Regulation of kinetochore recruitment of two essential mitotic spindle checkpoint proteins by Mps1 phosphorylation.
ABSTRACT Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphorylation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in kinetochore targeting and spindle checkpoint signaling.
- [show abstract] [hide abstract]
ABSTRACT: The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.Cell cycle (Georgetown, Tex.) 03/2013; 12(8). · 5.24 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The spindle assembly checkpoint (SAC) is a surveillance system that ensures the timely and accurate transmission of the genetic material to offspring. The process implies kinetochore targeting of the mitotic kinases Bub1, BubR1 and Mps1 which is mediated by the N-terminus of each kinase. Here, we report the 1.8 Å structure of the tetratricopeptide repeat (TPR) domain in the N-terminal region of human Mps1. The structure reveals an overall high similarity to the TPR motif of the mitotic checkpoint kinases Bub1 and BubR1, and a number of unique features that include the absence of the binding site for the kinetochore structural component KNL1, and determinants of dimerization. Moreover, we show that a stretch of amino acids at the very N-terminus of Mps1 is required for dimer formation, and that interfering with dimerization results in mislocalization and misregulation of kinase activity. Our results provide important insight into the molecular details of Mps1 mitotic functions including features that dictate substrate selectivity and kinetochore docking.Biochemical Journal 10/2012; · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.The Journal of Cell Biology 04/2013; · 10.82 Impact Factor
Molecular Biology of the Cell
Vol. 20, 10–20, January 1, 2009
Regulation of Kinetochore Recruitment of Two Essential
Mitotic Spindle Checkpoint Proteins by Mps1
Quanbin Xu,*†Songcheng Zhu,*†‡Wei Wang,* Xiaojuan Zhang,* William Old,*
Natalie Ahn,*§and Xuedong Liu*
*Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309;§Howard Hughes
Medical Institute, University of Colorado, Boulder, CO 80309; and‡Institutes of Biomedical Sciences, Fudan
University, Shanghai 200032, China
Submitted March 28, 2008; Revised September 26, 2008; Accepted October 3, 2008
Monitoring Editor: Tim Stearns
Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of
tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset
until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and
kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The
mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint
activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required
for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphory-
lation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces
recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in
kinetochore targeting and spindle checkpoint signaling.
Faithful segregation of chromosomes is essential for genome
stability and organism development (Lengauer et al., 1997;
Nicklas, 1997; Nasmyth, 2002). Aberrant chromosome seg-
regation generates aneuploid cells, a hallmark frequently
associated with cancer cells (Lengauer et al., 1997). It has
been speculated that aneuploidy may be a driving force for
cellular transformation. Aneuploidy is primarily caused by
errors during mitosis. In normal cells, correct segregation of
chromosomes is ensured by an evolutionarily conserved
surveillance signal transduction pathway called the mitotic
spindle checkpoint (McIntosh, 1991). Defects in chromosome
separation elicit checkpoint signal(s) to delay the onset of
anaphase until every chromosome has successfully attached
to the spindles (Amon, 1999; Yu, 2002; Draviam et al., 2004;
Weaver and Cleveland, 2005). The spindle checkpoint re-
sponse is highly robust because a single unoccupied kinet-
ochore is sufficient to cause mitotic arrest until proper at-
tachment of microtubules is reestablished (Rieder et al.,
The molecular components of the spindle checkpoint
pathway were first identified in yeast through various ge-
netic screens (Hoyt et al., 1991; Li and Murray, 1991; Weiss
and Winey, 1996; Amon, 1999). Subsequent studies revealed
that most of the key checkpoint proteins are conserved from
yeast to vertebrate systems. Homologues of these compo-
nents in mammalian cells include Bub1, BubR1, Bub3, Mad1,
Mad2, and Mps1 (Wassmann and Benezra, 2001; Kops et al.,
2005). Delay of mitotic progression upon triggering of the
spindle checkpoint is apparently achieved by inhibition of
the anaphase promoting complex/cyclosome (APC/C), an
E3 ubiquitin ligase that is responsible for ubiquitination and
degradation of securing and cyclin B (Nasmyth, 2001). Deg-
radation of securin activates the separase protease, which
removes the cohesion protein Scc1 from the held sister chro-
matids, allowing their subsequent separation in anaphase
(Uhlmann et al., 1999; Nasmyth, 2001). The current paradigm
for turning on spindle checkpoint signaling invokes produc-
tion of diffusible inhibitors of CDC20, an activator and sub-
strate specificity selector for APC/C (Yu, 2002). Inhibitors of
the APC may include activated Mad2, BubR1, or Bub1 or a
complex of Cdc20, Mad2, BubR1, and Bub3 (Fang et al., 1998;
Sudakin et al., 2001; Tang et al., 2004). The inhibition is
released upon proper attachment of kinetochores to the
spindle, although the molecular mechanism(s) underlying
extinguishment of the checkpoint signal remains to be elu-
Mps1 is among the several protein kinases implicated in
transducing the checkpoint signal. Originally identified as a
dual-specificity kinase whose levels are elevated in a variety
of tumor cell lines (Mills et al., 1992; Lindberg et al., 1993),
Mps1 seems to be an essential mitotic kinase that regulates
normal mitotic progression, chromosome congression, and
cytokinesis from yeast to vertebrate cells (Fisk et al., 2004;
Jelluma et al., 2008b). In yeast, Mps1 is essential for spindle
pole body duplication and has been implicated in centro-
some duplication in mammalian cells (Winey et al., 1991;
This article was published online ahead of print in MBC in Press
on October 15, 2008.
†These authors contributed equally to this work.
Address correspondence to: Xuedong Liu (xuedong.liu@colorado.
10 © 2008 by The American Society for Cell Biology
Fisk and Winey, 2001). Mps1 is distributed diffusely
throughout the cell and relocates to kinetochores in early
mitosis and upon activation of the spindle checkpoint
(Stucke et al., 2002, 2004; Liu et al., 2003). The protein kinase
activity of Mps1 is strongly elevated in mitosis and corre-
lates with increased autophosphorylation of Mps1 (Stucke et
al., 2002; Liu et al., 2003; Kang et al., 2007; Mattison et al.,
2007). Indeed, autophosphorylation of Mps1 at T676 of the
activation loop has been shown to contribute to the elevated
kinase activity (Kang et al., 2007; Mattison et al., 2007).
Whether autophosphorylation also regulates other aspects
of Mps1 biology remains unknown.
A common feature shared by the checkpoint proteins is
that they all localize to kinetochores upon activation of the
spindle checkpoint. Recruitment of checkpoint proteins to
kinetochores seems to be a hierarchical process (Martin-
Lluesma et al., 2002; Vigneron et al., 2004). For example,
kinetochore localization of Mad1 and Mad2 requires Mps1,
and Mad2 kinetochore localization depends on Mad1 but
not vice versa (Martin-Lluesma et al., 2002). This result is
consistent with the notion that Mps1 functions upstream in
the spindle checkpoint pathway (Abrieu et al., 2001). Kinet-
ochore localization of Mps1 requires Hec1/Ndc80, a core
component of the kinetochore outer plate essential for orga-
nizing microtubule attachment sites (DeLuca et al., 2005).
Recently, PRP4, a serine-threonine kinase, also has been
linked to kinetochore localization of Mps1, Mad1, and Mad2
(Montembault et al., 2007). In addition, other protein kinases
also have been implicated in Mps1 kinetochore recruitment
(Zhao and Chen, 2006; Montembault et al., 2007).
