A Dissertation
entitled
Regulation and Post-translational modifications of Borealin
by
Dipali A. Date
Submitted to the Graduate Faculty as partial fulfillment of the requirements for The Doctor of Philosophy Degree in Biology
______
Dr. William R. Taylor, Committee Chair
______
Dr. Patricia Komuniecki, Dean
College of Graduate Studies
The University of Toledo
August 2010
ii
An Abstract of
Regulation and Post-translational modifications of Borealin
by
Dipali A. Date
As partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology
The University of Toledo August 2010
Cancer occurs when normal regulation of the cell division cycle is disrupted by genetic or environmental factors. Hence, understanding the molecular mechanisms that regulate cell division is essential for the development of anti-cancer therapeutics.
Borealin/Dasra B/CDCA8 (Cell Division Cycle Associated 8) is a member of the chromosomal passenger complex (CPC) also composed of Aurora B, INCENP and
Survivin. The CPC exhibits a dynamic pattern of localization during mitosis and plays important roles in chromosome segregation, spindle assembly checkpoint (SAC) and cytokinesis.
We identified Borealin to be an E2F/Rb target; several genes repressed by Rb dependent pathways are highly expressed in various cancers. We observed that Borealin expression was elevated in lymphomas, brain, and colon cancers and down regulated in response to DNA damage in a p53 dependent manner. Borealin is regulated in a cell cycle dependent manner and we show that it is degraded by the 26S proteasome during the cell cycle. We identified a putative stability region between amino acids 141-168 that protects
Borealin from proteolytic degradation. Further, over expression of CDH1, an activator of the Anaphase promoting complex (APC/C) caused a minimal decrease in Borealin levels.
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Therefore Borealin may be targeted by an E3 ligase other than APC/C.
Post-translational modifications of the passenger proteins are essential in the regulation of the CPC. We observed that Borealin is phosphorylated in vivo, in response to increased expression of constitutively active Cdk1/Cyclin B1. In addition, we observed a reduced level of slow migrating phosphorylated Borealin species upon treatment with a combination of purvalanol (Cdk1 inhibitor) and ZM447439 (Aurora B inhibitor).
However, Borealin was inefficiently phosphorylated by CDK1 in vitro. We further investigated candidate phosphatases belonging to the Cdc14 family. Immunofluorescence analysis revealed that Cdc14B and Borealin co-localize to the nucleolus of interphase cells. Conversely, overexpression of Cdc14B only caused a subtle decrease in mobility shift characteristic of Borealin phosphorylation. Also, Borealin was still dephosphorylated in cells lacking Cdc14B. However, shRNA mediated depletion of
Cdc14A did induce phosphorylation of endogenous Borealin. Hence, Borealin maybe dephosphorylated by Cdc14A, while CDK1 and Aurora B may exert a combinatorial effect to induce phosphorylation of mitotic Borealin.
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Acknowledgments
I am perpetually grateful to my advisor Dr. William R. Taylor for guiding me through every step of acquiring this degree. I am thankful to my committee members for their guidance towards the completion of my degree. I am especially thankful to Dr. Deborah
Chadee and Dr.Song-Tao Liu for helpful discussions about my project. I would like to express my sincere gratitude towards my friends and lab mates Megan Drier and Michael
Beiker for helping me conduct the experiments and survive through the ones that failed.
In addition, I would like to acknowledge the past lab members Cara Jacobs and Harpreet
Kaur for their work on the projects that I later continued. I would also like to acknowledge all the undergraduate students for making the laboratory work a fun experience. Finally, this dissertation would not have been possible without the love and support of my parents Mr. and Mrs. Ashok and Vaishali Date. A special thanks to my fiancé Preshit Gawade for being my support and motivation through this journey.
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Table of Contents
Abstract iii
Acknowledgement v
Table of contents vi
List of Figures ix
List of Abbreviations xi
I. Introduction
1. Cell division and Cell Cycle Checkpoints 1
2. Chromosomal Passenger Complex 5
3. Regulation of Borealin 12
4. Post translational modifications of Borealin 16
II. Hypothesis 25
III. Materials and Methods
1.Cell lines and culture conditions 26
2.Drug treatments 26
3. Antibodies 27
4. Western Blotting 28
4. Transient Transfections 28
5. Immunofluoresence 29
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6. Invitro kinase Assay 30
7. Generation of recombinant adenoviruses 31
8. Immunoprecipitation 31
9. Generation of Cdc14 depleted cell lines 32
10. Analysis of DNA synthesis 33
IV. Results
1. Regulation of Borealin
1.1 Borealin is downregulated upon DNA damage 34
1.2 Borealin is downregulated in a p53 dependent manner 37
1.3 Borealin is downregulated in a Rb dependent manner 39
1.4 Effect of proteasome inhibition on the levels of endogenous 41
Borealin
1.5 Association of Borealin with Ubiquitin 46
2. Phosphorylation of Borealin
2.1 Borealin is phosphorylated in response to CDK1 51
overexpression in vivo
2.2 Borealin is not an optimal substrate for CDK1 in vitro 55
2.3 Effect of kinase inhibitors on Borealin phosphorylation 59
2.4 Effect of PLK1 overexpression on Borealin phosphorylation 62
3. Dephosphorylation of Borealin
3.1 Kinetics of Borealin dephosphorylation 64
3.2 Borealin and Cdc14B co-localize to the nucleolus of 66
interphase cells
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3.3 Effect of Cdc14 overexpression on Borealin 68
3.4 Cdc14B does not mediate proteasome mediated degradation of 71
Borealin
3.5 Analysis of Cdc14 depletion on the status of Borealin 74
phosphorylation
V. Discussion 76
VI. Conclusion 87
VII. References 89
VIII. Appendix
1. Analysis of Borealin phosphorylation with a phospho specific 96
2. Analysis of Borealin phosphorylation in cells over expressing 96
Borealin
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List of Figures
1. Stages of Cell Cycle 4
2. Structure of the CPC 7
3. Localization of the CPC 11
4. Potential degradation sites in Borealin 15
5. Phosphorylation sites mapped in Borealin 19
6. The effects of DNA damage on the levels of p53 and Borealin 35
7. The effect of DNA damage on Borealin levels in HT1080 cells 36
8. p53 and p21/waf1 are required for Borealin down-regulation in response 38
to DNA damage
9. Borealin is down-regulated in a Rb-dependent manner 40
10. Effect of proteasome inhibition on the levels of Borealin 42
11. Effect of proteasome inhibition on the levels of Borealin 44
12. Association of Borealin with ubiquitin 47
13. Effect of APC co-activators on Borealin 49
14. Characterization of recombinant adenoviruses 53
15. The effect of CDK1 on phosphorylation of Borealin in vivo 54
16. Borealin is not an optimal CDK1 substrate in vitro 56
17. CDK1 does not phosphorylate a Borealin peptide encompassing S219 58
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18. Effect of kinase inhibitors on Borealin phosphorylation during S-phase 60
19. Effect of kinase inhibitors on the phosphorylation of Borealin during 61
mitosis
20. Effect of PLK1 over expression on Borealin phosphorylation 63
21. Kinetics of Borealin phosphorylation 65
22. Borealin and Cdc14B co-localize to the nucleolus of interphase cells 67
23. Effect of Cdc14B overexpression on Borealin phosphorylation 69
24. Cdc14B does not mediate proteasome- mediated degradation of 73
Borealin
25. Effect of Cdc14 knock down on Borealin phosphorylation 75
26. Borealin down -regulation in response to DNA damage by an indirect 81
multi-component system
27. Analysis of Borealin phosphorylation with a phospho-specific antibody 98
28. Analysis of Borealin phosphorylation in cells overexpressing Borealin 100
x
List of Abbreviations
APC/C Anaphase Promoting Complex/Cyclosome ATM Ataxia-telangiectasia ATR Ataxia -Rad3-related protein ATP Adenosine triphosphate BIR domain baculovirus IAP repeat domain BPB Bromo phenol blue BrDu Bromodeoxyuridine BSA Bovine serum albumin BUB1/ 3 budding uninhibited by benzimidazole 1/3 BUBR1 budding uninhibited by benzimidazole related 1 CBF CCAAT-binding Factor CDK Cyclin dependent kinase CDK1-AF Cyclin dependent kinase T14A Y15F CDCA8 Cell Division Cycle Associated 8 CDC Cell Division Cycle CHK1/2 Csk homologous kinase 1 and 2 Clp1 Cdc14-related protein phosphatase 1 CMV Cytomegalovirus CPC Chromosomal Passenger Complex DAPI 4’, 6- Diamidino-2-phenylindole D box Destruction box DNA Deoxyribonucleic acid DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl Sulfoxide DTT Dithiothreitol ECT2 Epithelial cell transforming gene 2 EDTA Ethylenediaminetetraacetic acid ERK1/ 2 Extracellular signal related kinases 1 and 2 E2F E2F promoter binding factor FBS fetal bovine serum GFP Green Fluorescent Protein GST Glutathione S transferase HA Hemagglutinin hDM2 Human double minute 2 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HP-1 Heterochromatin protein-1
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HRP Horse radish peroxidase HU Hydroxyurea IAP inhibitor of apoptosis INCENP Inner Centromeric Protein JNK c-Jun N terminal kinase MAD 1/2 mitotic arrest deficient 1 and 2 MAPK Mitogen activated protein kinase MCAK Mitotic centromere-associated kinesin MEK 1/2 MAPK/Erk kinase 1 and 2 MEF Mouse embryonic fibroblasts MgcRacGAP Male germ cell Rac-GTPase-activating protein MgCl2 Magnesium chloride MKLP1/ 2 Mitotic Kinesin-Like Protein 1 and 2 MOI Multiplicity of infection MPS1 Multipolar Spindle 1 Myt1 Membrane-associated inhibitory kinase NaCl Sodium Chloride NaF Sodium fluoride NB1 Nuclear Cyclin B1 Nek2A NIMA-related kinase 2A NF-Y nuclear transcription factor Y NOC Nocodazole PBS Phosphate buffered saline PLK1/2/3 Polo like kinase 1/2/3 PMSF Phenyl methane sulfonyl fluoride PPP1-7 Phospho Protein Phosphatase 1-7 PVDF Polyvinyldifluoride Raf Kinase Ras-activated factor kinase RanBP2 RAN binding protein 2 Rb Retinoblastoma Rho GEF Rho- Guanine nucleotide exchange factor Rho GTP Rho- Guanosine triphosphate SCF Skp1-Cull-Fbox SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SENP3 SUMO1/sentrin/SMT3 specific peptidase 3 shRNA Short hairpin ribonucleic acid S phase Synthesis phase SV40 Simian virus 40 SUMO Small Ubiquitin-like Modifier TKO Triple knock out TTA Tetracycline transactivator WD40 domain Tryptophan -Aspartate dipeptide domain WTB1 Wild type Cyclin B1 OA Okadaic acid µCi Micro curie
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Introduction
Cancer is the second leading cause of death in the United States of America. Half of all men and one-third of all women in the USA will develop cancer during their lifetimes (ACS 2009). Cancer is characterized by uncontrolled cell division. The cell division cycle requires the coordination of a wide variety of processes that in turn, depend on the activity of a family of protein kinases, the cyclin dependent kinases
(CDKs). Cancer occurs when the normal regulation of the cell cycle is disrupted by genetic or environmental factors. Hence, understanding the molecular mechanisms that regulate the cell division cycle is essential for the development of targeted anti-cancer therapeutics.
1. Cell division and Cell Cycle Checkpoints
The cell cycle is the ordered series of events required for the faithful duplication of one eukaryotic cell into two genetically identical daughter cells. The cell cycle can be divided into Interphase, composed of DNA replication (S) and Gap phases G1 and G2.
During G1 phase, cells grow and synthesize enzymes and metabolites required for DNA synthesis that takes place during the S phase. G2 is the second gap phase and an important control point before mitosis. Multiple DNA damage checkpoints within G1 and
G2 ensure the fidelity of DNA replication within the S phase. For example, in diverse organisms from yeast to mammals, a rapid G2 arrest is mediated by Ataxia- telangiectasia/Ataxia -Rad3-related protein (ATM/ATR) which inactivates cyclin-
1 dependent kinase-1 (CDK1) (O'Connell et al., 2000). The ATM/ATR kinase phosphorylates and activates the Csk homologous kinases (CHK ) 1 and 2, which phosphorylate and inactivate the phosphatase CDC25. The CDC25 phosphatase is responsible for dephosphorylating and activating CDK1 at the G2/M boundary. In animals as diverse as humans and the nematode C elegans tumor suppressor protein p53 imposes additional points of cell cycle control in response to DNA damage. Prominent among these is the G1 arrest which occurs primarily as a result of transcriptional induction of the CDK inhibitor p21/waf1 by the transactivation function of p53. p53 also mediates a long term arrest in G2 by down-regulating a large number of genes that encode proteins essential to enter into and progress through mitosis (Jackson et al., 2005).
The importance of these checkpoints to normal physiology is underscored by the fact that mutations in many checkpoint proteins contribute to cancer. For example, germ line mutations in p53 are responsible for the cancer susceptibility syndrome first described by
Li and Fraumeni. A syndrome of similar clinical presentation is caused by mutations in
CHK1. Somatic mutations in p53 also occur in more than half of all human cancer.
