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INTRODUCTION |
The centrosome, a major microtubule-organizing center of the
animal cells, directs the formation of bipolar mitotic spindles, which
is essential for accurate chromosome segregation to daughter cells (for
reviews, see Refs. 1-3). Since each daughter cell inherits one
centrosome upon cytokinesis, the centrosome must duplicate prior to the
next mitosis and do so only once. Thus, centrosome duplication must
take place in coordination with other cell cycle events including DNA
synthesis. In mammalian cells, the centriole, the core component of the
centrosome, initiates duplication at the G1/S boundary
(reviewed in Refs. 4-6). Activation of cyclin-dependent
kinase 2 (CDK2)1-cyclin E has
recently been found to be essential for the centrosome to initiate
duplication (7, 8). The activity of CDK2-cyclin E is regulated by the
temporal expression of cyclin E, which normally occurs in late
G1 (9, 10), and it has been known that active CDK2-cyclin E
complexes are required for initiation of DNA replication (11, 12).
These observations indicate that the late G1-specific activation of CDK2-cyclin E plays a key role for the coordinated initiation of centrosome and DNA duplication. Indeed, we have shown
that constitutive activation of CDK2-cyclin E by cyclin E
overexpression in cultured mammalian cells results in uncoupling of the
initiation of centrosome and DNA duplication; in these cells, the
centrosomes initiate duplication in early G1 long
before the onset of DNA synthesis (13). Unlike the initiation of DNA synthesis, which can only be triggered by CDK2-cyclin E after completion of a series of necessary events (14, 15), the initiation of
centrosome duplication appears to depend primarily on the activation of
CDK2-cyclin E. Thus, the late G1-specific activation of
CDK2-cyclin E may serve as a checkpoint control for timely initiation
of centrosome duplication.
We have recently identified nucleophosmin (NPM/B23) as a substrate of
CDK2-cyclin E in the initiation of centrosome duplication (16).
NPM/B23, also called numatrin or NO38, was originally identified as a
major nucleolar phosphoprotein localized in granular regions of the
nucleolus and has been shown to be associated with preribosomal
particles (17-19). To date, NPM/B23 has been implicated in several
distinct cellular functions, including assembly and/or intranuclear
transport of preribosomal particles, cytoplasmic/nuclear trafficking,
the regulation of DNA polymerase
activity, and centrosome
duplication (16-21). NPM/B23 has also been shown to possess molecular
chaperoning activities, including preventing protein aggregation,
protecting enzymes during thermal denaturation, and facilitating
renaturation of chemically denatured proteins (22). We have shown that
NPM/B23 associates specifically with unduplicated centrosomes, and this
association is controlled by CDK2-cyclin E-mediated phosphorylation, in
which NPM/B23 loses its affinity to centrosomes in its phosphorylated
form (16). Dissociation of the centrosomal NPM/B23 is essential for the
centrosome to initiate duplication (16). For instance, microinjection
of the anti-NPM/B23 monoclonal antibody, which blocks the CDK2-cyclin E-mediated phosphorylation of NPM/B23, inhibits centrosome duplication. Moreover, ectopic expression of this NPM/B23 deletion mutant
(NPM(
186-239)), which is unable to be phosphorylated by
CDK2-cyclin E, results in suppression of centrosome duplication. These
results demonstrate that dissociation of centrosomal NPM/B23 by
CDK2-cyclin E-mediated phosphorylation is critical for initiation of
centrosome duplication and that the site(s) of NPM/B23 phosphorylated
by CDK2-cyclin E lies within the sequence between amino acid residues
186 and 239.
We here show that Thr199 of NPM/B23 is specifically
phosphorylated by CDK2-cyclin E in vitro, and this
phosphorylation is also observed in vivo. When NPM/B23
mutant with a substitution of this specific threonine residue to
alanine (nonphosphorylatable) is expressed in cells, it affects
centrosome duplication in a dominant negative fashion, resulting in
suppression of centrosome duplication. These observations provide
direct evidence that the CDK2-cyclin E-mediated phosphorylation of
NPM/B23 on Thr199 is critical for dissociation of
centrosomal NPM/B23 and initiation of centrosome duplication.
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EXPERIMENTAL PROCEDURES |
Cells and Transfection--
Swiss 3T3 and HeLa cells were
maintained in complete medium (Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, penicillin (100 units/ml),
and streptomycin (100 µg/ml)) in an atmosphere containing 10%
CO2.
For generation of HeLa cells overexpressing cyclin E, a plasmid
encoding a human cyclin E gene was co-transfected with a plasmid encoding a neomycin-resistant gene into HeLa cells by the calcium phosphate protocol. As a negative control, a vector was transfected. G418-resistant colonies that arose in the medium containing G418 (800 µg/ml) at 2-3 weeks after transfection were subcloned and analyzed for cyclin E expression. One cell line that overexpressed cyclin E (HeLa/CycE) and one vector-transfected G418-resistant cell
line (HeLa/Vec) were maintained.
For transient transfection of wild-type and mutant NPM/B23
sequences, Swiss 3T3 cells were co-transfected with plasmids encoding either a FLAG-tagged wild-type or substitution mutant
(Thr199
Ala) NPM/B23 with a puromycin
resistance gene plasmid (pBabe/puro) at a molar ratio of 20:1 by the
calcium phosphate protocol. After transfection in 37 °C for 8 h, cells were fed with fresh complete medium for 16 h. The cells
were then treated with complete medium containing puromycin (4 µg/ml)
for 36 h. The puromycin-resistant cells were pooled and replated
on coverslips and further cultured in fresh complete medium for 24 h.
Plasmid Construction and Purification of GST-NPM/B23--
Mutant
as well as wild-type NPM/B23 cDNA sequences were fused
to glutathione S-transferase using a two-step polymerase
chain reaction as described (23). The polymerase chain
reaction-amplified products were inserted in frame into a pGEX-4T-1
vector using BamHI and EcoRI restriction sites.
