMAN O00566 1 async LKRVTFALPD

Figure 3.5: “RVxF” motifs from mitosis-specific and asynchronous-specific clusters reveal subtle differences.

The “RVxF” motif sequences from proteins in A. mitosis-specific and B. asynchronous specific clusters were used to generate a weblogo (Crooks, Hon et al. 2004) for comparison between the respective binding motifs.

Figure 3.6: Clustering of PP1 binding proteins identified shows isoform-specific clustering in mitotic and asynchronous GFP-TRAP eluates.

Significantly enriched proteins (SAINT AvgP score  0.9) in all of the three isoforms of

PP1 were clustered based on both isoform-specificity and cell cycle specificity. Red in the heat map indicates the presence of the protein and black indicates the absence of the protein.

3.3.3 Validation of mass spectrometry results

Mass spectrometry data analysis revealed multiple novel potential PP1 interacting partners. Since we were interested in analyzing the cell cycle dependent interactions, we chose 9 proteins with critical roles in the cell cycle to further validate with immunoblotting. Mitotic and asynchronous input cell extracts and proteins eluted from

GFP-TRAP pull downs were immunoblotted with antibodies against the proteins of interest (Figure 3.7). The immunoblotting confirmed the interaction between PP1 and all the proteins selected (Aurora B, Aurora A, TPX2, CDCA2 (RM), TACC3, GCN2,

DBC1, BRCA1, and RIF1) in both mitotic and asynchronous extracts. Mitosis-specific enrichment and isoform-specific enrichment was also observed for some of these interactions.

CDCA2 (RM), a known mitotic PP1 binding protein, was used as a positive control in the experiment. Although CDCA2 (RM) was enriched in both asynchronous and mitotic eluates from GFP-PP1 expressing cells compared to the GFP only expressing cells, the comparison between asynchronous and mitotic eluates shows a 1000-fold greater binding in mitotic eluates (Figure 3.7 and 3.8). Similarly, Aurora B, Aurora A,

TPX2, GCN2 and TACC3 were highly enriched in mitotic eluates when compared to asynchronous eluates (Figure 3.7 and 3.8). BRCA1, another known PP1 binding partner whose interaction with PP1 is not dependent on cell cycle, was also used as a positive control and was confirmed to interact with PP1 in both mitotic and asynchronous cell lysates (Figure 3.7 and 3.8). DBC1 and RIF1 proteins were also enriched in both mitotic and asynchronous eluates confirming their interaction with PP1 (Figure 3.7 and 3.8).

Figure 3.7: Validation of mass spectrometry results by immunoblotting.

GFP-TRAP eluates from mitotic and asynchronous GFP-control, GFP-PP1, GFP-PP1 and GFP-PP1 expressing cells were run on SDS-PAGE, transferred onto nitrocellulose membranes and blotted with the indicated antibodies. Mass markers in kDa are shown on right.

Figure 3.8: Quantification of immunoblotting results for the three PP1 isoforms.

Immunoblots obtained using all the antibodies were quantified using ImageJ and were normalized to the respective controls and log2 transformed to calculate the fold-change for each protein in GFP-TRAP pull down from mitotic and asynchronous extracts of each isoform-PP1 (A), PP1 (B) and PP1 (C). For each isoform, the fold-change was normalized with the amount of GFP pulled-down in the respective isoform TRAP to account for changes in GFP-PP1, GFP-PP1 and GFP-PP1 protein expression.

3.4 Discussion

PP1 functions in conjunction with multiple regulatory proteins that control the function of PP1 in diverse signaling pathways. Close to 200 interacting partners have been identified for PP1 among which numerous proteins play roles in regulating the cell division cycle (Figure 3.1). Given the highly dynamic and transient nature of protein interactions during cell cycle, my main objective in this study to was to characterize the different interactomes of PP1 in mitotic versus asynchronous cell populations. This will help us gain an in-depth understanding of how PP1 regulates the cell cycle as well how the cell cycle is controlled by PP1.

Using quantitative mass spectrometry, mitosis-specific interacting proteins, asynchronous-specific interacting proteins or proteins interacting in both mitotic and asynchronous extracts were identified (Figure 3.3). The mass spectrometry data confirmed the significant (SAINT AvgP score  0.9) enrichment of numerous known PP1 binding proteins including ASPP1, CDCA2 (RM), YLPM1, NEB1, Taperin (TPRN),

KNL1 (CASC5), MYPT1 and Phostensin (PPP1R18) among others (Table 3.1). The data highlighted that the specificity of PP1 binding depends not only on the cell cycle phase but also on the PP1 isoform. For example, previous studies have shown that the association of protein KNL1 (CASC5) with PP1 reduces during mitosis in an Aurora B- dependent manner (Liu, Vleugel et al. 2010). In this study, KNL1 (CASC5) shows an

AvgP score of 1 in asynchronous extracts and 0.5 in mitotic extract suggesting decreased association with PP1 in mitosis consistent with previous reports (Appendix B). In contrast, CDCA2 (RM), which binds PP1 specifically during mitosis showed an AvgP

score of 1 in mitotic extracts which reduced to 0.5 in asynchronous extracts, suggesting decreased association with PP1 during interphase (Qian, Beullens et al. 2015) (Appendix

B). Similarly, known isoform-dependent interactions were also confirmed in the data presented here. MYPT1, a PP1-specific interactor, showed a 100-fold enrichment in

PP1 extracts (both mitotic and asynchronous) when compared using the average iBAQ areas for each PP1 isoform.

Protein interactors from each cluster which possess the ‘RVxF’ motif and have critical roles in cell cycle regulation were validated by immunoblotting. Of specific interest were mitotic kinases Aurora A and Aurora B, both of which share common substrates with the phosphatase. The immunoblotting data corresponds to the mass spectrometry data in that Aurora A, along with its regulator TPX2, are highly enriched in mitotic immunoprecipitations while Aurora B interacts with PP1 in both asynchronous and mitotic extracts (Figure 3.7). Of note here is the fluctuating levels of these mitotic proteins during the cell cycle (Lindon, Grant et al. 2015). The protein expression of

Aurora A, Aurora B, and TPX2 peaks during mitosis but these proteins degrade at the end of mitosis. This increase in protein expression might explain the increased interaction with PP1 observed during mitosis. In this experiment, no difference was seen in the level of Aurora B expression in mitotic cells which might have affected the interaction results.

Among other validated proteins is TACC3, a centrosomal protein which plays a major role in mitotic spindle assembly and chromosome segregation (Hood and Royle

2011). TACC3 belongs to the transformed acidic coiled coil (TACC) protein family and binds microtubules to promote microtubule stability during mitosis (Nwagbara, Faris et al. 2014). TACC3 is known to be regulated by phosphorylation by protein kinase Aurora

A which is critical for its accumulation at the mitotic spindle (Lioutas and Vernos 2013,

Burgess, Peset et al. 2015). Here we identified TACC3 as a potential PP1 binding partner with a perfect ‘KVTF’ motif. Both mass spectrometry results and immunoblotting results confirmed the presence of TACC3 in GFP-PP1 immunoprecipitations. Furthermore, there was 10 fold more TACC3 in mitotic eluate as compared to asynchronous eluate.

Comparison between eluates from different isoforms of PP1 suggests 2-fold more binding to the PP1 isoform which shares the same localization space (centrosome) as

TACC3 during mitosis. Reciprocal TACC3 immunoprecipitations should be tested for

PP1 interaction to confirm this result. Moreover, to test for direct interaction between

TACC3 and PP1, TACC3 mutant with mutated RVxF motif (RARA) should be expressed and tested for PP1 binding.

GCN2 (EIF2AK4) is a protein kinase which can sense nutritional stress (specifically low amino acid levels) by binding uncharged tRNAs thereby repressing global protein translation by phosphorylating eIF2 (Dong, Qiu et al. 2000, Zhang, McGrath et al.

2002, Dever and Hinnebusch 2005). GCN2 has also been known to regulate G1/S transition by coordinating DNA replication initiation timing during S phase with growth and stress signals (Hamanaka, Bennett et al. 2005, Grallert and Boye 2007). The immunoblotting data shows that GCN2 is enriched in GFP-PP1 immunoprecipitations

(mitotic and asynchronous). Interestingly, small band shifts were observed in mitotic and asynchronous GFP-PP1 eluates, suggesting binding of PP1 to phosphorylated GCN2

(Figure 3.7).

DBC1 was initially identified by cloning a chromosomal region which was homozygously deleted in multiple breast cancers (Nishiyama, Hornigold et al. 1999).

DBC1 controls gene expression by negatively regulating human deacetylases sirtuin 1

(SIRT1) and histone deacetylase 3 (HDAC3) (Kim, Chen et al. 2008). Depletion of

DBC1 has been shown to cause a G2/M delay in the cell cycle (Tanikawa, Wada-Hiraike et al. 2013). Here we were able to demonstrate that DBC1 is enriched in both mitotic and asynchronous GFP-PP1 immunoprecipitations correlating with the mass spectrometry results where DBC1 has an AvgP score of greater than 0.9 for both cell populations.

As described previously (Section 2.4), RIF1/PP1 interaction in yeast has been implicated in regulating replication origin firing (Dave, Cooley et al. 2014, Mattarocci,

Shyian et al. 2014). In Section 2, we identified phosphorylation within the ‘RVSF’ motif of RIF1 and this phosphorylation could be attributed to protein kinase Aurora B. Here, we were able to confirm the interaction between RIF1 and PP1 in both asynchronous and mitotic GFP-PP1 immunoprecipitations. Moreover, there was at least a 5-fold enrichment of RIF1 in asynchronous immunoprecipitations as compared to mitotic immunoprecipitations (Figure 3.8). This is consistent with the results from Section 2 as we would expect less association of PP1 with RIF1 when it is phosphorylated within the

‘RVSF’ motif during mitosis. To confirm and validate each of these interactions with

PP1, reciprocal immunoprecipitations with the respective interactor need to be performed. Mutation within the RVxF motif of these interactors will help determine if these interactions with PP1 are direct or they are part of the same complex without directly interacting with each other.

3.5 Conclusion

PP1 plays a critical role in driving cell cycle progression and achieves that through multiple interacting partners. Although the expression of PP1 remains constant throughout the cell cycle, its activity is regulated by binding to diverse cell cycle phase- specific docking proteins. Here we have identified 173 asynchronous-specific and 78 mitosis-specific potential PP1 binding partners through quantitative mass spectrometry of

GFP-tagged PP1 expressing cells. We were able to validate biochemically 9 of these interactions in asynchronous and mitotic cell populations. These novel interactions can help us decipher the functional pathways which are regulated by the phosphatase.

CHAPTER 4. VALIDATION AND CHARACTERIZATION OF INTERACTION

BETWEEN CENTROSOMAL PROTEIN CEP192 AND PP1

4.1 Introduction

Centrosomes form the major microtubule-organizing centers in mammalian cells and are critical for the processes of spindle formation, spindle organization, and chromosome segregation. Each centrosome consists of two orthogonally placed barrel shaped centrioles, which precisely duplicate only once during each division cycle (Nigg and

Stearns 2011, Bornens 2012). These centrioles are surrounded by highly ordered protein rings called the peri-centriolar material (PCM). Centrioles act as platforms for the assembly of new centrosomes, flagella, and primary cilia while the PCM nucleates the microtubules (Bettencourt-Dias and Glover 2007, Nigg and Raff 2009).

The centrosome cycle is a highly regulated process largely governed by reversible protein phosphorylation. As a cell enters into S-phase, the process of centriole duplication starts with the formation of a pro-centriole. A new daughter centriole is formed perpendicular to each mother centriole and the new centrioles grow and elongate through

S and G2 phases of cell cycle. Prior to the onset of mitosis, the centrosomes grow dramatically mostly by the recruitment of additional PCM components (centrosome maturation). At this point, the two centrosomes separate and move to opposite poles of a cell to form spindle poles during mitosis. At the end of mitosis, the mother, and daughter centriole are disengaged but are connected through a proteinaceous linker during interphase until the next mitosis (Wang, Soni et al. 2011).

Centrosome maturation and duplication are regulated by serine/threonine protein kinases, including Aurora A, PLK1 and Nek2A (Hannak, Kirkham et al. 2001, Berdnik and Knoblich 2002, Tsai and Zheng 2005, Archambault and Glover 2009, Lee and Rhee

2011). Major players involved in this cycle of centrosomal phosphorylation include the pericentrin, a protein that forms the PCM inner layer with a radially oriented outer matrix of other crucial centrosomal proteins including -tubulin ring complex, centrosomal protein of 192 kDa (CEP192) and centrosomal protein of 215 kDa (CEP215), among others (Brito, Gouveia et al. 2012). All these proteins couple the centrosomal cycle with the cell cycle, and is crucial for accurate cell division (Figure 4.1A).

CEP192 is a centrosomal protein with a critical function in centrosome maturation and centriole duplication as well as bipolar spindle formation (Gomez-Ferreria, Rath et al. 2007, Joukov, De Nicolo et al. 2010). This protein not only determines the centrosome size but is also essential for the recruitment of other PCM components including the - tubulin ring complex through its interaction with multiple centrosomal proteins (Figure

4.1B) (O'Rourke, Gomez-Ferreria et al. 2014). CEP192 has been proposed to act as a scaffold for nucleation of microtubules and other regulatory factors during mitosis.

CEP192 is localized in the inner layer of peri-centriolar material (Figure 4.1C) and interacts with CEP152 to recruit protein kinase PLK4 to the centrosome which regulates centriole biogenesis during the cell cycle (Sonnen, Gabryjonczyk et al. 2013). CEP192 has been demonstrated to be regulated by protein kinases Aurora A and PLK1 and, in turn, regulates their activity at the centrosome (Joukov, Walter et al. 2014, Asteriti, De

Mattia et al. 2015, Yang and Feldman 2015). CEP192 is a critical player in the kinase

Figure 4.1: Regulation of the centrosome.

(A) Each centrosome consists of two orthogonal centrioles surrounded by peri-centriolar matrix (PCM), which duplicate during the S-phase of the cell cycle (centrosome duplication). In the preparation of cell division, the PCM grows by the recruitment of more proteins (centrosome maturation) which eventually allow the centrosomes to separate prior to the onset of mitosis (centrosome separation). Once separated, the centrosomes form the two spindle poles and act as the major microtubule organizing centers during mitosis. (B) The known protein-protein interaction data for the centrosomal protein CEP192 from STRING database (Szklarczyk, Franceschini et al. 2015). (C) CEP192 is localized to the inner layer of peri-centriolar material as seen in the cross-section of a mother centriole with ninefold symmetry.

activation cascade of Aurora A and PLK1 at the centrosomes, and acts as a scaffold to recruit and activate the centrosomal Aurora A via autophosphorylation at the T-loop

Thr288 residue. Once activated, Aurora A can trigger the activation of PLK1 by phosphorylating its T-loop Thr210 residue. This active PLK1 can then dock to CEP192

Thr44 and phosphorylate the protein on several serine residues (Joukov, Walter et al.

2014). These phosphorylated residues act as a recruiting platform for other PCM proteins such as the -tubulin ring complex which controls microtubule nucleation. Loss of

CEP192 in mammalian cells leads to numerous centrosomal abnormalities due to incorrect centrosome segregation caused by impaired centrosome separation and centrosome maturation highlighting the importance of CEP192 as a critical regulator of centrosome function.

CEP192 is a heavily phosphorylated protein with 67 phosphorylated serine, threonine or tyrosine residues identified in different phospho-proteomic data sets derived from

PhosphoSitePlus (Figure 4.2) (Joukov, Walter et al. 2014, Asteriti, De Mattia et al.

2015). Given the dynamic nature of phosphorylation, it can be presumed that these sites need to be dephosphorylated once they transfer the signal to the downstream effectors.

Very little is known about phosphatases working to dephosphorylate CEP192. In this section, I hypothesize that CEP192 interacts with PP1 via its ‘KHVTF’ motif and propose that this interaction is critical for the dephosphorylation and the function of CEP192 at the centrosome.

Figure 4.2: Regulation of CEP192 by phosphorylation.

The known phosphorylation sites in the human CEP192 proteins were identified using PhosphoSitePlus (http://www.phosphosite.org/proteinAction.action?id=13438) (Hornbeck, Zhang et al. 2015). The letters and numbers refer to amino acids (serine (S), threonine (T), tyrosine (Y)) and their position in CEP192 sequence. Numbers on the bar below refer to the amino acid residue position in the sequence.

4.2 Materials and Methods

4.2.1 Conservation of potential PP1 binding motif among eukaryotes

The eukaryotic protein orthologs of CEP192 were extracted from the genomic sequences of organisms with full genome sequence available using EggNOG4.5 (Huerta-

Cepas, Szklarczyk et al. 2016). Smith-Waterman algorithm along with composition based score adjustment was used to calculate sequence similarities. The sequences from all orthologous proteins identified were aligned using Clustal Omega to determine the conservation of the potential PP1 binding ‘KHVTF’ motif among these organisms

(Sievers, Wilm et al. 2011). Trimmed part of this alignment containing the ‘KHVTF’

motif was used to create a web logo using Weblogo (Schneider and Stephens 1990,

Crooks, Hon et al. 2004).

4.2.2 Molecular cloning and site-directed mutagenesis of CEP192

pEGFP-C1-CEP192 construct generated using human CEP192 was obtained from

Dr. Kyung S. Lee at the National Cancer Institute, Bethesda, MD. Residues 507-1065

(containing the ‘KHVTF’ motif) from this construct were amplified by PCR using

Gateway compatible primers and inserted into pDONR201 via BP ligation reaction (Life

Technology). This construct was sub-cloned into bacterial expression vector pDEST42 to create a C-terminal V5 and 6XHis tagged fusion construct (CEP192-WT507-1065). The putative PP1 binding motif ‘KHVTF’ in this construct was mutated to ‘KHATA’

(CEP192-RARA507-1065) by site-directed mutagenesis as per manufacturer’s protocol. The constructs were sequence verified by Eurofin MWG Operon LLC sequencing.

4.2.3 Expression and purification of human CEP192 and its RARA mutant

CEP192-WT507-1065 and CEP192-RARA507-1065 constructs were transformed into

BL-21 (DE3) E. coli cells and were grown at 37°C until they reached an O.D. 600 of 0.4.

The cells were induced with 0.1 mM IPTG at 22°C for 16 h. Bacteria were pelleted at

4,000 rpm for 20 min, re-suspended in 1X extraction buffer (50 mM Hepes-NaOH pH

7.5, 150 mM NaCl, 5% (v/v) glycerol and 10 mM imidazole) and frozen at -80°C until further use. For protein purification, cells were thawed with the addition of 1% (v/v)

Tween-20, 20 mM imidazole, 1 mM phenylmethanesulfonylfluoride (PMSF), 1 mM benzamidine, 2 µg mL-1 leupeptin, 5 µg mL-1 pepstatin and lysed using sonication (6-8

cycles of 30 sec pulse each). Crude lysates were clarified at 20,000 rpm for 30 min at

4oC. Supernatants were removed, and an additional 1 mM PMSF and 1 mM benzamidine were added prior to incubation with Ni-NTA agarose matrix (Qiagen) end-over-end at

4oC for 1.5 h. Matrix was captured in a column via gravity sedimentation and washed with 500 column volumes of Buffer A (1 x extraction buffer with 1 M NaCl, 1% (v/v)

Tween-20, and 20 mM imidazole followed by 100 column volumes of Buffer B (1 x extraction buffer). Ni-NTA bound proteins were eluted in 1x extraction buffer containing

300 mM imidazole, pH 7.5. Protein eluates were concentrated using a 30,000 Da molecular weight cutoff Amicon concentrator (EMD Millipore).