Furthermore, inactivating germ line mutations in ATM are responsible for the cancer predisposition syndrome ataxia telangiectasia. Thus, control of the cell cycle in response to DNA damage is essential to suppress tumorgenesis. Progression through the cell cycle culminates at M phase or mitosis, which includes nuclear division plus cytokinesis. The spindle assembly checkpoint (SAC) is a prominent cell cycle control that operates during mitosis (Nicklas, 1997). Major components of the SAC include the proteins mitotic arrest deficient (MAD)1 and 2,budding uninhibited by benzimidazole (BUB) 1 and 3, budding uninhibited by benzimidazole related 1(BUBR1), CDC20, multipolar spindle 1
2
(MPS1) kinase and Aurora B kinase. During prometaphase the SAC proteins concentrate at the kinetochores and monitor kinetochore- microtubule attachments. The SAC negatively regulates the anaphase promoting complex (APC/C) activator CDC20. The
APC/C is a multisubunit E3- ubiquitin ligase that targets Securin and Cyclin B1 for degradation mediated by the 26S proteasome. When chromosome bi-orientation is achieved, CDC20 can activate APC/C to cause degradation of securin that activates the enzyme separase resulting in separation of the sister chromatids that promotes a cell’s entry into anaphase. APC/C mediated degradation of Cyclin B1 causes CDK1 inactivation and ultimately mitotic exit (Vader et al., 2008) . The chromosomal passenger complex (CPC) plays a crucial role during chromosome alignment. The CPC recognizes incorrect chromosome-spindle interactions and generates unattached chromosomes that activate the SAC. The CPC is a multi subunit complex composed of the proteins Aurora
B and regulatory subunits inner centromere protein (INCENP), Survivin and Borealin.
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Second Gap Gap Phase Phase
Fig 1: Stages of the cell cycle. The cell cycle is broadly divided in two phases the interphase which is composed of first growth phase (G1) followed by DNA replication
(S) and the second growth phase (G2). The cells prepare for division in the interphase and undergo division during the M phase composed of mitosis (nuclear division) and cytokinesis (cytoplasmic division). Mitosis can be further broken down into several distinct phases namely prophase, prometaphase, metaphase, anaphase and telophase.
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Chromosomal Passenger Complex
2.1 Structure of the CPC
The CPC is an essential regulator of mitotic events. The enzymatic core of the
CPC is Aurora B kinase, Aurora B interacts with three regulatory subunits INCENP,
Survivin and Borealin. Aurora B is a highly conserved serine/threonine kinase with homologs in S.cerevesiae, C.elegans, D.melanogaster and several mammalian species.
Three Aurora kinases A, B and C are present in vertebrates with different functions and tissue specificity. Aurora B binds INCENP via a conserved IN box at the C- terminus of
INCENP. This binding activates Aurora B which phosphorylates INCENP at two conserved serine residues at the C-terminus which further activates the kinase in a feedback loop (Vader et al., 2008). Aurora C also appears to function as a part of the
CPC; however it is expressed most abundantly in the testis. Aurora A is widely expressed and plays various roles in centrosome maturation and ultimately affects formation of a bipolar spindle. INCENP was the first member of the CPC to be identified in a screen for novel components of the mitotic chromosomes (Cooke et al.,
1987). INCENP is a large, multi domain microtubule binding protein that binds and regulates Aurora B. INCENP binds Survivin and Borealin through its helical N terminus to form a stable quaternary CPC (Jeyaprakash et al., 2007). Survivin is a multifunctional protein with a domain characteristic of the inhibitor of apoptosis (IAP) family, the baculovirus IAP repeat (BIR) domain (Vader et al., 2006a).
Borealin also known as CDCA8/Dasra B is the most recently described component of the CPC in vertebrates. Borealin was identified in a proteomic screen for novel human proteins associated with histone depleted mitotic chromosomes (Gassmann
5 et al., 2004) and simultaneously in a screen for X.laevis chromosome binding proteins
(Sampath et al., 2004). Borealin is highly conserved in vertebrates and encoded by a single gene in mammals. X.laevis has two Borealin orthologs Dasra A and B, while
C.elegans contains a single ortholog CSC-1. Recently, a novel Borealin homolog Nbl1 was identified in fission yeast (Bohnert et al., 2009). Borealin is a basic protein composed of 280 amino acids that encompass two putative nuclear localization signals at the amino and carboxy terminus each. Borealin is essential to target endogenous CPC to the centromere, and the N- terminal half of Borealin interacts with the CPC in vivo (Vader et al., 2006b). The N-terminus of Borealin is sufficient to bind Survivin and INCENP and localize the CPC components to the spindle midzone and midbody and rescue cytokinetic defects. The C- terminal half of Borealin was characterized as a novel dimerization motif essential for efficient centromeric localization of the protein (Bourhis et al., 2009).
Borealin also binds DNA and, along with Survivin may act as a scaffold to recruit
INCENP bound Aurora B to the centromeres.
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Aurora B Survivin N C N INCENP C N Borealin IN Box
C
Fig 2: Structure of CPC. The CPC is composed of enzymatic core Aurora B and three regulatory subunits INCENP, Survivin and Borealin. Aurora B binds INCENP via the conserved IN box at the C-terminus of the large scaffold protein. The other two regulatory subunits Survivin and Borealin interact with each other and bind the helical N- terminus of INCENP to form the quaternary CPC.
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2.2 Localization and function of CPC
The chromosomal passenger proteins exhibit a dynamic localization pattern during mitosis. Initially they are present throughout the chromatin. However, during prometaphase they are displaced to the inner centromeric chromatin followed by their location to the microtubules of the central spindle at the metaphase-anaphase transition.
Finally, the chromosomal passenger proteins concentrate at the midbody during telophase and cytokinesis (Earnshaw and Bernat, 1991).The precise mechanism for this characteristic localization pattern of the CPC is unknown. The passenger proteins are mutually interdependent for their localization and stability in vivo since depletion of any one of the subunits results in diffuse cytoplasmic localization and degradation of the others (Musacchio and Salmon, 2007). The distinct localization pattern of the CPC correlates with the diverse functions of the complex. The CPC is targeted to the chromosome arms via interaction of INCENP with heterochromatin protein-1 (HP-1)
(Ainsztein et al., 1998). Displacement of HP-1 from mitotic chromosomes is mediated by Aurora B dependent phosphorylation of Histone H3 (Fischle et al., 2005);(Hirota et al., 2005). While at the centromeres, the passenger proteins play a major role in activation of the SAC by destabilizing incorrect kinetochore-microtubule interactions. Aurora B can phosphorylate kinetochore-localized-microtubule capture proteins like Ndc80 and influence the localization and function of the centromere associated kinesin MCAK
(Cheeseman et al., 2002);(Andrews et al., 2004). Aurora B selectively phosphorylates its substrates at incorrect kinetochore-microtubule attachments. Inhibition of Aurora B causes a failure to correct syntelic and merotelic attachments, which are characterized by a lack of tension at the kinetochore-microtubule interface. Recently, a spatial separation
8 model has been proposed to explain the role of Aurora B in the SAC. This model implicates the physical distance of Aurora B from its kinetochore substrates to be a major determinant in regulating microtubule stability. In the presence of incorrect kinetochore- microtubule attachments, a lack of tension allows phosphorylation of kinetochore substrates that are in close proximity to Aurora B kinase at the inner centromere.
Phosphorylation reduces affinity of substrates for microtubules, causing destabilization of incorrect attachments and subsequent activation of SAC. Upon bipolar attachment, kinetochores are moved away from the centromeres as a result of the spindle pulling forces. Under these conditions, Aurora B is unable to phosphorylate kinetochore proteins thereby stabilizing the bipolar attached state (Liu et al., 2009).
Localization of the CPC to the spindle midzone and midbody is essential for the proper function of the contractile ring and helps to ensure cytoplasmic division (Vader et al., 2008).Relocalization of the CPC from centromeres to central spindles at the metaphase-anaphase transition requires dynamic microtubules. INCENP binds to polymerized microtubules via its coiled-coil domain and interacts with β-tubulin via the amino terminal domain; this may play a role in localization of the CPC to the spindle midzone and midbody (Mackay et al., 1993). During anaphase, the CPC interacts with
MKLP1 and MKLP2, members of the kinesin super family. Aurora B mediated phosphorylation of centralspindlin, a complex of MKLP1 and male germ cell Rac-
GTPase activating protein (MgcRacGAP) recruits epithelial cell transforming gene
(ECT2) a Rho-guanine nucleotide exchange factor (RHO-GEF) to the cleavage plane.
ECT2 then catalyzes the formation of Rho-guanosine triphosphate (RHO- GTP) which accumulates at the cleavage plane. Further, RHO-GTP activates citron kinase; myosin
9 light chain kinase, a downstream target of citron kinase activates myosin to begin contraction of the cleavage furrow (Minoshima et al., 2003). The CPC also interacts with vimentin; Aurora B mediated phosphorylation of vimentin is required for cleavage furrow formation (Goto et al., 2006).
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Prometaphase Metaphase
Early Anaphase Anaphase Telophase
Blue : DNA Red: Borealin
Fig3: Localization of CPC. The passenger proteins exhibit a dynamic pattern of localization; initially decorating the chromosome arms at pro-metaphase, followed by their localization to the inner centromeres of a metaphase cell. The CPC finally concentrates at the spindle midzone during anaphase followed by their translocation to the midbody structure of a telophase cell.
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2. Regulation of Borealin
Several proteins involved in the G2/M phase of the cell cycle are regulated
transcriptionally, many of which are targets of the E2 promoter binding factor (E2F)
family of DNA-binding transcription factors (Harbour and Dean, 2000). The
Retinoblastoma gene (Rb) was the first tumor suppressor to be identified. The function
of Rb depends on interactions with the E2F family of transcription factors. The Rb
family consists of three members p130, p107 and p105Rb. These proteins play
overlapping roles in regulating the transcription of E2F target genes. The E2F sites are
present on promoters of many genes essential for cell cycle progression and Rb
functions to repress the transcription of these genes by interacting with E2F (Harbour
and Dean, 2000). The E2F family contains six classical E2Fs (E2F1-6) and two atypical
E2Fs (E2F7-8).The six classical E2Fs bind DNA as a heterodimer with the related
proteins DP1 and DP2. In addition, E2F1-3 appear to be mainly involved in activating
transcription whereas E2F4-6 repress transcription of E2F targets. The atypical E2Fs
bind DNA independently of DP proteins and lack both a Rb binding domain and a
transactivation domain. These atypical E2Fs appear to repress their targets in a Rb
independent manner likely by competing with activating E2F in binding to target
sequence(Cam and Dynlacht, 2003).
A number of genes repressed by Rb dependent pathways are highly expressed in
various cancers, for example plk1 and ki67 (Brown and Gatter, 2002). Further,
chromosomal passenger proteins Aurora B and Survivin are highly expressed in a
number of cancers. Borealin was also found to be over-expressed in gastric cancer
(Chang et al., 2006). We identified Borealin to be an E2F/Rb target via an AffymetrixR
12 microarray carried out to search for genes that failed to be repressed in cells lacking the
Rb family proteins p107 and p130. Similar to several other E2F targets, we found that
Borealin was over-expressed to varying levels in brain cancers, lymphoma and colon cancer (Date et al., 2007).
Borealin is regulated in a cell cycle dependent manner with increased levels of expression during G2 and mitosis. A higher level of Borealin protein was found in transiently transfected cells after they were arrested in mitosis with nocodazole. The transfected borealin is transcriptionally controlled by a constitutive CMV promoter, which shows constant activity throughout the cell cycle. Also levels of endogenous
Borealin increase when cells are blocked in mitosis with nocodazole (Kaur et al., 2007).
These results indicate that the Borealin may be stabilized during mitosis. Borealin was identified as a mitotic substrate of the small ubiquitin-like modifier (SUMO) system.
The sumoylation was cell cycle regulated, with Borealin-SUMO2/3 conjugates prominent during metaphase and progressively decreasing on anaphase onset. Borealin was specifically modified by the SUMO E3 ligase RanBP2 and desumoylated by the
SUMO specific isopeptidase SENP3. A mutant Borealin with all 25 lysine residues mutated to non-sumolyatable arginine interacted with other CPC components and showed similar subcellular localization pattern as wild type Borealin (Klein et al., 2009).
The SUMO and ubiquitin systems may concomitantly modify and regulate the stability of a target protein.
The ubiquitin system tags proteins for degradation mediated by the 26S proteasome. The process of ubiquitination involves an activating enzyme E1, a carrier enzyme E2 and a ubiquitin ligase E3. Two major classes of cell cycle regulated E3
13 ligases are the Skp1-Cul-Fbox (SCF) complexes that regulate entry into S phase and the
APC/C that regulates mitotic exit (Nandi et al., 2006). The APC/C links polyubiquitin chains in an ATP-dependent manner to its substrate molecules and targets them for degradation. The APC/C is composed of 13 subunits and is regulated by the binding of co-activator proteins and by phosphorylation. The APC/C targets different proteins for degradation at different times in the cell cycle. For example, Nek2A and Cyclin A are the early set of substrates for APC/C in the cell cycle. CDC20 and CDH1 are APC/C co-activators that belong to a family of WD40 repeat proteins (Passmore, 2004). CDC20 is a mitosis specific activator of the APC/C, while CDH1 is essential to maintain active
APC/C during late mitosis and early G1 phases. Phosphorylation of APC/C core subunits is essential to allow CDC20 to bind and activate APC/C. Phosphorylation of
CDH1 by CDK1 prevents binding and activation of APC/C by CDH1.The activators target distinct sets of substrates depending on the presence of specific recognition sequences. The CDC20 bound APC/C ubiquitinates substrates with a well-defined destruction box (D box) (R-X-X-L), whereas CDH1-APC/C recognizes a KEN box (K-
E-N) on the substrate. Ubiquitination plays an important role in several biological processes including cell cycle regulation. The role of ubiquitination in Borealin regulation has not been investigated.
