Human Mps1 Kinase is required for the Mitotic Spindle Checkpoint,
but not for Centrosome Duplication
Dissertation
zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.)
der Fakultät für Biologie der Universität Konstanz
Vorgelegt von
Volker Matthias Stucke
Martinsried/Konstanz 2003
TABLE OF CONTENTS...I-III
ACKNOWLEDGEMENTS... 1
ZUSAMMENFASSUNG ... 3
SUMMARY... 6
INTRODUCTION... 8
The cell cycle and checkpoints - a general overview... 8
Chronology of M phase events... 10
Regulation of M phase progression: protein phosphorylation and proteolysis ... 11
Identification of the mitotic spindle checkpoint components ... 13
The mitotic spindle checkpoint senses kinetochore-microtubule attachment and/or tension ... 18
The checkpoint signaling pathway... 19
Mps1-like protein kinases and the mitotic spindle checkpoint ... 22
S. cerevisiae Mps1p and spindle pole body duplication ... 25
Mps1-like protein kinases and centrosome duplication in vertebrates ... 28
AIM OF THE WORK ... 32
RESULTS... 33
Characterisation of the human protein kinase hMps1 ... 33
Sequence analysis of hMps1 ... 33
Recombinant human Mps1 is an active enzyme ... 35
Characterisation of monoclonal anti-hMps1 antibodies... 36
hMps1 is a cell cycle-regulated protein kinase ... 38
hMps1 localizes to kinetochores during mitosis ... 39
An amino-terminal fragment of hMps1 is sufficient for kinetochore localization . 42 Conclusions ... 44
hMps1 is not required for centrosome duplication ... 45
hMps1 does not localize to centrosomes... 45
Microinjection of anti-hMps1 antibodies does not block centrosome duplication . 46 Silencing of hMps1 by siRNA does not inhibit centrosome duplication ... 49
Overexpression of hMps1 does not interfere with centrosome duplication... 50
Conclusions ... 52
hMps1 is an essential component of the mitotic spindle checkpoint ... 53
hMps1 is specifically phosphorylated upon mitotic spindle checkpoint activation 53 Microinjection of anti-hMps1-antibodies interferes with the mitotic spindle checkpoint ... 54
Silencing of hMps1 by siRNA inactivates the mitotic spindle checkpoint... 57
hMps1 is required for kinetochore recruitment of Mad1/Mad2 complexes... 60
hMps1 is not required for kinetochore recruitment of Hec1, hBub1, hBubR1, CENP-E, CENP-F and CENP-B ... 62
Kinetochore localization of hMps1 is dependent on Hec1 and partially on hMad1, but not on the other Mad/Bub and CENP-E/F proteins ... 64
Conclusions ... 66
DISCUSSION ... 67
hMps1 is a cell-cycle regulated protein kinase ... 67
hMps1 localizes to kinetochores, but not to centrosomes ... 69
hMps1 is not required for regulation of centrosome duplication ... 71
hMps1 is an essential component of the mitotic spindle checkpoint in human cells ... 73
hMps1 is implicated in kinetochore recruitment of hMad1/hMad2 complexes... 75
hMps1 is not required for kinetochore-association of Hec1, hBub1, hBubR1, CENP-E, CENP-F and CENP-B ... 77
Kinetochore localization of hMps1 is dependent on Hec1 and partially on hMad1, but not on hMad2, hBub1, hBubR1 and CENP-E/-F... 79
Model for hMps1 function in the mitotic spindle checkpoint ... 81
MATERIALS AND METHODS ... 83
Chemicals and materials... 83
hMps1 cDNA and plasmid constructions... 83
Expression of recombinant His6-hMps1WT and His6-hMps1D663A in Sf9 and High5 insect cells ... 84
Antibody production ... 85
Cell culture, synchronization, in vivo labeling and transfections... 85
Establishment of stable cell lines expressing myc-hMps1WT and myc- hMps1D663A ... 87
Cell extracts, immunoblotting and immunoprecipitation... 87
In vitro kinase assays... 88
Immunofluorescence microscopy ... 89
Antibody microinjection I (for mitotic spindle checkpoint assay)... 89
Antibody microinjection II (for centrosome duplication assay) ... 90
Centrosome duplication assay... 91
siRNA experiments ... 92
Flow cytometric analysis... 92
LIST OF ABBREVIATIONS ... 93
REFERENCES ... 95
Acknowledgements
I would like to acknowledge all the people who contributed to this work over the last years.
I am grateful to Prof. E.A. Nigg for giving me the opportunity to work in his laboratory. I appreciated having access to many scientific and technical resources within the laboratory. In addition, I am thankful to him for reviewing this manuscript.
I also would like to thank Prof. H.-W. Hofer and Prof. D. Malchow, both University of Konstanz, for agreeing to be members of the Thesis Committee and for dedicating some time to evaluate the presented work. I am especially indebted to Prof. H.-W.
Hofer for his continuous help and advice on the organization of the examinations.
I acknowledge the many contributions of the Department of Cell Biology at the Max- Planck-Institute: I would like to express my gratitude to all members of the lab and I appreciated to work with all of you. In particular, I am thankful to Herman Silljé, my supervisor during the thesis, who helped me to find my way within the lab. He gave me a lot of technical advices and was always open for interesting discussions. Special thanks I would like to address to Alicja Baskaya, who did an excellent job in helping me raising monoclonal antibodies. I also would like to acknowledge Elena Nigg for technical assistance during that time. In addition, I would like to thank Silvia Martin- Lluesma for the numerous discussions we had on the mitotic spindle checkpoint.
Many thanks to all the people for reviewing this manuscript and for giving me comments and suggestions: Herman Silljé, Ulrike Grüneberg, Thomas Kufer, Tim Holmström, Christoph Baumann, Thomas Mayer, Roman Körner and Janina Karres. I am very thankful to Evelyn Fuchs for her help and patience in my fight against computers. Furthermore, I am very grateful to my PhD colleagues Arno Alpi, Christian Preisinger and Thomas Kufer for having shared with me joy and pain of lab- life throughout the last years.
In particular to Thomas Kufer I would like to express my special thanks. Thomas, as a colleague and friend, I really appreciated the scientific and non-scientific sessions we had in and on the Lake of Starnberg together with Bier, Brezen and Obatzter.
I also would like to express thanks to my friends for their continuous support, above all Steffi and Markus together with Florin. To Kathrin I am thankful for always being present in difficult times and to Ali I am grateful for his help and encouragements throughout all the years.
Zum Schluss möchte ich mich bei meinen Eltern für die Unterstützung über all die Jahre hinweg bedanken.
Zusammenfassung
Im Zellzyklus von eukaryotischen Organismen laufen eine Reihe komplexer Vorgänge ab, die letztendlich zur Verdoppelung der Chromosomen und zur Verteilung einer Kopie eines jeden Chromosoms auf die beiden Tochterzellen führen.
Dabei ist es von entscheidender Bedeutung, dass die einzelnen Ereignisse im Zellzyklus präzise durch speziell eingerichtete Kontrollmechanismen aufeinander abgestimmt werden (Nasmyth, 2002). Bei einem Versagen dieser Kontrollmechanismen kann es zu schwerwiegenden Folgen kommen und Erkrankungen wie z.B. Krebs können entstehen. Eine zentrale Rolle bei der Kontrolle und Regulation der Zellproliferation kommt der Proteinphosphorylierung durch Proteinkinasen zu (Nigg, 2001). Deshalb ist die Untersuchung der Funktionen der verschiedenen Proteinkinasen im Verlauf des Zellzyklus von grosser Wichtigkeit.