In this report, we investigate the functional relevance of
Mps1 kinase activity and autophosphorylation in kineto-
chore localization of spindle checkpoint proteins. We found
that the kinase activity of Mps1 is essential for its kinet-
ochore recruitment and autophosphorylation of Mps1 at
T12 and that S15 is necessary for kinetochore targeting
and subsequent recruitment of other spindle checkpoint
MATERIALS AND METHODS
Cell Culture, Transfections, and Antibodies
SW480 and 293T cells were purchased from American Type Culture Collec-
tion (Manassas, VA) and were maintained at 37°C in a 5% CO2atmosphere in
DMEM supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA),
penicillin, streptomycin (100 IU/ml and 100 mg/ml, respectively), and l-
glutamine. Mirus transfection reagent (Mirus, Madison, WI) was used for
transfection of 293T cells and retroviral packaging. Small interfering RNA
(siRNA) transfection was performed using SiLentFect (Bio-Rad, Hercules,
CA). The 21-nucleotide RNA duplexes targeting Mps1 were purchased from
Dharmacon RNA Technologies (Lafayette, CO). Antibodies against Mps1
(C19) or Mps1-NT were from Santa Cruz Biotechnology (Santa Cruz, CA) or
Millipore (Billerica, MA). Anti-phospho-Smad2 was a gift of Drs. P. Ten Djike,
C. Heldin, and A. Moustakas. Anti-Mad2 and CREST antibodies were pur-
chased from Covance Research Products (Princeton, NJ) and Antibodies
(Davis, CA), respectively.
DNA Manipulation and Stable Cell Line Generation
Mammalian Mps1 and Smad2 expression vectors have been described previ-
ously (Zhu et al., 2007). Point mutations in Mps1 were constructed using the
QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Deletion mutants of
Mps1 were constructed by polymerase chain reaction (PCR), and the relevant
fragments of Mps1 were subcloned into pREX-IRES-Hygromycin (Hygro), a
derivative of the bicistronic retroviral vector pREX-IRES-GFP described pre-
viously (Liu et al., 2000). The Mps1 siRNA-insensitive allele (Mps1R) was
created by scrambling the coding sequence using QuikChange mutagenesis
with a pair of oligonucleotides (oligos) (5?-CAAGAGCCAGATGATGCCA-
GAGATTATTTTCAAATGGCCAGAGC-3? and 5?-GCTCTGGCCATTTGA-
AAATAATCTCTGGCATCATCTGGCTCTTG-3?). All constructs and mu-
tations were confirmed by DNA sequencing. Retroviral expression constructs
were transfected into 293T cells with the pCL amphotrophic helper plasmid
(Naviaux et al., 1996). Forty-eight hours after transfection, virus-containing
supernatant was collected and used to infect SW480 cells as described previ-
ously (Liu et al., 1997). Cells expressing a defined level of green fluorescent
protein (GFP) were isolated using a MoFlo cell sorter (Dako Colorado, Fort
Collins, CO) as described previously (Liu et al., 1997).
Mass Spectrometry Protein Sequencing Analysis
Eight micrograms of GST-TEV-6xHis-Mps1 purified from insect cell were
autophosphorylated under the conditions described previously (Zhu et al.,
2007). The reaction mixture was processed with standard dithiothreitol re-
duction, iodoacetimide alkylation, and in-solution tryptic digestion. The di-
gestion mixture was fractionated by a microcapillary reverse phase high-
performance liquid chromatography column directly coupled to an electron
ion-spray mass spectrometer (QSTAR Pulsar; Applied Biosystems/MDS
Sciex, Foster City, CA). Phosphopeptides were analyzed using a polarity
switching method to alternate between detection and sequencing of the
phosphopeptides in the same run (Williamson et al., 2006). First a negative
mode precursor ion scan is acquired, monitoring for the marker ion PO3?at
?79 m/z, over a mass range of 500-1800 m/z, with Q1 set to low resolution
and Q3 set to unit resolution. When the signal intensity of the precursor ion
scan is above a threshold of 1000 cps, polarity is switched to positive mode,
and a high-resolution scan is acquired for charge determination and accurate
mass measurement of the three most intense ions, followed by positive mode
MS/MS sequencing. Tandem mass spectrometry (MS/MS) was searched with
MASCOT version 2.0 (MatrixScience, London, United Kingdom) by using a
small database of 50 standard proteins, including the sequence of the MPS1
fusion protein. Parent mass tolerance was 1.2 Da, MS/MS tolerance was 0.6
Da, with fixed modifications set to carbamidomethyl on cysteine, and variable
modifications set for methionine oxidation and phosphorylation on Ser, Thr,
and Tyr. MS/MS identifications with mascot scores above 20 were the man-
ually validated for quality and phosphorylation site determination.
Orthophosphate Labeling of Cells and Two-dimensional
(2D) Phosphopeptide Mapping
For 2D phosphopeptide analysis of Mps1, pEXL-FLAG-Mps1 and pEXL-
FLAG-Mps1T12S153AAwere transient transfected into 293T cells. Forty-eight
hours after transfection, wild-type and mutant kinases were immunoprecipi-
tated with the FLAG antibody from cell lysates and labeled with [?-32P]ATP
under the autophosphorylation conditions before SDS-polyacrylamide gel
electrophoresis (PAGE). Radiolabeled Mps1 or Mps1T12S153AAwas digested
with trypsin and subjected to 2D phosphopeptide mapping as described
previously (Boyle et al., 1991). SW480 cells stably expressing yellow fluores-
cent protein (YFP)-Mps1 and YFP-Mps1T12S153AAgrown in 10-cm plates
were synchronized by double thymidine block. After washing twice with
phosphate-free media (DME; Invitrogen), cells were released in 4 ml of
phosphate-free media with 10% dialyzed fetal bovine serum and 0.1 ?g/ml
nocodazole for 12 h plus 1 mCi/ml [32P]orthophosphate (PerkinElmer Life
and Analytical Sciences, Boston, MA) for 4 h. Cells were harvested and
YFP-Mps1 or YFP-Mps1T12S153AAwere immunoprecipitated with an Mps1
CT antibody (Millipore) in radioimmunoprecipitation assay buffer and re-
solved by 12% SDS-PAGE gel before transferring to a nitrocellulose mem-
brane. Radiolabeled YFP-Mps1 and YFP-Mps1T12S153AAwere analyzed as
siRNA Knockdown and Immunofluorescence Microscopy
Mps1 knockdown was achieved using the siRNA (GCACGUGACUACUUU
CAAAUU) synthesized by Dharmacon RNA Technologies. To achieve high
knockdown efficiency, SW480 cells were transfected twice with 20 nM Mps1
siRNA with SiLentFect (Bio-Rad) every 24 h according to the manufacturer’s
protocol. For immunostaining, cells were grown on coverslips and washed
three times with Dulbecco’s phosphate-buffered saline (D-PBS) before fixing
for 10 min in D-PBS plus 1% paraformaldehyde at room temperature. After
fixation, the cells were blocked with 5% nonfat dry milk in PBS/Tween 20 for
45 min and probed with primary antibody diluted in 5% nonfat dry milk for
90 min. Anti-Mps1, NT (Millipore), and anti-Mad2 (Covance Research Prod-
ucts) were used at 1:100 dilution. Kinetochores were identified by staining
cells with the human autoimmune serum CREST (Antibodies) at 1:300 dilu-
tion. After extensive wash, the secondary antibodies conjugated with Alexa
Fluor 596 (Invitrogen) were applied with 1:600 dilution. The slides were
prepared according to standard procedure.
Image Acquisition and Analysis
Pictures of immunofluorescence-stained cells were taken on a TE2000-S mi-
croscope (Nikon, Tokyo, Japan) equipped with MetaMorph image analysis
software (Molecular Devices, Sunnyvale, CA). Acquired images were sized,
scaled, pseudocolored, and overlaid by using MetaMorph software. For quan-
titation of the relative amount of YFP-Mps1 and related mutants on the
kinetochores, a method described by Hoffman et al. (2000) was adopted, with
minor modifications. The primary 16-bit images were analyzed using ImageJ
software (http://rsbweb.nih.gov/ij/). Briefly, the kinetochores were centered
by a circle with 3-pixel radius (Rin) (0.86 ?m in diameter, which is large
enough to cover a majority of kinetochore fluorescence in SW480 cell), and the
Mps1 Kinetochore Targeting
Vol. 20, January 1, 2009 11
total integrated fluorescence counts within this region (Fin) were measured.
To subtract the background within this area, an outer circle with 4 pixel radius
(Rin) was centered on the same kinetochore and the integrated fluorescence
counts (Fout) was obtained (a detailed illustration of the method is described
in figure 3 of Hoffman et al., 2001). The background of fluorescence (Fback-
ground) can be calculated as Fbackground? (Fout? Fin)(? Rin2/?(Rout? Rin)2).