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165 - 18 42 135 141 N C RXXL D BoxD D BoxD RXXL D BoxD Motif Motif Stabilization RXXLXXXX/D
Fig.4: Putative degradation and stabilization motifs identified in Borealin. The APC co-activators CDC20 and CDH1 bind to recognition sequences in the target protein known as the D box. The amino acid sequence of Borealin has two partial (RXXL) D boxes at residues 18-21 and 135-138 in addition to one complete (RXXLXXXX/D/N/E)
D box between amino acids 42-50, the influence of these motifs in Borealin regulation have not been tested. The sequence between amino acids 141-165 appears to contain a putative stabilization motif that protects Borealin from proteolytic degradation.
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4. Post translational modifications of Borealin
4.1 Phosphorylation of Borealin
Post-translational modifications of the chromosomal passenger proteins are essential in the regulation of the CPC. Phosphorylation is an important form of post- translational modification that regulates protein stability, function and localization. For example, phosphorylation of INCENP at its coiled-coil domain by CDK1 during metaphase and dephosphorylation by Cdc14 during anaphase is essential for localization of the CPC to the central spindles in yeast (Pereira and Schiebel, 2003). Also, phosphorylation at S197 of INCENP is essential for midbody localization of the protein during mitosis (Yang et al., 2007). Borealin is expressed in a cell cycle dependent manner and the levels of endogenous Borealin significantly increase during mitosis.
Human cells express two forms of Borealin, a slow migrating form and a faster moving electrophoretic species. Treatment of mitotic cells with a phosphatase led to the disappearance of the slower migrating electrophoretic species, while treatment with a phosphatase and phosphatase inhibitor restored the slow moving form confirming that the mobility shift of Borealin was caused by phosphorylation and this modification occurs during mitosis (Kaur et al., 2007).
More than 20 potential phosphorylation sites have been identified in Borealin
(Fig. 5). Ser 154, Ser 219, Ser 275 and Thr 278 of Borealin were found to be phosphorylated by Aurora B kinase in vitro. Aurora C could phosphorylate Borealin at
Ser 154 and Ser 165 in vitro (Hayama et al., 2007) ;( Slattery et al., 2008). Bioactive cell permeable peptides corresponding to the four Aurora B phosphorylation sites on
Borealin were added to an in vitro kinase assay of recombinant Borealin and Aurora B.
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Treatment with these peptides reduced the phosphorylation of Borealin mediated by
Aurora B in vitro. Further, treatment of cells with bioactive peptide corresponding to
Borealin 261-280 affected the stability of the protein (Hayama et al., 2007). However, treatment of cells with Aurora B inhibitor ZM447439 or VE465 did not eliminate or decrease the slow migrating phosphorylated form of mitotic Borealin. This suggests that
Aurora B is not responsible to generate the phosphorylated form of mitotic Borealin
(Kaur et al., 2007). Borealin was phosphorylated at T88, T94, T169 and T230 by Mps1 kinase in vitro. Severe defects in chromosome alignment seen upon Borealin depletion were rescued upon expression of a phosphomimetic mutant Borealin- 4TD mutant.
Also, expression of Borealin-4TD but not Borealin- WT was efficient in restoring chromosome alignment defects caused by Mps1 depletion (Jelluma et al., 2008).
Mass spectrometric analysis of microtubule associated proteins identified
Borealin S219 to be phosphorylated in vivo (Nousiainen et al., 2006). Further, mutation of S219 to non-phosphorylatable S219A eliminated the slow migrating phosphorylated form of mitotic Borealin. This indicates that phosphorylation of S219 is responsible for the mitotic mobility shift of Borealin (Nigg, 2001). Expression of S219A mutant was unable to efficiently rescue the multinucleated phenotype that occurs upon depletion of endogenous Borealin (Kaur and Taylor, submitted). Also, phosphorylation at S219 was found to be essential for complete activation of the spindle assembly checkpoint in response to the spindle poison taxol (Kaur and Taylor, submitted). Aurora B is essential for correction of chromosome misattachments at the centromeres. S219 phosphorylation of Borealin may be essential in localization or activity of Aurora B at the centromeres and hence activation of the SAC. Thus, phosphorylation of Borealin at S219 may be
17 required for chromosome segregation and completion of cytokinesis (Kaur and Taylor, submitted).
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*S23 *T199 **S165 ** S215 ** S219 *S194 *S154 S180 N T106 C S15 S110 T169 T204 T189 Y212 S224 S244 S274 S164
Fig5: Phosphorylation sites mapped in Borealin. Phosphorylation sites were identified by searching online databases (Phosida and Phosphosite).These sites catalogue phosphorylated peptides identified from cell lysates using mass spectrometry. * Sites that are conserved in mammals, fish and frogs. ** Also conserved in C.elegans. In addition to these sites T88, T94, T169 and T230 of Borealin that are phosphorylated by Mps1 kinase in vitro. S154, S215, S219 and T278 of Borealin were found to be phosphorylated by
Aurora B kinase. Aurora C could also phosphorylate Borealin at S154 and S165 in vitro.
19
4.2 Proline directed Serine/ Threonine kinases
The S219 residue of Borealin is immediately followed by a proline. One of the candidate proline directed kinases that may phosphorylate Borealin at S219 is CDK1.
The CDKs are a family of serine/threonine kinases that exert their effects on cell-cycle events by phosphorylating a large number of proteins in the cell. These CDK substrates are typically phosphorylated at serine (S) or threonine (T) residues followed by a proline
(P), hence the phosphorylation site for CDKs is [S/T] PX [K/R]. Activation of CDK1 is critical for the initiation of mitosis in human cells, and this requires association of the catalytic subunit CDK1 with the regulatory subunit Cyclin B1. Activated CDK1-Cyclin
B1 complexes phosphorylate nuclear lamins, kinesin-related motors and condensins among other proteins. These events are essential for nuclear envelope breakdown, centrosome separation, spindle assembly and chromosome condensation respectively
(Nigg, 2001). Cyclin levels fluctuate during the cell cycle rising through the S and G2 phases and peaking in mitosis. In contrast, CDK levels are unchanged throughout the cell cycle. CDK1 is held inactive before mitosis by inhibitory phosphorylation at Thr14 and
Tyr15 catalyzed by Wee1 and Myt1 kinases. In G2, rapid dephosphorylation at these inhibitory sites mediated by Cdc25 results in the activation of CDK1. Cyclin-CDK complexes are regulated by the proteolysis of cyclins at specific stages. Inactivation of
CDK1-Cyclin B1 is essential for mitotic exit and occurs via ubiquitin dependent proteolysis of Cyclin B1. Other potential Borealin kinases include the Mitogen Activated
Protein Kinase (MAPK). The MAP kinases comprise of a family of ubiquitous proline- directed serine/threonine kinases. They are major components of pathways controlling embryogenesis, cell differentiation, cell proliferation and cell death (English and Cobb,
20
2002). They are also regulated by phosphorylation cascades. Some of the most well studied members of the MAP kinase family include Extracellular signal related kinases
(ERK) 1 and 2, MAP/ERK kinases (MEK) 1 and 2, c-Jun N-terminal kinase (JNK) and p38 kinases. Raf Kinase Inhibitory Protein regulates the mitotic spindle assembly checkpoint by controlling Aurora B Kinase activity in Raf/MEK/ERK signaling dependent manner (Rosner, 2007). Another important family of mitotic kinases is Polo like kinases (Plks) which are serine/threonine kinases conserved in all eukaryotes. Plk has homologs in S.cerevisiae (Cdc25p), S.pombe (Plo1p), X.laevis (Plx1) and mammals
(Plk1-3). Of the three mammalian homologs, Plk1 plays roles in multiple mitotic events, where as the functions of Plk2 and Plk3 are not well defined. The activity of Plk1 is cell cycle regulated with an increase during mitosis. Plk1 is known to interact with Survivin during mitosis. Further, Plk1 mediated phosphorylation of Survivn at S20 is essential for accurate chromosome alignment and cellular proliferation (Colnaghi and Wheatley,
2010). Also, during mitosis Plk1 forms a complex with INCENP phosphorylated by
Aurora B and CDK1. The complex formation mediates PLK1 localization to the kinetochores and is important for chromosomal dynamics (Goto et al., 2006). Plk1 exhibits dynamic intracellular localization through the cell cycle. It is known to associate with spindle poles, kinetochores, central spindle, midzone and the midbody. Plk1 is regulated by phosphorylation and may be subject to proteolytic degradation mediated by
APC/C/C (Nakajima et al., 2003).
21
4.3 Dephosphorylation of Borealin
Borealin is phosphorylated during mitosis, dephosphorylation occurs two hours post mitotic exit as confirmed by a decrease in the slow migrating phosphorylated species and CyclinB1 degradation (Kaur et al., 2007). Treatment of asynchronous cells blocked in S phase with cyclohexamide induced the phosphorylation of Borealin. Further, treatment of interphase cells with a broad spectrum phosphatase inhibitor, sodium fluoride (NaF) induced an increase in the phosphorylated form of Borealin. This indicates that Borealin may be constantly phosphorylated during the cell cycle; however it appears to be rapidly dephosphorylated potentially by a labile phosphatase active only during the interphase. Phospho Protein Phosphatase 1-7 (PPP1-PPP7) is a family of okadaic acid sensitive phosphatases. PPP’s play an important role in mitotic spindle assembly and their activity is cell cycle regulated (Tournebize et al., 1997). However treatment of asynchronous or S phase cells with okadaic acid or cyclosporine did not have any effect on the migration of Borealin. This suggests that Borealin is not dephosphorylated by members of this family of phosphatases (Kaur et al., 2007).
Cdc14 belongs to a family of dual specificity Serine/Threonine phosphatases, active during interphase and inactive during mitosis (Wan et al., 1992). In yeast, Cdc14 is essential for multiple anaphase events and most importantly for the inactivation of CDK substrates during exit from mitosis (Visintin et al., 1998).Cdc14 related phosphatase-1
(Clp1), a Cdc14 homolog of fission yeast is a bona fide interacting partner of the CPC, which co-localizes with the chromosomal passengers during mitosis (Bohnert et al.,
2009). There are two Cdc14 homologs detected in humans Cdc14A and Cdc14B, whose function in cell cycle progression is not completely understood. Cdc14A and Cdc14B
22 share a high sequence homology, with a highly conserved N-terminal catalytic domain and a non-conserved C-terminal domain. The protein levels as well as the phosphatase activity of the Cdc14’s fluctuate slightly throughout the cell cycle (Kaiser et al., 2002).
Cdc14A shows centrosomal localization during interphase, and plays a role in the regulation of centrosome cycle (Kaiser et al., 2002). Cdc14A dephosphorylates Cdh1, thereby activating APC/C Cdh1, which ubiquitinates cyclins resulting in mitotic exit
(Bembenek and Yu, 2001). Much less is known about the function of Cdc14B, which localizes to interphase nucleoli and to the spindle apparatus during mitosis. The dramatic differences in their localization profiles suggest that Cdc14A and Cdc14B perform different tasks in the cell (Hansen et al., 2008). Both Cdc14A and Cdc14B have been reported to interact with and dephosphorylate tumor suppressor p53, specifically at a
CDK phosphorylation site serine 315 (Li et al., 2000). Cdc14B displays microtubule bundling and stabilizing activities and may play a role in modulating spindle dynamics during mitosis (Cho et al., 2005). By analogy to yeast Cdc14, in mammalian cells
Cdc14B may regulate mitotic exit by dephosphorylating CDK1 substrates (North and
Verdin, 2007). Despite these intriguing initial observations the roles of Cdc14A and B have been recently called into question. Homozygous deletion of Cdc14B did not produce spindle defects or increase the mitotic index. Further, Cdc14B null cells degraded securin and cyclin B and completed mitosis normally (Berdougo et al., 2008).
23
In the present study we demonstrate that Borealin is down–regulated in response to DNA damage by an indirect multi-component system. The regulation of Borealin is mediated by the Rb/E2F pathway in a p53 and p21/waf1 dependent manner. Further, we reveal that Borealin is subjected to proteasome mediated degradation and an E3 ligase other than APC/C may be responsible for targeting Borealin to the proteasome. We also provide evidence that Borealin is phosphorylated in vivo in response to CDK1 over- expression, however in vitro Borealin is not an optimal CDK1 substrate. In addition,
CDK1 may exert an indirect effect to modify the phosphorylation status of mitotic
Borealin. We also show that dephosphorylation of Borealin occurs after mitotic exit and may be mediated by Cdc14 A phosphatase. Overall, these studies have revealed that
Borealin is subjected to various forms of regulation and post translational modifications.
24
II. Hypothesis
We predict that Borealin is regulated by an Rb dependent pathway and down- regulation of the protein in response to DNA damage is mediated by tumor suppressor p53. Since Borealin is stabilized during mitosis, we hypothesize that it is a target of the
26S proteasome which may be ubiquitinated prior to degradation. Borealin is phosphorylated at S219 during mitosis to produce a slow migrating electrophoretic species. We theorize that the phosphorylation at S219 is mediated by a proline directed serine/threonine kinase which may be active during mitosis. To identify the enzyme, we carried out in vitro kinase assays and tested the effect of overexpression as well as inhibition of various mitotic kinases on the phosphorylation status of mitotic Borealin.
Borealin is dephosphorylated upon mitotic exit; we predict the Cdc14 families of dual specificity phosphatases mediate this dephosphorylation of Borealin.
25
III. Materials and Methods
1. Culture conditions and cell lines
Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Cellgro) with 10% fetal bovine serum (FBS) and penicillin/streptomycin in a humidified atmosphere of 10% CO2 and a temperature of 37°C. Two types of mouse embryonic fibroblasts (MEF) were used, wild type and a cell line lacking all the three Rb family proteins p130, p107 and p105. The other cell lines were primarily derived from various human tissues. HCT116 cell lines were derived from human colon carcinomas. Four
HCT116 strains were used. Two parental cell lines with wild type p53 (p53+/+) and wild type p21 (p21+/+), two additional cell lines with both p53 alleles (p53-/-) and both p21
(p21-/-) alleles knocked out by homologous recombination. The HT1080 cell line was derived from a fibrosarcoma and contains wild type p53. HEK293 cell line is an embryonic kidney derived cell line, while HeLa M was derived from cervical cancer tissue.