Die Familie der Mps1-Proteinkinasen (Monopolar spindle 1) hat mehrere wichtige Aufgaben im Zellzyklus (Winey and Huneycutt, 2002). Mps1p wurde zuerst in Saccaromyces cerevisiae identifiziert und näher charakterisiert. Untersuchungen an MPS1-Mutanten gaben Aufschluss über die Funktion dieser Proteinkinase. Zum einen ist Mps1p an der Verdoppelung des Spindel-Polkörperchens beteiligt (Winey et al., 1991) und zum anderen ist die Kinase eine Komponente im mitotischen Spindelkontrollpunkt (Weiss and Winey, 1996). Das Spindel-Polkörperchen ist das Centrosomäquivalent in Hefezellen und somit das Mikrotubuli-Organisationszentrum (MTOC) in diesen Zellen (Adams and Kilmartin, 2000). Zu Beginn der Mitose dient das MTOC zum Aufbau des mitotischen Spindelapparates, der aus Mikrotubuli besteht. Mikrotubuli zeigen in dieser Phase eine hohe Dynamik, was durch Hinzufügen und Entfernen der sie aufbauenden Einzelbausteine, der Tubulindimere, erklärt wird. Während des Zellzyklus muss sich das Spindel- Polkörperchen/Centrosom verdoppeln, um zu Beginn der Mitose eine bipolare Spindel aufbauen zu können. Hefemutanten des MPS1-Gens bilden dagegen eine monopolare Spindel, die durch einen Defekt während der Verdoppelung des Spindel- Polkörperchens verursacht wird (Winey et al., 1991). Die Untersuchung von MPS1- Mutanten liess aber auch die zweite, bedeutende Funktion von Mps1p im Zellzyklus erkennen: die Kinase spielt im mitotischen Spindelkontrollpunkt eine wichtige Rolle.
Metaphase zur Anaphase zu einer gleichmässigen Verteilung der Chromosomen auf die Tochterzellen kommt (Millband et al., 2002). Beim Eintritt in die Mitose binden die Mikrotubuli des mitotischen Spindelapparates an die replizierten Chromosomen, auch Schwesterchromatiden genannt. Die Bindestellen der Mikrotubuli auf den Schwesterchromatiden werden als Kinetochoren bezeichnet. Dabei handelt es sich um spezifische Proteinkomplexe an den Centromeren von Chromosomen, die die Mikrotubuli einfangen und stabilisieren (Kitagawa and Hieter, 2001). Der mitotische Spindelkontrollpunkt verzögert nun die Trennung der Schwesterchromatiden solange bis alle Kinetochoren an Mikrotubuli gebunden sind. Die Hauptkomponenten des mitotischen Spindelkontrollpunkts lokalisieren an Kinetochoren, um den Zustand der Mikrotubulibindung zu überprüfen (Musacchio and Hardwick, 2002). Die Proteine Mad1p, Mad2p, Mad3p und Bub1p, Bub2p und Bub3p sowie die Proteinkinase Mps1p bilden die Hauptkomponenten des mitotischen Spindelkontrollpunktes in S.
cerevisiae, der in allen eukaryotischen Organsimen konserviert ist (Amon, 1999).
Diese Proteine erkennen Defekte während der Chromosomensegregation und führen über einen komplexen Signaltransduktionsweg zu einem mitotischen Arrest. Erst wenn alle Kinetochoren mit Mikrotubuli verbunden sind, wird das inhibitorische Zellzyklussignal ausgeschaltet, und die Zelle kann mit dem Ablauf der Mitose weitermachen.
In diversen Organismen wurden Proteinkinasen identifiziert, die Homologie zu Mps1p von S. cerevisiae aufweisen. Für Schizosaccaromyces pombe (He et al., 1998) und auch für Xenopus laevis (Abrieu et al., 2001) wurde eine Funktion der Mps1 Proteinkinase im mitotischen Spindelkontrollpunkt gezeigt. Demgegenüber steht eine Studie in Mus musculus, in der mMps1 für die Duplikation der Centrosomen notwendig ist (Fisk and Winey, 2001).
Wir haben eine detailierte Untersuchung über die Funktion der humanen Mps1 Proteinkinase durchgeführt. Diese Kinase wurde ursprünglich als TTK (Mills et al., 1992) oder PYT (Lindberg et al., 1993) bezeichnet, und wurde jetzt als hMps1 umbenannt (Fisk and Winey, 2001). Wir zeigen, dass hMps1 im Verlauf des Zellzyklus reguliert wird. Proteinexpression und Kinaseaktivität sind während der Mitose am höchsten. Wird zusätzlich der mitotische Spindelkontrollpunkt induziert ist hMps1 maximal phosphoryliert und aktiviert. Mehrere, hochspezifische monoklonale Antikörper gegen hMps1 zeigen, dass das Protein in frühen Phasen der Mitose an den Kinetochoren lokalisiert, wofür die amino-terminale, regulatorische Domäne von
hMps1 notwendig ist. Wir finden keine Lokalisierung von hMps1 an Centrosomen in Interphase oder an den Spindelpolen während der Mitose. Zudem geben mehrere funktionelle Analysen wie die Überexpression von Wild-typ-Kinase oder katalytisch inaktiver Mutante, Antikörpermikroinjektionen und siRNA-Experimente keine Hinweise auf eine Funktion von hMps1 in der Duplikation von Centrosomen.
Stattdessen wird aber durch Antikörpermikroinjektionsstudien und siRNA- Experimente gezeigt, dass hMps1 eine essentielle Funktion in der Etablierung und/oder in der Aufrechterhaltung eines aktiven mitotischen Spindelkontrollpunktes in humanen Zellen hat. Wird die Funktion von hMps1 ausgeschaltet, können die Zellen nicht mehr den Spindelkontrollpunkt aktivieren und somit auch nicht mehr im Zellzyklus in der frühen Mitose arretieren, wenn Spindeldefekte vorliegen.
Schliesslich wird noch der Mechanismus untersucht, wie hMps1 im Spindelkontrollpunkt funktioniert. Wir zeigen, dass hMps1 wichtig ist für die Kinetochorlokalisierung von spezifischen Spindelkontrollpunktproteinen. hMps1 ist einerseits notwendig für die Bindung von hMad1/hMad2-Proteinkomplexen an Kinetochoren, während andererseits die Lokalisierung der Spindelkontrollpunktproteine hBub1/hBubR1 und CENP-E nicht von hMps1 abhängen. Demgegenüber ist das Protein Hec1 entscheidend für eine effiziente Lokalisation von hMps1 an den Kinetochoren.
Aus diesen Resultaten folgern wir dass die Proteinkinase hMps1 für den mitotischen Spindelkontrollpunkt essentiell ist, nicht aber für die Duplikation der Centrosomen. Diese Schlussfolgerung steht im Widerspruch zur gegenwärtigen Annahme, dass Mps1-Kinasen evolutionär konservierte duale Funktionen haben. Wir schlagen vor, dass die primäre Funktion von Mps1 Proteinkinasen im mitotischen Spindelkontrollpunkt liegt.
Summary
The founding member of the Mps1 kinase family, Mps1p of Saccharomyces cerevisiae, has been implicated in the regulation of many cell cycle-related processes, including the duplication of the spindle pole body (SPB) (Winey et al., 1991) and the correct segregation of chromosomes during cell division (Weiss and Winey, 1996).
Mutants in the MPS1 gene give rise to monopolar spindles (Winey et al., 1991; Schutz and Winey, 1998) and mps1 mutants are unable to arrest in mitosis in response to spindle damage, indicating a function in the mitotic spindle checkpoint.
In this study, we have undertaken a functional analysis of the putative Mps1 homolog in human cells. This kinase originally was designated as TTK (Mills et al., 1992) or PYT (Lindberg et al., 1993), but recently has been renamed hMps1 (Fisk and Winey, 2001). We show that hMps1 is a cell cycle-regulated protein kinase, displaying maximal kinase activity during mitosis. In addition, the activity and phosphorylation state increase upon activation of the mitotic spindle checkpoint.