GST-NPM fusion proteins were bacterially purified according to the
protocol provided by the manufacturer (Amersham Pharmacia Biotech).
Briefly, cells were induced with
isopropyl-1-thio-
-D-galactopyranoside for 4 h
before harvesting. Clarified bacterial lysates were passed over a
Sepharose 4B column, and GST-NPM proteins were eluted. The
concentration of the eluted GST-NPM was estimated by comparison with
bovine serum albumin with a known concentration run in parallel on
SDS-PAGE.
Immunoblot Analysis--
Cells were lysed in SDS/Nonidet P-40
lysis buffer (1% SDS, 1% Nonidet P-40, 50 mM Tris (pH
8.0), 150 mM NaCl, 4 mM Pefabloc SC (Roche
Molecular Biochemicals), 2 µg/ml leupeptin, 2 µg/ml aprotinin). The
lysates were boiled for 5 min and then cleared by a 10-min
centrifugation at 20,000 × g at 4 °C. The
supernatant was further denatured at 95 °C for 5 min in sample
buffer (2% SDS, 10% glycerol, 60 mM Tris (pH 6.8), 5%
-mercaptoethanol, 0.01% bromphenol blue). Samples were resolved by
SDS-PAGE and transferred onto Immobilon-P (Millipore Corp.) sheets. The
blots were first incubated in blocking buffer (5% (w/v) nonfat dry
milk in Tris-buffered saline plus Tween 20) for 1 h. The blots
were then incubated with primary antibody for 2 h, followed by
incubation with horseradish peroxidase-conjugated secondary antibody
for 1 h. All of the procedures were performed at room temperature. The antibody-antigen complex was visualized by ECL chemiluminescence (Amersham Pharmacia Biotech).
Indirect Immunofluorescence--
Cells grown on coverslips were
fixed with 10% formalin, 10% methanol for 20 min at room
temperature. The cells were permeabilized with 1% Nonidet P-40 in
phosphate-buffered saline for 5 min, followed by incubation with
blocking solution (10% normal goat serum in phosphate-buffered saline)
for 1 h. Cells were then probed with primary antibodies for 1 h, and antibody-antigen complexes were detected with either rhodamine-
or FITC-conjugated goat secondary antibody by incubation for 1 h
at room temperature. The samples were washed three times with
phosphate-buffered saline after each incubation and then counterstained
with 4',6-diamidino-2-phenylindole (DAPI).
For co-immunostaining of
- and
-tubulins to examine centriole
pairs within centrosomes, cells were first placed on ice for 30 min to
destabilize microtubules nucleated at the centrosomes. The cold-treated
cells were then subjected to brief extraction (~30 s) with cold
extraction buffer (0.75% Triton X-100, 5 mM PIPES, 2 mM EGTA (pH 6.7)), briefly washed in cold
phosphate-buffered saline, and fixed with 10% formalin, 10% methanol.
Cells were immunostained with anti-
-tubulin monoclonal (DM1A) and
anti-
-tubulin polyclonal (24) antibodies. The antibody-antigen
complexes were detected with FITC-conjugated goat anti-rabbit IgG and
rhodamine-conjugated goat anti-mouse IgG antibodies.
In Vitro Kinase Assay--
For examination of CDK2-cyclin E
activity in cyclin E-overexpressing HeLa cells, cell lysates were
subjected to immunoprecipitation using anti-cyclin E antibody (sc-198;
Santa Cruz Biotechnology, Inc.). The antibody-antigen complexes were
collected with protein A-agarose and tested for a histone H1 kinase
activity as described previously (13).
For in vitro phosphorylation of NPM/B23 by CDK2-cyclin E,
CDK2-cyclin A, or CDK1-cyclin B, GST, GST-NPM/wt, or GST-NPM/T199A was
incubated with baculovirally purified active CDK2-cyclin E or
CDK2-cyclin A complexes (25) or the immunoprecipitated CDK1-cyclin B
from mitotically arrested Swiss 3T3 cells by nocodazole treatment using
agarose-conjugated anti-cyclin B monoclonal antibody (GNS1; Santa Cruz
Biotechnology) (26). The in vitro kinase reactions were
performed in 10 mM PIPES buffer in the presence of
[
-32P]ATP at 25 °C for 15 min and at 32 °C for
an additional 15 min. The samples were resolved by SDS-PAGE, and the
gel was dried and autoradiographed.
For both histone H1 and GST-NPM kinase assays, 32P
incorporation was quantitated by scanning with a Fuji 1000 phosphoimager.
BrdUrd Incorporation Assay--
The assay was performed using
the BrdUrd labeling kit (Roche Molecular Biochemicals) according
to the manufacturer's instructions. Briefly, cells were fixed in 70%
ethanol in 50 mM glycine (pH 2.0) for 20 min at
20 °C,
incubated in the blocking buffer for 1 h at room temperature, and
then probed with anti-
-tubulin polyclonal and anti-BrdUrd monoclonal
antibodies for 30 min at 37 °C. Antigen-antibody complexes were
detected by FITC-conjugated sheep anti-mouse IgG and
rhodamine-conjugated goat anti-rabbit IgG antibodies. The preparation
of cells is described in the legend to Fig. 5.
Phosphoamino Acid Analysis--
GST-NPM/wt phosphorylated
in vitro by CDK2-cyclin E in the presence of
[32P-
]ATP was resolved by SDS-PAGE.
32P-Labeled NPM/B23 was eluted from the gel and subjected
to acid hydrolysis. The phosphorylated amino acids were separated by
two-dimensional electrophoresis on a thin layer cellulose gel plate as
described previously (27).
