MOLECULAR AND CELLULAR BIOLOGY, Dec. 2006, p. 9016–9034
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 23
Interaction between ROCK II and Nucleophosmin/B23 in the
Regulation of Centrosome Duplication?
Zhiyong Ma,1Masayuki Kanai,1Kenji Kawamura,1,2Kozo Kaibuchi,3
Keqiang Ye,4and Kenji Fukasawa1*
Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati,
Ohio 45267-05211; Department of Urology, Kanazawa Medical University, 1-1 Daigaku Uchinada, Ishikawa,
920-0293, Japan2; Nagoya University Graduate School of Medicine, Showa, Nagoya, Aichi 466-8500,
Japan3; and Department of Pathology and Laboratory Medicine, School of Medicine,
Emory University, Georgia 303224
Received 27 July 2006/Returned for modification 1 September 2006/Accepted 13 September 2006
Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23
localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr199by cyclin-
dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but
some NPM/B23 phosphorylated on Thr199remains at centrosomes. It has been shown that Thr199phosphor-
ylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the
centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a
protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively
active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression
results in the suppression of centrosome duplication, especially delaying the initiation of centrosome dupli-
cation during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome
localization activity-dependent manner. We further found that ROCK II kinase activity is significantly en-
hanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon
phosphorylation on Thr199. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in
association with CDK2/cyclin E activation and the emergence of Thr199-phosphorylated NPM/B23. All these
findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of
The centrosome is composed of a pair of centrioles and
surrounding protein aggregates known as pericentriolar mate-
rial. The centrosome, as a core component of the spindle pole,
plays a key role in the establishment of bipolar spindles during
mitosis, which is essential for the accurate segregation of chro-
mosomes to daughter cells (reviewed in references 10 and 13).
Upon cytokinesis, each daughter cell inherits only one centro-
some; hence, the centrosome must duplicate once in each cell
cycle prior to the next mitosis. In animal cells, centrosome
duplication proceeds in coordination with other cell cycle
events (i.e., DNA synthesis) (27): centrosome duplication be-
gins near the G1/S boundary and is completed at late G2.
Centrosome duplication begins with the physical separation of
paired centrioles, followed by procentriole formation in the
vicinity of each preexisting centriole. Initiation of centrosome
duplication is triggered by cyclin-dependent kinase 2 (CDK2)/
cyclin E (reviewed in reference 18), which is activated in late
G1primarily by temporal expression of cyclin E (reviewed in
references 28 and 35). Several targets of CDK2 in the initiation
of centrosome duplication have been identified, including nu-
cleophosmin (NPM)/B23, Mps1, and CP110 (7, 12, 32).
NPM/B23 is a multifunctional protein implicated in a
variety of cellular events, including ribosome assembly, pre-
rRNA processing (17, 39, 51), mRNA processing (34, 44),
DNA duplication through physical interaction with DNA
polymerase ? and RB (33, 42), nucleocytoplasmic protein
trafficking via binding to the nuclear localization signals of
target proteins (4, 40, 48), molecular chaperoning (41), and
centrosome duplication (15, 32, 38, 46, 49). Analysis of the
centrosomal association of NPM/B23 has revealed that
NPM/B23 localizes between the paired centrioles, and upon
phosphorylation on Thr199by CDK2/cyclin E, the majority
of the NPM/B23 proteins dissociate from centrosomes prior
to the initiation of centrosome duplication (separation of
paired centrioles), implicating NPM/B23 in the pairing of
centrioles (38). In this context, NPM/B23 acts as a suppres-
sor of centrosome duplication. Indeed, the down-regulation
of NPM/B23 results in the abnormal amplification of cen-
trosomes (15). However, some Thr199-phosphorylated pro-
teins remain at centrosomes and translocate toward a
mother centriole of the pair (38), suggesting that those
remaining NPM/B23 proteins may be involved in the regu-
lation of centrosome duplication in a manner different from
that used in centriole pairing. The interesting feature of
NPM/B23 is that it simultaneously exerts different functions
through affecting proteins of the different, often seemingly
antagonistic, biological pathways/processes. For instance,
NPM/B23 can act oncogenically as well as antioncogenically.
In cancers, NPM/B23 is frequently mutated or lost, suggest-
ing its role as a tumor suppressor, while NPM/B23 is equally
* Corresponding author. Mailing address: Department of Cell Biol-
ogy, University of Cincinnati College of Medicine, P.O. Box 670521
(3125 Eden Ave.), Cincinnati, OH 45267-0521. Phone: (513) 558-4939.
Fax: (513) 558-4454. E-mail: Kenji.Fukasawa@uc.edu.
?Published ahead of print on 2 October 2006.
frequently overexpressed, suggesting its oncogenic role (16).
Experimentally, the overexpression of NPM/B23 results in
cellular transformation (23), while partial depletion of
NPM/B23 in mice accelerates oncogenesis (15). Thus, it
would not be surprising if NPM/B23 is involved in the reg-
ulation of centrosome duplication in more than one path-
way, and such regulatory pathways could either promote or
suppress centrosome duplication.
Here, we identified ROCK II, also known as ROK? or
Rho(-associated) kinase, as a centrosomal protein that physi-
cally interacts with NPM/B23 with a strong affinity. ROCK II is
Ser/Thr kinase controlled by the small GTPase Rho (24, 26).
We found that ROCK II promotes centrosome duplication in
its kinase and centrosome localization activity-dependent man-
ner. Moreover, the kinase activity of ROCK II is markedly
enhanced by physical interaction with NPM/B23. Thr199-phos-
phorylated NPM/B23 has a higher binding affinity to ROCK II
than unphosphorylated NPM/B23, and ROCK II-NPM/B23
complex formation in vivo depends on the Thr199phosphory-
lation of NPM/B23. Thus, the effects of CDK2/cyclin E-medi-
ated phosphorylation of Thr199on the initiation of centrosome
duplication is twofold: removing the centriole pairing activity
of NPM/B23 and potentiating ROCK II to promote the initi-
ation of centrosome duplication.
MATERIALS AND METHODS
Cells and transfection. NIH 3T3 and mouse skin fibroblasts (MSFs) were
maintained in complete medium (Dulbecco’s modified Eagle’s medium supple-
mented with 10% fetal bovine serum, penicillin [100 units/ml], and streptomycin
[100 ?g/ml]) in an atmosphere containing 10% CO2. Transfection was performed
using either Lipofectamine 2000 (Invitrogen) or Fugene-6 (Roche) reagent.
Plasmids and antibodies. Generation of the ROCK II mutants (with the
catalytic domain [CAT], the coiled-coil domain [coil], RB, and the pleckstrin
homology domain [PH]) was previously described (2). Other ROCK II mutants
were generated by the PCR-based method. Glutathione S-transferase (GST)–
ROCK II and GST-CAT used for in vitro kinase assay were prepared from sf9
insect cells as described previously (26). GST-ROCK II (or -CAT) and His6?-
NPM/B23 proteins were bacterially purified using glutathione Sepharose 4B
(Pharmacia Biotech) and Ni-nitrilotriacetic acid resins (QIAGEN), respectively.
For generation of the plasmid encoding small interfering RNA (siRNA) specific
for ROCK II, the sequence 5?-GGAACTGCAAGACCAACTT-3? (correspond-
ing to cDNA sequence positions 2580 to 2598) was cloned into the pSUPER
vector (OligoEngine). For generation of the plasmid encoding siRNA specific for
NPM/B23, 5?-AGAACGGTCAGTTTAGGAG-3? (corresponding to cDNA se-
quence positions 133 to 151 ) was cloned into the pSUPER vector.