4.2.4 In-vitro PP1 binding assay

PP1 was purified as described in Section 2.2.4. PP1 (//) (6 µg) was pre- incubated with either CEP192-WT507-1065 or CEP192-RARA507-1065 in 500 µl binding buffer (25 mM Tris, pH 7.5, 5% (v/v) glycerol, 150 mM NaCl and 10 mM imidazole). To prevent non-specific binding, Ni-NTA beads were blocked with 1 mg mL-1 BSA solution in binding buffer for 1 h prior to incubation with PP1-CEP192 complex mix for 2 h at

4°C. The beads were washed with wash buffer (25 mM Tris, pH 7.5, 5 % (v/v) glycerol, 1

M NaCl and 10 mM imidazole) with 1% (v/v) Tween-20 followed by a wash with the binding buffer. The proteins were eluted off the beads using 2X SDS-PAGE cocktail and run on SDS-PAGE for further analysis.

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4.2.5 GFP-TRAP with U2OS-GFP-PP1 and HeLa-GFp-CEP192 cells

Stable cell lines integrated with a bacteria artificial chromosome containing the murine homolog of CEP192 tagged with GFP at the C-terminus (CEP192-GFP) were obtained from Dr. Laurence Pelletier at University of Toronto. The CEP192-GFP cells were cultured in DMEM supplemented with 10% (v/v) FBS, 100 U ml-1 penicillin- streptomycin and 400 µg mL-1 geneticin. Asynchronous lysates were prepared by scraping the cells in lysis buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 150 mM KCl) with protease inhibitor cocktail (Sigma) and phosphatase inhibitors (25 mM

NaF, 0.5 µM microcystin-LR) followed by sonication. The lysates were clarified by centrifugation at 14,000 rpm for 10 min. The protein concentration in the supernatant was determined by Bradford reagent using BSA as standard. To obtain mitotic cells, the cells were synchronized with 100 ng mL-1 nocodazole for 16 h followed by mitotic shake-off and release for 30 min. The cell pellet was suspended in lysis buffer and lysates prepared as described for asynchronous cells.

A GFP-TRAP experiment from this lysate was performed as described in

Section 3.2.2. Briefly, equal amount of cell lysate was incubated with GFP-TRAP beads

(Chromotek) for 1.5 h at 4°C followed by washing the beads three times with wash buffer

(25 mM Tris, pH 7.5, 5% (v/v) glycerol, 500 mM NaCl, 0.5% (v/v) NP-40). The bound proteins were eluted using 2X SDS-PAGE cocktail and run on SDS-PAGE to analyze for the presence of bound PP1.

GFP-TRAPs using U2OS cells expressing either GFP alone or the three isoforms of PP1 were performed as described in Section 3.2.2.

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4.2.6 Generation and validation of phospho ‘KHVTF’ CEP192 antibody

The phospho and dephospho versions of CEP192 peptide containing the potential

PP1 binding motif SEKHVTFENHK were synthesized with 98% purity at GL Biochem

(Shanghai) Ltd., China. The phospho-specific antibody was generated as described in

Section 2.2.2 and affinity purified using phospho-CEP192 peptide conjugated column as described in Section 2.2.3. The purified antibody (called p-CEP192 hereafter) was validated with dot blots using phospho and dephospho CEP192 peptides and western blots. To confirm the phospho-specificity, the antibody was quenched with 5 µg mL-1 of the antigen (SEKHVpTFENHK) before incubating the membrane with the antibody.

4.2.7 Kinase inhibitor assay

Kinase inhibitor assays using HeLa cells were performed as in Section 2.2.6.

4.2.8 Immunofluorescence and microscopy

HeLa cells were grown on poly-lysine coated coverslips, fixed with 3.7% (v/v) formaldehyde, permeabilized with 0.5% (v/v) Triton X-100 and blocked in 1% (w/v)

BSA in 1 x PBS. The coverslips were then incubated with either CEP192 antibody

(Bethyl, 1:1,000) or affinity purified phospho-CEP192 antibody (2 µg mL-1) for 2 h followed by Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-mouse secondary antibodies (Molecular Probes, Thermo Fisher Scientific,

1:500) for 1 h. Nuclei were counterstained with DAPI (Sigma Aldrich, 1 g ml-1) and images acquired using a Leica DMIRE2 microscope equipped with a digital charge-

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coupled device (CCD) camera (Hamamatsu, Photonics, K.K.). The co-localization images were acquired with Nikon A1R confocal microscope for better resolution.

4.3 Results

4.3.1 The potential PP1 binding ‘KHVTF’ motif in CEP192

Analysis of protein orthologs of human CEP192 showed that the potential PP1 binding ‘KHVTF’ sequence is highly conserved among higher eukaryotes. Among the 55 proteins screened, 32 (~ 60%) contain the ‘KHVTF’ motif. The residues critical for PP1 binding, valine and phenylalanine are completely conserved among the ‘KHVTF’ containing organisms (Figure 4.3A). A weblogo generated using sequences from all the orthologs (Figure 4.3B) confirms the consensus of ‘KHVTF’ motif among the eukaryotes with CEP192. Another striking observation is high conservation of two ‘SP’ sites few residues upstream of the ‘KHVTF’ motif that might be important in regulating

PP1 binding with CEP192.

4.3.2 CEP192 co-localizes with PP1

Centrosomal imaging of metaphase U2OS-GFP-PP1 cells showed staining of

CEP192 as expected (Figure 4.4A). This centrosomal signal co-localized with GFP-

PP1 signal suggesting that CEP192 and PP1 are in close vicinity, at least during metaphase (Figure 4.4A).

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Figure 4.3: Potential PP1 binding ‘KHVTF’ motif in CEP192.

(A) Clustal omega generated multiple sequence alignment using 55 human CEP192 orthologous eukaryotic protein sequences shows a high degree of conservation of the potential PP1 binding ‘KHVTF’ motif. The ‘KHVTF’ motif is highlighted in a red box and the consensus below shows the most conserved residues at each position. (B) The sequence highlighted in cyan in (A) was obtained from all the protein sequences and was used as the input to generate a weblogo (Crooks, Hon et al. 2004).

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4.3.3 CEP192 interacts with PP1

GFP-TRAP experiment using GFP-tagged CEP192 expressing cells showed an enrichment of PP1 in both the asynchronous and mitotic eluates (Figure 4.4B). This suggests CEP192 and PP1 may be a part of the same complex. This was also confirmed by reciprocal GFP-TRAP using GFP-tagged PP1 expressing U2OS cells where CEP192 was enriched in the TRAP eluates from all the three PP1 isoforms (Figure 4.4C).

To determine if the interaction between CEP192 and PP1 is direct, the eluates from the binding experiment using CEP192-WT507-1065 and CEP192-RARA507-1065 and

PP1 were immunoblotted for PP1. This blot showed an enrichment of PP1 in CEP192-

WT507-1065 assay as compared to the control suggesting a direct interaction (Figure 4.4D).

This interaction is reduced when CEP192-RARA507-1065 mutant is used for assay which suggests that the interaction of CEP192 and PP1 is via the ‘KHVTF’ motif (Figure

4.4D).

4.3.4 ‘KHVTF’ motif in CEP192 is phosphorylated during mitosis

PP1 binding showed preference towards dephosphorylated RV[S/T]F containing peptides derived from many proteins including CEP192 as compared to phospho-

RV[S/T]F peptides (Section 2.3.1). To determine the phospho-status of the ‘KHVTF’ motif in CEP192, phospho-specific ‘KHVTF’ antibody was generated and validated

(Figure 4.5A, B, and C). This antibody was used to analyze the phosphorylation within

‘KHVTF’ during the cell cycle. As shown by the immunoblot, the ‘KHVTF’ motif in

CEP192 is phosphorylated specifically during mitosis (Figure 4.6A). This result was also

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Figure 4.4: CEP192 interacts with PP1.

(A) Immunofluorescence using the CEP192 antibody shows co-localization with GFP- tagged PP1 expressing cells. The images of the two centrosomes were taken in two different planes. Blue indicates DAPI, green indicates GFP-PP1 and red indicates CEP192. Scale bar, 20 µm. (B) Input extracts and GFP-TRAP eluates from either control cells or GFp-CEP192 expressing cells were probed with the indicated antibodies. (C) Input extracts and GFP-TRAP eluates from control cells or GFP-PP1 or  or  expressing cells were probed with the indicated antibodies. (D) The elutes from in-vitro binding assay using PP1 // with either CEP192-WT507-1065 (V5 tagged) or CEP192- RARA507-1065 (V5 tagged) were probed with the indicated antibodies. Beads alone and PP1 alone act as a negative control for the assay.

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Figure 4.5: Validation of the phospho-KHVTF CEP192 antibody.

(A) Phospho or dephospho versions of KHVTF containing CEP192 peptide (SEKHVTFENHK) were spotted on nitrocellulose membrane at the indicated amounts in triplicate. The membrane was probed either with p-CEP192 antibody alone or with 5 µg mL-1 phospho-peptide or with 5 µg mL-1 dephospho-peptide to determine phospho- specificity. (B) Asynchronous or mitotic HeLa cell lysates made in the presence or absence of phosphatase inhibitors were probed with either CEP192 antibody or p- CEP192 antibody. (C) Asynchronous or mitotic HeLa cell lysates made in the presence or absence of phosphatase inhibitors were probed with p-CEP192 antibody alone or with 5 µg mL-1 phospho-peptide or with 5 µg mL-1 dephospho-peptide as indicated.

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confirmed by immunofluorescence of cells stained with p-CEP192 antibody which revealed centrosomal staining (Figure 4.6B) during mitosis.

4.3.5 PLK1 regulates the phosphorylation of the ‘KHVTF’ motif in CEP192

The phosphorylation within the ‘KHVTF’ motif in CEP192 might be a crucial regulator of its interaction with PP1. Inhibiting major mitotic kinases in the kinase inhibitor assay attributed this phosphorylation event to PLK1. Immunofluorescence of kinase-inhibited cells stained with phospho-KHVTF CEP192 antibody showed the inhibition of PLK1 reduced this phosphorylation in mitotic cells (Figure 4.6C).

4.4 Discussion

CEP192 is a crucial regulator of centriole duplication, maturation, and separation through its interaction with other centrosomal proteins (Figure 4.1B). CEP192 has been proposed to play a scaffolding role in the recruitment of PCM proteins at different times in the centrosomal cycle (Joukov, De Nicolo et al. 2010). This has been shown to be true in case of the recruitment of protein kinases Aurora A and PLK1, which are the major regulators of centrosomal protein phosphorylation. CEP192 binds both Aurora A and

PLK1 protein kinases and is essential for the sequential T-loop phosphorylation and therefore activation of both these protein kinases at the centrosomes (Joukov, Walter et al. 2014, Asteriti, De Mattia et al. 2015). Loss of interaction between CEP192 and Aurora

A and/or PLK1 results in centrosome and spindle assembly defects highlighting the importance of this signaling pathway (Joukov, Walter et al. 2014). However, the reverse

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Figure 4.6: Phosphorylation of the ‘KHVTF’ motif in CEP192.

(A) HeLa cells were synchronized in mitosis with thymidine-nocodazole block and released thereafter to collect cells at indicated times. The cell lysates were probed with the indicated antibodies including mitotic marker cyclin B and loading control tubulin. Mass markers are shown in kDa. (B) HeLa cells were stained with the indicated antibodies and imaged at different phases of the cell cycle as indicated. Blue indicates DAPI, red indicates tubulin and green indicates p-CEP192 antibody. Scale bar, 20 µm. (C) HeLa cells were treated with indicated protein kinase inhibitors and stained with the indicated antibodies to monitor the status of CEP192 ‘KHVTF’ phosphorylation. Scale bar, 20 µm. 109

dephosphorylation activity of CEP192 and other centrosomal has not been studied in detail with much less known about any centrosomal protein phosphatases.

PP1 binding proteins mediate their interaction with the PP1 catalytic subunit through docking motifs, primarily the RVxF motif (Section 1 and 2). CEP192 contains the classic PP1 binding motif ‘KHVTF’, which is highly conserved among eukaryotes

(Figure 4.3A). The association of PP1 with its regulatory proteins has also been shown to be regulated by phosphorylation in or around the PP1 docking RVxF motif. This phosphorylation in some cases has been attributed to CDK1 phosphorylation of a ‘S/TP’ site near the RVxF motif as in case of the regulatory protein CDCA2 (RM) (Qian,

Beullens et al. 2015). Interestingly, there are two conserved ‘SP’ sites C-terminal to the

‘KHVTF’ motif in CEP192 (Figure 4.3B) which might regulate the binding of PP1 with

CEP192 in a CDK1-dependent manner. These sequence-based observations make

CEP192 an ideal candidate for PP1 binding and regulation by PP1.

PP1 localizes to the centrosomes through its interaction with Nek2 (Helps, Luo et al. 2000, Mi, Guo et al. 2007) (Section 1.5.2). Apart from Nek2, no centrosomal targeting subunits for PP1 have been identified as yet. This localization of PP1 was confirmed here using GFP-tagged PP1 expressing U2OS cells. In addition, this PP1 co-localizes with centrosomal CEP192 (Figure 4.4A). With GFP-tagged CEP192 expressing HeLa cells and GFP-tagged PP1 expressing U2OS cells, we could show that CEP192 and PP1 are a part of the same complex (Figure 4.4B, Figure 4.4C). Additionally, this interaction was direct as confirmed with the in-vitro binding assay and was reduced in the case of

CEP192-RARA507-1065 mutant (Figure 4.4.D), suggesting that the interaction occurs via

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KHVTF motif and the valine and the phenylalanine residues are crucial for this interaction.

Phosphorylation within the RVxF motif in PP1 regulatory proteins plays a role in controlling the association of PP1 with its regulatory proteins (Section 2). Here, I could show that the threonine within the ‘KHVTF’ motif of CEP192 is phosphorylated specifically during mitosis (Figure 4.6) and that PLK1 protein kinase regulates the phosphorylation of this site (Figure 4.7). Although the ‘KHVTF’ sequence does not align well with the PLK1 consensus sequence (D/E-X-S/T-Φ-X-D/E (X, any amino acid; Φ, a hydrophobic amino acid), the phosphorylation of this site is directly or indirectly regulated by PLK1 as shown by the data presented here. Although there has been evidence that PLK1 has such a strong preference for hydrophobic amino acid (F) at +1 position that it can phosphorylate sequences even with a basic residue (K/R) at -1 or -2 position (Kettenbach, Schweppe et al. 2011). PLK1 mediated phosphorylation of CEP192 is essential for the recruitment of other proteins like -tubulin ring complex, which may regulate the phosphorylation of this motif through additional protein kinases.

Many recent studies have been able to identify a binding motif for the phosphatase

PP2A-B56 (Hertz, Kruse et al. 2016, Wang, Wang et al. 2016). The motif “LSPI” has been shown to directly interact with the B56 regulatory protein of PP2A in substrates like

CDCA2 (RM) and BUBR1 (Xu, Raetz et al. 2013, Qian, Beullens et al. 2015).

Interestingly, we also noted the presence of this “LSPV” motif in CEP192, suggesting that CEP192 might also help in the recruitment of PP2A-B56 to the centrosomes. More experiments will be required to show that CEP192 may act as a scaffold protein to recruit

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the major phosphorylation regulatory proteins like Aurora A, PLK1, PP1 and PP2A-B56 to the centrosomes and control the centrosomal phosphorylation turn over.

4.5 Conclusion

The data presented here reveals a novel interaction between the centrosomal protein

CEP192 and PP1. This interaction occurs via the highly conserved ‘KHVTF’ motif in

CEP192 that, by analogy with other RVxF motifs, probably binds in the hydrophobic binding pocket of PP1. Furthermore, my work also shows that the valine and phenylyalanine in the ‘KHVTF’ motif are critical for binding to PP1 and mutations in these residues reduces the binding between CEP192 and PP1. Phosphorylation within the

‘KHVTF’ might play a role in PP1 docking, and based on previous results it can be assumed that the phosphorylation within ‘KHVTF’ motif abolishes CEP192 binding to

PP1 (Section 2). Interestingly this motif is phosphorylated during mitosis suggesting that

CEP192-PP1 interaction might be cell cycle dependent.

Although many recent phospho-proteomic studies identified multiple proteins that are phosphorylated (Müller, Schmidt et al. 2010, Habermann, Mirgorodskaya et al. 2012,

Barretta, Spano et al. 2016), dephosphorylation at the centrosomes has not been studied in detail. Unveiling this interaction suggests that PP1 at the centrosomes might have more centrosomal substrates and thereby control protein phosphorylation balance at the centrosomes.

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CHAPTER 5. PERSPECTIVES AND FUTURE DIRECTIONS

5.1 PP1 as the master controller of cell cycle

PP1, along with PP2A, is responsible for 90% of all the dephosphorylation reactions in a eukaryotic cell (Virshup and Shenolikar 2009). Through its interactions with multiple

“RVxF” containing cell cycle regulatory proteins, PP1 regulates several events in the cell cycle. The main goal of this thesis was to elucidate the mechanism of binding of PP1 with its regulatory proteins, specifically during mitosis (Chapter 2), and identify novel PP1 interacting partners which would provide more insight into the mitotic function of PP1

(Chapter 3 and 4). This thesis presents a novel mechanism for the regulation of PP1 and its targeting partners during mitotic phase of the cell cycle through phosphorylation within the “RVxF” motif. My work also uncovers many potential novel cell cycle dependent interactors of PP1 which might regulate the function of PP1 during this complex process.

5.1.1 Aurora B and PP1: the essential kinase-phosphatase equilibrium

The activation of mitotic kinase activity together with the repression of the counteracting phosphatase activity essentially drives mitotic progression. Studies in fission yeast have revealed an elegant relay mechanism between the protein kinase CDK1 and its counteracting phosphatases, PP1 and PP2A (Grallert, Boke et al. 2015). Elevated activity of CDK1 keeps the activity of both PP1 and PP2A in check during mitosis. At the mitotic exit, the reactivation of PP1 is essential for the sequential reactivation of

PP2A with its PP2A-B55 and PP2A-B56 regulatory subunits. In human cells, a similar

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relay between kinase and phosphatase activities was established recently in case of PP1 regulatory protein CDCA2 (RM) (Qian, Beullens et al. 2015). CDK1 dependent phosphorylation of CDCA2 (RM) prevents its association with PP1 until anaphase but facilitates its binding with PP2A-B56. As the CDK1 activity declines, PP1 can be recruited by CDCA2 (RM), and this PP1-CDCA2 (RM) complex can then counteract the activity of a localized pool of protein kinase Aurora B to initiate bulk dephosphorylation of phospho-substrates at the mitotic exit. I believe that a similar mechanism must exist with mitotic kinases and other regulatory subunits of phosphatases to drive mitosis in a timely manner and prevent futile cycles of phosphorylation and dephosphorylation, particularly during mitosis. The findings presented in this thesis suggest that there is a relay between CDK1 kinase, Aurora B and PP1 to control progression through mitosis

(Figure 5.1). CDK1 phosphorylates PP1 at Thr320 to keep it inactive until anaphase. At the same time, Aurora B kinase phosphorylates RV[S/T]F motifs which dissociates the phosphatase from its regulatory proteins keeping the phosphatase activity in check during mitosis. The decline in the activity of CDK1 leads to auto-dephosphorylation and hence partial activation of PP1, which can then dephosphorylate RV[S/T]F motifs and re- associate with its partners and carry out bulk dephosphorylation of mitotic phospho- proteins. This suggests a novel mechanism of regulation of PP1 activity during mitosis and at mitotic exit. Various studies have shown that PP1 antagonizes Aurora B activity during spindle assembly checkpoint and mitotic exit through its interaction with multiple regulatory proteins (Kim, Holland et al. 2010, Liu, Vleugel et al. 2010, Lampson and

Cheeseman 2011, Meadows 2013).