The integrated intensity of YFP-Mps1 or its related mutants on a given
kinetochore was obtained using the equation Fkinetochore? Fin? Fbackground.
Because the expression levels of the fusion protein in each given cell could
affect the fluorescence intensity of the kinetochore, cells with similar overall
fluorescence intensity were chosen for quantitation. In addition, the average
values of kinetochore fluorescence was normalized to the relative expression
levels of YFP-Mps1 proteins in the cytosol in each cell, which was calculated
as a ratio of average cytoplasmic fluorescence intensity of a given cell (Bi)
(calculated by an average of fluorescence intensity of 3-pixel radius circles
randomly chosen in the cytoplasmic region with the number of circles picked
equaling to the number of kinetochores quantified in a given cell) versus
average cytoplasmic fluorescence from at least ten cells (B?). Finally, kineto-
chore fluorescence intensity of YFP-Mps1 or related mutants in a given cell is
normalized to the average fluorescence staining intensity of CREST in the
same cell (Ci). Thus relative fluorescence intensity of YFP-Mps1 or related
mutants is defined as F’kinetochore? (Fin? Fbackground)/Ki, where Ki?
(Bi/B?) ? Ci. For the endogenous Mps1 kinetochore localization, average
values of kinetochore fluorescence of Mps1 were normalized to the CREST
fluorescence intensity. The statistical analysis was performed using GraphPad
software (GraphPad Software, San Diego, CA).
Identification of Mps1 Autophosphorylation Sites by
Mps1 is hyperphosphorylated during mitosis with a con-
comitant increase in kinase activity (Stucke et al., 2002; Liu et
al., 2003; Kang et al., 2007). Phosphorylation could be a
compelling mechanism to regulate Mps1 function during
mitosis. To determine the role of Mps1 phosphorylation, we
expressed and purified recombinant Mps1 from insect cells
by using the baculoviral expression system. To identify
Mps1 phosphorylation sites in vitro, purified Mps1 kinase
was autophosphorylated by incubation with cold ATP and
subjected to tryptic digestion. Multiple electrospray ioniza-
tion-liquid chromatography/tandem mass spectrometry runs
were carried out to identify the phosphopeptides. With
?90% coverage of Mps1 from three independent runs, we
were able to identify eight distinct phosphopeptides, with a
total of 14 serines or threonines that were phosphorylated
(Table 1 and Supplemental Figure 1A). The phosphorylation
sites occupied in vitro are predominantly in the N- and
C-terminal regions of Mps1 in the activation loop of the
kinase domain. Two types of Mps1 autophosphorylation
sites were detected. The first type is phosphoserine or phos-
phothreonine (pS/T) followed by a hydrophobic residue
(e.g., Ile, Leu, or Val). We have previously found that Mps1
can transphosphorylate two distal serine residues of
Smad2/3 at the carboxyl terminal SSXS motif (Zhu et al.,
2007). Thus, this type of site can be targeted for either trans-
or autophosphorylation by Mps1. The second type of phos-
phorylation site for Mps1 is phosphoserine or phospho-
threonine (pS/T) followed by a negatively charged residue
(e.g., D or E).
One phosphopeptide containing S821 near the carboxy
terminus of Mps1 does not fall into these two categories.
Because insect cells are eukaryotes, there is a possibility that
some of the sites identified are independent of Mps1 kinase
and phosphorylated by other kinases in insect cells. To
address this issue, we also purified catalytically inactive
Mps1 kinase (Mps1KD) from insect cells. As shown in our
previous study (Zhu et al., 2007), purified Mps1KD can
neither autophosphorylate itself nor phosphorylate other
substrates, such as Smad2, in vitro. Because no label can be
incorporated in the purified Mps1KD in the presence of
labeled ATP, it is unlikely there are other kinases capable of
phosphorylating Mps1 copurified with Mps1KD in vitro.
Mps1KD was incubated with or without ATP and subjected
to mass spectrometry analysis by using identical conditions
as wild-type Mps1. Four phosphopeptides were identified
(Supplemental Figure 1B) in both samples, suggesting no
additional phosphorylation in vitro with purified Mps1KD.
All of them except one feature phosphoserine or phospho-
threonine (pS/T) followed by a proline residue. Interest-
ingly, all of these sites except S821 were not detected in
wild-type Mps1 in vitro, suggesting that kinase-dead Mps1
is preferentially targeted by other kinases in insect cells. The
only phosphopeptide shared by the wild-type and the ki-
nase dead Mps1 is the one containing pSer821. This type of
site is known to be targeted by mitogen-activated protein
(MAP) kinase (Zhao and Chen, 2006; Cui and Guadagno,
2008). Thus, by comparing the phosphorylation profile of
wild-type and kinase-dead Mps1, we can definitively as-
sign Mps1 autophosphorylation sites in vitro. Identifica-
tion of autophosphorylation sites of Mps1 suggests that
Mps1 exhibits a rather broad spectrum of specificity of
phosphorylation site choice. This begs the question of
whether any of these autophosphorylation sites are phys-
Cell Cycle-dependent Mps1 Subcellular Localization
Subcellular localization of Mps1 is tightly regulated during
the cell cycle (Fisk and Winey, 2001; Stucke et al., 2002, 2004;
Liu et al., 2003). In the G1 phase of the cell cycle, endogenous
Mps1 is distributed diffusely throughout cells and relocates
to the nucleus and centrosomes during the G2/M transition.
During prophase and prometaphase, Mps1 is targeted to the
kinetochores; it comes off the kinetochores in metaphase. On
completion of mitosis, Mps1 returns to the cytoplasm. To
determine whether Mps1 phosphorylation plays a regula-
tory role for cell cycle-dependent dynamic localization, we
stably expressed a YFP-Mps1 fusion protein in SW480 colon
cancer cells. Localization of YFP-Mps1 during cell cycle
progression was tracked by fluorescence microscopy of live
cells. As shown in Figure 1A, YFP-Mps1 is targeted to the
kinetochore, identified by staining with CREST antisera,
during prophase and prometaphase. The staining pattern of
YFP-Mps1 is in excellent agreement with previously de-
scribed endogenous Mps1 localization (Fisk and Winey,
2001; Stucke et al., 2002, 2004; Liu et al., 2003), suggesting that
YFP-Mps1 is a valid system to study Mps1 localization
properties during mitosis.
Previous studies suggested that the N-terminal 301 amino
acids of Mps1 are sufficient for targeting Mps1 to the kinet-
Table 1. Summary of Mps1 phosphopeptides
T12 and S15
T33 and S37
T360, S362, S363
Q. Xu et al.
Molecular Biology of the Cell 12
ochore (Liu et al., 2003; Stucke et al., 2004). Subsequent
studies further confirmed the importance of the N-terminal
region of Mps1 in mediating kinetochore localization of
Mps1. To further confirm the essential role of the N-terminal
region of Mps1 in targeting full length Mps1 to the kineto-
chore, we expressed a small deletion mutant of Mps1
(? amino acids [aa] 12-95) in SW480 cells. Kinetochore local-
ization of YFP-Mps1 was determined by treating cells ex-
pressing wild-type or mutant YFP-Mps1 with nocodazole,
which causes prometaphase arrest by activating spindle
checkpoint signaling. Although YFP-Mps1 shows robust ki-
netochore localization, removal of aa 12-95 does not affect
the expression levels of the fusion protein but almost abro-
gates Mps1 targeting to kinetochores (Figure 1, B–D). Even
though this deletion represents a relatively small perturba-
tion in Mps1, there is a possibility that such a truncation may
disrupt Mps1 structure and result in a misfolded protein. To
rule out this possibility, we examined centrosome localiza-
tion of wild-type and mutant Mps1. As shown in Figure 1D,
mutant YFP-Mps1 is indistinguishable from wild-type Mps1
in centrosome localization, suggesting that it is unlikely that
such a deletion causes the protein to be misfolded. This
result also suggests that the kinetochore targeting signal of
Mps1 is separable from the centrosome targeting signal.