The antibodies used in this study were anti-ROCK II polyclonal (07-443;
Upstate Biotechnology), anti-ROCK II monoclonal (610623; BD Biosciences),
anti-ROCK I polyclonal (sc-5560; Santa Cruz), anti-NPM/B23 monoclonal (51),
anti-NPM/B23 polyclonal (generated in our laboratory ), anti-GST (Z-5)
polyclonal (sc-459; Santa Cruz), anti-FLAG monoclonal (M2; Sigma), anti-?-
tubulin monoclonal (GTU-88; Sigma), anti-?-tubulin polyclonal (generated in
our laboratory), anticentrin monoclonal (36), anti-GFP monoclonal (1814460;
Roche), anti-phospho-Ser19MLC 2 polyclonal (3671; Cell Signaling), anti-MLC
2 polyclonal (sc-15370; Santa Cruz), anti-His tag monoclonal (Ab-1; Oncogene
Science), antivimentin polyclonal (AB994; Chemicon), anti-cyclin E polyclonal
(M-20; Santa Cruz), and anti-?-tubulin monoclonal (TUB2.1; Sigma) antibodies.
GST-NPM/B23 affinity purification of centrosomal proteins. Centrosomes
were isolated as described previously (29, 32). Centrosomes are denatured in 9
M urea to dissociate centrosomal components and then subjected to renaturation
by dialysis against the buffer solution (20 mM Tris-Cl [pH 7.4], 25 mM NaCl, 5
mM MgCl2, 1 mM EDTA, 1 mM ATP, 1 mM GTP, 0.01% NP-40, 2 ?g/ml
leupeptin, 2 ?g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride [PMSF]) at
4°C. At 6 h of dialysis, bacterially purified GST-NPM/B23 or a GST control was
added, and the samples were further dialyzed for 12 h. GST-NPM/B23 and
associating proteins were pulled down using glutathione Sepharose 4B, washed
in the buffer (20 mM Tris-Cl [pH 7.4], 80 mM NaCl, 5 mM MgCl2, 1 mM EDTA,
10% glycerol) five times, resolved in a 6 to 15% gradient of sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and silver stained.
The bands of interest were excised, and subjected to mass spectrometric analysis
as described previously (32).
In vitro protein binding assay. His6?-NPM/B23 and GST-ROCK II (wild-type
and mutant) proteins were incubated in the binding buffer (50 mM Tris-Cl [pH
7.5], 1 mM MgCl2, 0.1 mM ATP, 10% glycerol, 0.1 ?g/?l bovine serum albumin,
2 ?g/ml leupeptin, 2 ?g/ml aprotinin, 1 mM PMSF) for 2 h at 4°C. GST affinity
beads were added and incubated for an additional 2 h. After extensive washes
with the wash buffer (50 mM Tris-Cl [pH 7.5], 1 mM MgCl2, 0.1 mM ATP, 50
mM NaCl, 10% glycerol, 1% Tween 20), the precipitates were resolved by
SDS-PAGE and subjected to immunoblot analysis.
Immunoblotting and immunoprecipitation. For immunoblot analysis, cells
were lysed in SDS–NP-40 lysis buffer (1% SDS, 1% NP-40, 50 mM Tris [pH 8.0],
150 mM NaCl, 2 ?g/ml leupeptin, 2 ?g/ml aprotinin, 1 mM PMSF). The lysates
were boiled for 5 min and cleared by centrifugation at 4°C. The samples were
denatured in sample buffer (2% SDS, 10% glycerol, 60 mM Tris [pH 6.8], 5%
?-mercaptoethanol, 0.01% bromophenol blue), resolved by SDS-PAGE, and
transferred onto an Immobilon-P membrane (Millipore). The blots were incu-
bated in blocking buffer (5% [wt/vol] nonfat dry milk in Tris-buffered saline plus
Tween 20) for 1 h and incubated with primary antibodies for 16 h at 4°C. After
extensive washing in Tris-buffered saline plus Tween 20, the blots were incubated
with horseradish peroxidase-conjugated secondary antibodies for 1 h at room
temperature. The antibody-antigen complex was visualized by ECL chemilumi-
nescence (Amersham Pharmacia). Quantification was performed with Quantity
One software (Bio-Rad). For immunoprecipitation, cells were lysed in NP-40
lysis buffer (50 mM Tris-HCl [pH 7.5], 50 mM ?-glycerophosphate, 150 mM
NaCl, 5 mM MgCl2, 10 mM EGTA, 5 mM NaF, 1% Na deoxycholate, 1% NP-40,
2 ?g/ml leupeptin, 2 ?g/ml aprotinin, 1 mM PMSF). The lysates were precleared
by incubation with protein G-agarose beads for 1 h at 4°C, and incubated with
antibodies overnight at 4°C. The immuno-complexes were collected by protein G
agarose beads, washed in ice-cold NP-40 lysis buffer, boiled in sample buffer, and
resolved by SDS-PAGE.
In vitro kinase assay. GST-ROCK II or GST-CAT purified from sf9 cells or
immunoprecipitates from cells transfected with GFP-ROCK II mutants using
anti-GFP antibody were subjected to a kinase reaction in a buffer (50 mM Tris-Cl
[pH 7.5], 1 mM MgCl2, 5 mM NaF, 2 ?g/ml leupeptin, 2 ?g/ml aprotinin, 1 mM
PMSF) with a substrate (0.15 ?g/?l of vimentin) in the presence of [?-32P]ATP
and incubated for 30 min at 30°C. For examination of the kinase activities of
CDK2/cyclin E, the lysates were subjected to immunoprecipitation using anti-
cyclin E antibody. The antibody-antigen complexes were collected with protein
A-agarose beads, and tested for a histone H1 kinase activity as described previ-
ously (30). The kinase activity was quantified using a Fuji phosphorimager.
Indirect immunofluorescence. Cells were fixed with 10% formalin–10% meth-
anol. For examination of the centrosomal localization of ROCK II, cells were
briefly extracted with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for
1 min, followed by extensive washing with PBS prior to fixation. Without a brief
extraction, ROCK II signals at centrosomes are heavily masked by the ubiquitous
presence of ROCK II, especially when GFP-tagged ROCK II is introduced. Cells
were then blocked by 10% normal goat serum in PBS for 1 h and incubated with
antibodies for 1 h. Cells were then incubated with secondary antibodies for 1 h
and counterstained for DNA with 4?, 6-diamidino-2-phenylindole (DAPI). Cells
were examined under a fluorescence microscope (Nikon Microphot-FX) using a
60? objective lens. The images were captured with a SPOT charge-coupled-
device camera (Diagnostic Instruments).
BrdU incorporation assay. The entire procedure was performed using a cell
proliferation assay kit (Amersham) as instructed by the supplier. Briefly, after
incubation in the labeling medium containing 5-bromo-2?-deoxyuridine (BrdU),
cells were washed with PBS and fixed with acetic acid-ethanol. After being
blocked with 3% bovine serum albumin in PBS plus 0.1% Tween 20), cells were
probed with anti-BrdU monoclonal antibody and detected with fluorescein iso-
thiocyanate-conjugated goat anti-mouse immunoglobulin G2a (IgG2a).
ROCK II binds directly to NPM/B23. To identify a centro-
somal protein(s) that interacts with NPM/B23, we isolated
centrosomes from NIH 3T3 cells by discontinuous sucrose
gradient centrifugation as described previously (29, 32). Since
the isolated centrosomes exist as “protein aggregates,” we first
dissociated centrosomal components in 9 M urea. The disso-
ciated centrosomal components were then renatured by dialy-
VOL. 26, 2006 CENTROSOME DUPLICATION CONTROL BY ROCK II AND NPM/B239017
FIG. 1. Physical interaction between NPM/B23 and ROCK II in vitro and in vivo. (A) Identification of ROCK II as an NPM/B23-interacting
centrosomal protein. Centrosomes isolated from NIH 3T3 cells were denatured in 9 M urea to dissociate centrosomal components, followed by
renaturation. During the renaturing process, the samples were incubated with either GST-NPM/B23 (lane 1) or GST (lane 2) and pulled down with
GST affinity beads. The protein complexes were resolved by SDS-PAGE and visualized by silver staining. The protein bands specific to
GST-NPM/B23 were excised, subjected to mass spectrometric analysis, and identified as ROCK II and vimentin. (B) Physical interaction between
9018 MA ET AL.MOL. CELL. BIOL.
sis. During the renaturation, GST-NPM/B23 (or GST as a
control) was added to the samples. GST-NPM/B23 (or GST)
and associating proteins were pulled down with GST affinity
beads, resolved by SDS-PAGE, and silver stained (Fig. 1A).