Several monoclonal antibodies raised against hMps1 show that hMps1 localizes to kinetochores, especially during early stages of mitosis and the kinetochore-targeting domain is located within the amino-terminal, regulatory domain of hMps1. However, hMps1 is localized neither to centrosomes in interphase nor to spindle poles in mitosis. Furthermore, various functional analyses - overexpression of wild-type and kinase dead alleles, antibody microinjections, and small interfering RNA (siRNA) - did not reveal any evidence for implicating hMps1 in centrosome duplication. In contrast, microinjection of highly specific antibodies, as well as silencing of hMps1 by siRNA demonstrated that hMps1 is required for the establishment and/or maintenance of the mitotic spindle checkpoint in human cells. Finally, we explore the mechanism through which hMps1 functions in checkpoint signaling. We show that hMps1 is required for the recruitment of hMad1/hMad2 complexes to kinetochores, whereas it does not affect kinetochore-association of the protein kinases hBub1/hBubR1 and the motor protein CENP-E, indicating that hMps1 functions through a specific subset of checkpoint proteins. Furthermore, we define the kinetochore protein Hec1 as an upstream component of hMps1, which is required to localize hMps1 efficiently to kinetochores. Our results lead us to conclude that hMps1 is required for the mitotic spindle checkpoint but not for centrosome duplication. This
conclusion upsets the current assumption that Mps1 kinases have evolutionarily conserved dual functions. We propose that the primary function of these kinases is related to the mitotic spindle checkpoint in all eukaryotes.
Introduction
The cell cycle and checkpoints - a general overview
“All cells are produced by the division of other cells.” This cell doctrine was supported and extended by the German pathologist Rudolf Virchow (1821-1902) and carries the important message that the only way to generate more cells is by division of those that already exist (Virchow, 1854). Ever since then the challenge has been to understand how cell division is regulated within different organisms.
The purpose of cell division is to produce two genetically identical daughter cells. Therefore, the DNA of eukaryotes has to be faithfully replicated, and the genetic material must then be accurately distributed into the two daughter cells so that each cell receives an identical copy of the parental genome. Additionally, in eukaryotes each daughter cell must receive one of the duplicated centrosomes and the appropriate complements of cytoplasm and organelles. To allow and ensure successful cell division, the eukaryotic cell cycle is divided into four stages or phases (Fig. 1). The G1 phase (G = gap) is the interval between the completion of M phase and the beginning of S phase. During G1, the cell grows and the gap phase provides time for the cell to monitor the internal and external conditions before entering the next stage.
If extracellular conditions are unfavorable, cells delay progression through G1 and may even enter a specialized resting state known as G0. If extracellular conditions are favorable and signals to grow and divide are present, cells progress through a commitment point near the end of G1 known as the restriction point in mammalian cells. After passing this point, cells enter S phase (S = Synthesis) during which the cell replicates its nuclear DNA. After DNA replication, the cell goes into the second gap phase, called G2. The G2 phase is the interval between the end of S phase and the beginning of M phase, in which the cell continues to grow. The most dramatic events take place in M phase (M = mitosis + cytokinesis). During mitosis, the division of the nucleus occurs by segregating the chromosomes, and the cytoplasm splits in two, a process called cytokinesis.
Figure 1: Illustration of the four different cell cycle stages in eukaryotic cells. Interphase is divided into G1-, S- and G2-phase, followed by mitosis (M phase). In M phase, the nucleus and then the cytoplasm divide. Image adopted from Purves et al., Life: The Science of Biology, 4th edition, 1995.
In a normal cell division, each set of replicated chromosomes is usually precisely distributed to each of the progeny. However, errors during DNA replication in S phase or chromosome segregation during mitosis can occur with catastrophic consequences. Loss or gain of gene function can lead to cell death or unregulated cell growth. To avoid these disastrous consequences cells employ surveillance mechanisms, so-called checkpoint pathways to ensure high-fidelity transmission of the genetic material. Ted Weinert and Leland Hartwell first defined checkpoints genetically with the isolation of mutants that are defective for the DNA damage checkpoint (Weinert and Hartwell, 1988; Hartwell and Weinert, 1989). These mutants are unable to arrest progression through the cell cycle upon DNA damage. Other checkpoints monitor the completion of DNA replication, spindle assembly, spindle pole position and spindle orientation (Clarke and Gimenez-Abian, 2000; Smith et al., 2002; Nigg, 2001). Most, if not all checkpoint pathways are conserved and can be found in lower eukaryotes like yeast as well as in higher eukaryotes such as in animal cells. All known checkpoints display a similar organization consisting of three parts, which are defined as: (1) the sensor, (2) the transducer and (3) the effector. Sensors look out for defects and emit a signal, while the transducers transmit the checkpoint signal throughout the cell or nucleus. Finally, the effectors regulate a target to delay cell cycle progression in order to allow for repair of defects. Alternatively, they can induce apoptosis (cell death).
Chronology of M phase events
Walther Flemming (1843-1905), a pioneer of mitosis research, was one of the first scientists to give a detailed description of the numerous events during cell division in animals and named the division of somatic cells “mitosis” (Flemming, 1882). In principle, mitosis can be divided into five distinct stages: prophase, prometaphase, metaphase, anaphase and telophase (Fig. 2).
Figure 2: Depiction of the important events that occur during progression through the distinct mitotic stages in eukaryotic cells. Image adopted from H.H.W. Silljé.
For progression through mitosis, the bipolar mitotic spindle apparatus plays an essential role. The mitotic spindle is constructed of microtubules, and during prophase a highly dynamic microtubule array (an aster) forms around each of the duplicated centrosomes in animal cells. Centrosomes are the major microtubule-organizing centers (MTOCs) in the cell, in both interphase and mitosis (Paoletti and Bornens, 1997; Kellogg et al., 1994; Doxsey, 2001). Furthermore, the two centrosomes separate to initiate the formation of the two spindle poles, thus determining spindle bipolarity (Raff, 2001; Rieder et al., 2001). Beside the establishment of the mitotic spindle, the replicated chromosomes, each consisting of two closely associated sister chromatids, start to condense during prophase. Prometaphase starts abruptly with the breakdown
of the nuclear envelope. Chromosomes can now attach to spindle microtubules via their kinetochores (specialized proteinaceous structure associated with centromeric DNA on mitotic chromosomes), and undergo active movement. At metaphase the chromosomes are aligned at the equator of the spindle, midway between the spindle poles, at the so-called metaphase plate. Each sister chromatid has achieved proper bipolar attachment to kinetochore microtubules emanating from the opposite poles of the mitotic spindle. At anaphase, the paired chromatids synchronously separate due to sudden loss in sister chromatid cohesion and each chromatid is pulled toward the spindle pole it faces. The kinetochore microtubules get shorter (Anaphase A) and the spindle poles also move apart (Anaphase B), both contributing to sister chromatid separation. During telophase, the chromosomes arrive at the poles of the spindle, nuclear envelopes reform around the daughter chromosomes, and chromatin decondensation begins. Cytokinesis, the division of the cytoplasm, starts with the contraction of an actomyosin-based contractile ring, which assembles at the site of the spindle midzone and pinches in the cell to create two daughters, each with one nucleus and one centrosome (Pines and Rieder, 2001).
Regulation of M phase progression: protein phosphorylation and proteolysis
Progression through the five different mitotic stages is mainly regulated by two post- translational mechanisms: protein phosphorylation and proteolysis. These two mechanisms are directly linked to each other: the proteolytic machinery is controlled by phosphorylation, whereas many mitotic kinases are downregulated by degradation (Nigg, 2001).
The master kinase in the regulation of mitosis is the cyclin-dependent kinase 1 (Cdk1), the founding member of the Cdk family of cell-cycle regulators (Murray, 1994; Nigg, 1995; Morgan, 1997). All Cdks consist of a catalytic subunit, which has to bind to a regulatory subunit to become enzymatically active. These regulatory subunits are called cyclins (Hunt, 1991; Pines, 1993a; Nigg, 1995), because, unlike the Cdks, their protein levels display a tightly controlled fluctuation during the cell cycle (Evans et al., 1983). Each Cdk family member interacts only with a specific
subset of cyclins during the course of one round of cell division, allowing the controlled and regulated progession through the cell cycle (Table 1).
Table 1: The major cyclins and Cdks for progression through the cell cycle in vertebrates.
Cdk1 interacts with two mitotic cyclins, cyclin A and cyclin B (Pines, 1993b).