2. Drug treatments
Chemicals were obtained for Sigma-Aldrich unless otherwise mentioned.
Nocodazole was used at a concentration of 200 ng/ml, hydroxyurea at 2 mM, and okadaic acid at 100 nM. The DNA damage agent Adriamycin® was used at a concentration of 0.2
µg/ml, Cisplatin at 1 µg/ml and Nutlin-3 (Calbiochem) at a concentration of 10µm. All
26 the three drugs were dissolved in dimethyl sulfoxide (DMSO). A lower concentration of nocodazole (100 ng/ml for 16 hours) was used in experiments to synchronize cells and was later removed by multiple washes with phosphate buffered saline (PBS). CDK inhibitor purvalanol (Alexis Biochemicals), Aurora-B kinase inhibitor ZM 447439 (Astra
Zeneca) and Mitogen Activated Protein Kinase (MAPK) inhibitor PD 98059 (Sigma-
Aldrich) were used at the following concentrations 10 µm, 2.5 µm, 50 µm and 10 µm respectively. Phosphatase inhibitor sodium fluoride (NaF) was dissolved in water and used at a concentration of 5 mM. Proteasome inhibitor MG132 was dissolved in DMSO and used at a concentration of 5 or 10 µm as required.
3. Antibodies
Antiserum to endogenous Borealin (Proteintech Group Inc.) was used at a dilution of 1:5000. Antibodies to PLK-1 and HRP conjugated Flag-tag Borealin (Bethyl
Laboratories) were used at a dilution of 1:1000 and 1:4000. Affinity-purified rabbit antiserum raised against a phospho-peptide encompassing S219 Borealin (Protein Tech
Inc.) was used at a dilution of 1:50. Antibodies to β-actin (Neomarkers) were used at a dilution of 1:4000 while antibodies to INCENP, Cyclin B1, CDK1, Cdc14A and HRP conjugated anti-p53 antibodies (Santa Cruz Biotechnologies) were used at a dilution of
1:1000, 1:200, 1:1000, 1:100 and 1:2000 respectively. Goat anti-mouse and goat anti- rabbit secondary antibodies conjugated to HRP from Santa Cruz Biotechnologies or Bio
Rad laboratories were used at a dilution of 1:1000 or 1:10,000 respectively.
27
4. Western blotting
Buffer composed of 50 mM Tris (pH 8.0), 150 mM NaCl, 1.0% NP-40, 1mM phenyl methane sulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), protease inhibitors
(1µg/ml aprotinin, 2 µg/ml leupeptin 1 µg/ml pepstatin) and phosphatase inhibitors (1 mM sodium fluoride and 1 mM sodium vanadate) was used to lyse cells on ice. Cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (12.5% acrylamide at a ratio of 37.5:1 acrylamide: bisacrylamide). For better separation of phosphorylated and unphosphorylated Borealin, cell lysates in certain experiments were separated using 12.5% acrylamide with a ratio of 29.2:0.8 acrylamide: bisacrylamide. The proteins were transferred on a polyvinyldifluoride (PVDF) membrane obtained from Millipore. Immunoblots were blocked in buffer composed of 0.05% (v/v)
Tween and 5 %(w/v) non-fat dry milk in PBS, antibodies for the immunoblots were diluted in the same buffer. The signal was detected by enhanced chemiluminesence
(Thermo Scientific).
5. Transient transfections
Transient transfections were carried out by transfecting HeLa M cells using
Fugene 6 ® (Roche) or Expressfect ® (Denville Scientific Inc). The transfection reagent and DNA were used at a ratio of 3:1 as per manufacturer’s instruction. DNA and transfection reagent was suspended in antibiotic and serum free DMEM to form DNA and polymer complexes respectively. The polymer complex was added to the DNA complex, the resulting solution vortexed and incubated at room temperature for 20 minutes before being added to the corresponding cells. The cells were incubated at 37°C;
28 complete DMEM was added 20 hours post transfection and cells incubated for another 24 hours prior to harvesting. Stable cell line expressing Flag-tagged wild type Borealin
(WT8) was generated previously in our laboratory (Harpreet Kaur, PhD thesis). Flag- tagged deletion mutants of Borealin (Bor-141, Bor-168, Bor-191 and Bor-221) were constructed formerly as mentioned in Harpreet Kaur’s PhD thesis.
6. Immunofluorescence
For immunofluorescence microscopy, HeLa M cells grown on coverslips were fixed with 4% formaldehyde and permeabilized with 1% sodium dodecyl sulfate (SDS) made in pre-warmed 1% PBS. The cells were blocked with PBS containing 0.1% bovine serum albumin (BSA) for 1 hour at room temperature. The cells were then incubated with a phospho S219 Borealin (1:50 dilution) and INCENP (1:1000 dilution) antibodies overnight at 4°C. Cells were then incubated with Alexafuor-568 conjugated goat anti rabbit (Santacruz Biotechnologies) and Alexafuor-488 conjugated goat anti mouse
(Sigma-Aldrich) at a dilution of 1:300 for 1 hour at room temperature. Nuclei were stained with 4’, 6- Diamidino-2-phenylindole (DAPI) and coverslips mounted using
Vectashield (Vector Laboratories). Images were analyzed using an Axiophot fluorescence microscope. In other experiments HeLa M cells were transiently transfected with constructs encoding either Flag-tagged wild type or S219A mutated versions of borealin.
24 hours post transfection cells were fixed, permeabilized and blocked as mentioned above. Blocked cells were incubated with a phospho S219-Flag (1:50 dilution) and M2 anti-flag (1:250 dilution) for 2 hours at 37°C. Cells were then incubated with Alexafuor-
568 conjugated goat anti rabbit and Alexafuor-488 conjugated goat anti mouse at a
29 dilution of 1:300 for 1 hour at room temperature. Nuclei were stained, cells mounted and analyzed as per the above mentioned procedure.
7. In vitro kinase Assays
Wild type Borealin and Borealin S219A were expressed and purified from E.coli as GST-Borealin and GST-S219A fusion protein using a pGEX-3X vector. Recombinant
GST-Borealin and GST- Borealin S219A were phosphorylated in vitro with 25 ng of purified CDK1/Cyclin-B1 (Cell Signaling Technology) in kinase buffer ( 20 mM HEPES pH 7.9, 5 mM MgCl2 ,10% glycerol ,100 mM DTT,10 mM ATP) in the presence 5-10
µCi of Ƴ (32P) ATP. The reaction was incubated at room temperature for 30 minutes and stopped by adding Stewart lamelli buffer (2% SDS, 100mM Tris, 0.05% BPB, 30% glycerol) and boiling for 5 minutes. The proteins were resolved by SDS-PAGE, stained with Comassie Blue, dried in a gel dryer and visualized using a Typhoon Phosphor
Imager®.
Amino terminal biotinylated peptides corresponding to Borealin S219
GNGSPLADAK, Borealin S219A GNGAPLADAK and CDK1 optimal substrate
HATPPKKKRK were obtained from Synthetic Biomolecules at a purity of 95%. Borealin
S219, Borealin S219A and CDK1 substrate at a concentration of 1 µm each were phosphorylated in vitro as per the protocol mentioned above. A PVDF membrane
(Millipore) was saturated with 500 µg of avidin followed by blocking in 0.5% BSA for 1 hour at room temperature each. The in vitro kinase reactions were spotted on pre-treated
PVDF membrane and incubated for 1 hour at room temperature. The PVDF membrane
30 was washed in an Isotemp incubator (Fischer Scientific) and reactions visualized by autoradiography.
8. Generation of recombinant adenoviruses
Recombinant adenoviruses encoding wild type Cyclin B1 (WTB1), nuclear
Cyclin B1 (NB1) and mutant Cyclin dependent kinase-1 (CDK1-AF) downstream of a tet operator , a minimal CMV promoter and a tetracycline transactivator (TTA) upstream of the CMV promoter were obtained (Taylor et al., 2000). Recombinant adenoviruses were amplified in HEK 293 cells and resuspended in 10% glycerol in PBS. The recombinant adenoviruses were serially diluted and used to infect HEK 293 cells seeded in a 96 well plate that was incubated at 37°C for 24 hours. The plaques formed were stained with 50% ethanol saturated with methylene blue; air dried and counted to determine the viral titre.
HeLa M cells were infected with the TTA virus plus the desired NB1, WTB1 or CDK1-
AF virus or viruses, each at a multiplicity of infection (MOI) of 50 plaque forming units per cell. The infected cells were incubated for 20-24 hours at 37°C prior to harvesting.
Wild type PLK-1 adenovirus and Pad CMV null adenovirus were obtained from Glaxo
SmithKline Pharmaceuticals Ltd. Recombinant adenoviruses were amplified and titrated as per the protocol mentioned above. HeLa M cells were infected with the WT PLK-1 or
CMV virus. The infected cells were incubated for 48 hours at 37°C prior to harvesting.
9. Immunoprecipitation
Asynchronous WT8 cells were lysed with buffer containing 50 mM Tris (pH 8.0),
400 mM NaCl, 0.5% NP-40, 0.1% deoxycholate, 1 mM PMSF, protease inhibitors (1
31
µg/ml aprotinin, 2 µg/ml leupeptin 1 µg/ml pepstatin) and phosphatase inhibitors (1 mM sodium fluoride and 1 mM sodium vanadate). 8 µg/ml anti ubiquitin antibodies and 2
µg/ml anti p53 antibodies (Santa Cruz Biotechnology) coupled to protein-G magnetic beads were added to the cleared lysates and incubated overnight at 4°C with constant agitation. The beads were washed thrice with lysis buffer and lysates separated via SDS-
PAGE. The transfer was probed with HRP conjugated antibody to flag-tagged Borealin
(Bethyl Laboratories).
Asynchronous WT8 cells treated with or without the proteasome inhibitor MG132 were lysed in buffer composed of 50 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl,
1% Triton X® -100, 10 mM N-ethylmalamide, protease inhibitors (1 µg/ml aprotinin, 2
µg/ml leupeptin 1 µg/ml pepstatin) and phosphatase inhibitors (1 mM sodium fluoride and 1 mM sodium vanadate). The lysates were immunoprecipitated with polyubiquitin affinity resin® or control resin (Calbiochem). Affinity matrix bound to the protein was resuspended in 2X gel loading buffer composed of 250mM Tris HCl, pH 6.8, 4% SDS,
10% β-mercaptoethanol, 20% glycerol and bromophenol blue. The immunoprecipitates were separated by SDS- PAGE and transfers probed with an HRP conjugated antibody to flag- tagged Borealin (Bethyl Laboratories).
10. Generation of Cdc14 depleted cell lines
For the production of lentiviral particles containing the shRNA directed against
Cdc14A, HEK293T cells were transfected with 3rd generation packaging system comprising of two packaging plasmids pRRE and pRSV-Rev, an envelope plasmid pCMV-VSVG and lentiviral plasmid Plko.1 using Expressfect®. The cells were grown in
32 a high serum growth media for 24 hours and lentiviral particles harvested 40 hours post transfection. For generation of Cdc14A depleted stable cell lines, HeLa M cells were infected with lentiviral particles encoding shRNA directed against Cdc14A using polybrene. Selection was begun 48 hours post infection with puromycin (1 µg/ml). Cells stably lacking the Cdc14A protein were pooled to obtain a Cdc14A knock down cell line two weeks post infection. Cdc14B knock out cell lines were obtained via homozygous deletion of the Cdc14B locus as described by Berdugo et al (Berdougo et al., 2008)
11. Analysis of DNA Synthesis
5-Bromo-2-deoxyuridine (BrdU; Sigma) was added to cells for 1.5 h. The cells were fixed in 70% ice-cold ethanol, incubated in 0.08% pepsin (Sigma) in 0.1 M HCl for
20 min at 37°C. Fixed cells were collected by centrifugation, incubated in 2 M HCl for
20 min at 37°C and in 0.1M sodium borate for 10 seconds and then incubated with anti-
BrdU-FITC (Becton Dickinson, San Jose, CA) for 30 min. Staining was analyzed by
Immunofluoresence microscopy.
33
IV. Results
1. Regulation of Borealin
1.1 Borealin is downregulated upon DNA damage
DNA damage induces high levels of p53 and mediates down-regulation of a large number of genes involved in the cell cycle. We wanted to analyze the effect of DNA damage on the regulation of Borealin. HCT116 cells with wild type p53 were treated with the DNA damaging agents adriamycin or cisplatin. We observe down-regulation of
Borealin within 48 hours of adriamycin treatment and a corresponding up regulation of p53 in HCT116 cells (Fig. 6A). Treatment of HCT116 cells with cisplatin induced p53 within 24 hours, however down-regulation of Borealin was observed only after 72 hours of treatment (Fig.6B). In contrast, HT1080 cells with wild type p53 required extensive adriamycin treatment (72 hours) for Borealin down-regulation, while cisplatin mediated a decrease in Borealin levels within 24 hours of treatment. A strong p53 induction was seen in response to adriamycin and cisplatin (24 hours) in HT1080 cells (Fig.7). The results indicate that DNA damaging agents have different abilities to down regulate Borealin expression, and these differences are cell type dependent.
34
(A) 0 24 48 72 Hours post Adriamycin p53
Borealin
β actin
(B) 0 24 48 72 Hours post CisCisplatin p53
Borealin
βactin
Fig 6: The effects of DNA damage on the levels of p53 and Borealin. HCT116 cells wild type for p53 were treated with DNA damaging agents. The lysates were separated on a 12.5% polyacrylamide gel and immunoblotted with rabbit antisera to endogenous
Borealin. The transfers were stripped and reprobed with HRP conjugated antibody to p53 and for β-actin as a loading control. (A) HCT116 p53+/+ cells were maintained as untreated control (0 hour) or treated with adriamycin (0.2 µg/ml) for 24, 48 and 72 hours respectively (B) HCT116 p53+/+ cells were maintained as untreated control (0 hour) or treated with cisplatin (1 µg/ml) for 24, 48 and 72 hours respectively.