This experimental procedure successfully screened the centro-
somal proteins that interacted with NPM/B23 with a high af-
finity and minimized the chaperoning activity-associated back-
ground protein binding of NPM/B23. There were two readily
distinguishable protein bands (?160 and ?55 kDa) unique to
GST-NPM/B23 (lane 1). These proteins were subjected to a
mass spectroscopic analysis and identified as ROCK II (160
kDa) and vimentin (55 kDa). ROCK II belongs to a ROCK
kinase family consisting of ROCK I and II, which share ?60%
We first tested the interaction between NPM/B23 and ROCK
II in vivo by coimmunoprecipitation assay using anti-ROCK II
and anti-NPM/B23 antibodies (Fig. 1B). Anti-ROCK II anti-
body coimmunoprecipitated NPM/B23 (Fig.1Ba, top panel,
lane 2), and anti-NPM/B23 antibody coimmunoprecipitated
ROCK II (Fig. 1Bb, middle panel, lane 1), demonstrating the
physical interaction of these two proteins in vivo. Since ROCK
I, another member of the ROCK kinase family, also localizes
to centrosomes (8), we tested the interaction between ROCK
I and NPM/B23. NPM/B23 was not precipitated with anti-
ROCK I antibody (Fig.1Ba, top panel, lane 1), and ROCK I
was not precipitated with anti-NPM/B23 antibody (Fig.1Bb,
top panel, lane 1). Thus, NPM/B23 does not interact with
To test whether NPM/B23 and ROCK II directly interact
with each other, an in vitro binding assay was performed using
bacterially purified NPM/B23 and ROCK II. Since ROCK II is
too large to be efficiently expressed in a bacterial system, a
series of GST-fused deletion mutants that cover distinct func-
tional domains (diagramed in Fig. 1C) were generated: GST-
CAT (kinase domain plus part of the coiled-coil domain),
GST-CAT/KD (GST-CAT in which Lys121at the ATP-binding
motif is replaced by Gly), GST-kinase domain (GST-kinase),
GST–coil-coil domain (GST-coil), GST-RB, and GST-PH.
These mutants were incubated with His6
cipitated with GST affinity beads. The precipitates were then
immunoblotted for NPM/B23 (Fig. 1D, top) and GST (bot-
tom). NPM/B23 was coimmunoprecipitated only when it was
incubated with GST-CAT (lane 1) and GST-CAT/KD (lane 2),
indicating that NPM/B23 directly binds to CAT domain
(amino acids [aa] 6 to 553) of ROCK II. Since GST-coil (aa
421 to 701) failed to bind to NPM/B23, the NPM/B23 binding
?-NPM/B23 and pre-
region likely resides within aa 6 to 420. Moreover, GST-kinase
(aa 6 to 372) failed to bind to NPM/B23. Thus, the sequence
comprising aa 373 to 420 may be critical for ROCK II to bind
to NPM/B23. Indeed, GST-CAT with aa 373 to 420 deleted
could no longer bind to NPM/B23 in vitro (Fig. 1D, lane 7).
Similarly, with green fluorescent protein (GFP)-tagged full-
length ROCK II with aa 373 to 420 deleted (GFP–ROCK
II?373-420), when expressed in NIH 3T3 cells, anti-GFP an-
tibody failed to coimmunoprecipitate NPM/B23 (Fig. 1E, sec-
ond panel, lane 3) and anti-NPM/B23 antibody failed to co-
immunoprecipitate GFP–ROCK II?373-420 (bottom panel,
Characterization of the centrosomal association of ROCK
II. We next examined whether ROCK II is present in the
isolated centrosomes. The cytoplasmic lysates containing cen-
trosomes prepared from NIH 3T3 cells were subjected to dis-
continuous sucrose gradient centrifugation, and the resulting
fractions were immunoblotted with anti-?-tubulin and anti-
ROCK II antibodies (Fig. 2A). The fractionation pattern of
ROCK II paralleled that of ?-tubulin, suggesting the presence
of ROCK II at the centrosomes.
We also examined the centrosomal localization of ROCK II
by coimmunostaining with anti-ROCK II and anti-?-tubulin
antibodies (Fig. 2B). We detected ROCK II at unduplicated
and duplicated centrosomes as well as spindle poles during
mitosis. To test the specificity of anti-ROCK II antibody, we
generated NIH 3T3 cells whose ROCK II expression was si-
lenced by siRNA. We found that ROCK II could be stably
silenced (?95%) (Fig. 2C). Cells whose ROCK II expression
was silenced by RNA interference (ROCK II-RNAi cells) and
control cells transfected with an siRNA vector with a random-
ized sequence were coimmunostained for ?-tubulin and ROCK
II. The ROCK II antibody-reactive signals were no longer detect-
able at centrosomes in ROCK II-RNAi cells (representative im-
munostaining images of ROCK II-RNAi and vector control cells
are shown in Fig. 2D), demonstrating the specificity of the anti-
ROCK II antibody.
To determine the region of ROCK II critical for centroso-
mal localization, the fragments of ROCK II described in the
legend to Fig. 1C were tagged with GFP (Fig. 3A), transfected
into NIH 3T3 cells, and immunostained for ?-tubulin. Inter-
estingly, several regions of ROCK II were found to localize to
centrosomes, including the kinase, coil, and PH mutants (im-
munostaining images of GFP-, GFP-CAT-, and GFP-PH-
transfected cells are shown in Fig. 3B). Since the C-terminal
PH domain constitutes an autoinhibitory region which folds
NPM/B23 and ROCK II in vivo. The lysates prepared from NIH 3T3 cells were subjected to immunoprecipitation using (a) either anti-ROCK I
(lane 1) or ROCK II (lane 2) antibodies (lane 3, a control IgG) and (b) anti-NPM/B23 antibody (lane 1) (lane 2, a control IgG). The
immunoprecipitates were then immunoblotted with anti-NPM/B23, anti-ROCK I, and anti-ROCK II antibodies. The cell lysates (5% of the
amount used for immunoprecipitation) were included in the analyses. (C) Diagram of ROCK II wild-type and deletion mutants. (D) Identification
of the sequence of ROCK II critical for NPM/B23 binding. Bacterially purified His6
GST-CAT (lane 1), -CAT/KD (lane 2), -kinase (lane 3), -coil (lane 4), -RB (lane 5), -PH (lane 6), and –CAT?373-420 (lane 7). The reaction
samples were subjected to precipitation using GST affinity beads and immunoblotted with anti-NPM/B23 antibody (upper panel) and anti-GST
antibody (lower panel). (E) In vivo demonstration of the critical sequence of ROCK II for NPM/B23 binding. NIH 3T3 cells were transfected with
either GFP-tagged full-length ROCK II or ROCK II with aa 373 to 420 deleted. A GFP vector was transfected as a control. Expression levels of
transfected ROCK II proteins were determined by immunoblot (IB) analysis using anti-GFP antibody (top). The lysates were also subjected to
coimmunoprecipitation (IP) assay using anti-GFP (middle) and anti-NPM/B23 (bottom) antibodies. The immunoprecipitates were immunoblotted
with anti-NPM/B23 as well as anti-GFP antibodies. wt, wild type.