Two-dimensional Tryptic Phosphopeptide Mapping--
For
preparation of in vivo 32P-labeled NPM/B23,
HeLa/CycE cells were labeled for 2.5 h in phosphate-free medium
containing 1 mCi/ml 32P-orthophosphate and 2% dialyzed
fetal bovine serum. Cells were lysed in lysis buffer (1% Triton X-100,
50 mM Tris (pH 8.0), 50 mM
-glycerophosphate, 50 mM sodium fluoride, 50 mM NaCl, 0.1% sodium deoxycholate, 4 mM
Pefabloc SC, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 4 µM Microcystin-LR). After preclearing with protein G-conjugated agarose, the lysates were subjected to immunoprecipitation with anti-NPM/B23 monoclonal antibody. Antibody-antigen complexes were
collected by protein G-conjugated agarose and were resolved by 10%
SDS-PAGE. The gel was dried and autoradiographed. The in vivo
32P-labeled NPM/B23 proteins were eluted from the gel.
In vitro 32P-labeled GST-NPM/wt fusion proteins
were prepared as described above for phosphoamino acid analysis.
Two-dimensional tryptic phosphopeptide mapping was performed as
described previously (27). Briefly, both in vitro labeled
GST-NPM/wt and in vivo labeled NPM/B23 proteins were
oxidized in performic acid and digested with tosylphenylalanyl
chloromethyl ketone-treated trypsin (Worthington). Each sample was
loaded onto a thin layer cellulose gel plate and run for 1 h at
1200 V at 4 °C. The plates were dried and subjected to
chromatography (37.5% n-butanol, 25% pyridine, 7.5%
acetic acid) in the vertical direction.
 |
RESULTS |
Identification of the Site of NPM/B23 Specifically Phosphorylated
by CDK2-Cyclin E in Vitro--
We have previously shown that NPM/B23
is a direct centrosomal protein substrate of a CDK2-cyclin E
serine/threonine kinase complex in centrosome duplication. NPM/B23
deletion mutant (
186-239) fails to be phosphorylated by CDK2-cyclin
E and acts as a dominant negative when expressed in cells (16),
indicating that the CDK2-cyclin E-mediated phosphorylation site(s) lie
between amino acids 186 and 239. The sequence analysis of human NPM/B23
revealed that there are several serine and threonine residues within
this region. By phosphoamino acid analysis, we first tested whether
serine or threonine residue(s) (or both) are phosphorylated by
CDK2-cyclin E in vitro. The in vitro kinase
reaction of wild-type NPM/B23 fused to GST (GST-NPM/wt) was performed
using baculovirally purified active CDK2-cyclin E in the presence of
[
-32P]ATP. As negative controls, GST proteins as well
as GST-NPM(
186-239) were used as substrates. The kinase reaction
samples were resolved by SDS-PAGE and autoradiographed (Fig.
1A). CDK2-cyclin E did not
phosphorylate the GST moiety (lane 1). As shown
previously, GST-NPM(
186-239) deletion mutant failed to be
phosphorylated by CDK2-cyclin E (lane 2), while
GST-NPM/wt was phosphorylated at a readily detectable level
(lane 3). The phosphorylated GST-NPM/wt proteins
were eluted from the gel and subjected to phosphoamino acid analysis
(Fig. 1B). We found that NPM/B23 was phosphorylated exclusively on threonine residue(s) in vitro by CDK2-cyclin
E.

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Fig. 1.
CDK2-cyclin E phosphorylates NPM/B23 in
vitro on threonine residue(s). A, wild-type
NPM/B23 and NPM mutant fused to GST (GST-NPM/wt and
GST-NPM( 186-239), respectively) as well as GST were subjected to
in vitro kinase reactions with CDK2-cyclin E. The reaction
samples were run on 10% SDS-PAGE (pH 8.8), and autoradiographed
(left panel). The right
panel shows the Coomassie blue-stained gel. Lane
1, GST; lane 2, GST-NPM( 186-239);
lane 3, GST-NPM/wt. B, the
32P-labeled GST-NPM/wt proteins as shown in A
were purified from the gel and subjected to acid hydrolysis. The
phosphorylated amino acids were then separated by two-dimensional
electrophoresis and visualized by autoradiography as described
previously (27). The positions of the migrations of phosphoserine
(p-Ser), phosphothreonine (p-Thr), and
phosphotyrosine (p-Tyr) standards, detected by ninhydrin
staining, are indicated by circles. +, the sample loading
origin.
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There are four possible CDK2 phosphorylation consensus sequences within
this region (Fig. 2A,
indicated by arrowheads). To identify which threonine
residue(s) are phosphorylated by CDK2-cyclin E, each of these four
threonine residues (Thr199, Thr219,
Thr234, and Thr237) was replaced with alanine
(nonphosphorylatable amino acid). These mutants fused to GST
(GST-NPM/T199A, GST-NPM/T219A, GST-NPM/T234A, and GST-NPM/T237A,
respectively) were subjected to an in vitro kinase assay
with CDK2-cyclin E (Fig. 2B, top
panels). GST and GST-NPM/wt were included as controls in the
experiment. Three GST-NPM mutants (T219A, T234A, and T237A) were
phosphorylated (lanes 4-6) at levels similar to
GST-NPM/wt (lane 2). However, GST-NPM/T199A
showed dramatically reduced phosphorylation (lane 3), suggesting that Thr199 is the primary
phosphorylation site of NPM/B23 by CDK2-cyclin E in
vitro.

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Fig. 2.