The Cdk1-cyclin A/B complex is enzymatically inactive when first formed, because Cdk1 is subject to negative regulation by phosphorylation on two inhibitory sites in the ATP-binding site (threonine 14 and tyrosine 15) by the kinases Wee1 and Myt1, respectively (Ohi and Gould, 1999) (Fig.3). Thus, activation of the Cdk1-cyclin A/B complex depends on the dephosphorylation of these two sites by the dual-specificity phosphatase Cdc25C (Coleman and Dunphy, 1994). Furthermore, Cdk1 activity is positively regulated by phosphorylation: a Cdk-activating kinase (CAK, MO15) (Harper and Adams, 2001) phosphorylates the T-loop of Cdk1 on threonine 161 (Nigg, 1996). As a consequence, the Cdk1 kinase activity is increased upon phosphorylation by CAK and structural changes within the kinase open the catalytic center for substrates (Fig. 3). Activated Cdk1-cyclin A/B complexes then phosphorylate numerous substrates. Most of the early mitotic events described in the previous part are triggered upon Cdk1-cyclin A/B-mediated phosphorylation. For example, phosphorylation of condensins is required for chromosome condensation, phosphorylation of nuclear lamins and microtubule-binding proteins for nuclear envelope breakdown and spindle assembly, respectively (Nigg, 1995; Kimura et al., 1998; Andersen, 1999a). Furthermore, Cdk1-cyclin A/B complexes contribute to the regulation of the anaphase-promoting complex/cyclosome (APC/C), the core component of the ubiquitin-dependent proteolytic machinery (a detailed description of the regulation of the APC/C will be given later). The APC/C controls the timely degradation of critical mitotic regulators, for example of cyclin B (Kramer et al., 2000; Peters, 2002). Thus, upon cyclin B destruction, Cdk1 becomes inactive, and
Cyclin-Cdk complex Cyclin Cdk partner G1-Cdk Cyclin D1 / D2 / D3 Cdk4, Cdk6
G1/S-Cdk Cyclin E Cdk2
S-Cdk Cyclin A / E Cdk2
M-Cdk Cyclin A / B Cdk1
Cdk1 substrates are dephosphorylated by counteracting phosphatases, which facilitates nuclear envelope reformation, spindle disassembly and chromosome decondensation, setting the stage for mitotic exit and cytokinesis.
Figure 3: The activity of Cdk1(=Cdc2)-cyclin B (= MPF, maturation-promoting factor) is regulated by phosphorylation and dephosphorylation on the residues threonine 14, tyrosine 15 and threonine 161.
The inhibitory phosphorylation of Wee1 kinase on Tyr-15 (and Myt1 kinase on Thr 14 and / or Tyr 15, not shown) is counteracted by the phosphatase Cdc25. CAK (MO15) kinase activity on Thr-161 leads to activation of Cdk1-cyclin B. Image adapted from Alberts et al., Molecular Biology of the Cell, 3rd edition, 1994.
Identification of the mitotic spindle checkpoint components
The goal of the mitotic cell cycle is to produce two genetically identical daughter cells from one parental cell. In addition to the tight control of Cdk1 activity and ubiquitin- mediated proteolysis of critical cell-cycle regulators during mitosis, the mechanism of chromosome segregation has to be precisely regulated and controlled. Chromosome segregation is an extremely complex, error-prone process, and defects can lead to aneuploidy (Jallepalli and Lengauer, 2001). In cancer, the outgrowth of aneuploid cells is a likely factor in the progression of tumor malignancy. In germ cell lines, errors that occur in the two chromosome segregation events of meiosis result in aneuploid gametes. These will produce embryos with abnormal chromosome contents, which might lead to developmental defects (Petronczki et al., 2003). But most commonly, these defects are lethal. To ensure fidelity of chromosome segregation, a checkpoint has evolved which is known as the spindle assembly checkpoint (Rudner and Murray, 1996; Wells, 1996), or mitotic spindle checkpoint (Li and Benezra, 1996). This checkpoint is also named kinetochore attachment checkpoint (Rieder et al., 1994), chromosome distribution checkpoint (Nicklas, 1997)
The molecular components of the mitotic spindle checkpoint were initially identified in Saccharomyces cerevisiae by mutants that failed to arrest in mitosis in the presence of spindle damaging agents such as the microtubule-depolymerizing drugs nocodazole or benomyl. The identified checkpoint components consist of Mad1p, Mad2p, Mad3p (mitotic arrest deficient) (Li and Murray, 1991) and Bub1p, Bub2p, Bub3p (budding uninhibited by benzimidazole) (Hoyt et al., 1991).
Subsequently, the dual-specificity kinase Mps1p (Monopolar spindle 1) was found to be an additional component of the spindle assembly checkpoint (Weiss and Winey, 1996). Together, these seven proteins are still viewed as the core components of the spindle assembly checkpoint. Homologs of many of these checkpoint proteins have been identified in Schizosaccharomyces pombe (He et al., 1997; He et al., 1998;
Bernard et al., 1998), Xenopus laevis (Chen et al., 1996; Chen et al., 1998), Drosophila melanogaster (Basu et al., 1998; Basu et al., 1999), Caenorhabditis elegans (Kitagawa and Rose, 1999), Mus musculus (Taylor and McKeon, 1997a;
Martinez-Exposito et al., 1999) and Homo sapiens (Li and Benezra, 1996; Jin et al., 1998; Cahill et al., 1998; Taylor et al., 1998; Chan et al., 1998; Chan et al., 1999). A detailed list of the characteristics and proposed functions of the core checkpoint proteins is shown in Table 2.
Recently, a number of previously characterised proteins such as Ndc80p/Hec1 (Martin-Lluesma et al., 2002; McCleland et al., 2003) and the protein kinase Aurora- B (Biggins and Murray, 2001; Kallio et al., 2002b; Murata-Hori and Wang, 2002;
Tanaka et al., 2002) have also been implicated in spindle checkpoint functioning.
Moreover, additional checkpoint proteins, for instance Zw10 (Zeste-white 10), Rod (Rough-deal) or CENP-E have been identified in higher eukaryotes, but not in yeast, suggesting that in these organsims checkpoint signaling is much more complex. These additional checkpoint components and their putative functions are summarized in Table 3.
With the exception of MPS1, the checkpoint genes are not essential for cell survival in budding yeast. However, deletion mutants lose chromosomes at a higher rate when exposed to low doses of spindle damaging agents. Although the mitotic checkpoint genes are non-essential in S. cerevisiae, complete loss of the mitotic checkpoint through the inactivation of Bub1 in D. melanogaster, Mad2 in C. elegans and Mad2 or Bub3 in mice leads to early embryonic lethality due to chromosome- missegregation events and associated apoptosis (Basu et al., 1999; Kitagawa and
Rose, 1999; Dobles et al., 2000; Kalitsis et al., 2000). Thus, it is believed that the mitotic checkpoint proteins in higher eukaryotes are required in every cell cycle to prevent missegregation of chromosomes. As indicated at the beginning of this part, aneuploidy can have severe consequences in adults by fostering tumor malignancy (Manchester et al., 1995; Jallepalli and Lengauer, 2001). In fact, mutations in or reduced expression of spindle assembly checkpoint components have been reported in some types of human cancer. For example, mutational inactivation of Bub1 has been implicated in human colorectal cancer (Cahill et al., 1998), and reduced expression of Mad2 has been implicated in human breast and ovarian cancers (Li and Benezra, 1996; Wang et al., 2002). Furthermore, haploinsufficiency of Mad2 resulted in a defective checkpoint in human cancer cells and primary mouse embryonic fibroblasts, and Mad2+/- mice are susceptible to lung cancer in later life (Michel et al., 2001).
Table 2: List of the core mitotic spindle checkpoint components in different eukaryotic organisms.