?-NPM/B23 proteins were mixed with bacterially purified
VOL. 26, 2006 CENTROSOME DUPLICATION CONTROL BY ROCK II AND NPM/B239019
back to interact with the N-terminal kinase domain (1, 6), it is
possible that GFP-kinase and GFP-PH may bind to the PH
and kinase domains of endogenous ROCK II, respectively. To
test this, GFP-tagged ROCK II fragments were transfected
into the ROCK II-RNAi cells and examined for their abilities
to localize to centrosomes (immunostaining images of GFP-
CAT and GFP-PH transfectants are shown in Fig. 3Ca to f). As
suspected, both GFP-kinase and GFP-PH failed to localize to
centrosomes in the ROCK II-RNAi cells, indicating that GFP-
kinase and GFP-PH localize to centrosomes via interaction
FIG. 2. Localization of ROCK II at centrosomes. (A) Centrosomes were isolated from NIH 3T3 cells by discontinuous sucrose gradient
centrifugation, and the resulting fractions were immunoblotted using anti-ROCK II (top) and anti-?-tubulin (bottom) antibodies. (B) Cells were
briefly extracted prior to fixation and coimmunostained with rabbit anti-ROCK II (green) and mouse anti-?-tubulin (red) antibodies. Cells were
also counterstained for DNA with DAPI (blue) and merged with the images of ROCK II and ?-tubulin immunostaining. (a to c) Cell with
unduplicated centrosomes; (d to f) cell with duplicated centrosomes; (g to i) mitotic cell. The arrows point to the positions of centrosomes. Scale
bar, 10 ?m. (C) siRNA-mediated silencing of ROCK II. NIH 3T3 cells were cotransfected with a pSUPER plasmid that encodes siRNA specific
for ROCK II (lane 1) and a plasmid encoding a puromycin-resistant gene at a 20:1 molar ratio. The vector (pSUPER with a random sequence of
the same nucleotide composition with ROCK II siRNA) was transfected as a control (lane 2). Puromycin-resistant colonies were pooled, and the
lysates from the transfectants were immunoblotted with anti-ROCK II (top) and anti-?-tubulin (bottom) antibodies. (D) The ROCK II-RNAi and
vector control cells described above were coimmunostained with anti-ROCK II (red) and anti-?-tubulin (green) antibodies. DAPI-stained images
(blue) were merged with the images of ?-tubulin immunostaining. (c and f) Merged images of ROCK II, ?-tubulin, and DNA staining. The arrows
point to the positions of centrosomes. Scale bar, 10 ?m.
9020 MA ET AL.MOL. CELL. BIOL.
FIG. 3. Characterization of the centrosome-binding activity of ROCK II. (A) Wild-type ROCK II and deletion mutants were tagged with GFP
and transfected into NIH 3T3 and ROCK II-RNAi cells. A GFP vector was transfected as a control. At 36 h posttransfection, cells were briefly
extracted, fixed and immunostained for ?-tubulin, and examined for the centrosomal localization of GFP-tagged ROCK II proteins, and results
are summarized to the right of the diagram. (B) Representative images of the GFP-, GFP-CAT-, and GFP-PH-transfected NIH 3T3 cells.
(C) GFP-CAT-, GFP-PH-, and GFP–?457-553-transfected ROCK II-RNAi cells. DAPI-stained images (blue) were laid over ?-tubulin- and
GFP-immunostained images. The arrows point to the positions of centrosomes. The panel to the right of each image shows the magnified image
of the area indicated by arrows. Scale bar, 10 ?m.
with endogenous ROCK II. In contrast, GFP-coil and GFP-
CAT were still able to localize at centrosomes, indicating that
the part of the coiled-coil domain shared by these two ROCK
II fragments (aa 421 to 553) may be critical for the centro-
some-binding activity of ROCK II. Further deletion within this
sequence narrowed it down to aa 457 to 553. For instance,
deletion of aa 457 to 553 of full-length ROCK II (GFP–?457-
553) as well as the CAT mutant (GFP–CAT?457-553), al-
though they localized to centrosomes in normal cells through
interaction with endogenous ROCK II, abolished the ability to
localize to centrosomes in the ROCK II-RNAi cells (immuno-
staining images of GFP–?457-553 are shown in Fig. 3Cg to i).
It should be noted here that GFP–?457-553 as well as GFP–
CAT?457-553 lack the sequence targeted by siRNA used for
silencing ROCK II expression. In addition, GFP-CAT/KD lo-
calizes to centrosomes (data not shown), indicating that cen-
trosome binding of ROCK II is independent of its kinase
activity. The centrosome-binding activities of wild-type and
mutant ROCK II in normal and ROCK II-RNAi cells are
summarized in Fig. 3A.
ROCK II promotes centrosome reduplication. We tested
whether ROCK II is involved in the regulation of centrosome
duplication by the centrosome reduplication assay. When cells
are exposed to DNA synthesis inhibitors such as aphidicolin
(Aph), centrosomes continue to duplicate without DNA syn-
thesis, resulting in centrosome amplification (the presence of
three or more centrosomes) (3). However, this phenomenon
preferentially occurs in cells with an impaired p53-dependent
checkpoint. In the presence of functional p53, p21 CDK inhib-
itor is up-regulated in a p53-dependent manner in response to
the stress associated with exposure to DNA synthesis inhibitors
(43), which in turn inhibits CDK2/cyclin E, a known initiator of
centrosome duplication. NIH 3T3 cells carried in our labora-
tory are partially defective in the p53-dependent checkpoint
function: they fail to up-regulate p21 upon exposure to Aph,
making cells permissive for centrosome reduplication during
Aph-induced arrest. ROCK II has an autoinhibitory C-termi-
nal region that binds to the N-terminal kinase region. The
kinase is activated when the negative regulatory interaction
between these two domains is disrupted by Rho binding to a
C-terminal region (1, 6, 19). Thus, deletion of the C-terminal
region constitutively activates ROCK II, as is the case for the
CAT mutant (Fig. 1C). We transfected GFP-CAT, GFP-CAT/
KD, and a control GFP vector into NIH 3T3 cells prearrested
by exposure to Aph for 24 h. Transfecting the pre-Aph-ar-
rested cells eliminates the possibility of misinterpretation of
data due to the potential alteration of cell cycle progression by
the forced expression of GFP-CAT. The GFP-CAT transfec-
tants showed a membrane blebbing typical of the constitutive
activation of ROCKs (Fig.4Bc), which is attributed to the
ROCK-mediated phosphorylation of myosin light chain 2
(MLC2) (9, 37). Such morphological change was not observed
in the control GFP vector (Fig.4Ba) or CAT/KD transfectants
(not shown). After transfection, cells continued to be exposed
to Aph for 36 h. Over 75% of the GFP-CAT-transfected cells
underwent centrosome reduplication, which is significantly
higher than that in the vector-transfected cells (?45%) (Fig.
4A; representative immunostaining images of GFP-CAT- and
vector-transfected cells are shown in Fig. 4Ba to d). Expression
of GFP-CAT/KD resulted in a substantial decrease in the
frequency of centrosome amplification (?30%) compared with
that in the vector-transfected cells, suggesting that GFP-
CAT/KD may act in a dominant negative manner. Further-
more, addition of the ROCK kinase inhibitor Y-27632 (21)
immediately after transfection suppressed the promotion of
centrosome reduplication by GFP-CAT (Fig. 4A; representa-
tive immunostaining images are shown in Fig. 4Be and f).
Thus, expression of the CAT mutant accelerates centrosome
reduplication under Aph-induced arrest in its kinase activity-
dependent manner. We also tested the centrosome duplication
regulatory role of ROCK II in U2OS human cells, and we
obtained similar results (data not shown). Thus, the involve-
ment of ROCK II in the regulation of centrosome duplication
is neither species nor cell type specific.