NPM/B23 is phosphorylated by CDK2-cyclin E on
Thr199 in vitro. A, amino
acid sequence of human NPM/B23 between residues 186 and 239. All
threonine residues in this region are indicated by
arrowheads. Each threonine residue is followed by proline,
which is known to comprise a CDK phosphorylation consensus sequence
(45, 46). By polymerase chain reaction-assisted mutagenesis, each
threonine residue was replaced by alanine as indicated. B,
GST-NPM/wt (Wt) and four point mutation NPM/B23 fusion
proteins (T199A, T219A, T234A, and T237A) were subjected to in
vitro kinase reactions with either CDK2-cyclin E (top
panels) or CDK2-cyclin A (bottom
panels). As a negative control, GST was used as a substrate
(lane 1). Among these mutants, GST-NPM/T199A
failed to be phosphorylated by both CDK2-cyclin E and CDK2-cyclin A
(lane 3). In contrast, other mutant GST-NPM
fusion proteins (lanes 4-6) were phosphorylated
by both CDK2-cyclin E and CDK2-cyclin A at an efficiency similar to
wild-type NPM (lane 2). Coomassie Blue staining
of the gel is shown on the right for each set. C,
CDK1-cyclin B phosphorylates NPM/B23 on Thr234 and
Thr237. Wild-type and mutant GST-NPM as well as GST-NPM
double alanine substitution mutant (T234A/T237A) were subjected to
in vitro kinase reactions with immunopurified CDK1-cyclin B
from mitotic Swiss 3T3 cells using anti-cyclin B antibody (GNS1; Santa
Cruz Biotechnology). The level of 32P incorporation was
quantitated by phospho-image analysis, and the result is
presented in arbitrary units with the level of 32P
incorporation observed in GST-NPM/wt as 1.0. The amounts of GST-NPM
proteins used in the reactions (2 µg/30 µl) were confirmed to be
approximately equal by Coomassie Blue staining of the gel (not
shown).
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CDK2 is known to be activated also by association with cyclin A, which
is up-regulated during S and G2 phases (9, 10, 28-30). We
thus examined whether CDK2-cyclin A could phosphorylate Thr199. GST-NPM fusion proteins were subjected to an
in vitro kinase assay with baculovirally purified
CDK2-cyclin A (Fig. 2B, bottom panels). CDK2-cyclin A could phosphorylate all of the
mutants except GST-NPM/T199A at an efficiency similar to GST-NPM/wt,
demonstrating that CDK2-cyclin A can also specifically phosphorylate
Thr199 in vitro.
It has previously been shown that CDK1-cyclin B, a CDK-cyclin complex
specifically activated during mitosis (reviewed in Ref. 31),
phosphorylates NPM/B23 (32). However, the phosphorylation target
site(s) of CDK1-cyclin B had not been identified. We thus tested
whether CDK1-cyclin B phosphorylates the same threonine residue that is
phosphorylated by CDK2-cyclin E. GST-NPM substitution mutants described
above as well as GST-NPM/wt were subjected to an in vitro
kinase assay with immunopurified CDK1-cyclin B (Fig. 2C).
CDK1-cyclin B phosphorylated GST-NPM/T199A (lane
2) at an efficiency similar to GST-NPM/wt (lane
1), indicating that Thr199 is not the target
site of CDK1-cyclin B. In contrast, the levels of 32P
incorporation of both GST-NPM/T234A (lane 4) and
GST-NPM/T237A (lane 5) were reduced to less than
50% of GST-NPM/wt. When both Thr234 and Thr237
were replaced with alanine residues (GST-NPM/T234A/T237A), the level of
32P-incorporation became almost undetectable
(lane 6). This result indicates that CDK1-cyclin
B phosphorylates both Thr234 and Thr237
in vitro. Moreover, CDK2-cyclin E and CDK1-cyclin B
phosphorylate different sites of NPM/B23.
Thr199 of NPM/B23 is phosphorylated in Vivo--
We
next examined whether Thr199 is phosphorylated in
vivo by two-dimensional tryptic peptide mapping of NPM/B23
prepared from metabolically labeled cells with
[32P]orthophosphate. Since CDK2-cyclin E is normally
activated only in late G1, we assumed that CDK2-cyclin
E-mediated phosphorylation of NPM/B23 might not be efficiently detected
if the exponentially growing cells are used. In addition, it is not
known whether CDK2-cyclin A-mediated phosphorylation of NPM/B23 on
Thr199 occurs in vivo in a similar manner as
in vitro. To circumvent these problems, we first generated
HeLa cells overexpressing cyclin E by transfecting human cyclin E
together with a plasmid encoding the neomycin resistance gene as a
selection marker. It has been shown that overexpression of cyclin E
results in constitutive activation of CDK2-cyclin E (9, 13-15). The
G418-resistant colonies were subcloned and examined for cyclin E
expression by immunoblot analysis, and one cell line that overexpressed
cyclin E (HeLa/Cyc E) was maintained for further experimentation (Fig.
3A). Consistent with the
previous studies (9, 13-15), the immunoprecipitates from HeLa/CycE
cells using anti-cyclin E antibody showed a histone H1 kinase activity
4-5-fold higher than in the vector-transfected control HeLa cells
(HeLa/Vec) (Fig. 3B).

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Fig. 3.