35
0 24 48 72 0 24 48 72 Hours post treatment Borealin
p53
β actin
Adriamycin Cisplatin
Fig 7: The effect of DNA damage on Borealin levels in HT1080 cells. HT1080 p53+/+ cells were treated with adriamycin (0.2 µg/ml) or cisplatin (1 µg/ml), with one untreated control (0 hour) each, followed by 24, 48 and 72 hours of treatment respectively. The lysates were separated a 12.5% polyacrylamide gel and immunoblotted with rabbit antisera to endogenous Borealin. The transfers were stripped and reprobed with HRP conjugated antibody to p53 and then with an antibody to β-actin to serve as a loading control.
36
1.2 Borealin is downregulated in a p53 dependent manner
To analyze if the down-regulation of Borealin in response to DNA damage was due to the high levels of p53 induced, we exposed HCT116 cells with wild type p53 to Nutlin 3, a small molecule inhibitor of the hDM2-p53 interaction. Nutlin 3 up-regulated p53 in
HCT116 cells leading to a rapid down-regulation of Borealin within 24 hours of treatment. Borealin was not down-regulated when p53-null HCT116 cells were exposed to Nutlin 3 confirming the specificity of this effect (Fig.8A). p53 can cause repression of cell cycle genes by inducing its downstream effector p21/waf1. Borealin was efficiently downregulated when HCT116 cells with wild type p21/waf1 were treated with Nutlin 3 for 24 hours. Borealin was not downregulated when HCT116 cells lacking p21 were treated with Nutlin 3 (Fig. 8B). These data confirm that down-regulation of Borealin in response to DNA damage is mediated by high levels of p53 and its target p21/waf1.
37
(A)
0 24 72 96 0 24 72 96 hours post Nutlin 3 Borealin
p53
β actin
HCT116 p53 +/+ HCT116 p53 -/-
(B)
0 24 48 0 24 48 hours post Nutlin3
Borealin
β actin
HCT116 p21+/+ HCT116 p21-/-
Fig 8: p53 and p21/waf1 are required for Borealin down-regulation in response to
DNA damage. To test the role of p53 in the down-regulation of Borealin, cells were treated with Nutlin 3 (10 µm). (A) HCT116 p53+/+ and p53-/- cells were treated with
Nutlin 3 for 24, 72 or 98 hours. The lysates were separated on a 12.5% polyacrylamide gel and immunoblotted with rabbit antisera to endogenous Borealin. The transfers were stripped and reprobed with antibodies to p53 and β-actin (B) HCT116 p21+/+ and p21-
/- cells were subjected to Nutlin 3 treatment for 24 and 48 hours. The cell lysates were resolved by SDS-PAGE and the membrane probed for Borealin and β-actin.
38
1.3 Borealin is downregulated in an Rb dependent manner
We identified Borealin as an E2F/Rb target by using an AffymetrixR microarray experiment carried out to search for genes that failed to be repressed in cells lacking the
Rb family proteins p107 and p130. To further investigate the role of the Rb family in the down-regulation of Borealin we treated wild type (WT) mouse embryonic fibroblasts and p130/107/105Rb-null (TKO) mouse embryonic fibroblasts with adriamycin. In WT cells,
Borealin was repressed within 24 hours of adriamycin treatment whereas repression in the TKO cells only occurred after 48 hours (Fig. 9). This suggests that the Rb family of protein is involved in the down-regulation of Borealin.
Thus, down-regulation of Borealin in response to DNA damage appears to be mediated by an indirect, multi-component system. DNA damage activates tumor suppressor p53; high levels of p53 induce its downstream effector p21/Waf1. p21/waf1 functions by inhibiting multiple CDK’s which would normally inactivate Rb family of proteins. In the poorly phosphorylated form, Rb/E2F complexes act as potent repressors of essential cell cycle genes, which may include Borealin. In support of this hypothesis, a global factor binding analysis has identified p130 and E2F4 bound to the borealin promoter in vivo (Alvarez et al., 2001). In addition, overexpression of a constitutively active form of CDK2 was able to abrogate the suppression of Borealin by high levels of p53 (Date et al., 2007).
39
0 24 48 0 24 48 hours post Adriamycin
Borealin
β- -actin
TKO WT
Fig 9: Borealin is down-regulated in a Rb-dependent manner. To study the role of the
Rb family in Borealin down-regulation we treated wild type (WT) and p130/107/105Rb- null (TKO) mouse embryonic fibroblasts with adriamycin (0.2 µg/ml) for 0, 24 and 48 hours. Cell lysates were separated by SDS-PAGE and the level of Borealin was determined by immunoblotting with antisera to endogenous Borealin. Transfers were stripped and reprobed for β-actin as a loading control.
40
1.4: Effect of proteasome inhibition on the levels of Borealin
The levels of endogenous Borealin increase when cells are blocked in mitosis, indicating that the Borealin may be stabilized during this stage of the cell cycle (Kaur et al., 2007). To analyze the factors involved in stabilization of mitotic Borealin, we treated cells with the proteasome inhibitor MG132. We observed a consistent increase in the levels of endogenous Borealin upon proteasome inhibition of S phase (HU) but not mitotic cells (NOC) (Fig. 10A). This suggests that Borealin is subjected to proteasome mediated degradation during interphase but may be protected from the proteasome during mitosis. To identify the region of Borealin that mediates its degradation by the proteasome, we tested the stabilization of several mutants upon proteasome inhibition.
We treated interphase cells over expressing WT- Borealin, Bor-141, Bor-168, Bor-191 and Bor- 221 with the indicated concentration of MG132 for 12 hours. All truncation mutants were up-regulated after adding MG132. Bor-141 showed a particularly dramatic up-regulation after proteasome inhibition. Interestingly, Bor-168 was upregulated to a similar extent as wild-type Borealin (Fig.10B). In another experiment, MG132 was added simultaneously with HU to block cells from entering mitosis. We obtained similar results when HU was added first for 8 hours before the addition of MG132 for 14 hours (Fig.
11A). In the latter case, there should be a minimal chance of trapping any cells in mitosis with MG132. These observations suggest that the region between amino acids 141 and168 protect the protein from proteolytic degradation.
41
(A)
HU NOC
DMSO MG132 DMSO MG132
Endo Borealin
β actin
(B)
UT WT-Bor Bor - 141 Bor-168 Bor-191 Bor--221 DMSO MG DMSO MG DMSO MG DMSO MG DMSO MG DMSO MG Hydroxyurea
Flag Borealin
β actin
Fig 10: Effect of proteasome inhibition on the levels of Borealin. (A) HeLa M cells were blocked in S phase with hydroxyurea (2 mM) or mitosis with nocodazole (200 ng/ml) with or without proteasome inhibitor MG132 (5 µM) for 14 hours. The lysates were separated on a 12.5% polyacrylamide gel and immunoblotted with rabbit antisera to endogenous Borealin. The transfers were stripped and reprobed with an antibody to β
42 actin. (B) HeLa M cells were transfected with flag-tagged WT-Borealin, Bor-141, Bor-
168, Bor-191or Bor-221.The cells were blocked in S phase with hydroxyurea (2 mM) and treated with proteasome inhibitor MG132 (5 µM) for 14 hours. The lysates were separated on a 12.5% polyacrylamide gel and immunoblotted with antibody to Flag-
Borealin followed by an antibody to β actin.
43
(A)
Bor-168 Bor-141 WT-Bor UT DMSO MG DMSO MG DMSO MG DMSO MG Hydroxyurea
Flag Borealin
β actin
(B)
6 5 4 3 2
Fold Induction Fold 1 0 WT-Bor Bor-141 Bor-168
Fig 11. Effect of proteasome inhibition on levels of Borealin (A) HeLa M cells were transfected with flag-tagged WT-Borealin, Bor-141 and Bor-168. The cells were blocked in S phase with hydroxyurea (2 mM) for 8 hours followed by treatment with
44 proteasome inhibitor MG132 (5 µM) for 14 hours. The lysates were separated on a
12.5% polyacrylamide gel and immunoblotted with antibody to Flag-Borealin followed by an antibody to β actin. (B) Quantification of Borealin stabilization upon proteasome inhibition
45
1.5 Association of Borealin with Ubiquitin
A number of proteasomal targets are subjected to ubiquitination prior to being degraded. To determine whether Borealin is ubiquitinated before degradation we analyzed WT8 cells which are HelaM cells stably transfected with flag-tagged wild type
Borealin. Asynchronous WT8 cells were transfected with HA-tagged ubiquitin, followed by immunoprecipitation with an anti-ubiquitin antibody. We did not observe the characteristic smear seen normally for ubiquitinated proteins. However, we noted the presence of a band in the immunoprecipitate lane corresponding to a higher molecular weight form of Borealin (Fig.12A). Similar results were obtained when asynchronous
WT8 cells pre- treated with MG132 were subject to immunoprecipitation with polyubiquitin affinity resin ®. The presence of a single band with a molecular weight between 40-50kd suggests that Borealin may be conjugated to one or more ubiquitin moieties prior to degradation (Fig.12B). However, the functional importance of this post translational modification of Borealin is unidentified. The amino terminus of Borealin posses three characteristic D box’s (RXXL) recognized by the APC/C co-activator Cdh1.
To analyze the role of APC/C in the proteolytic degradation of Borealin, we transfected cells with plasmids encoding Cdc20 and Cdh1. Transfection with the mitosis specific activator Cdc20 did not affect the levels of Borealin (Fig.13A). Transfection of varying concentrations of Cdh1 causes only a slight reduction in the levels of Borealin (Fig.13B).
Multiple attempts to detect the transfected protein failed. These results indicate that an E3 ligase other than APC/C may target Borealin for proteasomal destruction.
46
(A)
Input Borealin Input Borealin α Ubiquitin α p53 170 135 100
72
55
40 Flag Bor 33 Full length Bor
(B)
Input Control Polyubq MG132 170 beads beads 130 100 72 55
43 Flag Bor
34 Full length Bor 26
47
Fig 12: Association of Borealin with ubiquitin (A) Asynchronous WT8 cells were transfected with HA tagged ubiquitin. The lysates were immunoprecipitated using anti- ubiquitin or anti-p53 antibody as a negative control. The immunoprecipitates were separated via SDS-PAGE and transfers probed with antibody to flag-tag Borealin. (B)
Asynchronous WT8 cells treated with the proteasome inhibitor MG132 or DMSO were immunoprecipitated with polyubiquitin affinity resin ® or control resin (Calbiochem).
The immunoprecipitates were separated by SDS-PAGE and transfers probed with HRP conjugated antibody to flag-tagged Borealin (Bethyl Laboratories). Both A and B present results that were reproducible in at least two independent experiments.
48
(A)
UT Borealin Bor + Cdc20
Flag Borealin
Loading Control
(B)
Borealin + Cdh1 UT Borealin 1µg 3µg 5µg Flag Borealin
Loading Control
(C)
Borealin + CDH1 UT Borealin 1µ g 0.5µ g 0.25µg
Flag Borealin
Loading Control
Fig13: Effect of APC/C co-activators on Borealin (A) Asynchronous HelaM cells were transiently transfected with flag -tagged Borealin and GFP-Cdc20 (1 µg each). (B)
Asynchronous HEK 293 cells were transiently transfected with flag-tagged Borealin and myc-tagged Cdh1 at the indicated concentrations. (C) Asynchronous HEK 293 cells
49 were transiently transfected with flag- tagged Borealin and myc-tagged Cdh1 at the indicated concentrations. The lysates for all the three experiments were separated on a
12.5% polyacrylamide gel and immunoblotted with antibody to Flag-Borealin.
50
Phosphorylation of Borealin
2.1 Borealin is phosphorylated in response to CDK1 over expression in vivo.
Borealin is phosphorylated at S219 during mitosis; mutation of this residue to alanine eliminated the slow migrating phosphorylated form of mitotic Borealin. The residues following S219 of Borealin conform to a partial CDK1 consensus sequence
[S/T] P. CDKs exert their effects on cell-cycle events by phosphorylating a large number of proteins in the cell. This prompted us to analyze the role of CDK1 on the mitotic phosphorylation of Borealin.
We used a set of replication defective recombinant adenoviruses encoding wild type cyclin B1 (WTB1), nuclear cyclin B1 (NB1: a mutant of cyclin B1 fused to the nuclear localization signal of SV40 Large T antigen) and CDK1AF, a constitutively active T14A Y15F mutant resistant to inhibitory phosphorylation mediated by
Wee1/Myt1. These recombinant viruses provide an efficient method to induce the expression of foreign genes under the control of a tetracycline repressible promoter.
Expression from this promoter required co-infection with a recombinant adenovirus encoding the TTA tetracycline repressible transactivator. In our experiments we did not add tetracycline and instead constitutively expressed the exogenous genes. Asynchronous
HEK293 cells infected with recombinant WTB1 adenovirus showed expression of the exogenous protein at a MOI of 50, while exogenous NB1 and CDK1AF expression was detected at a MOI of 10 each (Fig.14). Further, to determine whether CDK1 triggers the phosphorylation of Borealin in vivo, S phase blocked HeLa M cells were infected with
TTA and a combination of the recombinant adenoviruses. Upon infection of cells with
NB1 plus CDK1AF, we observed a mobility shift characteristic of mitotic Borealin
51 phosphorylation (Fig.15). This mobility shift was also observed upon infection with TTA and CDK1AF virus, although not the same extent as when NB1 is included. These results demonstrate that Borealin is phosphorylated in response to increased expression of
CDK1/Cyclin B1 in vivo.