ROCK II requires its centrosome-binding activity to pro-
mote centrosome reduplication. The GFP-CAT mutant with
aa 457 to 553 deleted (GFP–CAT?457-553) can no longer
localize to centrosomes (Fig. 3A). To test whether GFP–
CAT?457-553 retains its kinase activity, NIH 3T3 cells were
transfected with the GFP vector control, GFP-CAT, or GFP–
CAT?457-553. The lysates prepared from the transfectants
were subjected to immunoprecipitation using anti-GFP anti-
body. The immunoprecipitated GFP, GFP-CAT, and GFP–
CAT?457-553 were then tested for their in vitro kinase activity
as described previously (20) using vimentin as the substrate,
which is one of the known physiological substrates of ROCK II
(14) (Fig. 5A). Similar levels of GFP-CAT and GFP–
CAT?457-553 were immunoprecipitated (Fig. 5A, top). The in
vitro kinase assay showed that both GFP-CAT and GFP–
CAT?457-553 phosphorylated vimentin at similar efficiencies
(Fig. 5A, middle), demonstrating that GFP–CAT?457-553 re-
tains full kinase activity.
We also tested whether the kinase activity of GFP–
CAT?457-553 is retained in vivo. It has been shown that
ROCK II phosphorylates MLC2 at Ser19, and Ser19phosphor-
ylation of MLC2 can serve as an indicator of the in vivo ROCK
II activity (1, 47), although the increase in the levels of phos-
pho-Ser19MLC2 may also be attributed to the inhibition of
MLC phosphatase by ROCK II (11, 22). We transfected GFP,
GFP-CAT, or GFP–CAT?457-553 into NIH 3T3 cells. Immu-
noblot analysis of the lysates prepared from the transfectants
with anti-GFP antibody showed that GFP-CAT and GFP–
CAT?457-553 were expressed at similar levels (Fig. 5B, top).
The lysates were then immunoblotted with anti-phospho-Ser19
MLC2 antibody as well as anti-MLC2 antibody, which detects
total MLC2 protein. There was no difference in the levels of
total MLC2 among the transfectants (Fig. 5B, bottom), while
the level of Ser19-phosphorylated MLC2 was noticeably in-
creased in the GFP-CAT-transfected cells (Fig. 5B, middle,
lane 1) compared with the level in control cells (lane 3). In the
cells transfected with GFP–CAT?457-553, the level of Ser19-
phosphorylated MLC2 was similar to those in cells transfected
with GFP-CAT (Fig. 5B, middle, lane 2). These results show
that the GFP–CAT?457-553 mutant retains full kinase activity
To address whether centrosomal localization is required for
ROCK II to promote centrosome duplication, we tested GFP–
CAT?457-553 by the centrosome reduplication assay. We
transfected GFP, GFP-CAT, and GFP–CAT?457-553 into
NIH 3T3 cells prearrested by Aph. After transfection, cells
9022MA ET AL.MOL. CELL. BIOL.
continued to be exposed to Aph for 36 h, and the centrosome
profiles of the GFP-positive cells were determined (Fig. 5C).
As shown in Fig. 4A, expression of GFP-CAT resulted in a
high frequency of centrosome amplification (?75%) compared
with that in the vector-transfected control cells (?45%). How-
ever, the expression of GFP–CAT?457-553 failed to promote
centrosome reduplication; the frequency of centrosome ampli-
fication was similar to that of the control cells, indicating that
centrosomal localization is critical for ROCK II to promote
ROCK II is required for the timely initiation of centrosome
duplication in normal cells. To obtain insight into the role of
ROCK II in the regulation of centrosome duplication in nor-
mal cells, the ROCK II-RNAi and control cells were exposed
to Aph for 60 h, and the centrosome profiles were determined
(Fig. 6A; representative immunostaining images are shown in
Fig. 6B). The control cells reduplicated centrosomes under
Aph-induced arrest, resulting in a frequency of centrosome
amplification of ?40% (in this assay, cells were not prearrested
by Aph; hence, the total Aph incubation time was shorter than
in the other centrosome reduplication assay [see, e.g., Fig.
4A]). In contrast, in ROCK II-RNAi cells, centrosome redupli-
cation was significantly suppressed (?20%). When GFP-CAT
which lacks the sequence targeted by siRNA was introduced into
the ROCK II-RNAi cells, the suppression of centrosome redu-
plication was no longer detected, demonstrating that the suppres-
sion of centrosome reduplication in the ROCK II-RNAi cells is
associated with the down-regulation of ROCK II.
In ROCK II-RNAi cells, centrosome reduplication during
Aph-induced arrest was suppressed but was not completely
FIG. 4. ROCK II promotes centrosome reduplication. (A) NIH 3T3 cells were first arrested by Aph (2 ?g/ml) treatment for 24 h. Cells were
then transfected with either the GFP-CAT or the GFP vector in the presence of Aph. After the transfection period (12 h), either the Y-27632
ROCK inhibitor (100 ?M) or dimethyl sulfoxide (DMSO) was added to the media in the duplicate GFP-CAT-transfected cell cultures. Cells were
fixed at 24 h posttransfection, immunostained for ?-tubulin, and counterstained for DNA with DAPI, and the centrosome profiles of the
GFP-positive cells were determined (?300 cells). The results are shown as averages ? standard errors from three independent experiments.
(B) Representative immunostained images of vector-transfected cells (a and b) and GFP-CAT-transfected cells (c and d) as well as GFP-CAT-
transfected cells in the presence of Y-27632 (e and f) are shown. The panels on the right show the magnified images of the areas indicated by
arrows. Scale bar, 10 ?m. It should be noted that in this experiment, the cells were directly fixed without preextraction to observe the membrane
blebbing associated with the constitutive activation of ROCK II. However, without preextraction, due to the ubiquitous presence of GFP-CAT,
the specific localization of GFP-CAT at the centrosomes is highly masked.
VOL. 26, 2006 CENTROSOME DUPLICATION CONTROL BY ROCK II AND NPM/B239023
inhibited, suggesting that ROCK II may be dispensable for
centrosome duplication per se, but it may be necessary for the
efficient initiation of centrosome duplication. Such a function
is essential for the coordinated initiation of centrosome dupli-
cation and DNA replication. We thus tested whether ROCK II
is involved in the timely initiation of centrosome duplication
during the cell cycle. For this experiment, we used primary
MSFs prepared from adult male mice, since initiation of cen-
trosome duplication and S-phase entry occur in a highly coor-
dinated fashion in these cells. We generated MSFs whose
ROCK II expression was silenced by the stable expression of
siRNA specific for ROCK II (Fig.6Ca). The control MSFs
(transfected with an siRNA vector with a randomized se-
quence) and ROCK II-RNAi MSFs were synchronized by se-
rum starvation, followed by serum stimulation. Every 3 h for a
period of 21 h, cells were examined for progression into S-
phase by BrdU incorporation and centrosome duplication by
immunostaining with anticentrin antibody (Fig.6Cb; an exam-
ple of anticentrin antibody immunostaining to detect undupli-
cated and duplicated centrosomes is shown on the left).
Among serum-starved quiescent MSFs, 5 to 8% of cells con-
tain already-duplicated centrosomes. Since centrosomes do
not duplicate in the quiescent state, the cells with duplicated
centrosomes are likely the ones which were in mid- to late G1
phase and had duplicated centrosomes but failed to proceed
through the cell cycle due to serum deprivation (43). The cell
cycle progression through G1to S was not altered by the
down-regulation of ROCK II: similar BrdU incorporation
FIG. 5. Centrosomal localization is required for ROCK II to promote centrosome reduplication. (A) NIH 3T3 cells were transfected with
GFP-CAT, GFP–CAT?457-533, or a GFP vector. The lysates were prepared from the transfectants at 18 h after transfection and immunopre-
cipitated with anti-GFP antibody. The immunoprecipitates were immunoblotted with anti-GFP antibody (top). The immunoprecipitates were also
subjected to in vitro kinase assay using vimentin as a substrate as described previously (20) (middle). The substrate band in the Coomassie blue
(CB) stain of the gel is shown in the bottom panel. IP, immunoprecipitation; IB, immunoblotting. (B) NIH 3T3 cells were transfected with
GFP-CAT, GFP-CAT?457-533, or a vector control. The lysates prepared at 24 h after transfection were immunoblotted with anti-GFP (top),
anti-Ser19phospho-MLC2 (middle), and anti-MLC2 (bottom) antibodies. Quantification of the levels of total and Ser19phospho-MLC2 are shown
in the graph at the bottom. (C) NIH 3T3 cells prearrested with Aph for 24 h were transfected with GFP-CAT, GFP–CAT?457-533, or a GFP vector
in the presence of Aph. The transfected cells were incubated for 36 h after transfection in the presence of Aph and immunostained for ?-tubulin,
and the centrosome profiles of the GFP-positive cells were analyzed (?300 cells). The results are shown as averages ? standard errors from three
9024MA ET AL.MOL. CELL. BIOL.
rates were observed between the ROCK II-RNAi and control
cells. In the control cells, the initiation of centrosome duplica-
tion and S-phase entry occur in a highly coordinated manner.