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Figure 5.1: Interplay between CDK1, Aurora B and PP1 activities during the cell cycle.

Elevated CDK1 activity at the G2/M transition is responsible for the phosphorylation of Thr320 in the catalytic subunit of PP1. This inhibitory phosphorylation reduces PP1 activity during mitosis. Additionally, here we report an increased phosphorylation of RV[S/T]F motifs during mitosis which can be attributed to the Aurora B activity. This signifies a novel targeting subunit based regulation of PP1 function in cell cycle. Furthermore, at the end of mitosis, decline in CDK1 activity partially activates PP1, which can then dephosphorylate these RV[S/T]F motifs, thereby increasing its association with regulatory proteins to carry out bulk dephosphorylation at mitotic exit.

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Through the data presented here, I believe that this antagonism between Aurora B kinase and PP1 regulatory proteins is a widespread phenomenon to control mitotic progression. This thesis provides evidence for this mechanism by implicating the phosphorylation of multiple RV[S/T]F motifs to protein kinase Aurora B (Chapter 2).

Among the proteins identified as phosphorylated within RV[S/T]F motif during mitosis,

65% were found to be regulated by the activity of Aurora B. Interestingly some of these proteins form critical components of spindle assembly checkpoint including UBR5,

SEH1, ELYS and ASPM (Chapter 2). Although Aurora B activity affects the phosphorylation of these RV[S/T]F motifs, it is important to determine if these are direct or indirect targets of Aurora B. Another crucial experiment would be to determine if the activity of Aurora B (phosphorylation within RV[S/T]F motif) would disrupt the formation of complexes between these proteins and PP1. I was able to show this in the case of RIF1 (at least in-vitro) (Chapter 2), but additional experiments will be needed to verify this in all other candidate proteins.

5.1.2 The novel cell cycle dependent PP1 interactome

The function of a protein can be best determined by studying its interacting partners. Another objective of this thesis was to find novel interacting partners of PP1 in asynchronous and mitotic cells to gain insight into its functions. This was achieved using quantitative mass spectrometry in collaboration with Dr. Arminja Kettenbach (Chapter

3). We were able to identify a total of 113 significantly enriched “RVxF” containing proteins in mitotic or asynchronous extracts (Table 3.1). Furthermore, seven novel PP1 binding partners were validated with immunoblotting. Specifically, protein kinases

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Aurora A and Aurora B, TPX2, GCN2, TACC3 were enriched in mitotic extracts while

DBC1 and RIF1 were enriched in asynchronous extracts (Figure 3.7). Although these interactions highlight novel roles for PP1, further experiments would uncover the functional role of PP1 in these associations. Reverse immunoprecipitations and “RVxF” mutation analysis (“RVxF” to PP1 non-binding “RARA” mutant) would confirm the direct interactions between the novel proteins enriched in GFP-PP1 immunoprecipitations.

Another novel PP1 interactor identified here is the centrosomal protein, CEP192

(Chapter 4). The function of CEP192 in mitotic spindle assembly and organization is regulated by Aurora A-dependent phosphorylation (Gomez-Ferreria, Rath et al. 2007). I was able to show that CEP192 and PP1 co-localize at the centrosomes and associate through the ‘KHVTF’ motif of CEP192. This interaction opens new avenues for regulation of centrosomal protein phosphorylation through the phosphatase activity of

PP1.

5.2 The therapeutic potential of PP1

The phosphorylation and dephosphorylation cycle acts as a dynamic switch to control the cellular response to internal and external cues. The stoichiometric balance between the activities of protein kinases and protein phosphatases is essential to maintain a healthy state in cells. The imbalance in either of these activities has been associated with a multitude of diseases such as cancer, diabetes, cardiovascular disorders and neurodegeneration (Zhang, Yogesha et al. 2013). The potential of protein kinases as druggable targets has been studied in-depth making them the second most important

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group of drug targets (Cohen 2002). The human genome encodes for about 400 serine/threonine protein kinases and in contrast only about 40 serine/threonine protein phosphatases (Virshup and Shenolikar 2009). This effectively translates to the fact that in order to counteract the activity of 10 serine/threonine kinases, there is a single phosphatase. This has led to the false belief that protein phosphatases are promiscuous enzymes and hence are not drug-targetable. However, multiple studies recently have shown evidence that protein phosphatases can be promising and important drug targets

(McConnell and Wadzinski 2009, Zhang, Yogesha et al. 2013).

PP1 is a major serine/threonine phosphatase in eukaryotic cells and can regulate diverse cellular processes like cell cycle progression, protein synthesis, gene transcription, muscle contraction, glycogen metabolism and synaptic plasticity

(Ceulemans and Bollen 2004). The catalytic subunit of the phosphatase is able to regulate these diverse processes by forming complexes with regulatory subunits, most of which possess the “RVxF” motif for binding to PP1. The binding site for the “RVxF” motifs is unique to PP1 and therefore, targeting this groove with compounds mimicking the

“RVxF” motif can provide the desired selectivity of only regulating PP1 among the PPP family members. To this end, the peptide called PDP1 (sequence:

RPKRKRKNARVTFAEAAEII), which contains the “RVxF” motif, antagonizes the binding of the PP1 endogenous inhibitor, inhibitor-2 to PP1 catalytic subunit (Chatterjee,

Beullens et al. 2012). This binding releases the inhibition of PP1 and thus has a pro- phosphatase activity in the cell.

Another example of PP1 being targeted for drug development is in the case of myosin light chain phosphatase (MLCP) which is a complex of PP1 catalytic subunit

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along with the myosin phosphatase targeting subunit 1 (MYPT1). This complex regulates muscle contraction and the interaction between actin and myosin, thereby aiding in cell motility (Xia, Stull et al. 2005). The inhibition of the phosphatase component MLCP increases the phosphorylation of the myosin light chain which has been shown to increase the motor activity of myosin and filament stability. One of the best MLCP inhibitors, compound 17e, has been shown to bind the catalytic subunit of PP1, increasing the phosphorylation of the myosin light chain and thereby disrupting the polymerization of actin and tubulin filaments. The administration of this compound has shown antiproliferative effects on prostate cancer cells by arresting the cells in G2/M phase and disrupting the formation of both microtubules and microfilaments (Grindrod, Suy et al.

2011). In fact, this compound has also been shown to initiate apoptotic cell death in

Burkitt’s Lymphoma cells when delivered at sub-micromolar concentrations (Grindrod,

Suy et al. 2011).

The loss of cell cycle control leads to genomic instability and has been recognized as a hallmark of cancer (Hanahan and Weinberg 2000). Most of the therapeutic strategies to target cell cycle in cancer have been against mitotic protein kinases like Aurora A,

Aurora B, PLK1, and CDK1. Due to lack of knowledge on the regulation of protein phosphatases during the cell cycle, they have been neglected for their potential as anti- mitotic drugs. A survey of the mutations in cBio Cancer Genomics Portal (Cerami, Gao et al. 2012, Gao, Aksoy et al. 2013), shows that one or multiple residues within the

“RVxF” motif in 35% of known PP1 regulatory proteins are mutated in cancer patients

(Table 5.1). This suggests the importance of this motif and PP1 binding in maintaining

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Table 5.1 List of known PP1 regulatory proteins with mutations in one or more residues within the PP1 binding “RVxF” motif.

Each of the known PP1 binding proteins from (Heroes, Lesage et al. 2013) was scanned for mutations in cBio Cancer Genomics Portal (Cerami, Gao et al. 2012, Gao, Aksoy et al. 2013) to reveal mutations within the “RVxF” motifs among all available cancer patient mutations.

PROTEIN RVXF SEQUENCE MUTATION TYPE CANCER

TYPE

AKAP149 KGVLF(153) F157S Missense

AKAP450 DKVSF(1052) S1055Y Missense Uterine

AURORA-A LKVLF(161), R343Q Missense Lung

SRVEF(346)

BCL-W RLVAF(95) R95H Missense Uterine

BRCA1 PKVTF(897) V899L Missense Stomach

CASC1 RSVRF(232) R226I Missense

F230L Missense

CASC5 RRVSF(57) R57C Missense

R58 Nonsense

CCDC128 KSVHF(677) K688T Missense

CENPE RRVTW(419) W423C Missense

FAM130A1 KNVRF(64) R62W Missense

FAM130A2 KNVHF(63) K63N Missense

FER KINASE LKIKF(605) F605L Missense Uterine

FLJ14744 KKVTF(639) K640N Missense

GL KRVSF(61) K61fs Deletion Stomach

R62W Missense Stomach

S64F/P Missense Stomach

GM RRVSF(63) S65L Missense Melanoma

GPR12 RTVTF(152) R152W Missense

V154A Missense

GLUTAREDOXIN RKVRF(14) R14Q Missense

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R17L Missense

IIIG9 RKVHF(129) R129W Missense

IRBIT KQIQF(40) K40 Splice

Q43 Nonsense

JARID1B RTVNW(1258) V1260M Missense

KCNA6 RRVRF(72) R72Q Missense Lung

adenocarcinoma

R75C Missense Head and neck

F76L Missense Uterine

LMTK1 KAVSF(1230) K1230 Deletion

A1231 Missense Melanoma

MAP1B RSVNF(1275) R1275H Missense

MCM7 RSVRF(673) R673 Nonsense

MKI67 RRVSF(504) R505H Missense

S507F Missense

MYPT 1 TKVKF(34) K37M Missense Bladder

MYPT 3 KQVLF(65) L68I Missense Stomach

N-COR RRVKF(413) R413K Missense Stomach

NIR SRVTF(199) S199fs Insertion Breast

NKCCL QRVSF(589) S592F Missense Melanoma

PMP22CD RHVTW(219) R219L/H/C Missense

V221I Missense

W223 Nonsense

R6 KRVVF(83) R84C Missense Stomach

RIMBP2 KSVHF(1046) K1046M Missense

H1049Y Missense

RPGRIP1L F1050L Missense

RRP1B KKVSF(973) S976F/L Missense

SACSIN VKVRF(786) R789W Missense Melanoma

SH2D4A RKVTF(951) T954A Missense

SOLUTE CARRIER 7-14 RKVGF(217), R309l/Q Missense Lung

LSVRF(306) adenocarcinoma

SPATA2 RRVQW(11) R11Q Missense

SPOCD1 KMVSF(160) K160N Missense

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SPRED1 KMVSF(1035) S1038F Missense

TMEM132D KKVKF(940) F944C Missense

TRPC4AP RKVTF(518) R518K Missense

TSKS RSVSW(1289) R1289S Missense

UBINUCLEIN 1 KAVSF(51) S54P Missense

ZAP3 KSVHF(1881) S1882F Missense

ZSWIM3 RVVHF(247) V249L Missense

genomic stability. The in-depth knowledge of the mechanism of PP1 binding during cell cycle can add significantly to the anti-mitotic therapeutic potential of PP1. The data presented in this thesis suggests that maintaining the phosphorylation of the “RV[S/T]F” containing proteins at the mitotic exit can prevent the binding of PP1 with these regulatory proteins. This disruption in PP1 binding at this stage might arrest the cells in mitosis and eventually lead to cell apoptosis presenting a potentially novel way to target

PP1 in the cell cycle.

PP1 has also been targeted in viral therapeutics. PP1 regulates the transcription of

HIV-1 by interacting with the viral Tat proteins through the “RVxF” motif. The PP1-

HIV-Tat complex could be disrupted by an 82-residue “RVxF” motif containing PP1 inhibitory protein (cdNIPP1). The binding of this protein was shown to inhibit viral transcription and replication (Ammosova, Yedavalli et al. 2011). This approach further led to the development of multiple compounds including 1E07 which could inhibit HIV transcription and replication with a low micromolar IC50. The use of these inhibitors was also extended to other viruses like Ebola virus. PP1 binds to the viral protein VP30 of the

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Ebola virus and the disruption of this complex by inhibitors was shown to affect viral replication (Ilinykh, Tigabu et al. 2014).

In conclusion, the understanding of the regulation of PP1 might provide additional routes for manipulating this protein as a therapeutic target. As described here, efforts have been made to target PP1 in different diseases including neurodegenerative diseases and cancer. Future research will need to address this possibility of taking protein phosphatases to the clinic, especially as anti-mitotic drugs and I believe that the work presented in this thesis is a step forward in that direction.

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139 APPENDIX A: MASS SPECTROMETRY DATA FROM P-RV[S/T]F IP

A.1. List of proteins specifically enriched in p-RV[S/T]F IP with their binding behavior upon Aurora B inhibition and SAINT scores Proteins which were specifically enriched in p-RV[S/T]F IP (SAINT score > 0.9) are listed with changes in binding to p-RV[S/T]F upon Aurora B inhibition. Fold change refers to the fold enrichment in p-RV[S/T]F IP versus the control. Proteins which were lost or reduced significantly (p < 0.05) upon Aurora B inhibition (Binding) are indicated.

GENE NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING UPON SAINT FOLD ACCESSION [VI]-[ST]- AURB INHIBITION SCORE CHANGE [FW] 1433E_HUMAN P62258 14-3-3 protein epsilon OS=Homo sapiens change not significant 0.92 2.36 GN=YWHAE PE=1 SV=1 1433S_HUMAN P31947 14-3-3 protein sigma OS=Homo sapiens GN=SFN change not significant 0.96 3.4 PE=1 SV=1 1433T_HUMAN P27348 14-3-3 protein theta OS=Homo sapiens change not significant 0.99 2.59 GN=YWHAQ PE=1 SV=1 ACACA_HUMAN Q13085 Acetyl-CoA carboxylase 1 OS=Homo sapiens lost 1 430 GN=ACACA PE=1 SV=2 ACACB_HUMAN O00763 dyglrRITFliaqe Acetyl-CoA carboxylase 2 OS=Homo sapiens lost 1 54 k GN=ACACB PE=1 SV=3 ACL6A_HUMAN O96019 Actin-like protein 6A OS=Homo sapiens change not significant 0.99 9 GN=ACTL6A PE=1 SV=1 ACLY_HUMAN P53396 ATP-citrate synthase OS=Homo sapiens GN=ACLY change not significant 1 624 PE=1 SV=3 ADDA_HUMAN P35611 Alpha-adducin OS=Homo sapiens GN=ADD1 PE=1 change not significant 1 148 SV=2 ADDG_HUMAN Q9UEY8 tsprtKITWmkae Gamma-adducin OS=Homo sapiens GN=ADD3 change not significant 1 68 ds PE=1 SV=1 AGAP3_HUMAN Q96P47 Arf-GAP with GTPase, ANK repeat and PH domain- lost 1 30 containing protein 3 OS=Homo sapiens GN=AGAP3 PE=1 SV=2 AGGF1_HUMAN Q8N302 Angiogenic factor with G patch and FHA domains 1 lost 0.99 14 OS=Homo sapiens GN=AGGF1 PE=1 SV=2 AHNK2_HUMAN Q8IVF2 KVPKVSFSST Protein AHNAK2 OS=Homo sapiens GN=AHNAK2 change not significant 1 154 KT PE=1 SV=2 AKP13_HUMAN Q12802 A-kinase anchor protein 13 OS=Homo sapiens change not significant 1 26 GN=AKAP13 PE=1 SV=2 ALKB5_HUMAN Q6P6C2 RNA demethylase ALKBH5 OS=Homo sapiens change not significant 1 36 GN=ALKBH5 PE=1 SV=2 AN13A_HUMAN Q8IZ07 hvlnaRITFgnvn Ankyrin repeat domain-containing protein 13A lost 1 24 gc OS=Homo sapiens GN=ANKRD13A PE=1 SV=3 ANC2_HUMAN Q9UJX6 Anaphase-promoting complex subunit 2 OS=Homo lost 1 14 sapiens GN=ANAPC2 PE=1 SV=1 ANK2_HUMAN Q01484 Ankyrin-2 OS=Homo sapiens GN=ANK2 PE=1 change not significant 0.97 18 SV=4

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] ANR28_HUMAN O15084 Serine/threonine-protein phosphatase 6 regulatory reduced 1 94 ankyrin repeat subunit A OS=Homo sapiens GN=ANKRD28 PE=1 SV=5 AP4B1_HUMAN Q9Y6B7 AP-4 complex subunit beta-1 OS=Homo sapiens lost 1 14 GN=AP4B1 PE=1 SV=2 AP4E1_HUMAN Q9UPM8 AP-4 complex subunit epsilon-1 OS=Homo sapiens lost 0.99 14 GN=AP4E1 PE=1 SV=2 APC_HUMAN P25054 Adenomatous polyposis coli protein OS=Homo change not significant 1 20 sapiens GN=APC PE=1 SV=2 APC1_HUMAN Q9H1A4 Anaphase-promoting complex subunit 1 OS=Homo change not significant 1 40 sapiens GN=ANAPC1 PE=1 SV=1 APC4_HUMAN Q9UJX5 Anaphase-promoting complex subunit 4 OS=Homo lost 1 30 sapiens GN=ANAPC4 PE=1 SV=2 APC5_HUMAN Q9UJX4 Anaphase-promoting complex subunit 5 OS=Homo change not significant 1 32 sapiens GN=ANAPC5 PE=1 SV=2 APC7_HUMAN Q9UJX3 Anaphase-promoting complex subunit 7 OS=Homo change not significant 1 94 sapiens GN=ANAPC7 PE=1 SV=4 ARGL1_HUMAN Q9NWB6 Arginine and glutamate-rich protein 1 OS=Homo lost 1 13 sapiens GN=ARGLU1 PE=1 SV=1 ARHG2_HUMAN Q92974 Rho guanine nucleotide exchange factor 2 OS=Homo lost 1 22 sapiens GN=ARHGEF2 PE=1 SV=4 ARHGH_HUMAN Q96PE2 afrvaKVSFpsyl Rho guanine nucleotide exchange factor 17 reduced 1 54 as OS=Homo sapiens GN=ARHGEF17 PE=1 SV=1 ASPM_HUMAN Q8IZT6 sanvsKVSFnek Abnormal spindle-like microcephaly-associated lost 1 46 av protein OS=Homo sapiens GN=ASPM PE=1 SV=2 ATAD2_HUMAN Q6PL18 ATPase family AAA domain-containing protein 2 change not significant 1 68 OS=Homo sapiens GN=ATAD2 PE=1 SV=1 ATD2B_HUMAN Q9ULI0 ATPase family AAA domain-containing protein 2B lost 1 54 OS=Homo sapiens GN=ATAD2B PE=1 SV=3 ATD3A_HUMAN Q9NVI7 ATPase family AAA domain-containing protein 3A reduced 0.99 5 OS=Homo sapiens GN=ATAD3A PE=1 SV=2 ATD3B_HUMAN Q5T9A4 ATPase family AAA domain-containing protein 3B reduced 0.92 5 OS=Homo sapiens GN=ATAD3B PE=1 SV=1 ATG9A_HUMAN Q7Z3C6 Autophagy-related protein 9A OS=Homo sapiens reduced 1 174 GN=ATG9A PE=1 SV=3 AURKA_HUMAN O14965 Aurora kinase A OS=Homo sapiens GN=AURKA change not significant 0.91 4.25 PE=1 SV=2 AUXI_HUMAN O75061 Putative tyrosine-protein phosphatase auxilin lost 0.97 14 OS=Homo sapiens GN=DNAJC6 PE=1 SV=3 AXIN1_HUMAN O15169 Axin-1 OS=Homo sapiens GN=AXIN1 PE=1 SV=2 lost 0.99 16