52
WT Cyclin B1 Nuclear Cyclin B1 Uninfected 10 50 100 200 MOI 10 50 Exogenous NB1
Endogenous CDK1 AF NB1
MOI 10 50 Uninfected Exogenous CDK1
Endogenous CDK1
MOI : Multiplicity of Infection Fig 14: Characterization of recombinant adenoviruses. Asynchronous HEK293cells were infected with recombinant adenovirus expressing Wild type Cyclin B1 at a multiplicity of infection (MOI) of 10, 50,100 and 200. Recombinant adenovirus expressing Nuclear Cyclin B1 and CDK1AF were used to infect cells at an MOI of 10 and 50 respectively, while one sample was maintained as an uninfected control. The cell lysates were resolved via SDS-PAGE and immunoblotted for Wild-type and Nuclear
Cyclin B1 with an anti-Cyclin-B1 (1:200) and for CDK1AF with anti-CDK1 (1:1000) antibodies respectively.
53
TTA
TTA NB1 CDK1AF NB1 +CDK1AF Hydroxyurea P Borealin
Borealin
Cyclin B1
CDK1
Fig 15: The effect of CDK1 on phosphorylation of Borealin in vivo. HeLa M cells were blocked in S phase with hydroxyurea (2 mM) for 16 hours. The cells were infected with the indicated combination of viruses at an MOI of 50 each.
The lysates were separated on a 12.5% polyacrylamide gel with a ratio of 29.2:0.8 acrylamide:bisacrylamide and immunoblotted with rabbit antisera to endogenous
Borealin. The transfers were stripped and reprobed with antibody to Cyclin B1,
CDK1 and β actin.
54
2.2 Borealin is not an optimal substrate for CDK1 in vitro
To determine whether CDK1 directly phosphorylates Borealin, we carried out an in vitro kinase assay with purified CDK1 and recombinant Borealin. We compared the phosphorylation of Borealin to Histone H1, an optimal CDK1 substrate. We observed that full length Borealin and the S219A mutant were phosphorylated by CDK1 in vitro
(Fig.16A). However, upon quantification we noted that Borealin phosphorylation was only ~1% that of Histone H1 (Fig.16C). We repeated the experiment multiple times with similar results, however the quantification is based on results from a single experiment.
Also, we can discount variability in the amount of substrates as a cause of decreased levels of phosphorylation, since we confirmed the generation of viable recombinant proteins which were equalized prior to carrying out the in vitro kinase assay (Fig.16B).
Further, to analyze the effect of CDK1 on Borealin phosphorylation at S219 we generated synthetic peptides encompassing S219 and compared these to an optimal
CDK1 peptide substrate. The peptides were subjected to in vitro kinase assay, however
CDK1 could not phosphorylate S219 peptide as compared to the control peptide which showed intense phosphorylation (Fig.17). These results indicate that Borealin is not an optimal substrate for CDK1 mediated phosphorylation in vitro. Also, the S219 mutation in full length Borealin did not cause a decrease in phosphorylation by CDK1. These observations suggest that the low level of Borealin phosphorylation that occurs in vitro may be at a residue other than S219.
55
(A) (B)
(C) 100
1.4
1.2
1
0.8
0.6
0.4
Percent phosphorylation 0.2
0 GST Borealin -Bor -S219A Histone H1
56
Fig 16: Borealin is not an optimal CDK1 substrate in vitro (A) Recombinant full length
GST tagged Borealin, Borealin S219A , histone H1 and GST control were phosphorylated in vitro with purified CDK1/Cyclin B1 in the presence of Ƴ(32P) ATP.
The proteins were resolved by 10% SDS-PAGE, stained with Comassie Blue, dried in a gel dryer and visualized by autoradiography. (B) Wild type Borealin and Borealin
S219A were expressed and purified from E.coli as GST fusion proteins. The proteins were separated on a 12.5% polyacrylamide gel and stained with Comassie blue. (C)
Quantification of Borealin phosphorylation. Radioactive gels were analyzed with a phosphorimager.
57
Borealin S219 Borealin S219A Cdk1 substrate GNGSPLADAK GNGAPLADAK HATPPKKKRK P- ATP 32 + P- ATP 32 -
Fig 17: CDK1 does not phosphorylate a Borealin peptide encompassing S219. N-terminal biotinylated peptides corresponding to Borealin S219, Borealin S219A and an optimal
CDK1 substrate (1µm) were phosphorylated in vitro by purified CDK1/CyclinB1 in the presence of Ƴ(32P) ATP. The in vitro kinase reactions were spotted on PVDF membrane saturated with avidin (500 µg) and incubated for an hour at room temperature. The PVDF membrane was washed thrice and reactions visualized by autoradiography.
58
2.3 Effect of kinase inhibitors on Borealin phosphorylation
To identify the kinase that may have an effect on the phosphorylation status of
Borealin, we treated interphase and mitotic cells with an array of kinase inhibitors and assayed for changes in the amount of Borealin phosphorylation. We observed no change in the mobility shift of phosphorylated Borealin when S phase cells, treated with a broad spectrum phosphatase inhibitor NaF were also exposed to Purvalanol (inhibitor of
CDK1), ZM447439 (inhibitor of Aurora B) or PD98059 (inhibitor of MAPK)
(Fig.18).This suggests that none of the kinases tested above modify the phosphorylation of Borealin during S phase. In a second type of experiment, we first blocked cells in mitosis using nocodazole, and then exposed to various kinase inhibitors in the presence of
MG132 to stop cells from exiting mitosis. During this short term treatment (~ 2 hours)
MG132 does not increase total Borealin levels. Treatment with ZM447439 (Aurora B kinase inhibitor) alone had no effect on the levels of mitotic Borealin. However, cells treated with a combination of purvalanol and ZM447439 consistently exhibited a reduced level of Borealin phosphorylation and increased levels of the faster migrating unphosphorylated species (Fig.19). We also exposed the nocodazole blocked cells to
BI2356, an inhibitor of PLK1 with mixed results. In one of these experiments BI2536 induced Borealin phosphorylation (data not shown). This potential effect of BI2536 needs to be further investigated. Overall, these results suggest that CDK1 and Aurora B may exert a combinatorial effect to modify the phosphorylation status of mitotic Borealin.
59
Borealin
β actin
Fig 18: Effect of kinase inhibitors on Borealin phosphorylation during S-phase.
HeLa M cells were blocked in S-phase with hydroxyurea (2 mM) for 16 hours.
The cells were treated with kinase inhibitors purvalanol (10µM), ZM447439 (2.5µM) and
PD98059 (10µM) for 1 hour. The cells were then treated with the phosphatase inhibitor
NaF (5 µm) for 2 hours. The lysates were separated on a 12.5% polyacrylamide gel with a ratio of 29.2:0.8 acrylamide: bisacrylamide and immunoblotted with rabbit antisera to endogenous Borealin. The transfers were stripped and reprobed for β actin as loading control
60
Nocadzole
Borealin
β actin
Fig 19: Effect of kinase inhibitors on the phosphorylation of Borealin during mitosis.
HeLa M cells were blocked in mitosis with nocodazole (200 ng/ml) for 16 hours, followed by treatment with the proteasome inhibitor MG132 (10 µM) and CDK1 inhibitor Purvalanol (10 µM), Aurora B inhibitor ZM447439 (2.5 µM) either alone or in combination for 4 hours at 37°C. The lysates were separated on a 12.5% polyacrylamide gel with a ratio of 29.2:0.8 acrylamide: bisacrylamide and immunoblotted with rabbit antisera to endogenous Borealin. The transfers were stripped and reprobed for β actin.
61
2.4 Effect of PLK1 overexpression on Borealin phosphorylation
As an additional test of the effect of PLK1 on Borealin phosphorylation, S-phase blocked HeLa M cells were infected with recombinant adenoviruses expressing wild-type
PLK1. We did not observe the mobility shift characteristic of mitotic Borealin phosphorylation upon PLK1 over expression (Fig.20). Therefore, unlike CDK1, PLK1 is unable to induce phosphorylation of Borealin at S219 upon overexpression.
62
HU
Uninfected CMV WTPlk1 NOC
Endo Borealin
Plk1
β actin
Fig 20: Effect of PLK1 overexpression on Borealin phosphorylation. HeLa M cells were blocked in S-phase with hydroxyurea (2 mM) or mitosis with nocodazole
(200ng/ml) for 16 hours. The cells were infected with indicated viruses. The lysates were separated on a 12.5% polyacrylamide gel with a ratio of 29.2:0.8 acrylamide: bisacrylamide and immunoblotted with rabbit antisera to endogenous Borealin. The transfers were stripped and reprobed with antibody PLK1 and β actin.
63
Dephosphorylation of Borealin
3.1 Kinetics of Borealin dephosphorylation
Borealin is phosphorylated during mitosis, dephosphorylation occurs after cells have exited mitosis. To verify the kinetics of Borealin dephosphorylation, we analyzed the phosphorylation status of Borealin in HeLa M cells synchronized in mitosis with nocodazole. Borealin was dephosphorylated within two hours of release from the mitotic block as seen by increase in the levels of faster migrating electrophoretic species
(Fig.21A). Under these conditions cells enter S phase at ~ 6-8 hours after release from the nocodazole block (Fig.21B). Protein phosphatases are a family of okadaic acid sensitive, serine/threonine phosphatases with essential mitotic functions. We observed that addition of okadaic acid had no effect on the kinetics of Borealin dephosphorylation as cells exited mitosis (Fig. 21A).This suggests that Borealin is not dephosphorylated by PP1, PP2A,
PP4 or PP5 which are the okadaic acid sensitive members of the protein phosphatase family. The enzyme dephosphorylating Borealin is proposed to be a labile phosphatase active during interphase and inactive during mitosis. One of the potential candidates is
Cdc14 that belongs to a family of dual specificity proline directed serine/threonine phosphatases. In yeast, Cdc14 plays an essential role in cell cycle regulation by dephosphorylation of CDK substrates. Cdc14A and Cdc14B are the two different Cdc14 isoforms in humans and we analyzed their role in the dephosphorylation of Borealin.
64
(A) Okadaic Acid Post Noc 0 hr 2 hr 4 hr 0 hr 2 hr 4 hr release Borealin
β actin
(B) 90 80 70 60 50 40 30 20 % Brdu PositivecellsBrdu % 10 0 T6 T8 T10 T12 T14 Hours post Noc release
Fig 21: Kinetics of Borealin phosphorylation. (A) HeLa M cells were synchronized in mitosis with nocodazole (100 ng/µl) for 16 hours. After release from the block, cells were treated with okadaic acid and lysates were collected at the indicated time points followed by separation on a 12.5% polyacrylamide gel. The transfer was immunoblotted with rabbit antisera to endogenous Borealin, stripped and reprobed for β actin. (B) HeLa
M cells synchronized in mitosis with nocodazole (100 ng/µl) for 16 hours. After release from the block, cells were treated with Brdu (10 µm), samples fixed at the indicated time points and analyzed for Brdu incorporation by immunofluorescence.
65
3.2 Borealin and Cdc14B co-localize to the nucleolus of interphase cells
To determine whether Cdc14 phosphatases play a role in the dephosphorylation of
Borealin, we analyzed the localization of Borealin and Cdc14A or Cdc14B in asynchronous cells. We transfected HeLa M cells with GFP-Cdc14A or GFP-Cdc14B followed by indirect immunofluorescence using anti-sera to endogenous Borealin. We observed that both Borealin and Cdc14B co-localized to the nucleolus of interphase cells, however we did not observe any staining for Cdc14A (Fig. 22A). Interestingly when overexpressed, Borealin is highly concentrated in the nucleolus. Our experiments show that endogenous Borealin in HeLa M cells can be found in the nucleolus, which may indicate a new function of Borealin at this location. We quantified pixel intensity in the nucleolus as a ratio of the average pixel intensity in the nucleoplasm. With this analysis we found that Cdc14B was enriched 2.25 in the nucleolus compared to the rest of the nucleus. Borealin was enriched 1.56 in the nucleolus (Fig.22B). These observations suggest that Cdc14B and Borealin may co-localize to the nucleolus of interphase cells.
66
(A) DNA CDC14B-GFP BOREALIN MERGE
(B)
300
250
200
150
100
50
Percent nucleolar Enrichment nucleolar Percent 0 Cdc14B Borealin DNA
Fig. 22: Borealin and Cdc14B co-localize to the nucleolus of interphase cells (A)
Asynchronous HeLa M cells were transiently transfected with GFP-Cdc14b. 24 hours post transfection, cells were blocked and stained with purified anti-sera to endogenous
Borealin. Blue depicts DNA, red depicts GFP-Cdc14B and green depicts endogenous
67
Borealin. (B) Average pixel intensity in the nucleus as a ratio of the average pixel intensity in the nucleoplasm. Repeated for many nucleoli to obtain an average ratio.
68
3.3 Effect of Cdc14 over expression on Borealin
To further investigate potential roles of Cdc14A and Cdc14B in the dephosphorylation of Borealin we co-transfected HeLa M cells with GFP-Cdc14A or
GFP-Cdc14B and full-length flag- tagged Borealin .The cells were then blocked in mitosis with nocodazole for 12 hours. The cell lysates were separated on a large 12.5% polyacrylamide gel and immunoblotted with an antibody to tagged Borealin. Over expression of both Cdc14A and Cdc14B resulted in a decrease in the total protein levels of Borealin (Fig.23A). We also analyzed the kinetics of Borealin dephosphorylation in the presence of Cdc14B. Cells co-transfected with flag- tagged Borealin and GFP tagged
Cdc14B were synchronized in mitosis. Over expression of Cdc14B slightly accelerated the dephosphorylation of Borealin under these conditions (Fig. 23B). Combined, these results indicate that overexpression of Cdc14 phosphatases has a minor effect on the phosphorylation status of Borealin.