The site of NPM/B23 phosphorylated in
vitro by CDK2-cyclin E is phosphorylated in
vivo. A, immunoblot analysis of cyclin E
expression in HeLa cells stably transfected with human cyclin E. HeLa
cells were co-transfected with a plasmid encoding human cyclin E (or an
empty vector as a control) and a plasmid encoding a neomycin-resistant
gene. The G418-resistant colonies from the cyclin E and the vector
transfection were subcloned (HeLa/CycE and HeLa/Vec, respectively). The
extracts prepared from these cells (50 µg of total protein) were
subject to immunoblot analysis using anti-human cyclin E polyclonal
antibody (sc-198; Santa Cruz Biotechnology). HeLa/CycE cells expresses
~5-fold more cyclin E proteins than HeLa/Vec cells. B,
histone H1 kinase activity of HeLa/CycE cells. The cell extracts
derived from exponentially growing HeLa/CycE and the control HeLa/Vec
cells were immunoprecipitated with anti-human cyclin E antibody, and
the immunoprecipitates were subjected to an in vitro histone
H1 kinase assay as described previously (13). C, tryptic
phosphopeptide mapping of in vitro phosphorylated NPM/B23 by
CDK2-cyclin E and in vivo phosphorylated NPM/B23 in
HeLa/CycE cells. GST-NPM/wt phosphorylated in vitro
by CDK2-cyclin E was prepared as described in the legend to Fig. 1. For
preparation of in vivo phosphorylated NPM/B23, HeLa/CycE
cells were metabolically labeled in the presence of
[32P]orthophosphate. The lysates were immunoprecipitated
with anti-NPM/B23 monoclonal antibody, and the immunoprecipitates were
resolved by 10% SDS-PAGE. 32P-Labeled NPM/B23 proteins
were eluted from the gel. The in vitro
32P-labeled GST-NPM/wt (a) and in
vivo 32P-labeled NPM/B23 (b) were oxidized
with performic acid and digested with trypsin as described previously
(27). Each tryptic digestion sample was loaded onto a thin layer
cellulose gel plate and subjected to electrophoresis (horizontal
dimension), followed by ascending chromatography. Mixed map was
generated by loading equal counts of trypsin-digested in vitro
32P-labeled GST-NPM/wt and in vivo
32P-labeled NPM/B23 (c). +, the origin of
the sample placement. The arrows indicate the
32P-labeled tryptic fragment that is observed both in the
in vitro and in vivo samples.
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HeLa/CycE cells were metabolically labeled in the presence of
[32P]orthophosphate. The cell lysates were
immunoprecipitated with anti-NPM/B23 monoclonal antibody. The
phospholabeled NPM/B23 proteins were purified from the gel after
fractionation of the immunoprecipitates in SDS-PAGE. The NPM/B23
proteins phosphorylated by CDK2-cyclin E in vitro were also
purified after fractionation by SDS-PAGE as described in the legend to
Fig. 1. The purified in vivo and in vitro labeled
NPM/B23 were subjected to tryptic digestion, followed by a
two-dimensional tryptic phosphopeptide mapping analysis. In
vitro phosphorylated NPM/B23 showed one specific spot (Fig. 3C, panel a). In vivo
phosphorylated NPM/B23 gave rise to five major phosphopeptides
(spots 1-5) (panel b).
This is not unexpected, since NPM/B23 has been shown to be
phosphorylated by other kinases at several residues, including
CDK1-cyclin B (32), casein kinase II (33), and nuclear kinase (N-II)
(34). However, one spot (spot 2 in
panel b, indicated by an arrow) showed
a migration similar to that observed for the in vitro
phosphorylated NPM/B23. To confirm that these spots represented
identical phosphorylation sites, a mixture of the in vivo
and in vitro labeled NPM/B23 was analyzed. We found that
these spots comigrated (panel c, indicated by an
arrow), demonstrating that the same tryptic peptide fragment was phosphorylated in vitro by CDK2-cyclin E and in
vivo in HeLa/CycE cells. When potential tryptic fragments were
deduced from the sequence, the primary fragment containing
Thr199 was found to be DTPAK, which contains
only one threonine residue. Thus, we concluded that NPM/B23 is
phosphorylated on Thr199 in vivo.
Suppression of Centrosome Duplication by Expression of
NPM/T199A--
We have previously shown that the mutant NPM/B23
(NPM(
186-239)) with a deletion of 54 amino acids, which includes
the Thr199 CDK2-cyclin E phosphorylation site, acts as a
dominant negative when expressed in cells, resulting in inhibition of
centrosome duplication (16). Although this observation strongly
suggests that the dominant negative activity of this mutant is due to
its inability to be phosphorylated by CDK2-cyclin E, it does not
exclude the possibility that the deleted sequence other than the
phosphorylation site may be also important for the regulation of
centrosome duplication. We thus examined whether the
nonphosphorylatable NPM/T199A mutant acts as a dominant negative in
centrosome duplication in fashion similar to NPM(
186-239). If
expression of NPM/T199A mutant results in suppression of centrosome
duplication, the phosphorylation of Thr199 is most likely a
sole event necessary for the NPM/B23-dependent control of
centrosome duplication. The FLAG epitope-tagged wild-type NPM/B23 (NPM/wt) and NPM/T199A were
placed in eukaryotic expression vectors and transfected into Swiss 3T3
cells together with a plasmid encoding a puromycin-resistant gene. As a
control, the vector was transfected. The puromycin-resistant cells
selected by puromycin treatment for 36 h were replated and
cultured for additional 24 h. Cells were first examined for the
level of expression of transfected NPM/B23 by immunoblot analysis using
anti-FLAG antibody (Fig. 4A).
Both NPM/wt and NPM/T199A transfectants expressed similar levels of
transfected NPM/B23.

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Fig. 4.
Initiation of centrosome duplication is
blocked by NPM/T199A expression. A, immunoblot analysis
of cells transfected with wild-type NPM/B23 (NPM/wt) and NPM/T199A.