AZI2_HUMAN Q9H6S1 5-azacytidine-induced protein 2 OS=Homo sapiens lost 0.97 12 GN=AZI2 PE=1 SV=1 BACD1_HUMAN Q8WZ19 BTB/POZ domain-containing adapter for CUL3- change not significant 0.97 12 mediated RhoA degradation protein 1 OS=Homo sapiens GN=KCTD13 PE=1 SV=1 BACD2_HUMAN Q13829 BTB/POZ domain-containing adapter for CUL3- change not significant 0.99 18 mediated RhoA degradation protein 2 OS=Homo sapiens GN=TNFAIP1 PE=1 SV=1 BACD3_HUMAN Q9H3F6 BTB/POZ domain-containing adapter for CUL3- change not significant 1 96 mediated RhoA degradation protein 3 OS=Homo sapiens GN=KCTD10 PE=1 SV=1 BARD1_HUMAN Q99728 BRCA1-associated RING domain protein 1 lost 1 22

141

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] BAZ2A_HUMAN Q9UIF9 Bromodomain adjacent to zinc finger domain protein change not significant 1 112 2A OS=Homo sapiens GN=BAZ2A PE=1 SV=4 BEGIN_HUMAN Q9BUH8 Brain-enriched guanylate kinase-associated protein lost 1 38 OS=Homo sapiens GN=BEGAIN PE=1 SV=1 BEND3_HUMAN Q5T5X7 BEN domain-containing protein 3 OS=Homo sapiens reduced 1 298 GN=BEND3 PE=1 SV=1 BLM_HUMAN P54132 Bloom syndrome protein OS=Homo sapiens change not significant 1 112 GN=BLM PE=1 SV=1 BOREA_HUMAN Q53HL2 Borealin OS=Homo sapiens GN=CDCA8 PE=1 change not significant 0.98 7.5 SV=2 BORG4_HUMAN Q9H3Q1 Cdc42 effector protein 4 OS=Homo sapiens lost 1 20 GN=CDC42EP4 PE=1 SV=1 BORG5_HUMAN Q00587 Cdc42 effector protein 1 OS=Homo sapiens reduced 1 30 GN=CDC42EP1 PE=1 SV=1 BRCA1_HUMAN P38398 Breast cancer type 1 susceptibility protein OS=Homo lost 1 54 sapiens GN=BRCA1 PE=1 SV=2 BUB1_HUMAN O43683 Mitotic checkpoint serine/threonine-protein kinase lost 1 56 BUB1 OS=Homo sapiens GN=BUB1 PE=1 SV=1 BUB1B_HUMAN O60566 Mitotic checkpoint serine/threonine-protein kinase lost 1 70 BUB1 beta OS=Homo sapiens GN=BUB1B PE=1 SV=3 BUB3_HUMAN O43684 Mitotic checkpoint protein BUB3 OS=Homo sapiens lost 1 46 GN=BUB3 PE=1 SV=1 C170B_HUMAN Q9Y4F5 Centrosomal protein of 170 kDa protein B OS=Homo change not significant 1 70 sapiens GN=CEP170B PE=1 SV=4 C170L_HUMAN Q96L14 Cep170-like protein OS=Homo sapiens change not significant 1 72 GN=CEP170P1 PE=5 SV=2 C1QBP_HUMAN Q07021 Complement component 1 Q subcomponent-binding lost 0.96 7 protein, mitochondrial OS=Homo sapiens GN=C1QBP PE=1 SV=1 CA226_HUMAN A1L170 Uncharacterized protein C1orf226 OS=Homo sapiens change not significant 1 16 GN=C1orf226 PE=1 SV=1 CAMP1_HUMAN Q5T5Y3 Calmodulin-regulated spectrin-associated protein 1 lost 1 28 OS=Homo sapiens GN=CAMSAP1 PE=1 SV=2 CAMP2_HUMAN Q08AD1 Calmodulin-regulated spectrin-associated protein 2 change not significant 1 110 OS=Homo sapiens GN=CAMSAP2 PE=1 SV=3 CAPON_HUMAN O75052 Carboxyl-terminal PDZ ligand of neuronal nitric change not significant 1 44 oxide synthase protein OS=Homo sapiens GN=NOS1AP PE=1 SV=3 CASC5_HUMAN Q8NG31 Protein CASC5 OS=Homo sapiens GN=CASC5 lost 1 70 PE=1 SV=3 CBX3_HUMAN Q13185 Chromobox protein homolog 3 OS=Homo sapiens change not significant 1 20 GN=CBX3 PE=1 SV=4

CDC16_HUMAN Q13042 Cell division cycle protein 16 homolog OS=Homo change not significant 1 52 sapiens GN=CDC16 PE=1 SV=2 CDC20_HUMAN Q12834 Cell division cycle protein 20 homolog OS=Homo change not significant 1 32 sapiens GN=CDC20 PE=1 SV=2 CDC23_HUMAN Q9UJX2 Cell division cycle protein 23 homolog OS=Homo change not significant 1 46 sapiens GN=CDC23 PE=1 SV=3 CDC27_HUMAN P30260 Cell division cycle protein 27 homolog OS=Homo change not significant 1 31 sapiens GN=CDC27 PE=1 SV=2

142

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] CDCA2_HUMAN Q69YH5 mrkrkRVTFged Cell division cycle-associated protein 2 OS=Homo reduced 1 132 lsp sapiens GN=CDCA2 PE=1 SV=2 CDK16_HUMAN Q00536 Cyclin-dependent kinase 16 OS=Homo sapiens reduced 1 106 GN=CDK16 PE=1 SV=1 CDK17_HUMAN Q00537 Cyclin-dependent kinase 17 OS=Homo sapiens change not significant 1 38 GN=CDK17 PE=1 SV=2 CDK18_HUMAN Q07002 Cyclin-dependent kinase 18 OS=Homo sapiens change not significant 1 34 GN=CDK18 PE=1 SV=3 CE170_HUMAN Q5SW79 Centrosomal protein of 170 kDa OS=Homo sapiens change not significant 1 478 GN=CEP170 PE=1 SV=1 CENPC_HUMAN Q03188 Centromere protein C 1 OS=Homo sapiens change not significant 1 70 GN=CENPC1 PE=1 SV=2 CENPF_HUMAN P49454 Centromere protein F OS=Homo sapiens change not significant 1 26.85 GN=CENPF PE=1 SV=2 CEP55_HUMAN Q53EZ4 Centrosomal protein of 55 kDa OS=Homo sapiens change not significant 1 9 GN=CEP55 PE=1 SV=3 CEP72_HUMAN Q9P209 Centrosomal protein of 72 kDa OS=Homo sapiens change not significant 1 26 GN=CEP72 PE=1 SV=2 CHTOP_HUMAN Q9Y3Y2 Chromatin target of PRMT1 protein OS=Homo lost 1 11 sapiens GN=CHTOP PE=1 SV=2 CLCN7_HUMAN P51798 anvskKVSWsgr H(+)/Cl(-) exchange transporter 7 OS=Homo sapiens reduced 1 82 drd GN=CLCN7 PE=1 SV=2 CMC2_HUMAN Q9UJS0 Calcium-binding mitochondrial carrier protein lost 0.96 4.25 Aralar2 OS=Homo sapiens GN=SLC25A13 PE=1 SV=2 CNDG2_HUMAN Q86XI2 Condensin-2 complex subunit G2 OS=Homo sapiens lost 0.98 14 GN=NCAPG2 PE=1 SV=1 CRYAB_HUMAN P02511 Alpha-crystallin B chain OS=Homo sapiens change not significant 0.91 5 GN=CRYAB PE=1 SV=2 CTNB1_HUMAN P35222 Catenin beta-1 OS=Homo sapiens GN=CTNNB1 change not significant 0.92 6 PE=1 SV=1 DDB1_HUMAN Q16531 DNA damage-binding protein 1 OS=Homo sapiens reduced 1 11.67 GN=DDB1 PE=1 SV=1 DDX20_HUMAN Q9UHI6 Probable ATP-dependent RNA helicase DDX20 lost 1 44 OS=Homo sapiens GN=DDX20 PE=1 SV=2 DDX50_HUMAN Q9BQ39 ATP-dependent RNA helicase DDX50 OS=Homo lost 1 19 sapiens GN=DDX50 PE=1 SV=1 DEN4C_HUMAN Q5VZ89 DENN domain-containing protein 4C OS=Homo change not significant 0.99 20 sapiens GN=DENND4C PE=1 SV=2 DHX15_HUMAN O43143 Putative pre-mRNA-splicing factor ATP-dependent change not significant 0.99 12 RNA helicase DHX15 OS=Homo sapiens GN=DHX15 PE=1 SV=2 DLG5_HUMAN Q8TDM6 Disks large homolog 5 OS=Homo sapiens GN=DLG5 lost 0.98 18 PE=1 SV=4 DLGP4_HUMAN Q9Y2H0 Disks large-associated protein 4 OS=Homo sapiens lost 0.97 8 GN=DLGAP4 PE=1 SV=3 DLGP5_HUMAN Q15398 Disks large-associated protein 5 OS=Homo sapiens lost 1 186 GN=DLGAP5 PE=1 SV=2 DNJB6_HUMAN O75190 DnaJ homolog subfamily B member 6 OS=Homo lost 0.98 12 sapiens GN=DNAJB6 PE=1 SV=2 DOCK7_HUMAN Q96N67 Dedicator of cytokinesis protein 7 OS=Homo sapiens lost 1 48 GN=DOCK7 PE=1 SV=4

143

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] DVL2_HUMAN O14641 rhtvnKITFseqc Segment polarity protein dishevelled homolog DVL-2 change not significant 0.98 16 yy OS=Homo sapiens GN=DVL2 PE=1 SV=1 DX39A_HUMAN O00148 ATP-dependent RNA helicase DDX39A OS=Homo change not significant 0.93 5.5 sapiens GN=DDX39A PE=1 SV=2 DYHC1_HUMAN Q14204 pdlcsRVTFvnft Cytoplasmic dynein 1 heavy chain 1 OS=Homo lost 1 26 vt sapiens GN=DYNC1H1 PE=1 SV=5 DYL1_HUMAN P63167 Dynein light chain 1, cytoplasmic OS=Homo sapiens change not significant 1 13.5 GN=DYNLL1 PE=1 SV=1 DYL2_HUMAN Q96FJ2 Dynein light chain 2, cytoplasmic OS=Homo sapiens reduced 0.94 5 GN=DYNLL2 PE=1 SV=1 ECHM_HUMAN P30084 Enoyl-CoA hydratase, mitochondrial OS=Homo change not significant 1 42 sapiens GN=ECHS1 PE=1 SV=4 ECT2_HUMAN Q9H8V3 Protein ECT2 OS=Homo sapiens GN=ECT2 PE=1 lost 1 76 SV=4 EIF3A_HUMAN Q14152 Eukaryotic translation initiation factor 3 subunit A change not significant 0.97 10 OS=Homo sapiens GN=EIF3A PE=1 SV=1 EIF3B_HUMAN P55884 Eukaryotic translation initiation factor 3 subunit B change not significant 0.98 20 OS=Homo sapiens GN=EIF3B PE=1 SV=3 EIF3D_HUMAN O15371 Eukaryotic translation initiation factor 3 subunit D lost 0.94 5 OS=Homo sapiens GN=EIF3D PE=1 SV=1 EIF3G_HUMAN O75821 Eukaryotic translation initiation factor 3 subunit G change not significant 1 20 OS=Homo sapiens GN=EIF3G PE=1 SV=2 EIF3H_HUMAN O15372 Eukaryotic translation initiation factor 3 subunit H lost 0.97 10 OS=Homo sapiens GN=EIF3H PE=1 SV=1 EIF3I_HUMAN Q13347 Eukaryotic translation initiation factor 3 subunit I change not significant 1 30 OS=Homo sapiens GN=EIF3I PE=1 SV=1 ELYS_HUMAN Q8WYP5 rlketRISFveedv Protein ELYS OS=Homo sapiens GN=AHCTF1 reduced 1 562 h PE=1 SV=3 EMD_HUMAN P50402 Emerin OS=Homo sapiens GN=EMD PE=1 SV=1 change not significant 1 5.17

ERC6L_HUMAN Q2NKX8 DNA excision repair protein ERCC-6-like OS=Homo change not significant 1 134 sapiens GN=ERCC6L PE=1 SV=1 EVI5_HUMAN O60447 Ecotropic viral integration site 5 protein homolog lost 1 40 OS=Homo sapiens GN=EVI5 PE=1 SV=3 F120B_HUMAN Q96EK7 Constitutive coactivator of peroxisome proliferator- reduced 1 22 activated receptor gamma OS=Homo sapiens GN=FAM120B PE=1 SV=1 F208A_HUMAN Q9UK61 Protein FAM208A OS=Homo sapiens lost 1 142 GN=FAM208A PE=1 SV=3 F261_HUMAN P16118 6-phosphofructo-2-kinase/fructose-2,6- reduced 1 26 bisphosphatase 1 OS=Homo sapiens GN=PFKFB1 PE=1 SV=3 F262_HUMAN O60825 6-phosphofructo-2-kinase/fructose-2,6- reduced 1 104 bisphosphatase 2 OS=Homo sapiens GN=PFKFB2 PE=1 SV=2 F263_HUMAN Q16875 6-phosphofructo-2-kinase/fructose-2,6- change not significant 0.98 16 bisphosphatase 3 OS=Homo sapiens GN=PFKFB3 PE=1 SV=1 FA21A_HUMAN Q641Q2 WASH complex subunit FAM21A OS=Homo change not significant 1 224 sapiens GN=FAM21A PE=1 SV=3 FA21B_HUMAN Q5SNT6 WASH complex subunit FAM21B OS=Homo sapiens change not significant 1 214 GN=FAM21B PE=1 SV=2 FA21C_HUMAN Q9Y4E1 WASH complex subunit FAM21C OS=Homo sapiens change not significant 1 214

144

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] FA21D_HUMAN Q5SRD0 Putative WASH complex subunit FAM21 OS=Homo reduced 1 42 sapiens GN=FAM21D PE=3 SV=2 FA50A_HUMAN Q14320 Protein FAM50A OS=Homo sapiens GN=FAM50A change not significant 1 26 PE=1 SV=2 FA54B_HUMAN Q9H019 Protein FAM54B OS=Homo sapiens GN=FAM54B reduced 1 104 PE=1 SV=2 FA83B_HUMAN Q5T0W9 Protein FAM83B OS=Homo sapiens GN=FAM83B change not significant 1 8.67 PE=1 SV=1 FABP5_HUMAN Q01469 Fatty acid-binding protein, epidermal OS=Homo lost 0.91 4 sapiens GN=FABP5 PE=1 SV=3 GCN1L_HUMAN Q92616 Translational activator GCN1 OS=Homo sapiens lost 1 27 GN=GCN1L1 PE=1 SV=6 GEMI4_HUMAN P57678 Gem-associated protein 4 OS=Homo sapiens lost 1 32 GN=GEMIN4 PE=1 SV=2 GLCI1_HUMAN Q86VQ1 Glucocorticoid-induced transcript 1 protein lost 0.97 18 OS=Homo sapiens GN=GLCCI1 PE=1 SV=1 GOGA2_HUMAN Q08379 Golgin subfamily A member 2 OS=Homo sapiens change not significant 1 226 GN=GOLGA2 PE=1 SV=3 GOGA4_HUMAN Q13439 Golgin subfamily A member 4 OS=Homo sapiens change not significant 1 58 GN=GOLGA4 PE=1 SV=1 GORS1_HUMAN Q9BQQ3 Golgi reassembly-stacking protein 1 OS=Homo change not significant 1 34 sapiens GN=GORASP1 PE=1 SV=3 GRAM3_HUMAN Q96HH9 GRAM domain-containing protein 3 OS=Homo change not significant 1 70 sapiens GN=GRAMD3 PE=1 SV=1 GRDN_HUMAN Q3V6T2 Girdin OS=Homo sapiens GN=CCDC88A PE=1 lost 0.97 12 SV=2 GRM1A_HUMAN Q96CP6 GRAM domain-containing protein 1A OS=Homo lost 0.99 12 sapiens GN=GRAMD1A PE=1 SV=2 GRM1B_HUMAN Q3KR37 GRAM domain-containing protein 1B OS=Homo change not significant 1 162 sapiens GN=GRAMD1B PE=1 SV=1 GSK3B_HUMAN P49841 Glycogen synthase kinase-3 beta OS=Homo sapiens change not significant 0.98 18 GN=GSK3B PE=1 SV=2 GWL_HUMAN Q96GX5 Serine/threonine-protein kinase greatwall OS=Homo change not significant 1 144 sapiens GN=MASTL PE=1 SV=1 GYS1_HUMAN P13807 Glycogen [starch] synthase, muscle OS=Homo change not significant 1 8 sapiens GN=GYS1 PE=1 SV=2 HAP28_HUMAN Q13442 28 kDa heat- and acid-stable phosphoprotein change not significant 1 72 OS=Homo sapiens GN=PDAP1 PE=1 SV=1 HAUS1_HUMAN Q96CS2 HAUS augmin-like complex subunit 1 OS=Homo lost 1 36 sapiens GN=HAUS1 PE=1 SV=1 HAUS3_HUMAN Q68CZ6 HAUS augmin-like complex subunit 3 OS=Homo lost 1 52 sapiens GN=HAUS3 PE=1 SV=1 HAUS4_HUMAN Q9H6D7 HAUS augmin-like complex subunit 4 OS=Homo lost 1 50 sapiens GN=HAUS4 PE=1 SV=1 HAUS5_HUMAN O94927 HAUS augmin-like complex subunit 5 OS=Homo lost 1 50 sapiens GN=HAUS5 PE=1 SV=2 HAUS6_HUMAN Q7Z4H7 HAUS augmin-like complex subunit 6 OS=Homo lost 1 100 sapiens GN=HAUS6 PE=1 SV=2 HAUS7_HUMAN Q99871 HAUS augmin-like complex subunit 7 OS=Homo reduced 1 30 sapiens GN=HAUS7 PE=1 SV=3 HAUS8_HUMAN Q9BT25 HAUS augmin-like complex subunit 8 OS=Homo lost 1 40 sapiens GN=HAUS8 PE=1 SV=3