69
(A) UT Bor Bor +14A Bor +14B Bor Asy HU Noc Noc Noc
Flag Borealin
β actin
(B) Nocadazole
HU T0 T6 T8 T10 T12 Hrs post Noc release - + -+ - + -+ - + - + Cdc14B Flag- Borealin
Actin
Fig.23: Effect of Cdc14B overexpression on Borealin phosphorylation (A) HeLa M were transiently transfected with flag-tagged Borealin and GFP-Cdc14A or GFP-
Cdc14B.The cells were blocked in mitosis with nocodazole (100 ng/µl) for 16 hours. (B)
HeLa M cells were co-transfected with Flag-Borealin and GFP-Cdc14B. The cells were blocked in mitosis with nocodazole (100 ng/µl) for 16 hours; cells were harvested at the indicated time points after release from the block. Cell lysates from both the experiments were separated on a 12.5% polyacrylamide gel with a ratio of 29.2:0.8 acrylamide: bisacrylamide and immunoblotted with antibody to Flag tagged Borealin.
The transfers were stripped and reprobed for β actin.
70
3.4 Cdc14B does not mediate proteasome mediated degradation of Borealin
In yeast, Cdc14 activates the E3 ubiquitin ligase APC by dephosphorylating the co-activator protein Cdh1 (Jaspersen et al., 1999). In humans, Cdc14B dephosphorylates
Skp2, a modular F box protein essential for the activity of SCF ligase that regulates cell cycle progression (Bassermann et al., 2008).The reduction of Borealin levels upon overexpression of Cdc14B suggested that this phosphatase might be triggering the proteasome-dependent degradation of Borealin. To test this idea, we overexpressed
Borealin and Cdc14B and exposed cells to MG132 to inhibit the proteasome. As before,
Cdc14B suppressed expression of the co-transfected Borealin (Fig. 24, compare lanes 3 and 5; band A and B). Simultaneous treatment with MG132 increased the amount of transfected Borealin (Fig .24, lanes 5 versus 6), however the level did not reach that in the absence of Cdc14B (Fig. 24, lane 3). More importantly, overexpression of Cdc14B did not reduce the level of endogenous Borealin (Fig. 24, compare lanes 7 or 9 to 11; band C). Together, these results suggest that Cdc14B does not target Borealin protein per se. Consistent with this idea we observed a decrease in the levels of another unrelated flag -tagged protein upon Cdc14A and Cdc14B over expression suggesting that the observed decrease in Borealin levels was due to a non-specific effect (data not shown).
One possibility is that over expression of Cdc14B suppresses the transcription of co- transfected genes under the control of otherwise constitutive promoters. These data indicate that Cdc14B does not regulate the stability of Borealin via proteolytic degradation during the cell cycle.
Further examination of our experiments with MG132 in combination with
Cdc14B reveal several interesting effects on Borealin regulation. Extended MG132
71 treatment increases the total amount of endogenous Borealin in HU treated cells (Fig. 24, compare lanes 1 to 2, 3 to 4, 5 to 6; band D) but not mitotic cells (Fig.24, compare lanes 7 to 8; band C and D). Also extended MG132 treatment appears to convert the phosphorylated endogenous Borealin to the under-phosphorylated form (Fig.24, compare lanes 7 to 8; shift from band C to D), this effect is not as evident with the exogenous
Borealin. Together these results suggest that the proteasome regulates the maintenance of
Borealin phosphorylation during mitosis.
72
Hydroxyurea (HU) Nocadazole (NOC) UT Borealin Bor + Cdc14B UT Borealin Bor + Cdc14B DMSO MG DMSO MG DMSO MG DMSO MG DMSO MG DMSO MG A B C D
β actin
1 2 3 4 5 6 7 8 9 10 11 12 A: Phospho Flag Borealin C: Phospho endogenous Borealin B: Non-phospho Flag Borealin D: Non- phospho endogenous Borealin
Fig. 24: Cdc14B does not mediate proteasome- mediated degradation of Borealin. HeLa
M cells were transiently transfected with Flag- Borealin and GFP-Cdc14B or Flag-
Borealin alone. The cells were blocked in S phase with hydroxyurea (2 mM) or mitosis with nocodazole (100 ng/ml) with or without proteasome inhibitor MG132 (5 µM) for 14 hours. MG132 was added simultaneously with either hydroxyurea or nocodazole. The lysates were separated on a 12.5% polyacrylamide gel with a ratio of 29.2:0.8 acrylamide: bisacrylamide and immunoblotted with antibody to Flag- tagged Borealin, followed by rabbit antisera to endogenous Borealin. The transfers were stripped and reprobed with an antibody to β actin.
73
3.5 Analysis of Cdc14 depletion on the status of Borealin phosphorylation
In humans, both Cdc14A and Cdc14B have been reported to interact with and dephosphorylate tumor suppressor p53 (Li et al., 2000). In addition, Cdc14A plays a role in the regulation of the centrosome cycle, while Cdc14B has been proposed to regulate mitotic exit by dephosphorylating CDK1 substrates (North and Verdin, 2007). However, homozygous deletion of Cdc14B failed to have an effect on the total protein levels and the phosphorylation status of endogenous Borealin when compared to a control cell line
(E121) wild type for Cdc14B (Fig.25A). To investigate the effect of Cdc14A depletion on endogenous Borealin, we generated a Cdc14A null cell line via use of lentiviral particles encoding four different shRNA’s directed against Cdc14A transcript. All the four cell lines lacked endogenous Cdc14A, depletion of which seemed to induce the mobility shift characteristic of Borealin phosphorylation in the 14-3A, 14-4A and 14-5A cells (Fig.25B). However, when we repeated the experiment using frozen stocks of the
14-2A, 14-3A, 14-4A and 14-5A cell lines the expression of endogenous Cdc14A was regenerated. We also attempted to perform a transient Cdc14A knock down, with no success. Thus, Cdc14A may be an important phosphatase that regulates the dephosphorylation of Borealin. However we need to regenerate a Cdc14A knock down cell line to confirm our preliminary observations.
74
(A)
E121 E120 E121 E120 Endo Borealin
β actin
(B)
UT 14-2A 14-3A 14-4A 14-5A Cdc14A
Endo Borealin
β actin
Fig.25: Effect of Cdc14 knock down on Borealin phosphorylation (A) Cdc14B wild type
(E121) and null (E120) cell lines were obtained from Berdugo et al., 2008. The cells were lysed and separated via SDS-PAGE. The transfers were probed with anti sera to endogenous Borealin and β actin as loading control. (B) Cdc14-2A, 3A, 4A and 5A cell lines were generated as mentioned in materials and methods. The cells were lysed and lysates separated by SDS-PAGE. The transfers were probed with antibody to endogenous
Cdc14A, stripped and reprobed with antibodies to endogenous Borealin and β actin.
75
V. Discussion
The CPC composed of Aurora B, INCENP, Survivin and Borealin plays essential roles in chromosome segregation, histone modification and cytokinesis. The CPC exhibits a highly dynamic pattern of localization throughout mitosis. Disruption of any one of the passenger proteins results in mislocalization of the complex causing severe defects in mitosis. Further, depletion of Borealin causes spindle abnormalities and defects in cytokinesis (Gassmann et al., 2004). Chromosomal passenger proteins Aurora B and
Survivin were found to be highly expressed in a number of cancers. Borealin was also found to be over-expressed in gastric cancer (Chang et al., 2006). Borealin levels were elevated by at least 50% in 45% of brain cancers, 34% of lymphomas and 61% of colon cancers analyzed when compared to the corresponding normal tissue (Date et al., 2007).
Hence it is essential to understand the transcriptional regulation of Borealin during cell cycle progression.
Post translational modifications of the chromosomal passenger proteins are essential to regulate CPC localization and function during mitosis. Phosphorylation is one of the most wide spread post translational modification affecting every elemental cellular process. Borealin is phosphorylated at S219 during mitosis; however the kinase phosphorylating this residue is unidentified. Phosphorylation at S219 is essential for centromeric localization of the protein. A non-phosphorylatable S219A mutant of
76
Borealin exhibits defective mitotic functions in addition to altered nuclear morphology
(Kaur and Taylor, submitted). It is important to identify the enzymes regulating the phosphorylation status and associated functions of Borealin.
1. Transcriptional regulation of Borealin in response to DNA damage
A number of important cell cycle genes including borealin are repressed in response to DNA damage caused by adriamycin (Date et al., 2007; Jackson et al., 2005).
In order to determine if DNA damage had a similar effect on the protein levels of
Borealin, we exposed HCT116 cells to adriamycin and cisplatin. Both treatments caused down-regulation of Borealin; however adriamycin was much more efficient at inducing this effect as compared to cisplatin. A contrasting trend was observed in HT1080 cells, in which exposure to adriamycin resulted in the down-regulation of Borealin, but not to the extent observed in response to cisplatin. Adriamycin and cisplatin use divergent modes of action to induce DNA damage. Adriamycin is an anthracycline antibiotic that functions via intercalating into the DNA double helix to stall the progression topoisomerase II. Adriamycin stabilizes the topoisomerase II complex after it has broken the DNA chain; preventing the double stranded DNA break from being resealed
(Momparler et al., 1976). Adriamycin also increases reactive oxygen species by several different mechanisms (Momparler et al., 1976). On the other hand, cisplatin forms a platinated coordination complex with DNA to inhibit DNA replication and transcription and further induces programmed cell death (Barnes and Lippard, 2004). Cisplatin adducts also occur on proteins with effects on many different cellular processes. Hence, the differential regulation of Borealin in the different cell lines may be related to the
77 divergent mode of actions of adriamycin and cisplatin in HCT116 and HT1080 cell lines.
Thus we may conclude that DNA damaging agents have different abilities to mediate down regulation of Borealin and these differences may be cell type dependent. Genomic damage results in the rapid induction of tumor suppressor p53. p53 triggers an essential pathway to cause down-regulation of a large number of genes that encode proteins needed to enter and progress through mitosis(Jackson et al., 2005). HCT116 cells treated with Nutlin-3 a small molecule inhibitor of the p53-hDM2 interaction, up regulated p53 leading to a rapid down-regulation in the levels of Borealin (Date et al., 2007). Borealin was not down regulated in HCT116 cells lacking either p53 or its transcriptional target p21. p21/waf1 encodes a protein that inhibits multiple CDK’s leading to dephosphorylation of Rb family of proteins (Boulaire et al., 2000). We observed defective repression of borealin upon over expression of a constitutively active cyclin
D1-CDK2 fusion capable of phosphorylating p105Rb (Date et al., 2007).
The Rb family proteins (p105, p107, p130) function primarily as regulators of the mammalian cell cycle progression, and suppressors of cellular growth and proliferation
(Macaluso et al., 2006). Borealin was repressed more efficiently in wild-type mouse embryonic fibroblasts than those lacking p130 and 107 after exposure to adriamycin.
Together these results suggest that p21/waf1 and the Rb family of proteins are required for the efficient down-regulation of Borealin in response to p53 signaling. Interestingly,
Borealin was still partially down regulated in MEF’s lacking all the three Rb family proteins upon adriamycin treatment. This observation indicates that an Rb independent pathway may additionally contribute to the transcriptional regulation of Borealin. For example, the Forkhead box (FOX) family of transcription factors play important roles in
78 regulating the expression of genes involved in cell growth, proliferation, differentiation, and longevity (Alvarez et al., 2001). Three members of the Forkhead transcription factor family FOXO4, FOXO1 and FOXO3a are regulated via phosphoinositide-3-kinase- protein kinase B (PI3K-PKB) mediated phosphorylation. Upon over production FOXO4,
FOXO1 and FOXO3a can induce cell cycle arrest or apoptosis. They regulate the transcription of p27/Kip1, increased levels of this CDK inhibitor causes cells to arrest in the G1 phase (Burgering and Kops, 2002). Further, Forkhead transcription factors control the cell cycle transition from G2-M via regulation of mitotic genes such as cyclin B1 and plk1 transcription. There mitotic transcriptional targets may also include borealin
(Alvarez et al., 2001).In addition, the CCAAT-binding Factor/ nuclear transcription factor Y (CBF/NF-Y) is known to bind promoters of Cyclin B1, CDK1, CDC25C and play a crucial role in their transcriptional activation at the G2/M phase. The CBF- binding site, a cell cycle-dependent element (CDE) and a cell cycle homology region
(CHR) is present in several genes activated at G2/M phase including borealin (Hu et al.,
2006). Thus Rb independent repression of Borealin may be mediated by transcription factors from the Forkhead or CBF/NF-Y family. Alternatively, the Rb independent repression of Borealin may be mediated by E2F7 or E2F8. These are the most recently discovered members of the E2F family, which can bind promoters normally bound by
E2F1, E2F3 and E2F4 to form potent repressor complexes (Chen et al., 2009).
Importantly, E2F7 and E2F8 do not bind to Rb family of proteins and may still be regulating Borealin in the Rb knock out cells.
Our studies propose an indirect, multi-component model for the regulation of
Borealin. DNA damage activates tumor suppressor p53 and induces transcriptional target
79 p21/waf1. This causes inhibition of multiple CDK’s resulting in the loss of Rb phosphorylation. Poorly phosphorylated Rb binds E2F to form a transcriptional repressor complex that inhibits transcription of multiple cell cycle genes which includes Borealin.
Consistent with this idea, a global factor binding analysis has identified p130 and E2F4 bound to the borealin promoter in vivo (O'Connell et al., 2000).