Swiss 3T3 cells were transiently transfected with plasmids encoding
FLAG epitope-tagged NPM/wt and NPM/T199A. As a control, an expression
vector used for construction of the plasmids was transfected. For each
transfection, a plasmid encoding a puromycin-resistant gene
(pBabe/puro) was co-transfected as a selection marker. Puromycin
was added to culture medium 16 h after transfection. Cells that
had been successfully transfected were selected within 36 h after
the addition of puromycin. The puromycin-selected cells were replated
and further cultured for an additional 24 h. The whole cell
lysates were prepared from the transfectants and probed with anti-FLAG
polyclonal antibody. NPM/wt and NPM/T199A transfectants expressed
similar levels of transfected NPM/B23. B and C,
the transfectants described for A were immunostained for
centrosomes (centrioles). After cold treatment and brief extraction
(see "Experimental Procedures"), cells were fixed and
co-immunostained with anti- -tubulin polyclonal and anti- -tubulin
monoclonal (DM1A) antibodies. Antigen-antibody complexes were detected
with FITC-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat
anti-mouse IgG antibodies. The nuclei were also counterstained with
DAPI. The representative immunostainings are shown in C. a and e, -tubulin immunostaining; b
and f, -tubulin immunostaining; c and
g, DAPI staining; d and h, overlay
images of -tubulin and -tubulin immunostainings.
I-VIII, approximately × 5 magnification of the
corresponding centrosome images indicated in b and
f. At a higher magnification, each single dot detected by
anti- -tubulin antibody was resolved to doublets (representing a pair
of centrioles) by anti- -tubulin antibody. Anti- -tubulin
antibody-reactive doublets (potentially duplicated centrosomes) are
indicated by arrowheads, and anti- -tubulin antibody
reactive singlets (unduplicated centrosomes) are indicated by
arrows. Scale bars for the images
shown in a-h, 20 µm. The number of centrosomes per cells
(n) were categorized into n = 1, n = 2, and n 3. For each transfectant, >400
cells were examined. The results shown in B are the average
centrosome profiles determined from three independent
experiments.
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Cells were examined for centrosomes by immunostaining of
-tubulin, a
major component of the pericentrial material of the centrosome
(reviewed in Ref. 35). The graph in Fig. 4B shows the
centrosome profiles of the vector, NPM/wt, and NPM/T199A transfectants. In the vector-transfected cells, ~40% of the cells contained one centrosome, and ~60% contained two centrosomes. The cells
transfected with NPM/wt showed centrosome profiles similar to the
vector transfectants. In contrast, the majority (>80%) of cells
transfected with NPM/T199A contained one centrosome, indicating that
ectopic expression of NPM/T199A results in suppression of centrosome duplication.
To verify whether the anti-
-tubulin antibody-reactive signals
(dots) represented intact centrosomes with a pair of
centrioles, cells were also immunostained for the centrioles. Since
-tubulin is one of the major constituents of centrioles,
immunostaining of
-tubulin allows visualization of a centriole pair
within the centrosome. Cells were subjected to cold treatment (which
depolymerizes microtubules nucleated at the centrosomes), followed by a
brief extraction prior to fixation (see "Experimental Procedures"), and co-immunostained with anti-
-tubulin polyclonal and
anti-
-tubulin monoclonal antibodies (Fig. 4C). Each dot
detected by anti-
-tubulin antibody (panels a
and e) was resolved to a pair of dots (representing a
centriole pair) by anti-
-tubulin antibody at a higher magnification (panels b and f, panels
I-VIII). All of the anti-
-tubulin antibody-reactive dots
were co-immunostained by anti-
-tubulin antibody as doublets. Thus,
the doublets detected by anti-
-tubulin antibody represent duplicated
centrosomes. The centrosome profiles determined by anti-
-tubulin
antibody were similar to those determined by anti-
-tubulin antibody
(data not shown).
To eliminate the possibility that inhibition of centrosome duplication
by NPM/T199A is due to a general cell cycle arrest, Swiss 3T3 cells
were transiently transfected with either a vector or a NPM/T199A mutant
plasmid along with a plasmid encoding a puromycin resistance gene as a
selection marker. The puromycin-resistant cells selected during 36-h
puromycin treatment were cultured for an additional 24 h. During
the final 3 h of culturing, BrdUrd was added to the medium
to monitor cell cycling. Cells were co-immunostained with
anti-
-tubulin polyclonal and anti-BrdUrd monoclonal antibodies (Fig.
5A). Approximately 10% of
vector-transfected and ~6% of NPM/T199A-transfected cells were
BrdUrd-positive, suggesting that the expression of NPM/T199A may be
partially cytotoxic. A similar observation was previously made for the
NPM(
186-239) deletion mutant (16). Examination of centrosomes
revealed that all of the BrdUrd-positive vector-transfected cells
contained duplicated centrosomes, while the majority (~80%) of
NPM/T199A-transfected BrdUrd-positive cells contained a single
centrosome (Fig. 5, B and C). Thus, dominant
negative activity of NPM/T199A specifically targets the centrosome
duplication process.

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Fig. 5.
Expression of NPM/T199A mutant specifically
inhibits centrosome duplication. Swiss 3T3 cells were transiently
transfected with either a plasmid encoding FLAG epitope-tagged
NPM/T199A or a control vector. For each transfection, pBabe/puro was
co-transfected as a selection marker. Puromycin was added to medium
16 h after transfection. Puromycin-resistant cells at 36 h
after the addition of puromycin were replated and further cultured for
24 h. During the final 3 h of culturing, BrdUrd was added to
the medium. Cells were then processed for co-immunostaining with
anti-BrdUrd monoclonal (B, panels a
and b) and anti- -tubulin polyclonal antibodies
(B, panels a' and b').
First, the percentage of cells that had incorporated BrdUrd was
determined through examination of >300 cells (A). In
vector-transfected cells, virtually all of the BrdUrd-positive cells
contained two anti- -tubulin antibody-reactive dots (duplicated
centrosomes) (B, panels a and
a', indicated by arrows), while the majority of
BrdUrd-positive NPM/T199A contained a single dot (unduplicated
centrosome) (B, panels b and
b', indicated by an arrow). The number of
centrosomes per cell in the BrdUrd-positive cells was scored by
fluorescence microscopy. For each transfectant, >100 BrdUrd-positive
cells were examined, and the results from three independent experiments
are shown in C.