Vertebrates S. cerevisiae S. pombe Main structural features
Comments References
Mad1 Mad1 Mad1 Coiled-coil Binds to Mad2 and
recruits Mad2 to kinetochores;
Phosphorylated by Mps1 and Bub1 in vitro
(Seeley et al., 1999;
Chen et al., 1998; Jin et al., 1998;
Hardwick and Murray, 1995;
Hardwick et al., 1996)
Mad2 Mad2 Mad2 Horma domain Binds to Mad1;
binds to Cdc20 and inhibits Cdc20-APC
activity
(Martin-Lluesma et al., 2002; Sironi et al., 2002; Millband and Hardwick, 2002;
Sironi et al., 2001;
Sudakin et al., 2001;
Howell et al., 2000;
Chen et al., 1999;
Chen et al., 1998;
Kallio et al., 1998;
Fang et al., 1998b;
Waters et al., 1998;
Li et al., 1997; Li and Benezra, 1996)
BubR1 Mad3 Mad3 Serine/threonine
kinase;
GLEBS-motif
Binds to Bub3;
binds to Cdc20 and inhibits APC in vitro;
binds to the mitotic motor CENP-E;
(Chen, 2002; Fang, 2002; Taylor et al., 2001; Sudakin et al.,
2001; Tang et al., 2001; Skoufias et al.,
2001; Chan et al., 1999; Millband and
Hardwick, 2002;
Fraschini et al.,
Table 2: List of the core mitotic spindle checkpoint components in different eukaryotic organisms.
Bub1 Bub1 Bub1 Serine/threonine
kinase;
GLEBS-motif
Binds to Bub3;
binds to Mad1 and Cdc20 in S. cerevisiae;
reported substrates include Bub3, hMad1, Adenomatous polyposis
coli
(Taylor et al., 2001;
Sharp-Baker and Chen, 2001; Olesen et al., 2001; Bernard et al., 2001; Brady and Hardwick, 2000;
Seeley et al., 1999;
Farr and Hoyt, 1998;
Taylor and McKeon, 1997b; Roberts et al., 1994; Kaplan et al.,
2001)
- Bub2 Cdc16 GAP (GTPase-
activating protein)
Regulates mitotic exit network in S. cerevisiae;
essential for cytokinesis in S. pombe
(Hu and Elledge, 2002; Lee et al., 2001; Pereira et al., 2000; Krishnan et al., 2000; Fraschini et al., 1999; Fesquet et al., 1999; Fankhauser et
al., 1993)
Bub3 Bub3 Bub3 Seven WD40
repeats
Interacts with Bub3- binding domains in Bub1
and BubR1
(Campbell and Hardwick, 2003;
Fraschini et al., 2001b; Sharp-Baker
and Chen, 2001;
Kalitsis et al., 2000;
Brady and Hardwick, 2000; Martinez- Exposito et al., 1999;
Taylor et al., 1998)
Mps1 Mps1 Mph1 Dual-specificity
kinase
Role in recruitment of checkpoint proteins to
kinetochores;
reported substrates include Mad1, Spc110 (in budding yeast also required for
spindle pole body duplication, but not in
fission yeast)
(Stucke et al., 2002;
Castillo et al., 2002;
Abrieu et al., 2001;
Fisk and Winey, 2001; Friedman et al., 2001; He et al., 1998; Weiss and Winey, 1996; Winey
et al., 1991)
Cdc20 Cdc20 Slp1 C-box;
D-box;
KEN-box;
WD40
Binds to Mad2, BubR1 (Mad3), APC;
activates APC;
phosphorylated by Cdk1 in mammals
(Kallio et al., 2002a;
Rudner and Murray, 2000; Kramer et al., 1998; Fang et al., 1998a; Hwang et al., 1998; Visintin et al., 1997; Kim et al., 1998; Sironi et al., 2001; Sudakin et al.,
2001; Tang et al., 2001) Vertebrates S. cerevisiae S. pombe Main structural
features
Comments References
Table 3: List of additional mitotic spindle checkpoint components mostly found in vertebrates.
Vertebrates S. cerevisiae S. pombe Main strucutral features
Comments References
CENP-E - - Kinesin-like plus-
end-directed motor
Interacts with microtubules and BubR1;
yeast kinesin homologues unclear
(Putkey et al., 2002;
Yao et al., 2000; Chan et al., 1999; Chan et al., 1998; Cooke et al.,
1997)
CENP-F - - Chromosome
segregation ATPase
Interacts with CENP-E and Bub1 in yeast-two
hybrid
(Chan et al., 1998)
Zw10 - - - Binds to Rod and Zwint-1;
no clear yeast homologue
(Scaerou et al., 2001;
Basto et al., 2000;
Scaerou et al., 1999;
Chan et al., 2000)
Rod - - - Binds to Zw10;
no clear yeast homologue
(Scaerou et al., 2001;
Basto et al., 2000;
Scaerou et al., 1999;
Chan et al., 2000)
Hec1 Ndc80 Ndc80 Coiled-coil Interacts with hSmc1
(cohesion);
required for chromosome segregation;
required to recruit hMad1/hMad2-complexes
to kinetochores
(Martin-Lluesma et al., 2002; Zheng et al., 1999; McCleland et al., 2003; Wigge and
Kilmartin, 2001)
Aurora-B Ipl1 Ark1 Serine/threonine
kinase
Ensures bipolar orientation;
proposed to sense tension at kinetochores
(Cheeseman et al., 2002; Kallio et al., 2002b; Murata-Hori
and Wang, 2002;
Biggins and Murray, 2001; Petersen et al., 2001; Tanaka et al.,
2002)
The mitotic spindle checkpoint senses kinetochore-microtubule attachment and/or tension
The kinetochore, a multi-layered proteinaceous complex that assembles on the centromeric DNA of each chromosome (Kitagawa and Hieter, 2001) is an integral part of the spindle assembly checkpoint (Fig. 4). During mitosis, the kinetochore mediates the interaction between the chromosome and spindle microtubules. In prometaphase, the kinetochores of a sister chromatid pair capture microtubules emanating from the opposite poles of the mitotic spindle.
Figure 4: Illustration of a metaphase chromosome showing its two sister chromatids attached to kinetochore microtubules. Each kinetochore forms a plaque on the surface of the centromere (red). The number of microtubules bound to a metaphase kinetochore varies from 1 in budding yeast to over 40 in some mammalian cells.
Then, the biorientated chromosomes move to the metaphase plate, a process called chromosome congression (Rieder and Salmon, 1994). In principle, two conditions are associated with the bipolar orientation of sister chromatids to the spindle: attachment, resulting from end-on docking of microtubules to the kinetochore, and tension, arising after bipolar attachment as an equilibrium between poleward and anti-poleward forces along the sister chromatids. The mitotic spindle checkpoint ensures that anaphase onset is only triggered when all the chromosomes are properly attached and aligned at the metaphase plate. This allows the separation of sister chromatids and their delivery to each spindle pole.
Elegant cell-biological studies have shown that a single unattached kinetochore is enough to inhibit the onset of anaphase throughout the cell. Laser ablation of this unattached kinetochore relieves the mitotic delay (Rieder et al., 1995).
Therefore, the checkpoint responds to the absence of microtubule occupancy at the unattached kinetochores (Hoffman et al., 2001). In addition, manipulations of chromosomes in insect spermatocytes showed that tension exerted across kinetochores during mitosis was enough to satisfy the spindle checkpoint (Li and Nicklas, 1995). However, to what extent lack of microtubule attachment and tension on kinetochores contribute to checkpoint activation in metazoan cells remains to be clarified (Millband et al., 2002; Musacchio and Hardwick, 2002). It has been proposed that the checkpoint proteins Mad2 and Bub1/BubR1 primarily sense attachment and tension, respectively (Hoffman et al., 2001; Skoufias et al., 2001;
Taylor et al., 2001; Zhou et al., 2002a). However, their respective roles in sensing attachment and tension remain to be confirmed and further analysed.
The checkpoint signaling pathway
Converging genetic, cell biological and biochemical studies have begun to shed some light on to how the mitotic spindle checkpoint components work at a molecular level.
As described, the spindle assembly checkpoint controls both the attachment of microtubules to kinetochores and the tension that is exerted at kinetochores upon bipolar attachment. In the absence of bipolar attachment, the spindle checkpoint proteins have to emit a global signal throughout the cell to inhibit anaphase onset.
How do the Mad/Bub proteins mediate cell cycle arrest and what is the target of the mitotic spindle checkpoint?