N-Terminal Autophosphorylation Sites of Mps1 Are
Required for Mps1 Kinetochore Targeting
Five phosphopeptides encompassing nine different Ser/Thr
residues identified from our mass spectrometry analysis of
Mps1 autophosphorylation sites in vitro are located in the
N-terminal region of Mps1 outside the kinase domain.
Given the important role of the N-terminal region of Mps1 in
kinetochore targeting, we hypothesized that phosphoryla-
tion of these sites may regulate Mps1 kinetochore recruit-
ment upon activation of spindle checkpoint signaling. To
test this hypothesis, we created YFP-Mps1NT0Pby changing
all nine Ser/Thr residues to alanines and stably expressed
this mutant in SW480 cells. As shown in Figure 2A, whereas
wild-type YFP-Mps1 shows robust kinetochore targeting
required for robust kinetochore targeting of Mps1. (A) Kinetochore
targeting of wild-type and N-terminal autophosphorylation site
mutant Mps1 (Mps1NT0P) in nocodazole-arrested mitotic cells.
Cells were treated and analyzed as described in Figure 1B. (B)
Asynchronous wild-type and mutant Mps1 cells were fixed with 1%
paraformaldehyde and stained with an anti-?-tubulin antibody and
Autophosphorylation of Mps1 at N-terminal residues is
cycle. (A) YFP-Mps1 is stably expressed in SW480 cells using the
bicistronic retroviral vector pREX-IRES-Hygro. YFP positive cells
were selected by fluorescence-activated cell sorting sorting. Asyn-
chronous cells were fixed with 1% paraformaldehyde and stained
with a CREST antiserum (Antibodies) to identify kinetochores (red)
and 4,6-diamidino-2-phenylindole (DAPI) to identify DNA. (B) De-
letion of aa 12-95 abrogates Mps1 kinetochore targeting in nocoda-
zole-arrested cells. YFP-Mps1?12-95 stably expressed in SW480 cells
was compared with YFP-Mps1 cells. Wild-type and mutant YFP-
Mps1 cells were treated with 0.1 ?g/ml nocodazole for 8 h after
release from synchronization with double thymidine treatment.
Cells were fixed with 1% paraformaldehyde and stained with a
CREST antiserum and DAPI. (C) Immunoblot of the endogenous
and YFP-Mps1 in SW480 cells. (D) Deletion of aa 12-95 of Mps1 does
not affect Mps1 centrosome localization in interphase cells. Asyn-
chronous wild-type and mutant Mps1 cells were fixed with 1%
paraformaldehyde and centrosomes were stained with an anti-?-
tubulin antibody and DAPI. Bar, 8 ?m.
Subcellular localization of YFP-Mps1 during the cell
Mps1 Kinetochore Targeting
Vol. 20, January 1, 2009 13
upon treatment with nocodazole, mutation of all nine N-
terminal autophosphorylation sites markedly reduces YFP-
Mps1 kinetochore localization. To determine whether
removal of these phosphorylation sites also perturbs centro-
some targeting of Mps1, we analyzed localization of wild-
type and mutant YFP-Mps1 on centrosomes in interphase
cells. No difference was observed between wild-type and
mutant Mps1 (Figure 2B), suggesting that these N-terminal
autophosphorylation sites are very important in regulating
Mps1 recruitment to the kinetochore but dispensable for
To further delineate which residues among the nine
serine/threonine sites identified above are crucial in medi-
ating kinetochore targeting of Mps1, we constructed three
additional Mps1 point mutants and stably expressed them in
SW480 cells. Because Mps11-301has been shown to be suffi-
cient for kinetochore targeting and deletion of Mps1 aa 12-95
abolishes Mps1 targeting (5 of 9 phosphorylation sites are
located within this region), we focused our efforts on the five
phosphorylation sites in the N-terminal region and did not
test the effects of T288, T360, S362, and S363 on kinetochore
localization of full-length Mps1. As shown in Figure 3A,
whereas mutation of T33, S37 has no discernible effects on
Mps1 kinetochore targeting, mutation of T12 and S15 to
alanines severely impairs Mps1 kinetochore localization and
displays a pattern almost identical to YFP-Mps1NT0P(Figure
3B). In contrast, mutation of S80 to alanine had only a minor
effect (Figure 3, A and B). Again, none of these mutations
affects Mps1 centrosome localization (data not shown). We
independently confirmed expression of wild-type and mu-
tant YFP-Mps1 fusions by immunoblotting analysis (Figure
3B). If phosphorylation of T12S15 is important, we would
expect that substituting T12S15 with aspartic acids should
partially mimic phosphorylation at these sites and the re-
sulting mutant should exhibit little defects in kinetochore
targeting. To test this hypothesis, YFP-Mps1T12S153DDwas
created and stably expressed in SW480 cells. As expected,
kinetochore targeting of this mutant is more resemble the
wild-type (Figure 3, B and C), which support the hypothesis
that phosphorylation at T12S15 is critical for Mps1 kineto-
There is a possibility that mutation of T12 and S15 could
have affected Mps1 kinase activity. We addressed this issue
by cotransfecting wild-type and mutant Mps1 with Smad2
in 293T cells followed by measuring Smad2 phosphorylation
by using a phospho-specific antibody. In agreement with
our previous result (Zhu et al., 2007), wild-type Mps1 phos-
phorylates Smad2 at SSMS motif, whereas little if any phos-
phorylation can be observed with kinase-dead Mps1 (Sup-
plemental Figure 2). Phosphorylation of Smad2 by T12 and
S15 mutants of Mps1 is as efficient as the wild-type Mps1,
suggesting that these mutants do not affect Mps1 kinase
activity. Taken together, our data suggests that phosphory-
lation of Mps1 at T12 and S15 is required for robust kinet-
ochore targeting of full-length Mps1 but dispensable for its
In Vitro and in Vivo Phosphorylation of T12 and S15
Even though Mps1 undergoes autophosphorylation at T12
and S15 in vitro and both residues prove to be critical for
kinetochore targeting of Mps1, it is still important to dem-
onstrate that these sites are also phosphorylated in cells. To
further demonstrate phosphorylation of Mps1 at T12S15
expressed in mammalian cells, FLAG-tagged Mps1 and
Mps1T12S153AAwere expressed in 293T cells. Wild-type and
mutant Mps1 were immunoprecipitated with the anti-FLAG
antibody, subsequently incubated with [?-32P]ATP under
sites absolutely required for Mps1 kinetochore targeting. (A) YFP-
Mps1T12S153AA, YFP-Mps1T33S373AA, and YFP-Mps1S803Awere stably
expressed in SW480 cells. Kinetochore localization of these Mps1 mutants
are analyzed as described in Figure 2. (B) Quantitation of fluorescent
density of kinetochores labeled by YFP-Mps1 and YFP-Mps1 mutants in
prometaphase cells. For YFP-Mps1, 115 kinetochores were determined in
six cells randomly selected prometaphase cells; for YFP-Mps1NTOP, 103
83 kinetochores were counted; for YFP-Mps1T12S153AA, 98 kinetochores
difference is statistically significant between YFP-Mps1, YFP-Mps1NTOP
(p ? 0.001) and so is YFP-Mps1 and YFP-Mps1T12S153AA. (C) Immuno-
blotting analysis of expression of wild-type and mutant Mps1 in SW480
cells. Cell lysates prepared from parental uninfected cells or cells stably
C19 (Santa Cruz Biotechnology).
Identification of the N-terminal Mps1 autophosphorylation
Q. Xu et al.
Molecular Biology of the Cell14
kinase reaction conditions before SDS-PAGE and blotting to
nitrocellulose membrane.32P-labeled Mps1 or Mps1 mutant
was excised and subjected to 2D tryptic mapping analysis
(Boyle et al., 1991). Shown in Figure 4, FLAG-tagged Mps1
and Mps1T12S153AAdiffers by only one major spot on the
thin layer chromatography (TLC) plate. The missing spot in
Mps1T12S153AAbut present in wild-type Mps1 is probably
the phosphopeptide containing T12 and S15. This result
further confirms the mass spectrometry data indicating that
T12 and S15 are targeted for phosphorylation.