80
DNA damage
Cdk
Borealin
Fig 26: Borealin down -regulation in response to DNA damage by an indirect multi- component system. DNA damage activates tumor suppressor p53 and induces p21/waf1. This causes the inhibition of multiple CDKs resulting in the loss of Rb phosphorylation. Poorly phosphorylated Rb binds E2F to form a transcriptional repressor complex that inhibits transcription of multiple cell cycle genes. An Rb independent pathway may also function to mediate Borealin repression
81
2. Proteasome mediated degradation of Borealin
Borealin is regulated in a cell cycle dependent manner with increased levels of expression during G2 and mitosis. Levels of endogenous Borealin increase when cells are blocked with nocodazole indicating that the protein may be stabilized during mitosis.
Upon inhibition of the proteasome with MG132, we observed increased levels of
Borealin in S-phase arrested cells. This suggests that Borealin is subjected to degradation via 26S proteasome during interphase. We analyzed the stabilization of several truncated forms of Borealin upon proteasome inhibition. A truncated form of Borealin extending from amino acids 1-141 (Bor-141) was dramatically stabilized as compared to Bor-168 and the other truncation mutants. This suggests that the region between amino acids 141 and 168 of Borealin confers protection from proteolytic degradation of the protein. There could be several explanations for the stabilization effect displayed by this region. The first is that the N-terminal 141 residues of Borealin are insufficient for tight binding to the CPC and the displaced Bor-141 is an easy target for the proteasome. Structural analyses reveal that the N-terminal 141 residues of Borealin are sufficient for interaction with its binding partner survivin and localizing the CPC to the spindle midzone and midbody (Jeyaprakash et al., 2007). It is possible that the C-terminus of Borealin stabilizes this interaction. Recently, the C-terminus of Borealin was found to contain a novel dimerization domain (Bourhis et al., 2009). Another possibility is that dimerization of Borealin protects it from the proteasome. Either way our results show that the C- terminus of Borealin controls the stability of the protein.
82
The vast majority of proteasome targets are subjected to ubiquitination in order to be targeted for degradation. We carried out a polyubiquitin affinity assay to determine if
Borealin is ubiquitinated. We detected a single high molecular weight species of Borealin as opposed to a characteristic smear of multiple ubiquitin conjugates. This observation suggests that Borealin may not be robustly ubiquitinated. Alternatively the ubiquitin conjugates of Borealin may be highly labile or dynamically deubiquitinated by an ubiquitin specific protease such as USP44. Borealin is subjected to a mitotic sumo conjugation-deconjugation cycle by RANBP2 and the SUMO specific isopeptidase
SENP3 (Klein et al., 2009). It has been observed that SUMO can act as a signal for the recruitment of E3 ubiquitin ligases leading to the ubiquitination and degradation of the modified substrate protein (Geoffroy and Hay, 2009). It will be interesting to determine if such an interplay of sumo-ubiquitin, conjugation-deconjugation cycle plays a role in the regulation of Borealin. The cell cycle profile of Borealin protein expression, a peak during mitosis and drop in G1 phase is reminiscent of APC/C cell cycle substrates.
APC/C is a multi subunit E3 ligase essential for mitotic progression that targets a number of cell cycle regulators for degradation. For example, Aurora B is degraded by the APC/C in a CDH1 dependent manner (Stewart and Fang, 2005). Two activator proteins participate in substrate recognition by the APC/C; CDC20 is a mitosis specific activator while CDH1 is essential for APC/C activity in G1 phase. We identified in the N-terminus of Borealin three potential degradation sequences (D-boxes RXXXL) characteristically recognized by APC/CDH1. Conversely over expression of the APC/C activator CDH1 had a minimal effect on the levels of Borealin. Thus we may conclude that Borealin is ubiquitinated by an E3 ligase other than APC/C during the cell cycle.
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3. Phosphorylation of Borealin
Post-translational modifications of the chromosomal passenger proteins are essential for regulating the localization and function of the CPC. For example, Survivin phosphorylation at T117 by Aurora B kinase is essential for centromeric localization of the protein (Colnaghi and Wheatley, 2010). Phosphoproteome analysis suggests that
Borealin is phosphorylated at more than 20 potential sites; including S219 (Nousiainen et al., 2006). Further, mutation of S219 to non-phosphorylatable S219A eliminated the slow migrating phosphorylated form of mitotic Borealin (Kaur and Taylor, submitted). The
S219 residue is succeeded by a proline; making the site a partial consensus sequence for
CDK1 mediated phosphorylation. Overexpression of CDK1 caused a mobility shift characteristic of mitotic phosphorylation of Borealin. However, Borealin was phosphorylated to a minimal level by CDK1 in vitro as compared to the optimal CDK1 substrate histone H1. In addition, CDK1 could not phosphorylate a synthetic peptide corresponding to Bor-S219 in vitro. The low level of Borealin phosphorylation in vitro could be due to the absence of a priming phosphorylation. For example, Plk3 mediated phosphorylation of Chk2 at S62 and S73 in vitro facilitates subsequent phosphorylation of Chk2 on T68 by ATM (Bahassi el et al., 2006). Thus phosphorylation or modification of an additional residue at the serine/threonine rich C-terminus of Borealin may be essential for CDK1 mediated phosphorylation of S219. Alternatively, the reduced levels of Borealin phosphorylation in vitro may arise because CDK1 preferentially phosphorylates Borealin present in a complex with the other CPC members rather than monomeric Borealin. Furthermore, a total lack of CDK1 mediated phosphorylation of a
Bor-S219 peptide may be caused by the absence of a binding site or priming residues
84 required by the kinase to bind Borealin and phosphorylate it at S219. For example, recognition of CDK substrates in vivo requires interaction of its binding partner cyclin with an RXL motif on the specific substrate (Ubersax and Ferrell, 2007). In addition, increased levels of Borealin phosphorylation upon CDK1 over expression in vivo may be caused indirectly due to activation of a downstream CDK1 kinase.
Borealin is phosphorylated by Aurora B kinase in vitro; however inhibition of
Aurora B alone had no effect on the levels of mitotic Borealin phosphorylation.
Conversely, inhibition of both Aurora B and CDK1 caused a decrease in the levels of slower migrating phosphorylated Borealin species. Overexpression of PLK1 did not mediate an increase in mitotic mobility shift of Borealin. To summarize, a combination of kinases, prime candidates being CDK1 and Aurora B may regulate the phosphorylation of mitotic Borealin and hence efficient functioning of the CPC during mitosis.
4. Dephosphorylation of Borealin
In budding yeast, the Cdc14 phosphatase dephosphorylates Sli15, an ortholog of
INCENP during anaphase leading to redistribution of the CPC from the kinetochores to midbody (Pereira and Schiebel, 2003). Hence, dephosphorylation of the chromosomal passengers may be an important mechanism governing the localization and function of the complex. Borealin is dephosphorylated two hours after mitotic exit as the cells enter the first gap phase. There are two Cdc14 homologs in humans Cdc14A and Cdc14B. We observed that Borealin co-localized with Cdc14B to the nucleolus of interphase cells.
Cdc14A shows centrosomal localization during interphase; however we did not observe any staining for Cdc14A with methanol or paraformaldehyde fixation methods. Upon
85 overexpression, both Cdc14A and Cdc14B mediated a decrease in the total levels of
Borealin protein. In addition, overexpression of Cdc14B accelerated the dephosphorylation of Borealin upon mitotic exit to a minor extent. The reduced levels of
Borealin upon Cdc14B overexpression indicate that the phosphatase might be triggering proteasome-dependent degradation of Borealin. To test this hypothesis, we overexpressed
Borealin and Cdc14B and exposed cells to MG132 to inhibit the proteasome. Cdc14B suppressed expression of the co-transfected Borealin; however overexpression of Cdc14B did not reduce the level of endogenous Borealin. Surprisingly, we observed a decrease in the levels of another unrelated flag- tagged protein upon Cdc14A and Cdc14B over expression suggesting that the observed decrease in Borealin levels was due to a non- specific effect. All combined, these results suggest that Cdc14B does not regulate the stability of Borealin via proteolytic degradation during the cell cycle. To ascertain the role of Cdc14 phosphatases in the regulation of Borealin, we studied the effect of their depletion on the status of Borealin phosphorylation. Deletion of the Cdc14B locus had no effect on the phosphorylation status of endogenous Borealin. One possible reason for this effect being that Cdc14A partially fulfills the function of Cdc14B when the latter is deleted. Further, knock down of Cdc14A via shRNA did seem to increase the mobility shift characteristic of Borealin phosphorylation. Thus similar to yeast Cdc14, vertebrate
Cdc14A may play an essential mitotic role by regulating the phosphorylation status and hence localization of chromosomal passenger Borealin. However, it may be essential to develop a cell line lacking both the Cdc14A and Cdc14B isoforms to obtain a definitive role for these phosphatases in regulating the phosphorylation status of Borealin.
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VI. Conclusion
The chromosomal passenger complex is an essential regulator of the cell cycle with functions in histone modification, correction of chromosome segregation defects and cytokinesis. The chromosomal passenger proteins are mutually interdependent for their distinctive localization pattern during mitosis. The passenger proteins are subjected to multiple post translational modifications that regulate their localization and function.
Borealin is the most recently described component of the CPC and not much is known about its regulation. Our studies confirm that Borealin is regulated in an Rb/E2F dependent manner. We also show that Borealin is down regulated in response to DNA damage in a p53 dependent manner. In addition, we provide evidence that Borealin is targeted for proteolytic degradation via the 26S proteasome. Furthermore, a putative stabilization motif between amino acids 141-168 appears to protect Borealin from proteolytic degradation. Our data suggests that in addition to being sumolyated, Borealin is ubiquitinated in an APC independent manner. Future studies will elucidate the E3 ligase that mediates ubiquitination and proteolytic degradation of Borealin.
Borealin is phosphorylated at S219 during mitosis. Our data from several in vitro kinase assays, kinase over expression and inhibition experiments illustrates that in vivo
CDK1 induces mitotic mobility shift characteristic of Borealin phosphorylation at S219.
However recombinant Borealin is a poor substrate for CDK1 in vitro. This suggests that
CDK1 may exert an indirect effect to mediate phosphorylation of Borealin. Further, we
87 provide evidence that the Cdc14B phosphatase does not mediate dephosphorylation of
Borealin; however Cdc14A may be involved in this process. Future studies will investigate the role of Cdc14A in dephosphorylating Borealin upon mitotic exit. Thus we have been able to identify important regulatory elements that phosphorylate (CDK1) and dephosphorylate (Cdc14A) Borealin during mitosis.
88
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VIII. Appendix
In order to further characterize the S219-phosphorylated form of Borealin, we raised a phospho-S219 peptide antibody (pS219). The antibody was raise against a phospho peptide encompassing S219 residue of Borealin. In this section we present data supporting the characterization of the phospho antibody.
1. Analysis of Borealin phosphorylation with a phospho-specific antibody
The pS219 antiserum detected a band that was of the appropriate size and was induced in cells arrested in mitosis with nocodazole (Fig. 27A).Immunofluorescence analysis of asynchronously growing HelaM cells indicated that the pS219 staining was observed in the region overlapping the chromosomes in metaphase cells. Several intense dots, in a similar position as the spindle poles were also observed (Fig. 27B). In early anaphase, faint pS219 staining was observed at the spindle midzone, and at telophase, prominent staining was observed at the midbody (Fig. 27B). Staining observed at the chromosomes, midzone and midbody overlapped to various extents with INCENP.
2. Analysis of Borealin phosphorylation in cells overexpressing Borealin
To correlate pS219 staining with the presence of total Borealin, we performed immunofluorescence with the pS219 antibody in cells overexpressing either wild-type or
S219A Flag-tagged Borealin. Cells were simultaneously stained with an antibody to the
96
Flag-tag. In cells overexpressing wild-type Borealin-Flag, pS219 staining was observed on the chromosomes, midzone and midbody as before (Fig. 28A). At these locations, pS219 staining overlapped with Flag-staining suggesting that these regions contain
Borealin. However, several bright foci of pS219A staining, including dots reminiscent of the spindle poles, contained no Flag-staining. This observation suggests that these spots represent non-specific binding of the pS219 antiserum to other cellular proteins.
In cells overexpressing the S219A form of Borealin, pS219 staining was depleted from the region containing chromosomes in metaphase. pS219 staining at the midzone was variable, with some cells showing very faint levels of staining (Fig. 28B).One explanation for these results is that the pS219 is unable to bind to the S219A mutant of
Borealin. When this mutant is overexpressed, some of it may localize to the chromosomes and displace endogenous pS219-reactive Borealin from this location.
Further experiments are planned to confirm that the binding of pS219 antiserum is phosphorylation specific.
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(A) P- S219 Asy Noc
(B) pS219 Borealin INCENP DNA MERGE METAPHASE ANAPHASE TELOPHASE
98
Fig27: Analysis of Borealin phosphorylation with a phospho-specific antibody. HelaM cells were subject to western and immunofluorescence analysis using an antibody, pS219, raised against a Borealin peptide phosphorylated at S219. (A) Western blotting with pS219. HelaM cells were either left untreated (UT) or exposed to nocodazole for
18 hours (NOC) and analyzed by western blotting. (B) Subcellular localization of phosphorylated Borealin. HelaM cells were analyzed by immunofluorescence using the pS219 antiserum in combination with a monoclonal antibody specific for INCENP.
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(A) pS219 Borealin a-FLAG (WT) DNA MERGE PRO- METAPHASE ANAPHASE
TELOPHASE (B) pS219 Borealin a-FLAG (S219A) DNA MERGE METAPHASE ANAPHASE TELOPHASE
100
Fig 28: Analysis of Borealin phosphorylation in cells overexpressing Borealin. HelaM cells were transfected with Flagged-tagged (A) wild-type or (B) S219A Borealin and analyzed by immunofluorescence. Transfected cells were simultaneously stained with the rabbit pS219 serum and a monoclonal antibody against the Flag tag.
101