145

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] HECD3_HUMAN Q5T447 E3 ubiquitin-protein ligase HECTD3 OS=Homo change not significant 1 152 sapiens GN=HECTD3 PE=1 SV=1 HERC2_HUMAN O95714 E3 ubiquitin-protein ligase HERC2 OS=Homo lost 1 86 sapiens GN=HERC2 PE=1 SV=2 HGS_HUMAN O14964 Hepatocyte growth factor-regulated tyrosine kinase reduced 1 44 substrate OS=Homo sapiens GN=HGS PE=1 SV=1 HMBX1_HUMAN Q6NT76 Homeobox-containing protein 1 OS=Homo sapiens change not significant 1 26 GN=HMBOX1 PE=1 SV=1 HMHA1_HUMAN Q92619 Minor histocompatibility protein HA-1 OS=Homo reduced 1 278 sapiens GN=HMHA1 PE=1 SV=2 HNRPK_HUMAN P61978 Heterogeneous nuclear ribonucleoprotein K reduced 1 4.92 OS=Homo sapiens GN=HNRNPK PE=1 SV=1 IF4B_HUMAN P23588 Eukaryotic translation initiation factor 4B OS=Homo change not significant 1 1172 sapiens GN=EIF4B PE=1 SV=2 IMA2_HUMAN P52292 Importin subunit alpha-2 OS=Homo sapiens reduced 1 13 GN=KPNA2 PE=1 SV=1 IMB1_HUMAN Q14974 Importin subunit beta-1 OS=Homo sapiens reduced 1 64 GN=KPNB1 PE=1 SV=2 INCE_HUMAN Q9NQS7 Inner centromere protein OS=Homo sapiens change not significant 0.98 4.4 GN=INCENP PE=1 SV=3 IQEC2_HUMAN Q5JU85 IQ motif and SEC7 domain-containing protein 2 lost 1 30 OS=Homo sapiens GN=IQSEC2 PE=1 SV=1 K0195_HUMAN Q12767 Uncharacterized protein KIAA0195 OS=Homo change not significant 1 28 sapiens GN=KIAA0195 PE=1 SV=1 K0528_HUMAN Q86YS7 Uncharacterized protein KIAA0528 OS=Homo lost 1 20 sapiens GN=KIAA0528 PE=1 SV=1 K1109_HUMAN Q2LD37 aspgpRVTFniq Uncharacterized protein KIAA1109 OS=Homo change not significant 0.98 10 dtf sapiens GN=KIAA1109 PE=1 SV=2 KANK1_HUMAN Q14678 KN motif and ankyrin repeat domain-containing lost 1 72 protein 1 OS=Homo sapiens GN=KANK1 PE=1 SV=3 KANK2_HUMAN Q63ZY3 KN motif and ankyrin repeat domain-containing reduced 1 78 protein 2 OS=Homo sapiens GN=KANK2 PE=1 SV=1 KCC2A_HUMAN Q9UQM7 Calcium/calmodulin-dependent protein kinase type II change not significant 1 31 subunit alpha OS=Homo sapiens GN=CAMK2A PE=1 SV=2 KCC2B_HUMAN Q13554 Calcium/calmodulin-dependent protein kinase type II change not significant 1 8.57 subunit beta OS=Homo sapiens GN=CAMK2B PE=1 SV=3 KCC2D_HUMAN Q13557 Calcium/calmodulin-dependent protein kinase type II change not significant 1 25.33 subunit delta OS=Homo sapiens GN=CAMK2D PE=1 SV=3 KCC2G_HUMAN Q13555 Calcium/calmodulin-dependent protein kinase type II change not significant 1 8.36 subunit gamma OS=Homo sapiens GN=CAMK2G PE=1 SV=3 KCTD3_HUMAN Q9Y597 BTB/POZ domain-containing protein KCTD3 change not significant 1 356 OS=Homo sapiens GN=KCTD3 PE=1 SV=2 KDM1A_HUMAN O60341 Lysine-specific histone demethylase 1A OS=Homo change not significant 0.99 14 sapiens GN=KDM1A PE=1 SV=2 KI67_HUMAN P46013 Antigen KI-67 OS=Homo sapiens GN=MKI67 PE=1 reduced 1 300.33 SV=2

146

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] KIF23_HUMAN Q02241 Kinesin-like protein KIF23 OS=Homo sapiens reduced 1 538 GN=KIF23 PE=1 SV=3 KIF2A_HUMAN O00139 Kinesin-like protein KIF2A OS=Homo sapiens change not significant 1 46 GN=KIF2A PE=1 SV=3 KIF2C_HUMAN Q99661 Kinesin-like protein KIF2C OS=Homo sapiens lost 1 26 GN=KIF2C PE=1 SV=2 KIF4A_HUMAN O95239 Chromosome-associated kinesin KIF4A OS=Homo reduced 1 596 sapiens GN=KIF4A PE=1 SV=3 KIF4B_HUMAN Q2VIQ3 Chromosome-associated kinesin KIF4B OS=Homo reduced 1 210 sapiens GN=KIF4B PE=1 SV=2 KIF5A_HUMAN Q12840 hsqkqKISFlennl Kinesin heavy chain isoform 5A OS=Homo sapiens change not significant 1 54 e GN=KIF5A PE=1 SV=2 KIF5C_HUMAN O60282 aaqkqKISFlennl Kinesin heavy chain isoform 5C OS=Homo sapiens change not significant 1 50 e GN=KIF5C PE=1 SV=1 KINH_HUMAN P33176 Kinesin-1 heavy chain OS=Homo sapiens change not significant 1 190 GN=KIF5B PE=1 SV=1 KLC1_HUMAN Q07866 Kinesin light chain 1 OS=Homo sapiens GN=KLC1 change not significant 0.99 16 PE=1 SV=2 KLC2_HUMAN Q9H0B6 Kinesin light chain 2 OS=Homo sapiens GN=KLC2 change not significant 1 34 PE=1 SV=1 KLH22_HUMAN Q53GT1 Kelch-like protein 22 OS=Homo sapiens lost 0.97 10 GN=KLHL22 PE=1 SV=2 KPCD3_HUMAN O94806 Serine/threonine-protein kinase D3 OS=Homo change not significant 1 32 sapiens GN=PRKD3 PE=1 SV=1 KPRA_HUMAN Q14558 Phosphoribosyl pyrophosphate synthase-associated change not significant 1 14 protein 1 OS=Homo sapiens GN=PRPSAP1 PE=1 SV=2 KPRB_HUMAN O60256 Phosphoribosyl pyrophosphate synthase-associated change not significant 1 56 protein 2 OS=Homo sapiens GN=PRPSAP2 PE=1 SV=1 LAS1L_HUMAN Q9Y4W2 Ribosomal biogenesis protein LAS1L OS=Homo reduced 1 46 sapiens GN=LAS1L PE=1 SV=2 LDB1_HUMAN Q86U70 LIM domain-binding protein 1 OS=Homo sapiens reduced 1 46 GN=LDB1 PE=1 SV=2 LIPB1_HUMAN Q86W92 Liprin-beta-1 OS=Homo sapiens GN=PPFIBP1 PE=1 reduced 1 180 SV=2 LIPB2_HUMAN Q8ND30 Liprin-beta-2 OS=Homo sapiens GN=PPFIBP2 PE=1 change not significant 0.99 14 SV=3 LMNA_HUMAN P02545 Prelamin-A/C OS=Homo sapiens GN=LMNA PE=1 change not significant 1 3.11 SV=1 LMO7_HUMAN Q8WWI1 LIM domain only protein 7 OS=Homo sapiens reduced 0.97 2.68 GN=LMO7 PE=1 SV=3 LRCH3_HUMAN Q96II8 Leucine-rich repeat and calponin homology domain- lost 0.98 12 containing protein 3 OS=Homo sapiens GN=LRCH3 PE=1 SV=2 LS14A_HUMAN Q8ND56 Protein LSM14 homolog A OS=Homo sapiens change not significant 0.99 16 GN=LSM14A PE=1 SV=3 LZTR1_HUMAN Q8N653 Leucine-zipper-like transcriptional regulator 1 reduced 1 42 OS=Homo sapiens GN=LZTR1 PE=2 SV=2 M2OM_HUMAN Q02978 Mitochondrial 2-oxoglutarate/malate carrier protein lost 1 22 OS=Homo sapiens GN=SLC25A11 PE=1 SV=3 M3K8_HUMAN P41279 Mitogen-activated protein kinase kinase kinase 8 lost 0.99 18

147

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] MAGD2_HUMAN Q9UNF1 Melanoma-associated antigen D2 OS=Homo sapiens lost 1 16 GN=MAGED2 PE=1 SV=2 MAP4_HUMAN P27816 Microtubule-associated protein 4 OS=Homo sapiens lost 0.99 14 GN=MAP4 PE=1 SV=3 MATR3_HUMAN P43243 Matrin-3 OS=Homo sapiens GN=MATR3 PE=1 change not significant 1 10.37 SV=2 MBB1A_HUMAN Q9BQG0 Myb-binding protein 1A OS=Homo sapiens change not significant 0.95 3.67 GN=MYBBP1A PE=1 SV=2 MFF_HUMAN Q9GZY8 Mitochondrial fission factor OS=Homo sapiens reduced 1 22 GN=MFF PE=1 SV=1 MICA3_HUMAN Q7RTP6 Protein-methionine sulfoxide oxidase MICAL3 change not significant 1 196 OS=Homo sapiens GN=MICAL3 PE=1 SV=2 MIO_HUMAN Q9NXC5 WD repeat-containing protein mio OS=Homo sapiens lost 0.97 10 GN=MIOS PE=1 SV=2 MITF_HUMAN O75030 Microphthalmia-associated transcription factor change not significant 1 54 OS=Homo sapiens GN=MITF PE=1 SV=2 MPP8_HUMAN Q99549 M-phase phosphoprotein 8 OS=Homo sapiens lost 1 20 GN=MPHOSPH8 PE=1 SV=2 MPRI_HUMAN P11717 qrfstRITFecaqis Cation-independent mannose-6-phosphate receptor reduced 1 80 OS=Homo sapiens GN=IGF2R PE=1 SV=3 MRCKA_HUMAN Q5VT25 Serine/threonine-protein kinase MRCK alpha reduced 1 118 OS=Homo sapiens GN=CDC42BPA PE=1 SV=1 MRCKB_HUMAN Q9Y5S2 Serine/threonine-protein kinase MRCK beta lost 0.97 10 OS=Homo sapiens GN=CDC42BPB PE=1 SV=2 MTOR_HUMAN P42345 Serine/threonine-protein kinase mTOR OS=Homo change not significant 1 90 sapiens GN=MTOR PE=1 SV=1 MUS81_HUMAN Q96NY9 Crossover junction endonuclease MUS81 OS=Homo lost 0.98 10 sapiens GN=MUS81 PE=1 SV=3 MY18A_HUMAN Q92614 Unconventional myosin-XVIIIa OS=Homo sapiens change not significant 1 19.13 GN=MYO18A PE=1 SV=3 MYO10_HUMAN Q9HD67 Unconventional myosin-X OS=Homo sapiens lost 1 67 GN=MYO10 PE=1 SV=3 MYO9B_HUMAN Q13459 Unconventional myosin-IXb OS=Homo sapiens reduced 1 104 GN=MYO9B PE=1 SV=3 NBEL1_HUMAN Q6ZS30 Neurobeachin-like protein 1 OS=Homo sapiens reduced 1 118 GN=NBEAL1 PE=2 SV=3 NFKB1_HUMAN P19838 Nuclear factor NF-kappa-B p105 subunit OS=Homo reduced 1 182 sapiens GN=NFKB1 PE=1 SV=2 NOL9_HUMAN Q5SY16 Polynucleotide 5'-hydroxyl-kinase NOL9 OS=Homo lost 0.99 20 sapiens GN=NOL9 PE=1 SV=1 NP1L1_HUMAN P55209 Nucleosome assembly protein 1-like 1 OS=Homo reduced 0.99 4 sapiens GN=NAP1L1 PE=1 SV=1 NP1L4_HUMAN Q99733 Nucleosome assembly protein 1-like 4 OS=Homo reduced 1 40 sapiens GN=NAP1L4 PE=1 SV=1 NS1BP_HUMAN Q9Y6Y0 Influenza virus NS1A-binding protein OS=Homo change not significant 1 24 sapiens GN=IVNS1ABP PE=1 SV=3 NSF1C_HUMAN Q9UNZ2 NSFL1 cofactor p47 OS=Homo sapiens reduced 1 190 GN=NSFL1C PE=1 SV=2 NU155_HUMAN O75694 Nuclear pore complex protein Nup155 OS=Homo change not significant 1 96 sapiens GN=NUP155 PE=1 SV=1 NU214_HUMAN P35658 Nuclear pore complex protein Nup214 OS=Homo lost 1 26 sapiens GN=NUP214 PE=1 SV=2

148

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] NUMA1_HUMAN Q14980 Nuclear mitotic apparatus protein 1 OS=Homo reduced 1 7.54 sapiens GN=NUMA1 PE=1 SV=2 NUMB_HUMAN P49757 QTIEKVSFCA Protein numb homolog OS=Homo sapiens change not significant 0.99 14 GN=NUMB PE=1 SV=2 NUP88_HUMAN Q99567 Nuclear pore complex protein Nup88 OS=Homo lost 1 20 sapiens GN=NUP88 PE=1 SV=2 NUP98_HUMAN P52948 Nuclear pore complex protein Nup98-Nup96 reduced 1 120 OS=Homo sapiens GN=NUP98 PE=1 SV=4 ODB2_HUMAN P11182 Lipoamide acyltransferase component of branched- change not significant 1 14 chain alpha-keto acid dehydrogenase complex, mitochondrial OS=Homo sapiens GN=DBT PE=1 SV=3 ODO1_HUMAN Q02218 2-oxoglutarate dehydrogenase, mitochondrial change not significant 0.99 14 OS=Homo sapiens GN=OGDH PE=1 SV=3 ODO2_HUMAN P36957 Dihydrolipoyllysine-residue succinyltransferase change not significant 1 8.33 component of 2-oxoglutarate dehydrogenase complex, mitochondrial OS=Homo sapiens GN=DLST PE=1 SV=4 ODP2_HUMAN P10515 Dihydrolipoyllysine-residue acetyltransferase change not significant 1 58 component of pyruvate dehydrogenase complex, mitochondrial OS=Homo sapiens GN=DLAT PE=1 SV=3 ODPA_HUMAN P08559 Pyruvate dehydrogenase E1 component subunit change not significant 1 112 alpha, somatic form, mitochondrial OS=Homo sapiens GN=PDHA1 PE=1 SV=3 ODPAT_HUMAN P29803 Pyruvate dehydrogenase E1 component subunit change not significant 1 28 alpha, testis-specific form, mitochondrial OS=Homo sapiens GN=PDHA2 PE=1 SV=1 ODPB_HUMAN P11177 Pyruvate dehydrogenase E1 component subunit beta, change not significant 1 88 mitochondrial OS=Homo sapiens GN=PDHB PE=1 SV=3 ODPX_HUMAN O00330 Pyruvate dehydrogenase protein X component, change not significant 1 24 mitochondrial OS=Homo sapiens GN=PDHX PE=1 SV=3 OGT1_HUMAN O15294 UDP-N-acetylglucosamine--peptide N- change not significant 1 20 acetylglucosaminyltransferase 110 kDa subunit OS=Homo sapiens GN=OGT PE=1 SV=3 OPTN_HUMAN Q96CV9 Optineurin OS=Homo sapiens GN=OPTN PE=1 change not significant 0.97 10 SV=2 OSBL3_HUMAN Q9H4L5 Oxysterol-binding protein-related protein 3 reduced 1 332 OS=Homo sapiens GN=OSBPL3 PE=1 SV=1 OSBL6_HUMAN Q9BZF3 Oxysterol-binding protein-related protein 6 change not significant 1 24 OS=Homo sapiens GN=OSBPL6 PE=1 SV=1 OSTM1_HUMAN Q86WC4 Osteopetrosis-associated transmembrane protein 1 reduced 1 22 OS=Homo sapiens GN=OSTM1 PE=1 SV=1 P5CR3_HUMAN Q53H96 Pyrroline-5-carboxylate reductase 3 OS=Homo lost 0.99 16 sapiens GN=PYCRL PE=1 SV=2 PABP4_HUMAN Q13310 Polyadenylate-binding protein 4 OS=Homo sapiens lost 0.96 8 GN=PABPC4 PE=1 SV=1 PACN3_HUMAN Q9UKS6 Protein kinase C and casein kinase substrate in change not significant 1 77.2 neurons protein 3 OS=Homo sapiens GN=PACSIN3 PE=1 SV=2

149

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] PAIRB_HUMAN Q8NC51 Plasminogen activator inhibitor 1 RNA-binding reduced 1 9.33 protein OS=Homo sapiens GN=SERBP1 PE=1 SV=2 PAN3_HUMAN Q58A45 PAB-dependent poly(A)-specific ribonuclease lost 0.97 8 subunit 3 OS=Homo sapiens GN=PAN3 PE=1 SV=3 PARN_HUMAN O95453 Poly(A)-specific ribonuclease PARN OS=Homo change not significant 0.98 12 sapiens GN=PARN PE=1 SV=1 PCH2_HUMAN Q15645 Pachytene checkpoint protein 2 homolog OS=Homo lost 0.98 14 sapiens GN=TRIP13 PE=1 SV=2 PCY1A_HUMAN P49585 Choline-phosphate cytidylyltransferase A OS=Homo reduced 1 124 sapiens GN=PCYT1A PE=1 SV=2 PCY1B_HUMAN Q9Y5K3 Choline-phosphate cytidylyltransferase B OS=Homo change not significant 1 42 sapiens GN=PCYT1B PE=1 SV=1 PDE4A_HUMAN P27815 cAMP-specific 3',5'-cyclic phosphodiesterase 4A reduced 1 36 OS=Homo sapiens GN=PDE4A PE=1 SV=3 PDE4B_HUMAN Q07343 cAMP-specific 3',5'-cyclic phosphodiesterase 4B reduced 1 30 OS=Homo sapiens GN=PDE4B PE=1 SV=1 PDE4D_HUMAN Q08499 cAMP-specific 3',5'-cyclic phosphodiesterase 4D reduced 1 180 OS=Homo sapiens GN=PDE4D PE=1 SV=2 PELP1_HUMAN Q8IZL8 Proline-, glutamic acid- and leucine-rich protein 1 reduced 1 70 OS=Homo sapiens GN=PELP1 PE=1 SV=2 PKCB1_HUMAN Q9ULU4 Protein kinase C-binding protein 1 OS=Homo sapiens change not significant 1 104 GN=ZMYND8 PE=1 SV=2 PKHA6_HUMAN Q9Y2H5 Pleckstrin homology domain-containing family A lost 1 16 member 6 OS=Homo sapiens GN=PLEKHA6 PE=1 SV=4 PKP3_HUMAN Q9Y446 Plakophilin-3 OS=Homo sapiens GN=PKP3 PE=1 change not significant 0.99 6.5 SV=1 PLK1_HUMAN P53350 Serine/threonine-protein kinase PLK1 OS=Homo reduced 1 74 sapiens GN=PLK1 PE=1 SV=1 PLPL6_HUMAN Q8IY17 mvrlqRVTFlalh Neuropathy target esterase OS=Homo sapiens lost 1 60 ny GN=PNPLA6 PE=1 SV=2 PLRKT_HUMAN Q9HBL7 Plasminogen receptor (KT) OS=Homo sapiens change not significant 1 120 GN=PLGRKT PE=1 SV=1 PMF1_HUMAN Q6P1K2 Polyamine-modulated factor 1 OS=Homo sapiens lost 0.98 10 GN=PMF1 PE=1 SV=2 PP6R1_HUMAN Q9UPN7 Serine/threonine-protein phosphatase 6 regulatory lost 1 22 subunit 1 OS=Homo sapiens GN=PPP6R1 PE=1 SV=5 PP6R2_HUMAN O75170 Serine/threonine-protein phosphatase 6 regulatory change not significant 1 14 subunit 2 OS=Homo sapiens GN=PPP6R2 PE=1 SV=2 PP6R3_HUMAN Q5H9R7 Serine/threonine-protein phosphatase 6 regulatory change not significant 1 53 subunit 3 OS=Homo sapiens GN=PPP6R3 PE=1 SV=2 PPHLN_HUMAN Q8NEY8 Periphilin-1 OS=Homo sapiens GN=PPHLN1 PE=1 reduced 1 29 SV=2 PPP6_HUMAN O00743 Serine/threonine-protein phosphatase 6 catalytic reduced 1 50 subunit OS=Homo sapiens GN=PPP6C PE=1 SV=1 PRC2B_HUMAN Q5JSZ5 Protein PRRC2B OS=Homo sapiens GN=PRRC2B change not significant 1 38 PE=1 SV=2 PRDX4_HUMAN Q13162 Peroxiredoxin-4 OS=Homo sapiens GN=PRDX4 change not significant 0.96 3.57 PE=1 SV=1