To determine whether Mps1 is phosphorylated at T12 and
S15 in mitotic-arrested cells, SW480 cells expressing either
YFP-Mps1 or YFP-Mps1T12S153AAwere left in nocodazole
for 12 h before labeling with [32P]orthophosphate for 4 h.
YFP-Mps1 or YFP-Mps1T12S153AAwas immunoprecipitated
from mitotic-arrested cells and blotted to nitrocellulose
membrane after resolution by SDS-PAGE. Both YFP-Mps1
and YFP-Mps1T12S153AAcan be labeled by [32P]orthophos-
phate in mitotic-arrested cells, although the intensity of YFP-
Mps1T12S153AAis lower than that of YFP-Mps1 (Supple-
mental Figure 3A). This could be a result of fewer occupied
sites due to mutations in the potential phosphorylation sites.
32P-Labeled YFP-Mps1 or YFP-Mps1T12S153AAbands from
mitotic cells were excised and subjected to 2D tryptic phos-
phopeptide analysis. Five major labeled phosphopeptides
are clearly visible in the YFP-Mps1 sample, and only four of
the five are detectable in YFP-Mps1T12S153AA. We interpret
the missing spot on the TLC plate as the T12, S15 containing
phosphopeptide (ELTIDSIMNK) because this spot is only
present in wild-type but not in the mutant Mps1. Hence,
phosphorylation of T12 and S15 is likely to be phosphory-
lated in mitotic-arrested cells.
The T12 and S15 Phosphorylation Sites Are Also Required
for Kinetochore Targeting of the N-Terminal Domain
Previous studies suggest that the amino-terminal 301 resi-
dues of Mps1 are both necessary and sufficient for its kinet-
ochore association (Liu et al., 2003; Stucke et al., 2004). Hav-
ing established that T12 and S15 are important for full-length
requires phosphorylation at T12 and S15. (A) Kinetochore localization
of YFP-Mps1, YFP-Mps11-301, and YFP-Mps11-301T12S153AAin promet-
aphase-arrested cells induced by nocodazole treatment (0.1 ?g/ml).
The kinetochores and chromosomes are identified by staining with
human CREST antisera and DAPI, respectively. (B) Quantitation of
fluorescent density of YFP-Mps1 and related mutants on the kineto-
chores of prometaphase cells. More than 52 kinetochores in at least
five randomly selected cells in each cell line were analyzed. YFP-
Mps11-301T12S153AAcannot relocalize to kinetochores in prometaphase
cells. (C) Immunoblotting analysis of expression of YFP-Mps11-301, and
YFP-Mps11-301T12S153AAin SW480 cells.
Kinetochore targeting of the N-terminal fragment of Mps1
S15 mutant Mps1 phosphorylation. FLAG-tagged Mps1 or
Mps1T12S153AAwere expressed in 293T cells by transient transfec-
tion. Forty-eight hours after transfection, FLAG-Mps1 or FLAG-
Mps1T12S153AAwere immunoprecipitated using an anti-FLAG an-
tibody and incubated in the presence of [?-32P]ATP under the in
vitro kinase reaction conditions before SDS-PAGE. Bands corre-
sponding to32P-labeled Mps1 or Mps1T12S153AAwere excised and
subjected to 2D phosphopeptide mapping analysis as described
previously (Boyle et al., 1991).
2D tryptic mapping analysis of wild-type or T12 and
Mps1 Kinetochore Targeting
Vol. 20, January 1, 200915
recruitment to the kinetochore, we also investigated whether
these two sites are also critical for the N-terminal fragment
of Mps1 to associate with the kinetochore. In agreement with
previous observations (Liu et al., 2003; Stucke et al., 2004),
YFP-Mps11-301relocates to the kinetochore in response to
nocodazole treatment when stably expressed in SW480 cells
(Figure 5A). This result confirms that the N-terminal 301
amino acid residues of Mps1 indeed contain the kinetochore
Even though the N-terminal fragment of Mps1 does not
contain the kinase domain, T12 and S15 can still be phos-
phorylated by Mps1 given that autophosphorylation of
Mps1 occurs through an intermolecular mechanism (Kang et
al., 2007; Mattison et al., 2007). In fact, the N-terminal domain
is an excellent substrate for Mps1 in vitro (data not shown).
To assess the role of T12 and S15 in the kinetochore targeting
of the N-terminal fragment of Mps1, we introduced point
mutations at these two positions into YFP-Mps11-301. The
resulting mutant (YFP-Mps11-301/T12S153AA) is defective in
kinetochore association (Figure 5A). Mutation of these phos-
phorylation sites has no effect on their expression levels
(Figure 5B). Together, these results suggest that Mps1 auto-
phosphorylation sites T12 and S15 are required for both the
full-length and the N-terminal region of Mps1 to associate
with the kinetochore upon activation of spindle checkpoint
Phosphorylation of T12 and S15 Is Required for Robust
Spindle Checkpoint Signaling and Recruitment of Mad2 to
Activation of spindle checkpoint signaling leads to recruit-
ment of checkpoint signaling components such as Mps1,
Bub1, BubR1, Mad1, and Mad2 to the kinetochore to delay
the onset of anaphase. Mps1 is required for kinetochore
localization of Mad2 (Martin-Lluesma et al., 2002; Howell et
al., 2004; Vigneron et al., 2004; Zhao and Chen, 2006).
Whether phosphorylation of Mps1 at T12 and S15 is re-
quired for the checkpoint signaling response is unknown.
Endogenous Mps1 can be depleted by treatment with a
siRNA duplex (Figure 6A), and knockdown of Mps1 inhibits
targeting of Mad2 to the kinetochore (Figure 6B). A siRNA-
resistant allele of Mps1 was constructed by scrambling the
targeted coding sequence (Mps1R). YFP-Mps1R can be effi-
ciently expressed in the presence of the siRNA targeting
endogenous Mps1 (Figure 6A). YFP-Mps1R can mediate
kinetochore recruitment of Mad2 upon depletion of the en-
dogenous Mps1 with the Mps1 siRNA (Figure 6C). To de-
termine whether phosphorylation of T12 and S15 is re-
quired for efficient Mad2 localization to the kinetochore in
response to spindle damage, we stably expressed YFP-
Mps1RT12S153AAand Mps1RNT0Pin SW480 cells (Figure
6, D and E). The resulting cell lines were transfected with
either control or Mps1 siRNA and treated with nocoda-
zole for 12 h. In cells treated with control siRNA, Mad2
localized to kinetochores in prometaphase arrested cells
due to the presence of the endogenous Mps1. In contrast,
in cells with the Mps1 siRNA, Mad2 was not recruited
to kinetochores, suggesting that neither YFP-Mps1RT12S153AA
nor Mps1RNT0Pis able to mediate kinetochore localization of
Mad2 in the absence of endogenous Mps1. These data sug-
recruitment of checkpoint protein Mad2. (A) Depletion of the endog-
enous but not YFP-tagged siRNA-resistant Mps1 (YFP-Mps1R) in
SW480 cells. Uninfected or YFP-Mps1R-expressing SW480 cells were
Mps1 or YFP-Mps1 was determined by immunoblotting with an Mps1
antibody. (B) Depletion of the endogenous Mps1 abrogates Mad2
kinetochore localization. Wild-type SW480 cells were transfected with
control or Mps1 siRNA. Two days after transfection, cells were treated
nocodazole for 12 h and fixed in 1% paraformaldehyde. Fixed cells
were stained with antibodies against Mps1 or Mad2 or DAPI. (C)
Autophosphorylation of Mps1 is required for kinetochore
of the endogenous Mps1 by siRNA. (D) Mutation of autophosphor-
ylation sites of Mps1 blocks kinetochore recruitment of Mps1 and
Mad2. (E) Phosphorylation of T12 and S15 of Mps1 is required for
kinetochore targeting of Mad2.
Q. Xu et al.
Molecular Biology of the Cell 16
gest that phosphorylation of the N-terminal region of Mps1
or, more specifically, of T12 and S15 is required for Mps1 to
activate spindle checkpoint signaling.