In contrast, in the ROCK II-RNAi cells, the initiation of cen-
trosome duplication was delayed significantly relative to that of
S-phase entry. Moreover, reintroduction of ROCK II, which
was engineered to be resistant to the siRNA, into ROCK
II-RNAi cells restored the coordinated initiation of centro-
some duplication and DNA replication. These observations
indicate that ROCK II plays a critical role in the timely initi-
ation of centrosome duplication (and thus the coupling of the
initiation of centrosome duplication and DNA replication).
Superactivation of ROCK II by NPM/B23. We next analyzed
the functional interaction between ROCK II and NPM/B23.
Since the kinase activity is essential for ROCK II to promote
centrosome duplication, we tested whether NPM/B23 binding
modulates the kinase activity of ROCK II. To this end, bacu-
lovirally purified GST–full-length ROCK II and GST-CAT
FIG. 6. Role of ROCK II in the timely initiation of centrosome duplication. (A) ROCK II-RNAi and control cells as well as ROCK II-RNAi
cells transiently transfected with GFP-CAT were subjected to centrosome reduplication assay. The cells were exposed to Aph for 60 h, and the
centrosome profiles were determined by ?-tubulin immunostaining (?300 cells). The results are averages ? standard errors from three experi-
ments. (B) Representative ?-tubulin-immunostained images of the vector control cells and ROCK II-RNAi cells after Aph treatment are shown.
Scale bar, 10 ?m. (C) MSFs were prepared from the abdominal skin of 8-week-old male mice. MSFs silenced for ROCK II expression and control
MSFs were generated by the method used for the generation of NIH 3T3 ROCK II-RNAi cells and control NIH 3T3 cells described in the legend
to Fig. 2. (a) Immunoblot analysis of ROCK II expression in ROCK II-RNAi cells and control MSFs. ROCK II-RNAi and control MSFs were
serum starved for 30 h and serum stimulated in the presence of BrdU for 21 h. To determine the rates of centrosome duplication and BrdU
incorporation, we carried out the experiment with a single cell culture as well as separately with duplicate cultures, which gave almost identical
results. For a determination of centrosome duplication, antibody against centrin, a known centriole marker (36), was used. An example of the
immunostained images for which anticentrin antibody was used to differentiate unduplicated and duplicated centrosomes is shown on the left. We
repeated the experiment twice, and we obtained almost identical results. The averages from two experiments are plotted in the graph.
VOL. 26, 2006 CENTROSOME DUPLICATION CONTROL BY ROCK II AND NPM/B239025
FIG. 7. Superactivation of ROCK II by NPM/B23 in vitro and in vivo and the functional interaction of ROCK II and NPM/B23 to promote
centrosome reduplication. (A) Baculovirally prepared GST-CAT and GST-ROCK II were subjected to in vitro kinase assay either in the absence
or in the presence of bacterially purified His6
by exactly the same method was used as a negative control. GST was also tested as a control. The bottom three panels show the immunoblots (IB)
of the kinase reaction samples with antivimentin (second panel), anti-His6
?-NPM/B23 (top) using vimentin as a substrate. His6
?-tagged mortalin (Mot330) bacterially prepared
?(third panel), and anti-GST (fourth panel) antibodies. wt, wild type.
9026 MA ET AL.MOL. CELL. BIOL.
mutant were subjected to an in vitro kinase assay in either the
presence or the absence of His6
a substrate (Fig. 7A). The His6
ROCK II or vimentin, was used as a negative control. Al-
though baculovirally purified GST-ROCK II (as well as GST-
CAT) is already active because of Rho proteins present in the
insect cells (see the results of control reactions with His6
Mot330 in Fig. 7A, lanes 2 and 4), the addition of NPM/B23
markedly increased (more than threefold) the kinase activities
of both GST-ROCK II and GST-CAT (lanes 1 and 3). Since
NPM/B23 binds to both GST-ROCK II and GST-CAT (Fig.
1), these results strongly suggest that NPM/B23 superactivates
ROCK II through direct binding.
To test the NPM/B23-mediated superactivation of ROCK II
in vivo, we cotransfected GFP-CAT and GFP-NPM/B23 into
cells. For the controls, cells were transfected with GFP-CAT,
GFP-NPM/B23, or the GFP vector. Immunoblot analysis with
anti-GFP antibody confirmed that comparable levels of GFP-
CAT and GFP-NPM/B23 were expressed (Fig. 7B, top). All
the transfectants expressed similar levels of total MLC2 (Fig.
7B, bottom), while the level of Ser19-phosphorylated MLC2
was increased in the GFP-CAT-transfected cells (Fig. 7B, mid-
dle, lane 1) compared with that in the control vector-trans-
fected cells (lane 4). However, the cotransfection of GFP-
NPM/B23 further increased the level of phospho-Ser19MLC2
(Fig. 7B, middle, lane 2). Thus, as occurs in vitro, NPM/B23
superactivates ROCK II in vivo.
To examine the physiological significance of the superacti-
vation of ROCK II by NPM/B23, we first attempted to silence
NPM/B23 by siRNA. However, NPM/B23 is essential for cell
survival, and we could only partially silence the expression of
NPM/B23 to ?30% of the normal level (Fig. 7C, top, lanes 3
and 4), which is consistent with the recent studies that at-
tempted to generate NPM/B23-deficient mice (15). Neverthe-
less, we examined whether partial silencing of NPM/B23 ex-
pression affects ROCK II activity. NPM/B23 RNAi cells and
control cells (transfected with an siRNA vector containing
randomized sequence) were transfected with either GFP-CAT
or the GFP vector. Similar levels of GFP-CAT (Fig. 7C, second
panel) and GFP (third panel) were expressed in the NPM/B23
RNAi and control cells. The levels of total MLC2 were com-
parable among all the transfectants (Fig. 7C, fifth panel), while
the level of Ser19-phosphorylated MLC2 was increased in the
GFP-CAT-transfected control cells (fourth panel, lane 2) com-
pared with that in the control GFP vector-transfected cells
?-NPM/B23 using vimentin as
?-tagged partial sequence of
?-Mot330) (25), which does not interact with
(lane 1). However, in the NPM RNAi cells, GFP-CAT expres-
sion only minimally increased the level of phospho-Ser19
MLC2 (Fig. 7C, fourth panel, lane 4). These findings further
demonstrate that NPM/B23 and ROCK II functionally interact
NPM/B23 has been shown to localize between the paired
centrioles within the centrosome proper and is likely involved
in the pairing of centrioles (38). Since separation of the paired
centrioles is the initial event of centrosome duplication, NPM/
B23 acts as a negative regulator of centrosome duplication in
this context. However, the findings that ROCK II promotes
centrosome duplication and NPM/B23 superactivates ROCK
II lead us to predict that NPM/B23 may also participate in the
regulation of centrosome duplication in a positive manner via
ROCK II. To test this possibility, we cotransfected GFP-CAT
and GFP-NPM/B23 into NIH 3T3 cells prearrested by Aph. As
a control, the GFP vector was transfected alone or with either
GFP-CAT or GFP-NPM/B23. After transfection, cells contin-
ued to be exposed to Aph for 36 h, and the centrosome profiles
of the GFP-positive cells were determined (Fig. 7D). The ex-
pression of GFP-NPM/B23 alone did not significantly alter the
frequency of centrosome reduplication significantly. As shown
earlier, GFP-CAT expression accelerated centrosome redupli-
cation in NIH 3T3 cell (?75%). However, the cotransfection
of NPM/B23 further accelerated centrosome reduplication
(?90%), demonstrating the functional interaction between
NPM/B23 and ROCK II to promote centrosome reduplication.