It is now clear that one main consequence of spindle checkpoint activation is the inhibition of the APC/C (Anaphase Promoting Complex/Cyclosome), a large multi-subunit E3 ubiquitin-protein ligase (Fig. 5) (Page and Hieter, 1999; King et al., 1996). An E3 ubiquitin ligase transfers ubiquitin to lysine residues in substrate proteins, and proteins modified in such a way are then recognized and degraded by the proteasome (Voges et al., 1999). The APC/C normally becomes active at the metaphase-anaphase transition, and its activity is required for anaphase entry. The
accessory factor Cdc20 and triggers the degradation of an anaphase-inhibiting protein called securin (Yanagida, 2000; Nasmyth et al., 2000). Polyubiquitin chains are added to securin by the APC/C, which leads to its destruction through 26S-proteasome- mediated proteolysis (Yamamoto et al., 1996; Cohen-Fix et al., 1996; Funabiki et al., 1996; Zou et al., 1999). Securin forms a tight complex with an evolutionarily conserved caspase-related protease termed separase (Funabiki et al., 1996; Ciosk et al., 1998; Kumada et al., 1998; Zou et al., 1999; Uhlmann et al., 2000; Waizenegger et al., 2000), thereby inhibiting separase´s activity. Thus, degradation of securin releases separase, which in turn must be phosphorylated, probably directly by cyclin- dependent kinases (Cdk1), in order to efficiently cleave its substrate (Stemmann et al., 2001). The substrate of separase is a subunit of a multiprotein complex termed cohesin, which creates physical links between sister chromatids (Waizenegger et al., 2000; Michaelis et al., 1997). Sister chromatid cohesion is first established during chromosome replication in S phase. Removal of the cohesion complex is regulated by two mechanisms: firstly, in higher eukaryotes the removal of cohesins from the chromosome arms is promoted by phosphorylation of the cohesion complex by Polo- like kinase 1 (Sumara et al., 2002). Residual cohesion at the centromeric region is enough to prevent sister-chromatid separation. Secondly, these remaining complexes must be disrupted through proteolytic cleavage of a cohesin subunit called Scc1 by separase (Uhlmann et al., 1999). This allows the sister chromatids to move poleward along the mitotic spindle, and anaphase is initiated (Fig. 5).
What is the nature of this “wait anaphase” signal that inhibits the activity of the APC/C and therefore prevents sister chromatid separation? The ubiquitin ligase activity of the APC/C towards securin requires association of APC/C with Cdc20, which activates the APC/C by direct binding (Visintin et al., 1997; Hwang et al., 1998; Shirayama et al., 1998; Fang et al., 1998a). Cdc20, the activating protein for the APC/C, is the molecular target of the mitotic spindle checkpoint, and two of the checkpoint proteins, Mad2 and BubR1 have been shown to interact with Cdc20, resulting in APC/C inhibition (Li et al., 1997; Fang et al., 1998b; Hwang et al., 1998;
Kim et al., 1998; Sudakin et al., 2001; Tang et al., 2001; Fang, 2002). In fact, two different checkpoint complexes have been purified from HeLa cells, which are capable of inhibiting the APC/C. One isolated checkpoint complex contains BubR1, Bub3 and substoichiometric amounts of Cdc20 (Tang et al., 2001). In addition, it was shown that recombinant BubR1 directly inhibits the activity of the APC/C and that the
kinase activity of BubR1 is not required for this inhibition (Tang et al., 2001).
Independently, another checkpoint complex has been isolated which is termed mitotic checkpoint complex (MCC). The MCC contains nearly stoichiometric amounts of BubR1, Bub3, Mad2 and Cdc20 (Sudakin et al., 2001), and the MCC is more potent than Mad2 alone at inhibiting the ubiquitin ligase activity of the APC/C. Importantly, the very same complex consisting of Mad3 (BubR1 homolog), Bub3, Mad2 and Cdc20 has been isolated from fission yeast (Millband and Hardwick, 2002) and budding yeast (Fraschini et al., 2001b). Future studies have to resolve the issue of whether Mad2 and BubR1 function independently or synergistically in transducing the anaphase-delaying signal by inhibiting APC/C activity.
Figure 5: The mitotic spindle checkpoint pathway. The checkpoint is activated by lack of microtubule attachment and tension at the kinetochores. The mitotic spindle checkpoint components are recruited to unattached kinetochores and might form several checkpoint protein complexes, e.g. the MCC, which is able to inhibit the APC/C. When all kinetochores achieve bipolar attachment to the mitotic spindle, the checkpoint is inactivated. The active APC/C is activated and ubiquitinates securin. Degradation of securin releases and activates separase, which cleaves Scc1, a subunit of the cohesion complex. Loss of sister chromatid cohesion triggers chromosome segregation and the onset of anaphase. Ub, ubiquitin.
Adopted and modified from (Yu, 2002).
The kinetochore is believed to act as a catalytic site for the production of this
“wait anaphase” signal that diffuses away to block the activity of the APC/C throughout the cell. In fact, it was shown that all of the vertebrate Mad and Bub checkpoint proteins localize to unattached kinetochores (Taylor et al., 2001; Chen et al., 1998; Sharp-Baker and Chen, 2001; Luo et al., 2002), consistent with the proposed role of kinetochores in generating the inhibitory checkpoint signal. In addition, along with the fact that Cdc20 is also enriched at kinetochores, it was suggested that the checkpoint complexes might be assembled at kinetochores (Fang et al., 1998b; Kallio et al., 1998). Furthermore, Cdc20 and several checkpoint proteins, including Mad2 and BubR1, turn over rapidly at the kinetochores in mammalian cells (Yu, 2002; Howell et al., 2000; Kallio et al., 2002a). Therefore, it is possible that unattached kinetochores catalyse the formation of the inhibitory checkpoint protein complexes, which then diffuse away to inhibit the APC/C. The diffusible signal might be the MCC and/or BubR1/Bub3/Cdc20. However, the MCC is present throughout the cell cycle and its formation does not require kinetochores (Sudakin et al., 2001).
Likewise, in yeast, functional kinetochores are not required for the formation of the MCC (Fraschini et al., 2001b). The exact role of the kinetochores in contributing to checkpoint signaling and in the formation of distinct checkpoint protein complexes has to be resolved by future studies.
Mps1-like protein kinases and the mitotic spindle checkpoint
A number of protein kinases are implicated in the mitotic spindle checkpoint, indicating that phosphorylation events play an important role in checkpoint signaling (Nigg, 2001). In addition to the checkpoint function exhibited by the kinases Bub1 (Sharp-Baker and Chen, 2001) and BubR1 (Chen, 2002), the family of Mps1-like protein kinases is emerging as an important regulator of mitotic progression.
Members of the Mps1 family of protein kinases are widely distributed in the eukaryotic world, but their functions are still poorly understood. Mps1p (Monopolar spindle 1) was first discovered in budding yeast S. cerevisiae (Winey et al., 1991), and kinases structurally related to Mps1p were described subsequently in S. pombe (Mph1p) (He et al., 1998), A. thaliana (PPK1) (Schutz and Winey, 1998), X. laevis (XMps1) (Abrieu et al., 2001) and mammals (mMps1/Esk in mouse,
hMps1/TTK/PYT in humans) (Douville et al., 1992; Fisk and Winey, 2001; Mills et al., 1992; Lindberg et al., 1993).
The founding member of the Mps1 family of protein kinases is budding yeast Mps1p. Analysis of the original mutant allele mps1-1, that carries a single point mutation in the kinase domain, revealed that cells failed in spindle pole body (SPB) duplication at the restrictive temperature, resulting in mitotic cells with a single SPB (Winey et al., 1991). In S. cerevisiae, the SPB is the centrosome equivalent organelle, thus contributing to the nucleation of microtubules and the formation of a bipolar mitotic spindle (Adams and Kilmartin, 2000). Because of the SPB duplication defect, the mps1-1 mutant cells formed a monopolar spindle (Winey et al., 1991). Normally, mutations that give rise to a monopolar spindle trigger the mitotic spindle checkpoint because of a disrupted mitotic spindle and cells arrest in mitosis (Weiss and Winey, 1996). However, unlike other yeast mutants that fail in SPB duplication and hence establish a monopolar spindle, mps1-1 cells do not show any cell cycle arrest (Weiss and Winey, 1996). Instead, these mutants missegregate their chromosomes and exit mitosis, leading to a drastic drop in viability (Winey et al., 1991). In addition, mps1-1 strains fail to arrest when microtubule-depolymerizing drugs like nocodazole destroy the spindle, but instead they exit mitosis as multibudded, polyploid cells (Weiss and Winey, 1996). This phenotype is common among mad and bub mutants (Li and Murray, 1991; Hoyt et al., 1991), and indicated that Mps1p acts in the mitotic spindle checkpoint. Consistent with this is the recent finding that Mps1p is localized to kinetochores (Castillo et al., 2002).