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Aberrant Mitoses with Monopolar Spindles Resulting from Expression
of NPM/T199A Mutant--
The finding that the expression of NPM/T199A
mutant results in the suppression of centrosome duplication but not DNA
duplication predicts that NPM/T199A-transfected cells should progress
through the cell cycle to mitosis without centrosome duplication. This should lead to mitosis with monopolar instead of bipolar spindles. To
test this prediction, Swiss 3T3 cells were transiently transfected with
either a vector or a NPM/T199A mutant plasmid along with a plasmid
encoding a puromycin resistance gene as a selection marker. The
puromycin-resistant cells were selected as described above (Fig. 4) and
immunostained with anti-
- and
-tubulin monoclonal antibodies and
anti-
-tubulin polyclonal antibody. The cells were also
counterstained with DAPI. Mitotic cells were identified by DAPI-stained
condensed chromosomes, and the number of spindle poles present in each
mitotic cell was determined. Virtually all of the vector-transfected
mitotic cells contained two spindle poles (Fig.
6A), forming bipolar mitotic
spindles (Fig. 6B, panels a-d). In
contrast, ~80% of the NPM/T199A-transfected mitotic cells contained
single spindle poles (Fig. 6A) with disorganized microtubule staining (Fig. 6B, panels e-h). These
observations further support the dominant-negative activity of the
NPM/T199A mutant, specifically inhibiting duplication of
centrosome.

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Fig. 6.
Expression of NPM/T199A mutant results in a
high frequency of monopolar mitosis. Swiss 3T3 cells were
transiently co-transfected with either a plasmid encoding FLAG
epitope-tagged NPM/T199A or a control vector along with pBabe/puro as a
selection marker. Puromycin-resistant cells were selected as described
in the legend to Fig. 4. Cells were immunostained with anti- - and
-tubulin antibodies (B, panels b
and f) and anti- -tubulin polyclonal antibody
(B, panels a and e). Cells
were also counterstained with DAPI (B, panels
c and g). Antigen-antibody complexes were
visualized by FITC-conjugated goat anti-mouse IgG and
rhodamine-conjugated goat anti-rabbit IgG antibodies. The mitotic cells
were first identified by condensed chromosomes under a fluorescence
microscope, and the number of spindle poles in each mitotic cell was
determined (A). For each transfectant, >50 mitotic cells
were examined. Representative immunostaining images are shown in
B. Panels d and h are
overlay images of panels a-c and
e-g, respectively. The arrows point to the
spindle pole. Scale bar, 10 µm.
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NPM/T199A Mutant Associates with the Centrosomes--
We have
previously shown that NPM/B23 dissociates from centrosomes upon
CDK2-cyclin E-mediated phosphorylation (16), which implies that the
NPM/T199A mutant should associate with centrosomes. We thus examined
the localization of transfected FLAG epitope-tagged NPM/T199A by
co-immunostaining with anti-
-tubulin polyclonal and anti-FLAG
monoclonal antibodies (Fig. 7). No
anti-FLAG antibody staining was observed in the vector-transfected
cells (Fig. 7B). In contrast, anti-FLAG antibody
detected a single dot adjacent to nucleus in the NPM/T199A-transfected
cells (Fig. 7F), which overlap with the dot detected by
anti-
-tubulin antibody (Fig. 7, E and H).
Thus, NPM/T199A mutant physically associates with centrosomes.

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Fig. 7.
NPM/T199A physically associates with
centrosomes. The control vector-transfected (A-D) and
FLAG epitope-tagged NPM/T199A-transfected cells (E-H)
described in the legend to Fig. 4 were examined for sublocalization of
transfected NPM/B23 mutant proteins by co-immunostaining with
anti- -tubulin (green; A and E) and
anti-FLAG monoclonal (red; B and F)
antibodies. Cells were also counterstained with DAPI (C and
G). D and H show the overlay images.
The arrow points to the centrosome. The arrow in
B was placed at the same position as shown in A. Scale bar, 10 µm.
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DISCUSSION |
Centrosome hyperamplification, which leads to formation of
aberrant mitotic spindles, is now well accepted as one of the major causes of chromosome instability in human cancers (36-39). In normal cells, the centrosome duplication cycle is tightly regulated. Coordinated initiation of centrosome and DNA duplication is one of the
major regulatory checkpoints for proper progression of the centrosome
duplication cycle, and it is established at least in part by the late
G1-specific activation of CDK2-cyclin E (7, 8).
Activation of CDK2-cyclin E plays a major role in the initiation of DNA
synthesis through phosphorylation of retinoblastoma susceptibility
protein (pRb). When pRb is phosphorylated, it releases the pRb-bound
E2F transcriptional factor, which then stimulates the transcription of
a number of genes required for DNA synthesis (for reviews, see Refs. 40
and 41). The requirement for E2F has also been implicated in the
initiation of centrosome duplication (42), suggesting that
E2F-dependent expression of specific protein(s) may be
needed for centrosome duplication. Indeed, it has been shown that, in
Chinese hamster ovary cells, synthesis of certain centrosomal proteins
during G1 is necessary for the initiation of centrosome
duplication (43). In addition, CDK2-cyclin E has been shown to directly
act on a centrosomal protein to initiate centrosome duplication.
NPM/B23 binds specifically to unduplicated centrosomes and loses its
centrosome binding activity when phosphorylated by CDK2-cyclin E. Dissociation of the centrosomal NPM/B23 appears to be a prerequisite
for the centrosomes to initiate duplication, since centrosome
duplication is blocked by microinjection of anti-NPM/B23 antibody,
which prevents CDK2-cyclin E-mediated phosphorylation and dissociation
of centrosomal NPM/B23 (16).