Thr199phosphorylation is important for NPM/B23 to super-
activate ROCK II in vivo. CDK2/cyclin E phosphorylates
NPM/B23 on Thr199, and this phosphorylation is critical for the
initiation of centrosome duplication (46). We next tested
whether the Thr199phosphorylation of NPM/B23 plays a role
in the superactivation of the ROCK II and ROCK II-depen-
dent promotion of centrosome duplication using the phospho-
mimetic mutant NPM/B23. For this particular experiment, the
standard centrosome reduplication assay may not be appropri-
ate, since cotransfection of wild-type NPM/B23 and CAT in-
duces centrosome reduplication in ? 90% of cells (Fig. 7D);
hence, further acceleration of centrosome duplication cannot
be confidently determined. To circumvent this problem, we
decided to shorten the duration of Aph exposure after trans-
fection. In the standard centrosome reduplication assay, cells
were exposed to Aph for total 72 h (24 h of prearresting plus
12 h of transfection plus 36 h of incubation). In this experi-
ment, the final incubation time was shortened to 18 h. FLAG
epitope-tagged wild-type NPM/B23, the T199A mutant (un-
(B) Cells were transfected with GFP-CAT plus the GFP vector, GFP-CAT plus GFP-NPM/B23, GFP-NPM/B23 plus the GFP vector, or the GFP
vector alone. The lysates were prepared at 24 h posttransfection and immunoblotted with anti-GFP antibody (top), anti-phospho-Ser19MLC2
antibody (middle), and anti-MLC2 antibody (bottom). The quantifications of the levels of total and phospho-Ser19MLC2 are shown in the graph.
(C) Either a pSuper plasmid encoding the RNAi sequence targeted for NPM/B23 or a control plasmid with a randomized sequence with the same
nucleotide composition was cotransfected with a plasmid encoding a puromycin resistance gene as a rapid selection marker into NIH 3T3 cells.
After 3 days of puromycin selection, the drug-resistant cells were pooled and transfected with GFP-CAT. The lysates were prepared from the
transfectants at 24 h posttransfection and subjected to immunoblot analysis using anti-NPM/B23 (first panel), anti-GFP (second and third panels),
anti-phospho-Ser19MLC2 (fourth panel), and anti-MLC2 (fifth panel) antibodies. Quantifications of the levels of total and phospho-Ser19MLC2 are
shown in the graph. (D) NIH 3T3 cells prearrested by Aph treatment for 24 h were transfected with GFP-NPM/B23 plus the GFP vector,
GFP-CAT plus the GFP vector, GFP-CAT plus GFP-NPM/B23, or the GFP vector alone. After transfection, cells were exposed to Aph for 36 h,
and the centrosome profiles of the GFP-positive cells were determined (?300 cells). The results shown are averages ? standard errors from three
VOL. 26, 2006 CENTROSOME DUPLICATION CONTROL BY ROCK II AND NPM/B239027
phosphorylatable mutant NPM/B23; Thr1993Ala), the T199D
mutant (phospho-mimetic mutant NPM/B23; Thr1993Asp), or
a control vector was cotransfected with GFP-CAT into NIH
3T3 cells prearrested with Aph. After transfection, cells were
exposed to Aph, and the centrosome profiles of the GFP-
positive cells were determined (Fig. 8A). Under this protocol,
the expression of GFP-CAT resulted in a frequency of centro-
some reduplication of ?30%, and the cotransfection of wild-
type NPM/B23 and GFP-CAT resulted in the further induction
of centrosome amplification to ?60%. The cotransfection of
the NPM/B23 T199A mutant and GFP-CAT resulted in a
minimal induction of centrosome amplification (?20%) at
least in part due to the negative regulatory activity resulting
from the T199A mutation of centrosome duplication, as shown
FIG. 8. Thr199phosphorylation is important for NPM/B23 to superactivate ROCK II and to enhance the activity of ROCK II to promote
centrosome reduplication. (A) NIH 3T3 cells prearrested by Aph treatment for 24 h were cotransfected with FLAG-tagged wild-type (wt)
NPM/B23, the T199A mutant, the T199D mutant, or a vector control with GFP-CAT. After transfection, cells were incubated in the presence of
Aph for 18 h, and the centrosome profiles of the GFP-positive cells were determined (?200 cells). The results shown are averages ? standard
errors from three independent experiments. (B) NIH 3T3 cells were transfected with FLAG-tagged wild-type NPM/B23, the FLAG-tagged T199A
mutant, the FLAG-tagged T199D mutant, or a vector plasmid. The lysates were prepared at 36 h posttransfection and immunoblotted with
anti-FLAG (top blot), anti-phospho-Ser19MLC2 (middle blot), and anti-MLC2 antibodies (bottom blot). Quantification of the levels of total and
phospho-Ser19MLC2 are shown in the graph at the bottom. (C) ROCK II-RNAi cells (NIH 3T3 origin) and the vector control cells were
transfected with the FLAG-tagged T199D mutant. The lysates were prepared at 24 h posttransfection and immunoblotted with anti-ROCK II (first
blot), anti-FLAG (second blot), anti-phospho-Ser19MLC2 (third blot), and anti-MLC2 (fourth blot) antibodies. Quantifications of the levels of
total and phospho-Ser19MLC2 are shown in the graph at the bottom.
9028 MA ET AL.MOL. CELL. BIOL.
previously (46). In contrast, the cotransfection of the T199D
mutant and GFP-CAT resulted in a marked increase in the
frequency of centrosome amplification (?90%), indicating that
Thr199-phosphorylated NPM/B23 further enhances ROCK II
activity to promote centrosome reduplication.
We next tested the potential changes in ROCK II kinase
activity in vivo by expressing the T199A and T199D NPM/B23
mutants. NIH 3T3 cells were transfected with a control vector,
FLAG-tagged wild-type NPM/B23, or the T199A or T199D
NPM/B23 mutant and examined for levels of Ser19phosphor-
ylation of MLC2 by immunoblot analysis (Fig. 8B). Similar
levels of the FLAG-T199A mutant, the T199D mutant, and
wild-type NPM/B23 were expressed (Fig. 8B, top blot). There
was an approximately twofold increase in the level of phospho-
Ser19MLC2 in the wild-type NPM/B23 transfectants (Fig. 8B,
middle blot, lane 2) compared with the level in the control cells
(lane 1), However, the level of phospho-Ser19MLC2 was fur-
ther increased (approximately fivefold) in the T199D transfec-
tants (lane 4), while there was a noticeable decrease in the
level of phospho-Ser19MLC2 in the T199A transfectants (lane
3). Thus, phosphorylation on Thr199plays a role in the NPM/
B23-mediated superactivation of ROCK II in vivo, while the
expression of a nonphosphorylatable mutant may act in a dom-
inant negative manner. To exclude the possibility that the
T199D mutant may affect a kinase(s) other than ROCK II,
resulting in an increase in phospho-Ser19MLC2, we trans-
fected the FLAG-T199D mutant into ROCK II-RNAi and
control cells. The lysates from both transfectants were sub-
jected to immunoblot analysis (Fig. 8C). Both transfectants
expressed similar levels of the T199D mutant (Fig. 8C, second
blot). The level of Ser19phospho-MLC2 was up-regulated by
the expression of the T199D mutant in the control cells (Fig.