Previous studies suggest that Mps1 kinase activity is not
required for kinetochore targeting of Mps1 as GFP-
Mps1-KD (kinase dead) or GFP-Mps11-301without the ki-
nase domain can target to kinetochores (Liu et al., 2003;
Stucke et al., 2004). Given that Mps1 can undergo transau-
tophosphorylation, overexpression of GFP-Mps1-KD or
GFP-Mps11-301could still be phosphorylated by the endog-
enous Mps1. To further evaluate the requirement of Mps1
kinase activity in its kinetochore targeting, we stably ex-
pressed siRNA-resistant YFP-Mps1R-KD or YFP-Mps1R1-301
in SW480 cells. As expected, both of them can target to the
kinetochore as described in cells transfected with control
siRNA (Figure 7, A and B). However, when endogenous
Mps1 was depleted by treatment with siRNA, neither YFP-
Mps1R-KD nor YFP-Mps1R1-301can accumulate at kineto-
chores. Consistent with the defects of Mps1 kinetochore
targeting, Mad2 localization to the kinetochore was also
abolished (Figure 7C). Thus, these results suggest that ki-
nase activity-deficient Mps1 is incapable of relocating to the
kinetochore in the absence of endogenous Mps1. Mps1 ki-
nase activity is required for kinetochore localization of spin-
dle checkpoint proteins including Mps1 itself.
Phosphorylation of S821 Is Important for Kinetochore
Targeting of Full-Length Mps1
Previous studies suggest that phosphorylation of Xenopus
Mps1 at S844 by MAP kinase is essential for kinetochore
targeting in Xenopus egg extracts. The equivalent site of S844
in human Mps1 is S821. Our mass spectrometry data sug-
gests that S821 is phosphorylated in insect cells by unknown
kinases. To address the significance of this phosphorylation
in mammalian cells, we constructed a stable cell line ex-
pressing the S821A mutant of YFP-Mps1. In agreement with
the Xenopus system results, kinetochore localization of YFP-
Mps1S8213Ais decreased by at least 50% compared with the
wild-type control, suggesting that phosphorylation of this
site play a significant role in kinetochore recruitment of
Mps1 in mammalian cells (Figure 8, A and C). To determine
whether S821 also affects centrosome localization of Mps1,
we compared centrosome staining of YFP-Mps1 and YFP-
Mps1S8213Ain interphase cells. No significant difference is
observed between control and the mutant. Thus, phosphor-
ylation of S821 seems to play a role in regulating kinetochore
but not centrosome localization of Mps1.
We report here that autophosphorylation of T12 and S15 at
the N-terminal domain of Mps1 is a key regulatory event
required for Mps1 kinetochore targeting and subsequent
recruitment of Mad2 to the kinetochore upon activation of
spindle checkpoint signaling. We showed that phosphory-
lation of T12 and S15 occurs in mitotic-arrested cells and that
mutation of T12 and S15 abrogates Mps1 kinetochore asso-
ciation. We propose that phosphorylation of T12 and S15
may either create a recognition motif to interact with cellular
machinery to transport Mps1 to the kinetochore or cause
allosteric changes in Mps1 to expose the kinetochore target-
ing signal(s) embedded in the N-terminal region of Mps1.
Hyperphosphorylation of Mps1 has been well docu-
mented in mitotic cells (Stucke et al., 2002, 2004; Liu et al.,
2003; Fisk et al., 2004). The phosphorylation sites of Mps1 in
mitotic cells have just begun to emerge (Zhao and Chen,
2006; Kang et al., 2007; Mattison et al., 2007; Cui and Guad-
agno, 2008). It seems that Mps1 is targeted by both auto-
phosphorylation and transphosphorylation by other ki-
nases. Consistent with previous observation, we found that
the activation loop of Mps1 is targeted by autophosphory-
lation (T675, S678, and T686) (Kang et al., 2007; Mattison et
al., 2007). Autophosphorylation of these sites has been
shown to enhance Mps1 kinase activity by four- to sevenfold
and enhances kinetochore targeting of Mps1 and Bub1
ing and recruitment of Mad2 upon activation of the mitotic spindle
checkpoint in SW480 cells. (A) Kinetochore localization of the N-
terminal fragment of Mps1 requires endogenous Mps1. SW480 cells
stably expressing YFP-Mps1R1-301were transfected with control or
Mps1 siRNA. Localization of YFP-Mps1R1-301was determined by im-
munofluorescence microscopy as described in text. B is the same as A
except kinase-deficient Mps1 mutant (YFP-Mps1R-KD) was used. (C)
Mps1 kinase activity is required for kinetochore recruitment of Mad2.
SW480 cells stably expressing YFP-Mps1R-KD were transfected with
control or Mps1 siRNA. Cells were fixed and stained with an antibody
against Mad2 and with DAPI. (D) Quantitation of fluorescent density
of kinetochores labeled by YFP-Mps1 and related mutants in control or
Mps1 siRNA treated prometaphase cells. At least 42 kinetochores from
prometaphase cells in each cell line were scored and differences be-
tween control and Mps1 siRNA treated samples from YFP-Mps1R-KD
or YFP-Mps1R1-301are statistically significant (p ? 0.05).
Mps1 kinase activity is required for its kinetochore target-
Mps1 Kinetochore Targeting
Vol. 20, January 1, 2009 17
(Kang et al., 2007; Mattison et al., 2007). Mps1 purified from
mitotic HeLa extracts showed phosphorylation at T33, S37,
S80, S281, T436, T453, T468, T360, S363, T371, S382, and S821
(Kang et al., 2007; Kasbek et al., 2007; Cui and Guadagno,
2008; Jelluma et al., 2008a). Using Mps1 purified from insect
cells, which possess robust kinase activity, we demonstrated
that some of these sites are primarily Mps1 autophosphor-
ylation sites (e.g., T33, S37, S80, T360, S363, T675, T676, T678,
and T686). T12 and S15 phosphorylation has not been de-
scribed in other studies. There is a possibility that these two
sites are phosphorylated at low stoichiometry compared
with other sites and only associate with active Mps1 local-
ized to the kinetochores. Given that kinetochore targeting of
Mps1 is a highly dynamic process and only a small fraction
of Mps1 bound to kinetochores with the residence time of
Mps1 being ?10 s (Howell et al., 2004), dynamic or revers-
ible phosphorylation of T12S15 would be consistent with its
role in regulating Mps1 kinetochore recruitment.
Phosphorylation of the TP or SP sites in Mps1 can be
attributed to MAP kinase (Zhao and Chen, 2006; Kang et al.,
2007) or Cdk2 kinase activity (Kasbek et al., 2007; Cui and
Guadagno, 2008), although a recent study using Mps1 pu-
rified from insect cells suggests that S821 may be an auto-
phosphorylation site., It has been proposed that phosphor-
ylation of these sites increases the stability of Mps1 during
mitosis (Kasbek et al., 2007; Cui and Guadagno, 2008). How-
ever, S821 is also phosphorylated in Mps1 kinase-dead mu-
tant purified from insect cells, suggesting that this site is
more likely to be targeted by other kinases rather than an
autophosphorylation site. The fact that TP or SP sites are
readily detectable in mammalian cells with hyperactive
MAP kinase pathway and Xenopus extracts with elevated
MAP kinase activity suggests that the MAP kinase pathway
may cross talk with the Mps1 pathway through hyperphos-
phorylation of Mps1 at the canonical MAP kinase phosphor-
Hyperphosphorylation of Mps1 at multiple sites occurs
both in vitro and in vivo (Kang et al., 2007; Kasbek et al.,
2007; Mattison et al., 2007; Cui and Guadagno, 2008; Jelluma
et al., 2008a). There are considerable variations in the num-
ber of phosphorylation sites reported in the literature. For
example, there are far more autophosphorylation sites with
recombinant Mps1 purified from Escherichia coli than from
insect cells. This observation may suggest the heterogeneity
of Mps1 phosphorylation, which poses significant chal-
lenges to address the function of each individual site in vivo
if functional redundancy exists among these phosphoryla-
tion sites. Throughout our studies we use the T12S15 double
mutant to address the potential function of these sites in
Mps1 kinetochore relocalization, it is very possible that only
one of these sites is occupied in vivo for a given Mps1
molecule. Consistent with this notion, T12 singly phosphor-
ylated peptide is more readily detectable than the T12S15
doubly phosphorylated peptide, suggesting T12 is a prefer-
able phosphorylation site in wild-type Mps1. Because of the
potential redundancy of S15 and presence of T12S15 double
phosphorylated species in vivo, it is necessary to use double
mutant to address the function of T12 S15 phosphorylation
in Mps1 kinetochore recruitment.