We have previously shown that NPM/B23 deletion mutant
(NPM(
186-239)), which fails to be phosphorylated by CDK2-cyclin E, acts as a dominant negative when expressed in cells, resulting in
suppression of centrosome duplication (16). In this study, we
identified Thr199 as the CDK2-cyclin E phosphorylation
target site of NPM/B23 both in vitro and in vivo.
The NPM/B23 mutant with an alanine substitution at this site
(NPM/T199A) acts as a dominant negative when expressed in cells and
suppresses centrosome duplication, similar to the NPM(
186-239)
deletion mutant. The initial stage of centrosome duplication consists
of a series of distinct steps: loss of orthogonal configuration and
physical separation of the centriole pair, which is followed by
synthesis of a procentriole next to each preexisting centriole.
Co-immunostaining of centrosomes in the NPM/T199A-transfected cells
with anti-
-tubulin and anti-
-tubulin antibodies detected anti-
-tubulin antibody-reactive doublets (a centriole pair) within the anti-
-tubulin antibody-reactive dot (pericentriolar material), suggesting that the duplication of centrosomes in the
NPM/T199A-transfected cells is blocked in the early stage. This is
consistent with our earlier finding using cells expressing
NPM(
186-239), the nonphosphorylatable deletion mutant, by thin
section transmission electron microscopy (16). In these cells,
unduplicated centrosomes are physically intact with the orthogonal
configuration of the centriole pair, which is typical of those found in
early G1 phase of the cell cycle. Thus, similar to the
nonphosphorylatable deletion mutant, expression of NPM/T199A mutant
blocks the early step of centrosome duplication. Dominant negative
activity of NPM/T199A on centrosome duplication is further evidenced by
the high frequency of aberrant mitoses with monopolar spindles in the
NPM/T199A-transfected cells, resulting from cell cycle progression to
mitosis, without centrosome duplication. These observations provide
direct evidence that CDK2-cyclin E-mediated phosphorylation of NPM/B23
comprises one of the key events in the initiation of centrosome
duplication. At present, the molecular basis of the centrosome-binding
property of NPM/B23 (i.e. which centrosomal protein(s) the
unphosphorylated form of NPM/B23 associates with) is unknown. The
identification of the CDK2-cyclin E-mediated phosphorylation site will,
however, expedite the elucidation of the particular centrosomal
component(s) with which NPM/B23 directly associates.
Another important issue of the inhibition of centrosome duplication by
expression of NPM/T199A mutant is the consequence of monopolar spindle
formation. Considering the role of the centrosomes (spindle poles) in
cytokinesis (47), it is safe to assume that monopolar mitotic cells do
not undergo cytokinesis. If these cells enter the next cell cycle
without cytokinesis, we should expect an increase in the number of
cells with abnormal amplification of genome. However, the flow
cytometric analysis of the NPM/T199A-transfected cells failed to detect
any noticeable increase in the number of cells with abnormally
amplified genome (data not shown), suggesting that formation of
monopolar spindles probably leads to cell death. However, the mechanism
of how cell death is induced in the monopolar mitotic cells remains to
be clarified.
NPM/B23 associates with and dissociates from centrosomes in a cell
cycle stage-specific manner (16, 44). During early to middle
G1, NPM/B23 associates with the unduplicated centrosomes. In late G1, NPM/B23 dissociates from the centrosomes upon
phosphorylation by CDK2-cyclin E. During S and G2 phases,
association of NPM/B23 with the duplicated centrosomes is not detected.
However, during mitosis, NPM/B23 reassociates with the centrosomes.
This cell cycle stage-dependent dissociation and
reassociation of NPM/B23 with centrosomes may be controlled by
differential phosphorylation by CDK-cyclin complexes. CDK2-cyclin E
activity peaks during late G1, triggering dissociation of
the centrosomal NPM/B23. Upon entry into S phase, cyclin E expression
becomes halted. Since cyclin E is intrinsically unstable, cyclin
E-dependent CDK2 activity becomes minimal during S phase
(9, 10). In contrast, the level of cyclin A is low in late
G1 but increases during S and G2 phases
(27-30). Thus, during S and G2 phases of the cell cycle, CDK2-cyclin A activity is high. We found that CDK2-cyclin A could also
phosphorylate NPM/B23 specifically on Thr199 in
vitro at an efficiency similar to CDK2-cyclin E. Thus, it is
possible that the continual presence of active CDK2-cyclin A is
responsible for preventing the reassociation of NPM/B23 to centrosomes
during S and G2. Moreover, NPM/B23 has previously been
shown to be phosphorylated by CDK1-cyclin B, a mitotic CDK-cyclin complex (32). We found that CDK1-cyclin B specifically phosphorylates Thr234 and Thr237 in vitro, which
are different from CDK2-cyclin E (and cyclin A)-mediated
phosphorylation sites. It remains to be investigated whether
phosphorylation of Thr234 and/or Thr237 by
CDK1-cyclin B is required for reassociation of NPM/B23 with the
centrosomes during mitosis. These questions are currently addressed in
our laboratory.
NPM/BNPM/B23 has been shown to participate in various cellular events
that are to all appearances unrelated to each other, including ribosome
assembly, intracellular trafficking, DNA polymerase activity, and
centrosome duplication. These diverse functions of NPM/B23 are perhaps
attributed to its molecular chaperoning activity as reported previously
(22). All of the cellular events in which NPM/B23 has been shown to
function involve either large multiprotein complexes or organelles
consisting of many different proteins in a crowded condition. Thus,
association/dissociation of NPM/B23 may dramatically influence the
centrosome proper and thus determine the structural as well as
functional state of the centrosome. Moreover, the CDK2-cyclin
E-mediated functional modification of NPM/B23 may target other cellular
event(s) as well, since the BrdUrd incorporation assay showed that
expression of NPM/T199A mutant partially blocks (or slows down) the
cell cycle progression.