Beside the phenotypes associated with the mps1-1 mutant, overexpression studies of Mps1p gave a second clue for the involvement of the kinase in the mitotic spindle checkpoint. Overexpression of Mps1p in budding yeast led to a cell cycle arrest in metaphase in the absence of any apparent spindle damage (Hardwick et al., 1996). The metaphase arrest upon Mps1p overexpression is dependent on all the other checkpoint proteins (Mad1, 2, 3 and Bub1, 3), indicating that the arrest requires an intact spindle assembly checkpoint. Overexpression of a dominantly acting BUB1-5 allele also blocks cell cycle progression in mitosis with undamaged spindles and this delay is also dependent upon the functions of the other checkpoint genes, including Mps1p (Farr and Hoyt, 1998). Therefore, the cell cycle arrest caused by MPS1 and BUB1-5 overexpression is interdependent upon the function of each other and
Mps1p and Bub1p (Hardwick et al., 1996; Farr and Hoyt, 1998). In addition, it was recently shown that the checkpoint-dependent arrest caused by Mps1p overexpression requires Ipl1p, the budding yeast member of the Aurora protein kinase family (Biggins and Murray, 2001). Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and to segregate their chromosomes normally, indicating that Ipl1 is required for maintaining this checkpoint arrest (Biggins and Murray, 2001). The important roles of kinetochores in checkpoint signaling have already been mentioned.
However, an interesting observation is that cell cycle arrest induced upon Mps1p overexpression does not require intact kinetochores. In an ndc10-1 strain, which lacks functional kinetochores, overexpression of Mps1p still causes an arrest, suggesting that despite the absence of intact kinetochores the mitotic spindle checkpoint can be activated (Fraschini et al., 2001b).
In budding yeast, activation of the mitotic spindle checkpoint triggered upon spindle damage can be monitored by the phosphorylation state of Mad1p (Hardwick and Murray, 1995). Mad1p is also hyperphosphorylated in wild-type and in all of the mad and bub mutants after Mps1p overexpression, whereas in mps1-1 cells no modification of Mad1p was detected (Hardwick et al., 1996). Despite the cell cycle arrest upon overexpression of the BUB1-5 allele no hyperphosphorylation of Mad1p was detected, suggesting that Mad1p phosphorylation does not correlate with mitotic arrest (Farr and Hoyt, 1998). However, Mps1p purified from yeast was able to phosphorylate an amino-terminal and carboxyl-terminal fragment of Mad1p, at least in in vitro kinase assays (Hardwick et al., 1996). Additional in vivo substrates of Mps1p as well as the mechanism by which Mps1p is activated in the mitotic spindle checkpoint are currently unknown.
In fission yeast S. pombe, the MPS1 ortholog, called mph1(Mps1p-like pombe homolog), was identified in a screen for fission yeast genes that arrest cells in metaphase when overexpressed (He et al., 1998). Fission yeast mph1 could complement the spindle checkpoint defect of a budding yeast mps1-1 mutant, indicating that it represents a bona fide functional homolog (He et al., 1998). The mph1 gene is not essential, but the deletion mutant shows defects in the mitotic spindle checkpoint response. Importantly, overexpression of Mph1p mimics activation of the checkpoint and imposes a metaphase arrest, which is dependent on the checkpoint protein Mad2p (He et al., 1998). Recently, it was shown that Mph1p, together with Bub1p and Bub3p, is required for kinetochore recruitment of Mad3p
(Millband and Hardwick, 2002). However, contrary to budding yeast Mps1p, Mph1p is not required for spindle pole body duplication. This aspect will be discussed in more detail later.
Initial characterisations have also been reported for putative vertebrate homologs of yeast Mps1p. Most recently, Mps1 kinase has been implicated in the mitotic spindle checkpoint in Xenopus egg extracts (Abrieu et al., 2001). XMps1 was shown to localize to kinetochores and to be necessary for the establishment and maintenance of a mitotic spindle checkpoint-mediated arrest reconstituted in Xenopus egg extracts (Abrieu et al., 2001). Furthermore, kinetochore recruitment of the checkpoint proteins XMad1, XMad2 and CENP-E was dependent on the kinase activity of XMps1, consistent with the yeast data that Mps1 kinases must act early in the checkpoint pathway (Abrieu et al., 2001). Mammalian Mps1 family members are expressed in all proliferating cells and tissues (Mills et al., 1992; Hogg et al., 1994), consistent with a proposed function in cell cycle progression. Furthermore, gene expression of hMps1/TTK is under the tight control of the transcription factor E2F4, suggesting transcriptional induction during the G1 phase of the cell cycle (Ren et al., 2002) and gene expression of hMps1/TTK is induced upon IL-2 (Schmandt et al., 1994) and TNF-α treatment (Ah-Kim et al., 2000). Functional analysis of mammalian Mps1 kinases and their involvement in the mitotic spindle checkpoint have not been performed so far. However, murine Mps1 was reported to localize to kinetochores in mitosis (Fisk and Winey, 2001).
S. cerevisiae Mps1p and spindle pole body duplication
Besides a function in the mitotic spindle checkpoint, S. cerevisiae Mps1p was shown to be essential for the duplication of the spindle pole body (SPB) (Winey et al., 1991).
SPBs have a trilaminar disc-like structure (Byers and Goetsch, 1974; Kochanski and Borisy, 1990) (Fig. 6A). The SPB is embedded in the nuclear envelope throughout the cell cycle, allowing it to nucleate simultaneously nuclear and cytoplasmic microtubules (Byers and Goetsch, 1974). SPBs duplicate only once in each cell cycle and function to nucleate microtubules that will form the mitotic spindle. A proper bipolar mitotic spindle is essential for the accurate segregation of chromosomes.
Figure 6: The spindle pole body of S. cerevisiae.
(A) Electron microscopy of a thin section of a budding yeast cell in mitosis showing two spindle pole bodies (SPBs), a half-bridge (HB), nuclear pore complexes (NPCs) and the nuclear membrane (NM), which remains intact during mitosis. The three layers within the SPB are also shown: inner plaque (IP), central plaque (CP) and outer plaque (OP). Bar, 0.1µm. Adopted from (Adams and Kilmartin, 2000).
(B) Schematic illustration of the SPB-duplication cycle and the various functions of Mps1p in S.
cerevisiae. Mps1p acts in multiple steps in SPB duplication. The numerous mps1 mutants are listed with their terminal SPB morphology. Adopted from (Winey and Huneycutt, 2002).
The SPB duplication pathway in budding yeast was mainly described through electron microscopy analysis of wild type and mutant cells that fail at different stages of SPB duplication (Byers and Goetsch, 1975; Adams and Kilmartin, 1999). In brief, duplication starts in G1 of the cell cycle, with the formation of the satellite, an accumulation of SPB components on the cytoplasmic surface of the half-bridge, beside the existing SPB (Byers and Goetsch, 1975). The amorphous satellite appears to develop into a larger ordered structure called the duplication plaque (Adams and Kilmartin, 1999; O'Toole et al., 1999), which is thought to be the precursor of the new SPB. Insertion of the duplication plaque into the nuclear envelope occurs after assembly of the new SPB and then it associates with additional SPB components that will make up the inner (nuclear) plaque layers (Adams and Kilmartin, 1999).