The requirement for Mps1 kinase activity for its kineto-
chore recruitment has not been fully addressed. In agree-
ment with previous observations, the kinase-deficient Mps1
mutant can be recruited to kinetochores as is the N-terminal
region of Mps1 lacking the kinase domain (Stucke et al.,
2002, 2004; Liu et al., 2003). However, kinase-deficient Mps1
is defective in kinetochore targeting in Xenopus egg extracts
upon depletion of the endogenous Mps1 (Zhao and Chen,
2006). Given that Mps1 undergoes extensive intermolecular
autophosphorylation (Kang et al., 2007; Mattison et al., 2007),
it is quite possible that kinase-deficient Mps1 or the N-
terminal domain of Mps1 can be phosphorylated by the
endogenous Mps1 in mammalian cells. Our results showing
that endogenous Mps1 is required for kinetochore localiza-
tion of kinase-deficient Mps1 are consistent with this hy-
pothesis although it cannot be ruled out at this point that
another kinase whose activity depends on Mps1 is involved
in this process. It has been previously noted that inhibition
of Mps1 kinase activity abrogates spindle checkpoint re-
sponses in transform tumor cells but not normal cells
(Schmidt and Medema, 2006), suggesting that normal cells
may have multiple redundant pathways to ensure robust
checkpoint responses. This scenario could explain the ap-
parent differences in kinetochore targeting behavior of Mps1
kinase-deficient mutant between HeLa cells (Kang et al.,
2007) and SW480 cells (this study) when the endogenous
Mps1 is depleted. Whereas the spindle checkpoint is rela-
tively weakened in SW480 cells (Tighe et al., 2001), HeLa
cells possess a very robust spindle checkpoint control
(Schmidt and Medema, 2006). We speculate that there could
be an unidentified kinase(s) present in HeLa that is missing
recruitment of Mps1 but not for centrosome localization. (A) Kinet-
ochore targeting of YFP-Mps1 and YFP-Mps1S8213Ain nocodazole-
arrested mitotic cells. Cells were treated and analyzed as described
in Figure 1B. (B) Centrosome localization of YFP-Mps1 and YFP-
Mps1S8213Ain interphase cells. (C) Quantitation of fluorescent den-
sity of YFP-Mps1 and YFP-Mps1S8213Aon the kinetochores of
prometaphase cells. The differences between YFP-Mps1 and YFP-
Mps1S8213Aare statistically significant (p ? 0.001).
Phosphorylation of S821 is important for kinetochore
Q. Xu et al.
Molecular Biology of the Cell18
in SW480 cells and responsible for phosphorylating T12S15
when the autophosphorylation activity of Mps1 is disabled.
Future studies are needed to determine whether this is case.
In this report, we found that Mps1 is autophosphorylated
in vitro and in vivo at T12 and S15, and this phosphorylation
is required for kinetochore targeting of Mps1. Disagreement
exists in the literature as to the exact location of the Mps1
kinetochore targeting signal. Deletion mapping analyses
suggest that the target signal may be located in the N-
terminal region within the first 300 amino acids of Mps1
(Stucke et al., 2002, 2004; Liu et al., 2003). However, Chen and
colleagues recently demonstrated that phosphorylation of
S844 is crucial for kinetochore targeting of Mps1 and they
proposed that phosphorylation of S844 (equivalent to S821
of human Mps1) by MAP kinase may create a phospho-
epitope to serve as a kinetochore targeting signal (Zhao and
Chen, 2006). This raises the possibility that the C terminus of
Mps1 could generate a target signal upon phosphorylation.
Our mutation analysis agrees with the importance of S821
phosphorylation in kinetochore targeting of Mps1. How-
ever, our results are also consistent with the notion that the
N terminus of Mps1 contains a kinetochore localization sig-
nal and that this signal requires phosphorylation at T12 and
S15. More importantly, the C-terminal region of Mps1, in-
cluding the kinase domain, is insufficient for kinetochore
targeting (data not shown). To reconcile all these data, we
propose that kinetochore recruitment of Mps1 requires
phosphorylation at both the N terminus and C terminus of
the protein (Supplemental Figure 4). Whereas phosphoryla-
tion of the N terminus at T12 and S15 creates a kinetochore
target signal to be recognized by kinetochore-associated pro-
teins, phosphorylation of Mps1 at the C terminus at S821
causes allosteric changes in Mps1 conformation, which may
expose the kinetochore targeting signal. Further experiments
are needed to identify the cellular components that recog-
nize the kinetochore targeting signal and whether a signifi-
cant conformational change occurs in Mps1 upon phosphor-
ylation and dephosphorylation.
We thank Dr. Mark Winey for advice and sharing unpublished results and
Katheryn Resing for mass spectrometry advice. We thank Drs. David Clarke
and Kristen Barthel for critical readings of the manuscript. This work was
supported by National Institutes of Health grant CA-107098 and a Pilot grant
from the Colorado Cancer Center (to X. L.).
Abrieu, A., Magnaghi-Jaulin, L., Kahana, J. A., Peter, M., Castro, A., Vigneron,
S., Lorca, T., Cleveland, D. W., and Labbe, J. C. (2001). Mps1 is a kinetochore-
associated kinase essential for the vertebrate mitotic checkpoint. Cell 106,
Amon, A. (1999). The spindle checkpoint. Curr. Opin. Genet. Dev. 9, 69–75.
Boyle, W. J., van der Geer, P., and Hunter, T. (1991). Phosphopeptide mapping
and phosphoamino acid analysis by two-dimensional separation on thin-layer
cellulose plates. Methods Enzymol. 201, 110–149.
Cui, Y., and Guadagno, T. M. (2008). B-Raf(V600E) signaling deregulates the
mitotic spindle checkpoint through stabilizing Mps1 levels in melanoma cells.
Oncogene 27, 3122–3133.
DeLuca, J. G., Dong, Y., Hergert, P., Strauss, J., Hickey, J. M., Salmon, E. D.,
and McEwen, B. F. (2005). Hec1 and nuf2 are core components of the kinet-
ochore outer plate essential for organizing microtubule attachment sites. Mol.
Biol. Cell 16, 519–531.
Draviam, V. M., Xie, S., and Sorger, P. K. (2004). Chromosome segregation
and genomic stability. Curr. Opin. Genet. Dev. 14, 120–125.
Fang, G., Yu, H., and Kirschner, M. W. (1998). The checkpoint protein MAD2
and the mitotic regulator CDC20 form a ternary complex with the anaphase-
promoting complex to control anaphase initiation. Genes Dev. 12, 1871–1883.
Fisk, H. A., Mattison, C. P., and Winey, M. (2004). A field guide to the Mps1
family of protein kinases. Cell Cycle 3, 439–442.
Fisk, H. A., and Winey, M. (2001). The mouse Mps1p-like kinase regulates
centrosome duplication. Cell 106, 95–104.
Hoffman, D. B., Pearson, C. G., Yen, T. J., Howell, B. J., and Salmon, E. D.
(2001). Microtubule-dependent changes in assembly of microtubule motor
proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores. Mol.
Biol. Cell 12, 1995–2009.
Howell, B. J., Moree, B., Farrar, E. M., Stewart, S., Fang, G., and Salmon, E. D.
(2004). Spindle checkpoint protein dynamics at kinetochores in living cells.
Curr. Biol. 14, 953–964.
Hoyt, M. A., Totis, L., and Roberts, B. T. (1991). S. cerevisiae genes required for
cell cycle arrest in response to loss of microtubule function. Cell 66, 507–517.