150

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] PRKDC_HUMAN P78527 DNA-dependent protein kinase catalytic subunit change not significant 1 32 OS=Homo sapiens GN=PRKDC PE=1 SV=3 PRPS1_HUMAN P60891 Ribose-phosphate pyrophosphokinase 1 OS=Homo change not significant 1 70 sapiens GN=PRPS1 PE=1 SV=2 PRPS2_HUMAN P11908 Ribose-phosphate pyrophosphokinase 2 OS=Homo change not significant 1 44 sapiens GN=PRPS2 PE=1 SV=2 PRPS3_HUMAN P21108 Ribose-phosphate pyrophosphokinase 3 OS=Homo change not significant 1 40 sapiens GN=PRPS1L1 PE=1 SV=2 PRR5L_HUMAN Q6MZQ0 Proline-rich protein 5-like OS=Homo sapiens lost 0.98 10 GN=PRR5L PE=1 SV=2 PTN12_HUMAN Q05209 Tyrosine-protein phosphatase non-receptor type 12 lost 1 28 OS=Homo sapiens GN=PTPN12 PE=1 SV=3 PTPRK_HUMAN Q15262 Receptor-type tyrosine-protein phosphatase kappa change not significant 1 132 OS=Homo sapiens GN=PTPRK PE=1 SV=2 RABL6_HUMAN Q3YEC7 Rab-like protein 6 OS=Homo sapiens GN=RABL6 change not significant 1 132 PE=1 SV=2 RAE1L_HUMAN P78406 mRNA export factor OS=Homo sapiens GN=RAE1 change not significant 1 78 PE=1 SV=1 RAGP1_HUMAN P46060 Ran GTPase-activating protein 1 OS=Homo sapiens change not significant 1 106 GN=RANGAP1 PE=1 SV=1 RBP1_HUMAN Q15311 RalA-binding protein 1 OS=Homo sapiens lost 1 34 GN=RALBP1 PE=1 SV=3 RBP2_HUMAN P49792 E3 SUMO-protein ligase RanBP2 OS=Homo sapiens reduced 1 214 GN=RANBP2 PE=1 SV=2 REPS1_HUMAN Q96D71 RalBP1-associated Eps domain-containing protein 1 change not significant 1 82 OS=Homo sapiens GN=REPS1 PE=1 SV=3 REPS2_HUMAN Q8NFH8 RalBP1-associated Eps domain-containing protein 2 reduced 1 22 OS=Homo sapiens GN=REPS2 PE=1 SV=2 RFIP1_HUMAN Q6WKZ4 kktkkRVSFseql Rab11 family-interacting protein 1 OS=Homo sapiens reduced 1 78 ft GN=RAB11FIP1 PE=1 SV=2 RFIP2_HUMAN Q7L804 Rab11 family-interacting protein 2 OS=Homo sapiens change not significant 1 38 GN=RAB11FIP2 PE=1 SV=1 RGAP1_HUMAN Q9H0H5 Rac GTPase-activating protein 1 OS=Homo sapiens reduced 1 332 GN=RACGAP1 PE=1 SV=1 RGPD1_HUMAN P0DJD0 RANBP2-like and GRIP domain-containing protein 1 reduced 1 32 OS=Homo sapiens GN=RGPD1 PE=2 SV=1 RGPD2_HUMAN P0DJD1 RANBP2-like and GRIP domain-containing protein 2 reduced 1 32 OS=Homo sapiens GN=RGPD2 PE=2 SV=1 RGPD3_HUMAN A6NKT7 RanBP2-like and GRIP domain-containing protein 3 change not significant 1 46 OS=Homo sapiens GN=RGPD3 PE=2 SV=2 RGPD4_HUMAN Q7Z3J3 RanBP2-like and GRIP domain-containing protein 4 change not significant 1 44 OS=Homo sapiens GN=RGPD4 PE=2 SV=3 RGPD5_HUMAN Q99666 RANBP2-like and GRIP domain-containing protein change not significant 1 48 5/6 OS=Homo sapiens GN=RGPD5 PE=1 SV=3 RGPD8_HUMAN O14715 RANBP2-like and GRIP domain-containing protein 8 change not significant 1 48 OS=Homo sapiens GN=RGPD8 PE=1 SV=2 RHG29_HUMAN Q52LW3 Rho GTPase-activating protein 29 OS=Homo sapiens change not significant 1 100 GN=ARHGAP29 PE=1 SV=2 RHOA_HUMAN P61586 Transforming protein RhoA OS=Homo sapiens lost 0.98 14 GN=RHOA PE=1 SV=1 RICTR_HUMAN Q6R327 Rapamycin-insensitive companion of mTOR reduced 1 406 OS=Homo sapiens GN=RICTOR PE=1 SV=1

151

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] RIF1_HUMAN Q5UIP0 Telomere-associated protein RIF1 OS=Homo sapiens reduced 1 820 GN=RIF1 PE=1 SV=2 RL37_HUMAN P61927 60S ribosomal protein L37 OS=Homo sapiens change not significant 0.91 7 GN=RPL37 PE=1 SV=2 RL38_HUMAN P63173 60S ribosomal protein L38 OS=Homo sapiens change not significant 0.96 10 GN=RPL38 PE=1 SV=2 RRMJ3_HUMAN Q8IY81 pre-rRNA processing protein FTSJ3 OS=Homo change not significant 0.9 5 sapiens GN=FTSJ3 PE=1 SV=2 RRP1B_HUMAN Q14684 pssskKVTFglnr Ribosomal RNA processing protein 1 homolog B lost 1 20 nm OS=Homo sapiens GN=RRP1B PE=1 SV=3 RUVB1_HUMAN Q9Y265 RuvB-like 1 OS=Homo sapiens GN=RUVBL1 PE=1 change not significant 0.97 3.9 SV=1 RUVB2_HUMAN Q9Y230 RuvB-like 2 OS=Homo sapiens GN=RUVBL2 PE=1 change not significant 1 9.2 SV=3 S39A7_HUMAN Q92504 Zinc transporter SLC39A7 OS=Homo sapiens change not significant 0.97 10 GN=SLC39A7 PE=1 SV=2 S4A10_HUMAN Q6U841 Sodium-driven chloride bicarbonate exchanger change not significant 1 24 OS=Homo sapiens GN=SLC4A10 PE=1 SV=1 S4A7_HUMAN Q9Y6M7 kpvsvKISFedep Sodium bicarbonate cotransporter 3 OS=Homo reduced 1 292 rk sapiens GN=SLC4A7 PE=1 SV=2 S4A8_HUMAN Q2Y0W8 Electroneutral sodium bicarbonate exchanger 1 reduced 1 28 OS=Homo sapiens GN=SLC4A8 PE=1 SV=1 SAFB1_HUMAN Q15424 Scaffold attachment factor B1 OS=Homo sapiens change not significant 1 26.75 GN=SAFB PE=1 SV=4 SAFB2_HUMAN Q14151 Scaffold attachment factor B2 OS=Homo sapiens change not significant 1 17.75 GN=SAFB2 PE=1 SV=1 SAHH2_HUMAN O43865 Putative adenosylhomocysteinase 2 OS=Homo change not significant 1 4.83 sapiens GN=AHCYL1 PE=1 SV=2 SAHH3_HUMAN Q96HN2 Putative adenosylhomocysteinase 3 OS=Homo change not significant 1 4.2 sapiens GN=AHCYL2 PE=1 SV=1 SC16A_HUMAN O15027 Protein transport protein Sec16A OS=Homo sapiens reduced 1 22.67 GN=SEC16A PE=1 SV=3 SCAPE_HUMAN Q9BY12 S phase cyclin A-associated protein in the change not significant 1 120 endoplasmic reticulum OS=Homo sapiens GN=SCAPER PE=1 SV=1 SCRIB_HUMAN Q14160 Protein scribble homolog OS=Homo sapiens lost 0.99 18 GN=SCRIB PE=1 SV=4 SEC13_HUMAN P55735 Protein SEC13 homolog OS=Homo sapiens change not significant 0.91 6 GN=SEC13 PE=1 SV=3 SEH1_HUMAN Q96EE3 nsqvwRVSWnit Nucleoporin SEH1 OS=Homo sapiens GN=SEH1L lost 0.98 16 gtv, PE=1 SV=3 sgsvwRVTWah pefg SENP3_HUMAN Q9H4L4 Sentrin-specific protease 3 OS=Homo sapiens lost 1 16 GN=SENP3 PE=1 SV=2 SEP10_HUMAN Q9P0V9 Septin-10 OS=Homo sapiens GN=SEPT10 PE=1 reduced 0.97 14 SV=2 SEP11_HUMAN Q9NVA2 Septin-11 OS=Homo sapiens GN=SEPT11 PE=1 reduced 1 94 SV=3 SEPT2_HUMAN Q15019 Septin-2 OS=Homo sapiens GN=SEPT2 PE=1 SV=1 reduced 1 104

SEPT6_HUMAN Q14141 Septin-6 OS=Homo sapiens GN=SEPT6 PE=1 SV=4 reduced 1 40

152

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] SEPT8_HUMAN Q92599 Septin-8 OS=Homo sapiens GN=SEPT8 PE=1 SV=4 reduced 1 28

SEPT9_HUMAN Q9UHD8 Septin-9 OS=Homo sapiens GN=SEPT9 PE=1 SV=2 reduced 1 104

SET1A_HUMAN O15047 Histone-lysine N-methyltransferase SETD1A lost 0.98 16 OS=Homo sapiens GN=SETD1A PE=1 SV=3 SG223_HUMAN Q86YV5 Tyrosine-protein kinase SgK223 OS=Homo sapiens reduced 1 22 GN=SGK223 PE=1 SV=3 SGOL2_HUMAN Q562F6 Shugoshin-like 2 OS=Homo sapiens GN=SGOL2 lost 1 100 PE=1 SV=2 SHCBP_HUMAN Q8NEM2 SHC SH2 domain-binding protein 1 OS=Homo change not significant 0.99 14 sapiens GN=SHCBP1 PE=1 SV=3 SHKB1_HUMAN Q8TBC3 SH3KBP1-binding protein 1 OS=Homo sapiens change not significant 1 128 GN=SHKBP1 PE=1 SV=2 SI1L2_HUMAN Q9P2F8 Signal-induced proliferation-associated 1-like protein change not significant 0.91 5 2 OS=Homo sapiens GN=SIPA1L2 PE=1 SV=2 SI1L3_HUMAN O60292 Signal-induced proliferation-associated 1-like protein change not significant 0.91 4 3 OS=Homo sapiens GN=SIPA1L3 PE=1 SV=3 SIN1_HUMAN Q9BPZ7 Target of rapamycin complex 2 subunit MAPKAP1 reduced 1 116 OS=Homo sapiens GN=MAPKAP1 PE=1 SV=2 SK2L2_HUMAN P42285 Superkiller viralicidic activity 2-like 2 OS=Homo change not significant 1 144 sapiens GN=SKIV2L2 PE=1 SV=3 SLX4_HUMAN Q8IY92 Structure-specific endonuclease subunit SLX4 lost 1 20 OS=Homo sapiens GN=SLX4 PE=1 SV=3 SMC2_HUMAN O95347 Structural maintenance of chromosomes protein 2 lost 1 38 OS=Homo sapiens GN=SMC2 PE=1 SV=2 SMC4_HUMAN Q9NTJ3 Structural maintenance of chromosomes protein 4 lost 1 54 OS=Homo sapiens GN=SMC4 PE=1 SV=2 SMCA1_HUMAN P28370 Probable global transcription activator SNF2L1 reduced 1 68 OS=Homo sapiens GN=SMARCA1 PE=1 SV=2 SMCA5_HUMAN O60264 SWI/SNF-related matrix-associated actin-dependent reduced 1 170 regulator of chromatin subfamily A member 5 OS=Homo sapiens GN=SMARCA5 PE=1 SV=1 SMD2_HUMAN P62316 Small nuclear ribonucleoprotein Sm D2 OS=Homo lost 0.91 5 sapiens GN=SNRPD2 PE=1 SV=1 SMN_HUMAN Q16637 Survival motor neuron protein OS=Homo sapiens lost 0.99 22 GN=SMN1 PE=1 SV=1 SNX16_HUMAN P57768 Sorting nexin-16 OS=Homo sapiens GN=SNX16 lost 1 24 PE=1 SV=2 SON_HUMAN P18583 Protein SON OS=Homo sapiens GN=SON PE=1 change not significant 1 19 SV=4 SPAT5_HUMAN Q8NB90 Spermatogenesis-associated protein 5 OS=Homo lost 1 26 sapiens GN=SPATA5 PE=1 SV=3 SPE39_HUMAN Q9H9C1 dddleRVSWsge Spermatogenesis-defective protein 39 homolog lost 1 116 pvg OS=Homo sapiens GN=VIPAS39 PE=1 SV=1 SPT6H_HUMAN Q7KZ85 Transcription elongation factor SPT6 OS=Homo lost 1 70 sapiens GN=SUPT6H PE=1 SV=2 SRRM1_HUMAN Q8IYB3 Serine/arginine repetitive matrix protein 1 OS=Homo lost 0.97 8 sapiens GN=SRRM1 PE=1 SV=2 SRRM2_HUMAN Q9UQ35 Serine/arginine repetitive matrix protein 2 OS=Homo change not significant 1 76 sapiens GN=SRRM2 PE=1 SV=2 SRS10_HUMAN O75494 Serine/arginine-rich splicing factor 10 OS=Homo change not significant 0.95 6 sapiens GN=SRSF10 PE=1 SV=1

153

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] SSBP3_HUMAN Q9BWW4 Single-stranded DNA-binding protein 3 OS=Homo change not significant 1 30 sapiens GN=SSBP3 PE=1 SV=1 SSF1_HUMAN Q9NQ55 Suppressor of SWI4 1 homolog OS=Homo sapiens change not significant 0.97 10 GN=PPAN PE=1 SV=1 SSH3_HUMAN Q8TE77 Protein phosphatase Slingshot homolog 3 OS=Homo lost 0.98 20 sapiens GN=SSH3 PE=1 SV=2 SSRA_HUMAN P43307 Translocon-associated protein subunit alpha lost 0.91 5 OS=Homo sapiens GN=SSR1 PE=1 SV=3 STAM1_HUMAN Q92783 Signal transducing adapter molecule 1 OS=Homo change not significant 1 28 sapiens GN=STAM PE=1 SV=3 STK3_HUMAN Q13188 Serine/threonine-protein kinase 3 OS=Homo sapiens lost 0.98 18 GN=STK3 PE=1 SV=2 STRUM_HUMAN Q12768 WASH complex subunit strumpellin OS=Homo change not significant 1 208 sapiens GN=KIAA0196 PE=1 SV=1 SVIL_HUMAN O95425 Supervillin OS=Homo sapiens GN=SVIL PE=1 reduced 0.99 5.5 SV=2 SYNRG_HUMAN Q9UMZ2 Synergin gamma OS=Homo sapiens GN=SYNRG lost 0.98 16 PE=1 SV=2 TARA_HUMAN Q9H2D6 TRIO and F-actin-binding protein OS=Homo sapiens reduced 1 13 GN=TRIOBP PE=1 SV=3 TAXB1_HUMAN Q86VP1 Tax1-binding protein 1 OS=Homo sapiens reduced 1 26 GN=TAX1BP1 PE=1 SV=2 TBCD4_HUMAN O60343 svtprRISWrqrifl TBC1 domain family member 4 OS=Homo sapiens lost 1 126 GN=TBC1D4 PE=1 SV=2 TBL1R_HUMAN Q9BZK7 F-box-like/WD repeat-containing protein TBL1XR1 lost 1 32 OS=Homo sapiens GN=TBL1XR1 PE=1 SV=1 TBL1X_HUMAN O60907 F-box-like/WD repeat-containing protein TBL1X lost 0.98 10 OS=Homo sapiens GN=TBL1X PE=1 SV=3 TBL1Y_HUMAN Q9BQ87 F-box-like/WD repeat-containing protein TBL1Y lost 0.98 10 OS=Homo sapiens GN=TBL1Y PE=2 SV=1 TCOF_HUMAN Q13428 Treacle protein OS=Homo sapiens GN=TCOF1 PE=1 lost 1 12 SV=3 TERA_HUMAN P55072 Transitional endoplasmic reticulum ATPase reduced 1 67 OS=Homo sapiens GN=VCP PE=1 SV=4 TEX10_HUMAN Q9NXF1 Testis-expressed sequence 10 protein OS=Homo lost 1 26 sapiens GN=TEX10 PE=1 SV=2 TF3C1_HUMAN Q12789 rmtrlRVTWsm General transcription factor 3C polypeptide 1 change not significant 1 874 qedg OS=Homo sapiens GN=GTF3C1 PE=1 SV=4 TF3C2_HUMAN Q8WUA4 General transcription factor 3C polypeptide 2 reduced 1 248 OS=Homo sapiens GN=GTF3C2 PE=1 SV=2 TF3C3_HUMAN Q9Y5Q9 dylegKISFeefer General transcription factor 3C polypeptide 3 reduced 1 304 r OS=Homo sapiens GN=GTF3C3 PE=1 SV=1 TF3C4_HUMAN Q9UKN8 General transcription factor 3C polypeptide 4 reduced 1 314 OS=Homo sapiens GN=GTF3C4 PE=1 SV=2 TF3C5_HUMAN Q9Y5Q8 General transcription factor 3C polypeptide 5 reduced 1 360 OS=Homo sapiens GN=GTF3C5 PE=1 SV=2 TF3C6_HUMAN Q969F1 General transcription factor 3C polypeptide 6 reduced 0.98 16 OS=Homo sapiens GN=GTF3C6 PE=1 SV=1 TFE3_HUMAN P19532 Transcription factor E3 OS=Homo sapiens GN=TFE3 change not significant 1 66 PE=1 SV=4 TFEB_HUMAN P19484 Transcription factor EB OS=Homo sapiens change not significant 1 82 GN=TFEB PE=1 SV=3