In a screen for mutants in SPB duplication, the budding yeast Mps1p protein kinase was identified (Winey et al., 1991). The mps1-1 mutation results in a failure in SPB duplication at the restrictive temperature, and observation of the unduplicated SPB by electron microscopy suggested that Mps1p is required for a medial step in SPB duplication (Winey et al., 1991). These mutant cells show a single, large SPB with an enlarged and very prominent half-bridge structure (Fig. 6B). Therefore,
Mps1p is required for the transition from the satellite-bearing to side-by-side SPBs.
Subsequently, additional mutant alleles of MPS1 were identified in screens for mutants defective in SPB duplication (Schutz and Winey, 1998). One of these new alleles, mps1-737, showed a terminal morphology of the SPBs that is distinct from that of the other MPS1 mutations. This strain shows defects late in SPB duplication:
mutant cells assemble a second SPB that is structurally defective (Schutz and Winey, 1998) (Fig. 6B). Importantly, all mutants described so far contain a single point mutation in conserved residues within the kinase domain. Recently, another collection of new Mps1 alleles was created by PCR mutagenesis (Castillo et al., 2002). One allele isolated, mps1-8, which contains several mutations in the non-catalytic amino- terminal domain, is only defective in SPB duplication, but not in the mitotic spindle checkpoint (Castillo et al., 2002). The SPB observed at the restrictive temperature in these mutants has a short half-bridge, a SPB morphology not seen with other mps1 alleles (Fig. 6B). Thus, Mps1p is essential for SPB duplication, and it is required for a number of distinct steps in building a functional SPB (Winey and Huneycutt, 2002).
In line with this is the recent finding that Mps1p localizes to SPBs, suggesting that it may act at SPBs to control their assembly (Castillo et al., 2002). In addition, several of these alleles have been used to show that Mps1p is also required for SPB duplication in meiosis (Straight et al., 2000).
How does Mps1p regulate SPB duplication? Three components of the SPB have been shown to be Mps1p substrates in vitro: Spc98p, Spc110p and Spc42p (Castillo et al., 2002; Friedman et al., 2001; Pereira et al., 1998). Spc98p is a component of the 6S γ-tubulin complex in yeast that is responsible for the nucleation of microtubules (Knop and Schiebel, 1997). Spc110p binds to the 6S γ-tubulin complex at the nuclear side of the SPB (Knop and Schiebel, 1997; Sundberg and Davis, 1997), and Spc42p is a component of the core of the SPB (Donaldson and Kilmartin, 1996). Importantly, the phosphorylated forms of Spc98p, Spc110p, and Spc42p depend on Mps1p activity in vivo, and Mps1p and Spc42p were also found to bind each other by co-immuoprecipitation (Castillo et al., 2002). Overall, the Mps1p protein kinase in budding yeast is required for multiple steps in SPB duplication/assembly and it is likely to function by directly binding and phosphorylating SPB components in order to direct their assembly.
Mps1-like protein kinases and centrosome duplication in vertebrates
The analysis of Mps1p in budding yeast begs the question of whether the function of Mps1p in SPB duplication is conserved in other organisms, especially in vertebrates.
The possibility that Mps1 protein kinases are not general regulators for SPB- or centrosome duplication is provoked by the observation that the C. elegans genome lacks a clear Mps1p ortholog. Furthermore, fission yeast S. pombe Mph1p is clearly involved in the mitotic spindle checkpoint, but no function in SPB duplication could be observed (He et al., 1998).
During progression through the cell cycle, the centrosome needs to be duplicated once, and only once in animal cells. Normally, at the onset of mitosis, the duplicated centrosomes determine the bipolarity of the mitotic spindle (Raff, 2001).
However, deregulation of the centrosome duplication cycle leads to severe defects within a cell. Extra copies of centrosomes result in the formation of multipolar spindles, and a failure in centrosome duplication results in monopolar spindles. Both events will generally provoke abnormal chromosome segregation, leading to polyploid cells (Kramer and Ho, 2001; Nigg, 2002; Fisk et al., 2002). Importantly, many human tumor cells show aneuploidy and contain supernumerary centrosomes (Nigg, 2002; Lingle et al., 1998; Pihan et al., 1998; Weber et al., 1998). Therefore it is important to understand how centrosome duplication is regulated.
In animal cells, the centrosome was discovered and described almost simultaneously by Edouard van Beneden and Theodor Boveri, and it was depicted as a small body that seemed to control cell division (van Benenden, 1883; Boveri, 1887).
Electron microscopy revealed that the mammalian centrosome consists of two barrel- shaped centrioles that are embedded in a proteinaceous matrix of pericentriolar material (PCM) (Kellogg et al., 1994; Andersen, 1999b) (Fig. 7). Each centriole is composed of nine triplets of microtubules, thereby forming a 500nm long structure with a diameter of about 200nm. Importantly, the two centrioles can be distinguished from each other: the older of the two carries appendages that are close to its distal end. The appendages are implicated in anchoring microtubules (Bornens, 2002). The centrioles may contribute to PCM assembly and within the PCM many proteins such as γ-tubulin ring complexes are recruited that are essential for microtubule nucleation (Moritz and Agard, 2001) and for the formation of the mitotic spindle in prophase.
Figure 7: Structure of the centrosome in animal cells. The centrosome consists of two centrioles that are embedded in the PCM (pericentriolar material). Adopted from (Nigg, 2002)
At the end of mitosis each daughter cell inherits a single centrosome and by the start of the next mitosis each cell contains two centrosomes. Thus, during interphase, the centrosome has to be duplicated (Hinchcliffe and Sluder, 2001;
Stearns, 2001) (Fig. 8). In mammalian cells, this event starts in late G1/early S phase after loss of the orthogonal orientation of the two centrioles (Kuriyama and Borisy, 1981). The appearance of short daughter centrioles, so called pro-centrioles, at the proximal end of each parental centriole, indicates the beginning of centriole duplication at the beginning of S phase and during S phase (Kochanski and Borisy, 1990). Thus, centrosome duplication occurs by a semi-conservative mechanism (from the perspective of the entire centrosome). These procentrioles elongate during S and G2 phase, reaching mature length in mitosis and the following G1 (Kuriyama and Borisy, 1981; Lange et al., 2000). The completion of centrosome duplication takes place in G2 with the recruitment of several components of the PCM including γ- tubulin to increase microtubule-nucleation activity (Verde et al., 1992; Wolff et al., 1992; Lane and Nigg, 1996). Concomitantly, centrosome separation occurs with a pair of mother-daughter centrioles in each centrosome (Blangy et al., 1995; Walczak, 2000).
Figure 8: The centrosome duplication cycle.
Schematic illustration of the centrosome duplication cycle. The centrosome has to be duplicated once in every cell cycle to prevent chromosome missegregation or changes in ploidy. Modified and adopted from (Nigg, 2002).
On the molecular level, the coordination between DNA replication and centrosome duplication in S phase is mediated via the cyclin-dependent kinase 2 (Cdk2) (Hinchcliffe and Sluder, 2002). Cdk2 activity is required for both of these key S phase events. In addition, a common requirement for DNA replication and centrosome duplication is phosphorylation of the retinoblastoma gene product pRb and the subsequent release of the transcription factor E2F (Meraldi et al., 1999). E2F moves into the nucleus and switches on genes required for S phase, like cyclin A and E (Schulze et al., 1995; Ohtani et al., 1995). In some cell types like CHO or U2OS cells, the normal coordination between centrosome duplication and DNA replication can be disrupted by treating cells with hydroxyurea, which induces cell cycle arrest in S phase. If such cells are treated with hydroxyurea, DNA replication is blocked but centrosome duplication continues normally, leading to multiple copies (Balczon et al., 1995).
In Xenopus embryos, Cdk2 regulates centrosome duplication together with its binding partner cyclin E (Hinchcliffe et al., 1999; Lacey et al., 1999), whereas in mammalian somatic cells cyclin A has the predominant role in the Cdk2 complex (Meraldi et al., 1999; Balczon, 2001). Beside Cdk2, two other protein kinases have also been implicated in centrosome duplication. In the nematode Caenorhabditis elegans, the ZYG-1 kinase is required for centrosome duplication, and mutant