Jelluma, N., Brenkman, A. B., McLeod, I., Yates, J. R., 3rd, Cleveland, D. W.,
Medema, R. H., and Kops, G. J. (2008a). Chromosomal instability by inefficient
Mps1 auto-activation due to a weakened mitotic checkpoint and lagging
chromosomes. PLoS ONE 3, e2415.
Jelluma, N., Brenkman, A. B., van den Broek, N. J., Cruijsen, C. W., van Osch,
M. H., Lens, S. M., Medema, R. H., and Kops, G. J. (2008b). Mps1 phosphor-
ylates borealin to control aurora B activity and chromosome alignment. Cell
Kang, J., Chen, Y., Zhao, Y., and Yu, H. (2007). Autophosphorylation-depen-
dent activation of human Mps1 is required for the spindle checkpoint. Proc.
Natl. Acad. Sci. USA 104, 20232–20237.
Kasbek, C., Yang, C. H., Yusof, A. M., Chapman, H. M., Winey, M., and Fisk,
H. A. (2007). Preventing the degradation of mps1 at centrosomes is sufficient
to cause centrosome reduplication in human cells. Mol. Biol. Cell 18, 4457–
Kops, G. J., Weaver, B. A., and Cleveland, D. W. (2005). On the road to cancer:
aneuploidy and the mitotic checkpoint. Nat. Rev. Cancer 5, 773–785.
Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1997). Genetic instability in
colorectal cancers. Nature 386, 623–627.
Li, R., and Murray, A. W. (1991). Feedback control of mitosis in budding yeast.
Cell 66, 519–531.
Lindberg, R. A., Fischer, W. H., and Hunter, T. (1993). Characterization of a
human protein threonine kinase isolated by screening an expression library
with antibodies to phosphotyrosine. Oncogene 8, 351–359.
Liu, S. T., Chan, G. K., Hittle, J. C., Fujii, G., Lees, E., and Yen, T. J. (2003).
Human MPS1 kinase is required for mitotic arrest induced by the loss of
CENP-E from kinetochores. Mol. Biol. Cell 14, 1638–1651.
Liu, X., Constantinescu, S. N., Sun, Y., Bogan, J. S., Hirsch, D., Weinberg, R. A.,
and Lodish, H. F. (2000). Generation of mammalian cells stably expressing
multiple genes at predetermined levels. Anal. Biochem. 280, 20–28.
Liu, X., Sun, Y., Constantinescu, S. N., Karam, E., Weinberg, R. A., and Lodish,
H. F. (1997). Transforming growth factor beta-induced phosphorylation of
Smad3 is required for growth inhibition and transcriptional induction in
epithelial cells. Proc. Natl. Acad. Sci. USA 94, 10669–10674.
Martin-Lluesma, S., Stucke, V. M., and Nigg, E. A. (2002). Role of Hec1 in
spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2.
Science 297, 2267–2270.
Mattison, C. P., Old, W. M., Steiner, E., Huneycutt, B. J., Resing, K. A., Ahn,
N. G., and Winey, M. (2007). Mps1 activation loop autophosphorylation
enhances kinase activity. J. Biol. Chem. 282, 30553–30561.
McIntosh, J. R. (1991). Structural and mechanical control of mitotic progres-
sion. Cold Spring Harb. Symp. Quant. Biol. 56, 613–619.
Mills, G. B., Schmandt, R., McGill, M., Amendola, A., Hill, M., Jacobs, K., May,
C., Rodricks, A. M., Campbell, S., and Hogg, D. (1992). Expression of TTK, a
novel human protein kinase, is associated with cell proliferation. J. Biol.
Chem. 267, 16000–16006.
Montembault, E., Dutertre, S., Prigent, C., and Giet, R. (2007). PRP4 is a
spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2
localization to the kinetochores. J. Cell Biol. 179, 601–609.
Nasmyth, K. (2001). Disseminating the genome: joining, resolving, and sep-
arating sister chromatids during mitosis and meiosis. Annu. Rev. Genet. 35,
Nasmyth, K. (2002). Segregating sister genomes: the molecular biology of
chromosome separation. Science 297, 559–565.
Naviaux, R. K., Costanzi, E., Haas, M., and Verma, I. M. (1996). The pCL
vector system: rapid production of helper-free, high-titer, recombinant retro-
viruses. J. Virol. 70, 5701–5705.
Mps1 Kinetochore Targeting
Vol. 20, January 1, 2009 19
Nicklas, R. B. (1997). How cells get the right chromosomes. Science 275,
Rieder, C. L., Schultz, A., Cole, R., and Sluder, G. (1994). Anaphase onset in
vertebrate somatic cells is controlled by a checkpoint that monitors sister
kinetochore attachment to the spindle. J. Cell Biol. 127, 1301–1310.
Schmidt, M., and Medema, R. H. (2006). Exploiting the compromised spindle
assembly checkpoint function of tumor cells: dawn on the horizon? Cell Cycle
Stucke, V. M., Baumann, C., and Nigg, E. A. (2004). Kinetochore localization
and microtubule interaction of the human spindle checkpoint kinase Mps1.
Chromosoma 113, 1–15.
Stucke, V. M., Sillje, H. H., Arnaud, L., and Nigg, E. A. (2002). Human Mps1
kinase is required for the spindle assembly checkpoint but not for centrosome
duplication. EMBO J. 21, 1723–1732.
Sudakin, V., Chan, G. K., and Yen, T. J. (2001). Checkpoint inhibition of the
APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and
MAD2. J. Cell Biol. 154, 925–936.
Tang, Z., Shu, H., Oncel, D., Chen, S., and Yu, H. (2004). Phosphorylation of
Cdc20 by Bub1 provides a catalytic mechanism for APC/C inhibition by the
spindle checkpoint. Mol. Cell 16, 387–397.
Tighe, A., Johnson, V. L., Albertella, M., and Taylor, S. S. (2001). Aneuploid
colon cancer cells have a robust spindle checkpoint. EMBO Rep. 2, 609–614.
Uhlmann, F., Lottspeich, F., and Nasmyth, K. (1999). Sister-chromatid sepa-
ration at anaphase onset is promoted by cleavage of the cohesin subunit Scc1.
Nature 400, 37–42.
Vigneron, S., Prieto, S., Bernis, C., Labbe, J. C., Castro, A., and Lorca, T. (2004).
Kinetochore localization of spindle checkpoint proteins: who controls whom?
Mol. Biol. Cell 15, 4584–4596.
Wassmann, K., and Benezra, R. (2001). Mitotic checkpoints: from yeast to
cancer. Curr. Opin. Genet. Dev. 11, 83–90.
Weaver, B. A., and Cleveland, D. W. (2005). Decoding the links between
mitosis, cancer, and chemotherapy: the mitotic checkpoint, adaptation, and
cell death. Cancer Cell 8, 7–12.
Weiss, E., and Winey, M. (1996). The Saccharomyces cerevisiae spindle pole
body duplication gene MPS1 is part of a mitotic checkpoint. J. Cell Biol. 132,
Williamson, B. L., Marchese, J., and Morrice, N. A. (2006). Automated iden-
tification and quantification of protein phosphorylation sites by LC/MS on a
hybrid triple quadrupole linear ion trap mass spectrometer. Mol. Cell Pro-
teomics 5, 337–346.
Winey, M., Goetsch, L., Baum, P., and Byers, B. (1991). MPS1 and MPS2, novel
yeast genes defining distinct steps of spindle pole body duplication. J. Cell
Biol. 114, 745–754.
Yu, H. (2002). Regulation of APC-Cdc20 by the spindle checkpoint. Curr.
Opin. Cell Biol. 14, 706–714.
Zhao, Y., and Chen, R. H. (2006). Mps1 phosphorylation by MAP kinase is
required for kinetochore localization of spindle-checkpoint proteins. Curr.
Biol. 16, 1764–1769.
Zhu, S., Wang, W., Clarke, D. C., and Liu, X. (2007). Activation of Mps1
promotes transforming growth factor-beta-independent Smad signaling.
J. Biol. Chem. 282, 18327–18338.
Q. Xu et al.
Molecular Biology of the Cell 20