154

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] TFEC_HUMAN O14948 Transcription factor EC OS=Homo sapiens change not significant 1 20 GN=TFEC PE=1 SV=1 TIM13_HUMAN Q9Y5L4 Mitochondrial import inner membrane translocase change not significant 1 36 subunit Tim13 OS=Homo sapiens GN=TIMM13 PE=1 SV=1 TIM8A_HUMAN O60220 Mitochondrial import inner membrane translocase change not significant 0.98 16 subunit Tim8 A OS=Homo sapiens GN=TIMM8A PE=1 SV=1 TMF1_HUMAN P82094 TATA element modulatory factor OS=Homo sapiens change not significant 1 282 GN=TMF1 PE=1 SV=2 TNIP2_HUMAN Q8NFZ5 TNFAIP3-interacting protein 2 OS=Homo sapiens lost 1 66 GN=TNIP2 PE=1 SV=1 TOP2A_HUMAN P11388 DNA topoisomerase 2-alpha OS=Homo sapiens reduced 1 72 GN=TOP2A PE=1 SV=3 TOP2B_HUMAN Q02880 DNA topoisomerase 2-beta OS=Homo sapiens change not significant 1 336 GN=TOP2B PE=1 SV=3 TOP3A_HUMAN Q13472 DNA topoisomerase 3-alpha OS=Homo sapiens change not significant 0.98 10 GN=TOP3A PE=1 SV=1 TP53B_HUMAN Q12888 Tumor suppressor p53-binding protein 1 OS=Homo change not significant 1 84 sapiens GN=TP53BP1 PE=1 SV=2 TPD54_HUMAN O43399 Tumor protein D54 OS=Homo sapiens reduced 0.98 22 GN=TPD52L2 PE=1 SV=2 TR150_HUMAN Q9Y2W1 Thyroid hormone receptor-associated protein 3 change not significant 0.99 6.25 OS=Homo sapiens GN=THRAP3 PE=1 SV=2 TRAF6_HUMAN Q9Y4K3 TNF receptor-associated factor 6 OS=Homo sapiens change not significant 1 56 GN=TRAF6 PE=1 SV=1 TRAIP_HUMAN Q9BWF2 TRAF-interacting protein OS=Homo sapiens lost 0.98 10 GN=TRAIP PE=1 SV=1 TXTP_HUMAN P53007 Tricarboxylate transport protein, mitochondrial reduced 0.92 3.14 OS=Homo sapiens GN=SLC25A1 PE=1 SV=2 UBR5_HUMAN O95071 avhrvKVTFkde E3 ubiquitin-protein ligase UBR5 OS=Homo sapiens reduced 1 112 pg GN=UBR5 PE=1 SV=2 VAPA_HUMAN Q9P0L0 Vesicle-associated membrane protein-associated reduced 1 124 protein A OS=Homo sapiens GN=VAPA PE=1 SV=3 VAPB_HUMAN O95292 Vesicle-associated membrane protein-associated change not significant 1 52 protein B/C OS=Homo sapiens GN=VAPB PE=1 SV=3 VASP_HUMAN P50552 Vasodilator-stimulated phosphoprotein OS=Homo lost 1 20 sapiens GN=VASP PE=1 SV=3 VEZA_HUMAN Q9HBM0 Vezatin OS=Homo sapiens GN=VEZT PE=1 SV=3 lost 0.99 16

VP33B_HUMAN Q9H267 Vacuolar protein sorting-associated protein 33B reduced 1 152 OS=Homo sapiens GN=VPS33B PE=1 SV=2 VPRBP_HUMAN Q9Y4B6 spligRISFirerps Protein VPRBP OS=Homo sapiens GN=VPRBP lost 1 64 PE=1 SV=3 WASH1_HUMAN A8K0Z3 WAS protein family homolog 1 OS=Homo sapiens change not significant 1 70 GN=WASH1 PE=1 SV=2 WASH6_HUMAN Q9NQA3 WAS protein family homolog 6 OS=Homo sapiens change not significant 1 56 GN=WASH6P PE=1 SV=3 WASH7_HUMAN Q2M389 WASH complex subunit 7 OS=Homo sapiens change not significant 1 170 GN=KIAA1033 PE=1 SV=2 WDHD1_HUMAN O75717 WD repeat and HMG-box DNA-binding protein 1 reduced 1 474 OS=Homo sapiens GN=WDHD1 PE=1 SV=1

155

NAME UNIPROT [RK]-X(0,1)- PROTEIN DESCRIPTION BINDING SAINT FOLD ACCESSION [VI]-[ST]- SCORE CHANGE [FW] WDR47_HUMAN O94967 WD repeat-containing protein 47 OS=Homo sapiens reduced 1 48 GN=WDR47 PE=1 SV=1 WDR62_HUMAN O43379 WD repeat-containing protein 62 OS=Homo sapiens change not significant 1 190 GN=WDR62 PE=1 SV=4 WDR81_HUMAN Q562E7 WD repeat-containing protein 81 OS=Homo sapiens lost 1 68 GN=WDR81 PE=1 SV=2 WDR91_HUMAN A4D1P6 WD repeat-containing protein 91 OS=Homo sapiens reduced 1 56 GN=WDR91 PE=1 SV=2 WRIP1_HUMAN Q96S55 ATPase WRNIP1 OS=Homo sapiens GN=WRNIP1 change not significant 1 54 PE=1 SV=2 WRN_HUMAN Q14191 Werner syndrome ATP-dependent helicase lost 1 48 OS=Homo sapiens GN=WRN PE=1 SV=2 XPF_HUMAN Q92889 DNA repair endonuclease XPF OS=Homo sapiens lost 1 44 GN=ERCC4 PE=1 SV=3 XPO1_HUMAN O14980 Exportin-1 OS=Homo sapiens GN=XPO1 PE=1 lost 1 14 SV=1 YTDC1_HUMAN Q96MU7 YTH domain-containing protein 1 OS=Homo sapiens lost 0.98 14 GN=YTHDC1 PE=1 SV=3 YTDC2_HUMAN Q9H6S0 Probable ATP-dependent RNA helicase YTHDC2 lost 1 24 OS=Homo sapiens GN=YTHDC2 PE=1 SV=2 ZBED1_HUMAN O96006 mlvsnRVSWwg Zinc finger BED domain-containing protein 1 change not significant 1 52 stla OS=Homo sapiens GN=ZBED1 PE=1 SV=1 ZBTB5_HUMAN O15062 Zinc finger and BTB domain-containing protein 5 change not significant 1 52 OS=Homo sapiens GN=ZBTB5 PE=1 SV=1 ZC3H1_HUMAN O60293 Zinc finger C3H1 domain-containing protein change not significant 1 206 OS=Homo sapiens GN=ZFC3H1 PE=1 SV=3 ZFHX3_HUMAN Q15911 patskRISFpgsse Zinc finger homeobox protein 3 OS=Homo sapiens lost 1 32 s GN=ZFHX3 PE=1 SV=2 ZFHX4_HUMAN Q86UP3 Zinc finger homeobox protein 4 OS=Homo sapiens lost 0.98 10 GN=ZFHX4 PE=1 SV=1 ZFY16_HUMAN Q7Z3T8 Zinc finger FYVE domain-containing protein 16 lost 0.97 14 OS=Homo sapiens GN=ZFYVE16 PE=1 SV=3 ZN106_HUMAN Q9H2Y7 Zinc finger protein 106 OS=Homo sapiens reduced 1 86 GN=ZNF106 PE=1 SV=1 ZN318_HUMAN Q5VUA4 Zinc finger protein 318 OS=Homo sapiens change not significant 1 292 GN=ZNF318 PE=1 SV=2 ZN592_HUMAN Q92610 Zinc finger protein 592 OS=Homo sapiens change not significant 1 48 GN=ZNF592 PE=1 SV=2 ZN638_HUMAN Q14966 Zinc finger protein 638 OS=Homo sapiens reduced 1 20 GN=ZNF638 PE=1 SV=2 ZN687_HUMAN Q8N1G0 Zinc finger protein 687 OS=Homo sapiens change not significant 1 36 GN=ZNF687 PE=1 SV=1 ZO2_HUMAN Q9UDY2 Tight junction protein ZO-2 OS=Homo sapiens lost 0.99 14 GN=TJP2 PE=1 SV=2 ZWINT_HUMAN O95229 ZW10 interactor OS=Homo sapiens GN=ZWINT lost 0.97 8 PE=1 SV=2 ZZEF1_HUMAN O43149 dkwpkKVTFka Zinc finger ZZ-type and EF-hand domain-containing change not significant 1 392 gprl protein 1 OS=Homo sapiens GN=ZZEF1 PE=1 SV=6

156

APPENDIX B: MASS SPECTROMETRY DATA FROM PP1 GFP-TRAP

B.1. List of mitosis-specific or asynchronous-specific proteins enriched in PP1 immunoprecipitations from all the three isoforms. Proteins specifically enriched (SAINT score > 0.9 or known PP1 binding proteins) in GFP- TRAPs from either mitosis or asynchronous specific extracts are listed with their average iBAQ areas.

GENE NAME MITOSIS ASYNC MITOSIS ASYNC MITOSIS ASYNC MITOSIS ASYNC MITOSIS ASYNC SAINT SAINT AvG AvG control control       mitosis async average ibaq average average ibaq average ibaq average ibaq average ibaq average ibaq average um ibaq um um um um um um ibaq um PP1A_HUMAN 0.93 1 1803142.123 0 710939406.8 710939406.8 4043749.596 2148162.975 25215.33333 1745800.936

PP1B_HUMAN 0.64 1 146051.5921 0 195254.4912 2575341.039 190566050.3 190566050.3 0 1453569.122

PP1G_HUMAN 0.63 1 218889.4035 0 716891.9474 1243178.247 271105.2632 0 146101564.8 146101564.8

CD2B2_HUMAN 0 0 0 0 0 0 0 98483.37084 0 0

IF2B_HUMAN 0.12 0 702554.8409 461303.0455 241433.3636 0 0 0 0 0

WBP11_HUMAN 0.5 0 0 0 112680.069 0 0 57430.90777 113779.4483 0

SFPQ_HUMAN 0 0.13 2293718.144 2551596.188 1264976.438 2894707.807 394762.4766 641503.9712 63733.90625 976679.7959

RS28_HUMAN 1 0.2 6455038.083 0 56501037.78 0 97502256.33 0 19835523.17 0

CRKL_HUMAN 1 0.24 482703.7222 0 5915663.426 0 699634.1111 0 1309498.222 0

CSRP2_HUMAN 0.99 0.27 3739587.718 0 40002918.41 0 10070208.96 0 23467370.27 0

PAWR_HUMAN 0.99 0.27 0 0 2969806.893 0 1406001.821 0 359995.9286 0

TPX2_HUMAN 0.98 0.28 333492.0238 0 4807798.31 0 3485368.429 0 2097842.952 0

MSRB2_HUMAN 0.94 0.28 0 0 1165765.136 0 1258169.636 0 420168.3636 0

MYO1D_HUMAN 0.27 0.37 1397293.568 7210856.446 0 5398023.897 1150211.514 1679596.487 1430635.581 5789583.007

CHCH6_HUMAN 0 0.38 2964377.125 4250533.125 944544.25 256322.39 847417.0313 1440966.709 315232.3438 2266794.245

GLRX1_HUMAN 1 0.38 2580635.667 0 32382931.58 0 66271396.5 0 56321757.67 0

UB2L3_HUMAN 0.94 0.42 0 0 2147985.063 0 3800646.625 0 718837.75 0

PROF2_HUMAN 0.99 0.43 2335630.813 0 33440145.96 0 13175473.17 0 31807158.94 0

KI67_HUMAN 0.51 0.44 597704.3561 788517.0634 584702.7951 250509.507 167176.8171 3328.419705 21511.73659 76069.53084

MIF_HUMAN 1 0.45 125224408 0 3840701310 0 1715893823 0 2558731995 0

GSHR_HUMAN 0.96 0.45 250522.1481 0 1033110.852 0 0 0 255053.7407 0

AURKA_HUMAN 0.42 0.47 738603.75 0 376880.8194 0 86899.25 0 205622.125 0

PALLD_HUMAN 1 0.47 32982.73188 0 1059630.696 0 125744.2536 0 544072.3913 0

AAMDC_HUMAN 0.99 0.47 0 0 1818712.364 0 3533341.955 0 2828233.273 0

157

GENE NAME AvG AvG Control M Control A  M  A  M  A  M  A mitosis async SC24C_HUMAN 0.95 0.47 0 0 576310.5556 0 84437.71111 0 455472.1111 0

AURKB_HUMAN 0.45 0.49 741955.2727 0 248824.3182 0 940481.2727 0 765895.8636 210619.6617

MCM7_HUMAN 0.26 0.49 1136309.854 430494.8333 877324.6736 1824775.452 1273970.396 217834.6862 303389.5833 104487.7773

MTPN_HUMAN 0.98 0.49 400483.25 0 2213806.125 0 0 0 1607075.875 0

SAHH2_HUMAN 0.18 0.5 0 0 0 0 0 400110.8547 0 0

CDCA2_HUMAN 1 0.5 0 0 106512.3333 0 674033.9561 64325.52698 144561.7193 2067742.414

CSRN2_HUMAN 0.45 0.5 0 0 0 0 0 1908131.043 0 0

LMTK2_HUMAN 0 0.5 0 0 0 0 0 0 0 9107299.533

PPR37_HUMAN 0.5 0.5 0 0 448490.8387 161624.9087 3466646.484 1370436.809 0 0

MAP1B_HUMAN 0.45 0.5 0 0 119214.2478 104741.3612 0 0 0 0

STAU1_HUMAN 0.45 0.5 0 0 0 131955.6716 270022.2727 940823.4929 0 131969.8003

ZFY16_HUMAN 0 0.5 0 0 0 0 67392.54412 323980.2035 0 0

CRIP2_HUMAN 1 0.5 0 0 1048703.1 0 5405648.25 0 1816877.5 0

RTCB_HUMAN 0.98 0.5 543532.5345 0 1289300.379 0 130271.6897 0 207724.931 0

GUAA_HUMAN 0.98 0.5 131044.5349 0 389480.814 0 0 0 0 0

GRN_HUMAN 0.95 0.5 0 0 1813290.043 0 4568022.304 0 1494474.87 0

AZI1_HUMAN 0.94 0.5 0 0 125217.0563 0 22171.6338 0 0 0

PSMD9_HUMAN 0.94 0.5 0 0 1545016.227 0 199600.7273 0 0 0

ZN609_HUMAN 0.94 0.5 0 0 154941.2177 0 0 0 0 0

KITH_HUMAN 0.94 0.51 1327299.6 0 2037224.289 0 397670.0333 0 689927.5 0

NUFP2_HUMAN 0.94 0.51 0 0 499191.2419 0 352828.1613 0 281344.7742 0

THIO_HUMAN 1 0.52 7115093.393 0 75041764.52 0 22723346.76 0 36840610.29 0

PHF5A_HUMAN 0.98 0.61 534667.1875 0 5933784.792 0 7736963.25 0 24679514.75 0

SQSTM_HUMAN 0.98 0.61 0 0 1071488.563 0 1418694.813 0 0 0

HN1L_HUMAN 1 0.62 682494.5 0 10441579.75 0 9092201.095 0 8079895.071 0

TB182_HUMAN 0.99 0.65 0 0 332800.2849 0 129202 0 198727.7093 0

PRDX6_HUMAN 0.9 0.65 621573.8958 0 3639292.969 0 0 0 0 0

CX067_HUMAN 1 0.72 342696 0 3094542.128 0 468166.141 0 588354.5 0

PAIRB_HUMAN 1 0.73 1474630.957 0 24163789.57 0 22393437.55 0 6967315.12 0

TCPG_HUMAN 0.95 0.73 704540.0743 0 2636127.955 0 1187850.054 0 375329.527 0

CYTB_HUMAN 1 0.74 0 0 36125682.11 0 64618098.5 0 29440694.58 0

MAP4_HUMAN 1 0.76 241380.1667 0 3960836.121 0 1292679.769 0 1436332.384 0

TCPH_HUMAN 0.92 0.77 630724.1429 0 3075690.886 0 1180043.8 0 186360.6857 0

PDLI4_HUMAN 1 0.78 706050.2632 0 8282299.921 0 4696716.684 0 2237910.921 0

PRC2C_HUMAN 0.95 0.79 0 0 176853.8893 0 84221.61066 0 0 0

PTRF_HUMAN 1 0.8 585318 0 1996104.854 0 5997761 0 819994.875 0

SMTN_HUMAN 0.99 0.83 335153.1909 0 3981717.679 0 1390827.014 0 2323724.136 0

AL7A1_HUMAN 0.99 0.84 102791.4815 0 1118582.173 0 161057.0556 0 666625.7037 0

ANLN_HUMAN 0.96 0.84 426150.4143 0 2481792.214 0 956070.8952 0 1044949.857 0

RM14_HUMAN 0.99 0.86 0 0 4492202.4 0 1190516.15 0 475674.9 0

158

GENE NAME AvG AvG Control M Control A  M  A  M  A  M  A mitosis async BAG3_HUMAN 1 0.87 194018.3438 0 3927761.828 0 589952.0156 0 129604 0

TCPB_HUMAN 1 0.9 505111.0278 0 1819261.944 6668326.715 0 309970.1549 50148.61111 361385.0103

K1967_HUMAN 1 0.91 178807.9216 0 1350921.007 547650.2828 0 0 0 0

HCFC1_HUMAN 1 0.93 0 110106.303 476047.2652 370107.7154 86402.0303 61209.36247 280934.8788 105216.2115

HUWE1_HUMAN 0.99 0.94 21471.45854 88524.37073 77658.53496 325278.5249 0 0 0 0

SYNPO_HUMAN 1 0.96 1740195.293 555056.2935 4306973.272 3583128.482 3768759.44 2318587.478 2639537.141 20610458.1

TACC3_HUMAN 1 0.97 0 0 6988115.477 575954.8361 4142800.212 26140.28533 3928553.114 0

RASF8_HUMAN 0.99 0.97 0 0 343001.3656 1077957.6 156413.7419 0 0 0

MA7D1_HUMAN 0.99 0.97 0 0 676573.6111 261619.0121 94840.02222 176618.0224 0 1491976.829

RRP1B_HUMAN 0 0.97 0 0 138328.3194 369824.9261 171733.0648 1609257.811 0 339378.5354

TCOF_HUMAN 0.99 0.98 46562.11688 104357.5065 849188.6299 639016.553 296169.8442 541391.931 69354.92208 1131232.694

ZN185_HUMAN 1 0.98 585403.75 922047.5227 8034929.182 2900730.573 3332910.869 1047620.227 3512123.189 3524235.997

ATX2L_HUMAN 0.95 0.98 0 262448.9048 573261.8214 521705.5761 397508.1667 425903.7581 480784.6429 855368.6442

SON_HUMAN 0.95 0.98 0 188037.4815 151045.0535 155512.4884 126532.9588 161978.3987 55339.66667 633392.067

LPP_HUMAN 0.99 0.99 0 243899.4545 1110204.886 1404822.71 125518.2045 458893.3944 0 534243.05

MILK2_HUMAN 0.99 0.99 0 37433.71429 378409.2054 169644.1171 27806.55357 40038.02719 45892.83929 295968.4852

SC16A_HUMAN 1 0.99 0 28663.88312 381727.6494 278563.2382 59740.74026 51270.96399 0 136408.9657

IPP2L_HUMAN 0.95 0.99 0 0 0 0 0 0 0 0

CC85C_HUMAN 0.95 1 0 0 1149522.475 1066189.451 498619.75 1016399.726 0 0

CLCB_HUMAN 0.99 1 0 0 1042494.7 0 2255183.65 772329.0956 1848216.3 3678507.823

RASF7_HUMAN 1 1 0 0 2508336.619 3404897.348 0 76978.31088 0 0

TPRN_HUMAN 1 1 0 0 3440943.816 4548475.384 0 148717.994 490555.8684 0

SNW1_HUMAN 1 1 0 0 2953010.368 881054.0091 1086341.294 575800.3048 469602.1176 2065621.301

TCPE_HUMAN 0.99 1 469584.8684 133240.2368 2136093.351 5079879.767 143540.3421 518210.3253 184775.8947 432272.1252

ASPP1_HUMAN 1 1 0 0 448259.5034 968716.5578 1638106.279 762074.3055 0 0

NEB1_HUMAN 0.7 1 366133.1481 0 1611321.713 3443486.887 2590994.463 1310446.954 1260018.204 1128664.697

IPP2M_HUMAN 1 1 0 0 0 0 0 0 0 0

PP1RB_HUMAN 1 1 0 0 192500510.7 168032635.3 30990903.6 25189217.47 23389496.1 73962355.66

IPP2_HUMAN 1 1 0 0 7096164.75 15141617.08 8989775 3856131.023 6373687.25 39133196.68

ASPP2_HUMAN 1 1 0 0 5421108.686 9606166.804 2287254.906 812568.6723 0 0

PP1R8_HUMAN 1 1 0 0 32608734.47 25502462.67 116506241.1 92793546.46 129095660.2 111822955.7

PP1RA_HUMAN 1 1 0 0 3248826.543 4620743.248 6449777.777 2593039.637 1351949.261 6955732.887

RIF1_HUMAN 1 1 0 0 490953.9762 1863370.075 20146.42857 474305.0063 0 1786683.508

IASPP_HUMAN 1 1 0 0 32664634.45 38055868.01 1655523.026 6326196.486 87120.02632 473308.3054

PP1R7_HUMAN 1 1 0 0 145443733.4 141822727 26083863.52 31151410.67 19129975.45 100973744.5

PPR18_HUMAN 1 1 0 117588.25 59484621.48 79207410.42 793116.1563 3026426.181 1125662.563 2197241.402

YLPM1_HUMAN 1 1 0 0 4157571.072 7342903.96 2319241.309 1159706.785 1148236.172 11376873.09

NEB2_HUMAN 0.97 1 5925626.239 1372790.085 69449211.05 108920553.2 27743922.24 5559241.279 35784800.8 16850788.96

TOX4_HUMAN 0.99 1 0 0 1729120.971 1562911.884 4942124.529 2145993.067 1593603.412 3757912.675

RL38_HUMAN 0.92 1 2449128.778 0 14454957.67 2866336.406 2092362 5473813.8 15933515.33 2511619.881

159

GENE NAME AvG AvG Control M Control A  M  A  M  A  M  A mitosis async CWC15_HUMAN 0.99 1 0 0 2793085.15 1567189.021 2384713.95 1010040.303 1492280.4 4254981.021

PP16A_HUMAN 0.99 1 0 0 0 2512182.302 10871311.11 4031511.587 0 1202658.163

MYPT2_HUMAN 1 1 0 0 0 61804.40019 1383330.667 597450.3179 0 0

PP12C_HUMAN 1 1 0 0 0 7896252.827 13253684.79 9537544.705 0 0

SRRM2_HUMAN 1 1 41342.99564 0 672222.1111 234390.2131 703949.7952 1648621.575 60861.23965 4562203.336

MYPT1_HUMAN 1 1 654974.2642 144042.8679 897304.5943 39242185.7 292953787.5 155603666 906859.0755 354451.0109

PARD3_HUMAN 0.5 1 0 0 0 417481.1219 0 0 0 0

G6PI_HUMAN 0.31 1 0 0 0 0 0 1078941.13 0 2376416.122

SCRIB_HUMAN 0.86 1 0 127783.9759 0 843809.1672 0 97155.38024 0 80456.2956

TKT_HUMAN 0 1 0 0 0 0 0 3985849.985 0 3950928.57

PGK2_HUMAN 0.18 1 0 0 0 0 0 73815.04109 0 237635.7647

AHNK_HUMAN 0.01 1 0 124283.8656 0 984942.1363 0 338672.8119 0 688470.9984

ANXA5_HUMAN 0.34 1 0 0 0 0 0 845971.1273 0 1172654.905

CPSM_HUMAN 0.01 1 0 0 0 0 0 377283.3792 0 829869.7534

RL29_HUMAN 0.56 1 0 2707737.042 0 2946276.398 0 18294363.27 0 39332977.82

BCLF1_HUMAN 0.49 1 0 69887.29412 0 538236.3933 0 489622.9569 0 1183034.391

TR150_HUMAN 0.89 1 0 0 0 191735.4808 0 634440.6661 0 4394986.01

KCC2D_HUMAN 0.09 1 0 0 0 285613.6519 0 77142.70778 0 0

FSCN1_HUMAN 0.05 1 0 168930.6724 0 753565.0928 0 2213202.731 0 1002512.643

CT451_HUMAN 0 1 0 0 0 0 0 0 0 0

CT452_HUMAN 0 1 0 0 0 0 0 0 0 0

CT453_HUMAN 0 1 0 0 0 0 0 0 0 0

CT454_HUMAN 0 1 0 0 0 0 0 0 0 0

PDLI7_HUMAN 0.65 1 0 0 0 141197.0587 0 341539.7682 0 328431.8936

SRP14_HUMAN 0.33 1 0 0 0 1079838.545 0 1522404.625 0 3928790.784

1433G_HUMAN 0 1 0 0 0 0 0 118373.3845 0 553702.0335

MDHM_HUMAN 0.01 1 0 185096.75 0 0 0 1161788.481 0 1464761.776

1433E_HUMAN 0 1 0 0 0 0 0 977829.9694 0 1523729.972

H15_HUMAN 0.17 1 0 0 0 0 0 1773349.619 0 4733993.423

STRAP_HUMAN 0 0.99 0 0 0 0 0 579019.5641 0 223935.7171

HSPB8_HUMAN 0.5 0.99 0 0 0 2991713.292 0 0 0 496679.3173

RT12_HUMAN 0.85 0.99 0 0 0 1871679.561 0 476962.6926 0 1187846.63

FXR1_HUMAN 0.6 0.99 0 101135.5676 0 240224.9743 0 247340.1468 0 159965.4224

PFD2_HUMAN 0.83 0.99 0 0 0 2151719.587 0 1108724.754 0 1128265.603

PPBT_HUMAN 0.08 0.99 0 0 0 0 0 549231.7707 0 924660.0252

P5CS_HUMAN 0.08 0.99 0 0 0 0 0 484203.3282 0 542294.2819

C1TC_HUMAN 0.5 0.99 0 134807.2798 0 921909.4066 0 1028027.456 0 484511.1102

PGAM1_HUMAN 0.19 0.99 0 0 0 0 0 139147.7126 0 114644.008

K6PL_HUMAN 0.33 0.99 0 0 0 792864.8431 0 303000.8907 0 0

CNBP_HUMAN 0.74 0.99 0 407706.9333 0 644367.7417 0 851319.1855 0 1279354.946

160

GENE NAME AvG AvG Control M Control A  M  A  M  A  M  A mitosis async RL19_HUMAN 0.56 0.99 0 1681284.056 0 1832053.554 0 5389962.982 0 11816246.2

RLA2_HUMAN 0.18 0.99 0 0 0 4763769.298 0 5475080.978 0 7363229.893

CDC37_HUMAN 0.46 0.99 0 0 0 256039.2518 0 487294.7731 0 294697.486

IF4G1_HUMAN 0.35 0.99 0 0 0 0 0 100147.8974 0 211941.2588

PRS7_HUMAN 0.62 0.99 0 0 0 179017.5955 0 618363.8245 0 190502.5587

SF3B2_HUMAN 0.38 0.99 0 0 0 168646.0135 0 131636.3198 0 195993.1273

CT455_HUMAN 0 0.99 0 0 0 0 0 0 0 0

ENOG_HUMAN 0.13 0.99 0 0 0 0 0 0 0 70499.79736

IMA2_HUMAN 0.04 0.99 0 0 0 0 0 638112.5639 0 619880.6847

ANXA1_HUMAN 0.23 0.99 0 196278.1905 0 195333.1451 0 2680991.54 0 2117688.083

HS105_HUMAN 0.74 0.98 0 15618.34259 0 293018.1316 0 552456.0446 0 53969.93694

MAGD2_HUMAN 0.73 0.98 0 296210.2426 0 1992346.12 0 241089.5727 0 165838.7949

PDIA3_HUMAN 0.03 0.98 0 285907.6563 0 1126272.319 0 2224106.145 0 3524885.294

NAA15_HUMAN 0 0.97 0 0 0 0 0 97411.18416 0 76723.14495

PEX14_HUMAN 0.5 0.97 0 0 0 796873.2399 0 110288.4118 0 0

BAG6_HUMAN 0.49 0.97 0 0 0 408090.9269 0 51680.37405 0 0

DHE3_HUMAN 0.02 0.97 0 0 0 0 0 51496.37492 0 0

FKBP4_HUMAN 0.11 0.97 0 0 0 0 0 166355.9187 0 506392.4893

ES1_HUMAN 0.5 0.97 0 0 0 2138189.701 0 2681404.934 0 0

LYPA2_HUMAN 0.89 0.97 0 0 0 2639990.713 0 4745567.029 0 0

CO052_HUMAN 0 0.97 0 0 0 0 0 413592.1351 0 2288304.05

TYB10_HUMAN 0.01 0.97 0 0 0 0 0 2860798.795 0 5573686.564

SET_HUMAN 0.02 0.97 0 0 0 0 0 2376633.951 0 1857204.063

TRIP6_HUMAN 0.49 0.97 0 0 0 302142.9262 0 179411.0906 0 372173.9202

IF4B_HUMAN 0.78 0.97 0 583309.5882 0 1066086.488 0 755988.7095 0 1552327.549

PRS10_HUMAN 0.04 0.97 0 0 0 0 0 378439.3005 0 797454.2784

YTHD2_HUMAN 0 0.97 0 0 0 223667.9113 0 111597.7941 0 0

CBR1_HUMAN 0.21 0.97 0 0 0 0 0 995855.915 0 1343643.2

TYB4_HUMAN 0.01 0.97 0 0 0 0 0 1964618.452 0 5094975.783

PSA6_HUMAN 0.13 0.97 0 0 0 834405.0674 0 434304.8697 0 652826.4496

VINC_HUMAN 0.05 0.97 0 0 0 0 0 270160.734 0 264912.8957

RS19_HUMAN 0.07 0.97 0 2127932.5 0 2606672.145 0 2204354.632 0 3296860.505

WDR82_HUMAN 0.5 0.97 0 0 0 18249242.52 0 2897149.392 0 14895268.44

TARA_HUMAN 0.5 0.97 0 0 0 91599.99851 0 1207420.023 0 0

IDHC_HUMAN 0.85 0.97 0 0 0 166443.3599 0 75176.7397 0 263460.8681

KAD2_HUMAN 0.53 0.97 0 0 0 499642.5556 0 497302.1305 0 769542.1183

DBNL_HUMAN 0.36 0.97 0 0 0 228341.5496 0 1630484.923 0 0

RL34_HUMAN 0.08 0.97 0 5453308.958 0 3170565.573 0 6954843.503 0 7692001.168

CATA_HUMAN 0.12 0.97 0 0 0 0 0 109433.5093 0 277371.1693

RL37_HUMAN 0 0.97 0 0 0 361372.1729 0 1162244.817 0 6841927.674

161

GENE NAME AvG AvG Control M Control A  M  A  M  A  M  A mitosis async GLYM_HUMAN 0.08 0.97 0 0 0 239543.9449 0 400994.9367 0 833024.3641

PRC2A_HUMAN 0.45 0.97 0 0 0 151639.1573 0 65112.36592 0 465477.9757

HMGB1_HUMAN 0.02 0.97 0 0 0 0 0 1781035.427 0 325739.2017

RS14_HUMAN 0.06 0.96 0 15646785.21 0 29172923.72 0 14541246.35 0 12952099.39

CALR_HUMAN 0.08 0.96 0 535164.3194 0 1062695.429 0 2060911.526 0 2387547.502

TPIS_HUMAN 0 0.96 0 1873472.421 0 259321.9223 0 2754113.771 0 4376942.619

CBS_HUMAN 0.5 0.95 0 94429.93103 0 887942.0172 0 260696.0516 0 0

PGK1_HUMAN 0 0.95 0 515187.3833 0 0 0 1617602.21 0 2874128.62

RS20_HUMAN 0.33 0.95 0 14281554.42 0 29183926.72 0 22470451.59 0 18639698.1

PDIA1_HUMAN 0 0.95 0 512828.4615 0 1117590.87 0 731860.5649 0 1300687.574

ENOA_HUMAN 0 0.95 0 3988956.207 0 2018688.821 0 4485303.759 0 7820524.27

PLOD1_HUMAN 0 0.94 0 0 0 0 0 53095.14854 0 0

CYTA_HUMAN 0.5 0.94 0 0 0 2976846.6 0 197279.5422 0 0

FA83H_HUMAN 0.5 0.94 0 0 0 113005.3263 0 25809.95069 0 121174.66

AK1C1_HUMAN 0 0.94 0 0 0 0 0 0 0 0

PRDX5_HUMAN 0.04 0.94 0 0 0 0 0 970812.9985 0 1656289.472

AK1C3_HUMAN 0 0.94 0 0 0 0 0 22822.97936 0 0

MLF2_HUMAN 0.45 0.94 0 0 0 1838582.133 0 300568.4933 0 1371457.859

UBP5_HUMAN 0.86 0.94 0 0 0 388252.4809 0 1162238.335 0 0

ALDOA_HUMAN 0 0.94 0 1242278.478 0 0 0 3725765.202 0 5422326.784

S10AA_HUMAN 0.08 0.94 0 0 0 4527962.027 0 3019566.684 0 2571322.825

COPG1_HUMAN 0.11 0.94 0 0 0 0 0 28544.32913 0 75984.39404

RL8_HUMAN 0.13 0.94 0 4589634.271 0 4523117.641 0 7533567.174 0 8980420.494

PSME1_HUMAN 0.12 0.94 0 0 0 0 0 339333.657 0 580926.5417

PLIN3_HUMAN 0.31 0.94 0 0 0 0 0 116639.5177 0 192067.6174

PROF1_HUMAN 0 0.93 0 1849159.444 0 915709.0119 0 9514606.573 0 12223613.82

EIF3D_HUMAN 0 0.92 0 0 0 0 0 872848.5666 0 0

CTND1_HUMAN 0.05 0.92 0 107687.9259 0 238204.9596 0 71002.65073 0 96723.21973

MCM3_HUMAN 0.42 0.92 0 183448.7232 0 395012.6593 0 1130504.584 0 295942.0593

PSB5_HUMAN 0.08 0.92 0 0 0 0 0 876308.1149 0 468138.7045

BUB3_HUMAN 0.62 0.92 0 135178.625 0 913250.4914 0 1837575.784 0 1418546.153

SYG_HUMAN 0.45 0.92 0 0 0 162126.1061 0 484283.5693 0 188225.6946

RAB5C_HUMAN 0 0.92 0 0 0 0 0 0 0 0

LDHA_HUMAN 0 0.92 0 819048.125 0 433344.5466 0 10870936.6 0 13001923.04

TBCB_HUMAN 0.45 0.92 0 180835.5 0 250772.806 0 1171915.704 0 0

1433S_HUMAN 0.07 0.92 0 485374.5556 0 0 0 75099.32875 0 133708.0618

RL36_HUMAN 0.1 0.92 0 1792596.083 0 3749864.939 0 3262834.781 0 3901384.551

FWCH2_HUMAN 0.49 0.91 0 462081.25 0 1207826.261 0 346353.6055 0 0

SYK_HUMAN 0.08 0.91 0 0 0 0 0 312685.2878 0 245518.1008

ABCE1_HUMAN 0.36 0.91 0 133665.8235 0 0 0 163578.8797 0 244057.8989

162

GENE NAME AvG AvG Control M Control A  M  A  M  A  M  A mitosis async EIF3A_HUMAN 0.4 0.91 0 119762.6129 0 0 0 52126.42798 0 115234.7624

F120A_HUMAN 0.5 0.91 0 0 0 317412.8157 0 152922.3757 0 401786.6018

PGAM2_HUMAN 0.17 0.91 0 0 0 0 0 0 0 0

RS10L_HUMAN 0.24 0.91 0 0 0 0 0 0 0 0

DYL1_HUMAN 0.49 0.91 0 0 0 3143467.431 0 1052941.017 0 0

EIF3I_HUMAN 0.17 0.91 0 0 0 0 0 267596.3625 0 254767.4044

PRS4_HUMAN 0.15 0.91 0 215056.2222 0 315607.3973 0 266344.4927 0 339111.5363

ACTN4_HUMAN 0.29 0.91 0 235588.5369 0 866385.897 0 1080665.884 0 777911.8226

IF5AL_HUMAN 0.04 0.91 0 0 0 0 0 0 0 0

RL27_HUMAN 0.21 0.9 0 7047222.417 0 9349086.543 0 10736121.28 0 11922091.13

CAN2_HUMAN 0.37 0.9 0 1533578.222 0 0 0 1872946.385 0 387535.3566

CHCH3_HUMAN 0.18 0.29 0 15413589.58 0 389147.9098 0 8540854.793 0 16575213.46

CLCN7_HUMAN 0 0 0 0 0 0 0 75017.35324 0 0

ITPR3_HUMAN 0 0.47 0 0 0 0 0 0 0 0

MPP10_HUMAN 0 0.47 0 0 0 495704.2117 0 0 0 0

NOC2L_HUMAN 0 0.5 0 0 0 880442.3809 0 0 0 0

PCIF1_HUMAN 0 0 0 0 0 184942.987 0 0 0 0

S12A2_HUMAN 0 0.5 0 0 0 0 0 58129.00743 0 0

ZFYV9_HUMAN 0 0.5 0 0 0 0 0 117446.5872 0 0

163