8C, third blot, lane 2) but not in the ROCK II-RNAi cells (lane
1). Thus, the increase in the phospho-Ser19MLC2 level is due
to the enhanced kinase activity of ROCK II by the T199D
Cell cycle-dependent interaction between NPM/B23 and
ROCK II. Since NPM/B23 superactivates ROCK II through
direct physical interaction, we tested whether Thr199phosphor-
ylation affects NPM/B23’s ability to bind to ROCK II. NIH 3T3
cells were transfected with either the FLAG-tagged T199A
mutant or the T199D mutant, and the transfectants were sub-
jected to coimmunoprecipitation assay using anti-FLAG and
anti-ROCK II antibodies (Fig. 9A). Anti-ROCK II antibody
coimmunoprecipitated approximately threefold more of the
T199D mutant proteins than of the T199A mutant proteins
(Fig.9Aa). Similarly, anti-FLAG antibody coimmunoprecipi-
tated approximately threefold more ROCK II in the T199D
mutant-transfected cells than the T199A mutant-transfected
cells (Fig.9Ab), indicating that Thr199phosphorylation en-
hances the ROCK II binding affinity of NPM/B23.
We next performed an in vitro kinase assay using baculovi-
rally prepared GST-ROCK II in the presence of either the
concentrations, with vimentin as a substrate (Fig. 9B). At
higher concentrations, both the T199A and the T199D mutant
equally superactivated ROCK II. However, at lower concen-
trations, only the T199D transfectant could efficiently super-
?-tagged T199A mutant or the T199D mutant in various
ylation increases the ROCK II binding affinity of NPM/B23
and thus allows more-efficient superactivation of ROCK II.
The ability of a minimal concentration of Thr199-phosphor-
ylated NPM/B23 to superactivate ROCK II may be critical in
vivo, where the quantities of proteins are limited, raising the
possibility that only NPM/B23 phosphorylated on Thr199may
form a complex with ROCK II in vivo. To test this possibility,
NIH 3T3 cells were serum starved for 24 h and serum stimu-
lated. At every 3 h for a period of 18 h, cells were harvested
and examined for CDK2/cyclin E activity and ROCK II-NPM/
B23 complex formation (Fig. 9C). The in vitro histone H1
kinase assay of immunoprecipitated CDK2/cyclin E showed
the expected kinetics of CDK2/cyclin E activation during G1
progression (Fig. 9C, first panel). There was no significant
change in the levels of NPM/B23 and ROCK II during the cell
cycle progression (Fig, 9C, second and third panels, respec-
tively). As described previously (32, 46), NPM/B23 phosphor-
ylated on Thr199gradually accumulates during the G1progres-
sion in association with CDK2/cyclin E activation (Fig. 9C,
fourth panel). The lysates were also immunoprecipitated with
anti-ROCK II antibody, and the immunoprecipitates were im-
munoblotted with either anti-NPM/B23 (Fig. 9C, fifth panel)
or anti-phospho-Thr199NPM/B23 (sixth panel) antibodies. We
found that complex formation between ROCK II and NPM/
B23 becomes evident in mid- to late G1, and the kinetics of
ROCK II-NPM/B23 complex formation parallels those of
CDK2/cyclin E activation and the emergence of phospho-
Thr199NPM/B23. These observations strongly suggest that
complex formation between ROCK II and NPM/B23 in vivo
depends on the Thr199phosphorylation of NPM/B23.
The interaction between NPM/B23 and ROCK II at centro-
somes is required for the ROCK II-dependent promotion of
centrosome duplication. To test whether interaction between
ROCK II and NPM/B23 needs to occur at centrosomes for
ROCK II to promote centrosome duplication, we decided to
use the NPM/B23 missense mutant that can no longer localize
to centrosomes. We have identified Lys263of NPM/B23 as a
critical residue for NPM/B23 to localize to centrosomes. For
instance, the NPM/B23 K263R missense mutant (Lys2633Arg)
fails to localize to centrosomes (unpublished data). We first
examined whether the K263R mutant retains the ability to
interact with ROCK II. FLAG-tagged wild-type NPM/B23 and
the K263R mutant were transfected into NIH 3T3 cells. As a
control, a vector plasmid was transfected. The lysates prepared
from the transfectants were subjected to coimmunoprecipita-
tion assay using anti-ROCK II and anti-FLAG antibodies (Fig.
10A). Similar levels of ROCK II were coimmunoprecipitated
with FLAG–wild-type NPM/B23 and the K263R mutant (Fig.
10A, second panel), and similar levels of FLAG–wild-type
NPM/B23 and the K263R mutant were coimmunoprecipitated
with ROCK II (fourth panel). Thus, the K263R mutant binds
to ROCK II as efficiently as wild-type NPM/B23.
superactivate ROCK II. Bacterially purified His6
type and mutant NPM/B23 proteins (the T199D mutant, the
K263R mutant, and the T199D/K263R double mutant) were
tested for their activities in superactivating GST-CAT in vitro
(Fig. 10B). As with the results shown in Fig. 9B, wild-type NPM/
B23 (unphosphorylated NPM/B23) superactivated GST-CAT in
a concentration-dependent manner (Fig. 10B, lanes 2 and 3),
VOL. 26, 2006CENTROSOME DUPLICATION CONTROL BY ROCK II AND NPM/B23 9029
while both high and low concentrations of the T199D mutant
superactivated GST-CAT (lanes 4 and 5). The K263R mutant
superactivated GST-CAT in a concentration-dependent manner
(Fig. 10B, lanes 6 and 7), similar to what occurred with wild-type
NPM/B23, while both high and low concentrations of the T199D
K283R double mutant superactivated GST-CAT (lanes 8 and 9).
Thus, the K263R mutant retains full activity to superactivate ROCK
II, and its activity is influenced by phosphorylation on Thr199.
FIG. 9. Thr199phosphorylation of NPM/B23 is critical for NPM/B23-ROCK II complex formation in vivo. (A) NIH 3T3 cells were transfected with
either the FLAG-tagged T199D mutant or the FLAG-tagged T199A mutant. The lysates were prepared at 24 h posttransfection and subjected to
immunoprecipitation (IP) with either anti-ROCK II (a) or anti-FLAG (b) antibodies. The immunoprecipitates were then immunoblotted (IB) with
anti-FLAG and anti-ROCK II antibodies. (B) Baculovirally purified GST-ROCK II was subjected to in vitro kinase assay in the presence of various
concentrations of either the His6
kinase reaction mixtures were immunoblotted with antivimentin, anti-NPM/B23, and anti-GST antibodies. We also tested GST-CAT, and we obtained
similar results (data not shown). (C) NIH 3T3 cells were serum starved for 24 h and serum stimulated. Every 3 h for a period of 18 h, lysates were
prepared. The lysates were immunoprecipitated with anti-cyclin E antibody, and the immunoprecipitates were subjected to in vitro histone H1 kinase
assay (first panel). The lysates were also immunoblotted with anti-NPM/B23 (second panel), anti-ROCK II (third panel), and anti-phospho-Thr199
NPM/B23 (fourth panel) antibodies. The lysates were also immunoprecipitated with anti-ROCK II antibody, and the immunoprecipitates were
immunoblotted with anti-NPM/B23 antibodies (fifth panel) as well as anti-phospho-Thr199NPM/B23 antibodies (bottom panel).
?-tagged T199A mutant or the His6
?-tagged T199D mutant with vimentin as a substrate (top panel). Aliquots of the
9030 MA ET AL.MOL. CELL. BIOL.
FIG. 10. CentrosomelocalizationiscriticalforNPM/B23toaugmentROCKIIactivitytopromotecentrosomereduplication.(A)NIH3T3cellswere
transfected with FLAG-tagged wild-type (Wt) NPM/B23, the K263R mutant, or a vector plasmid. At 24 h posttransfection, the lysates were prepared and
subjected to immunoprecipitation (IP) using either anti-FLAG or anti-ROCK II antibody. The immunoprecipitates were then immunoblotted (IB) with
wild-type NPM/B23, the T199D mutant, the K263R mutant, or the T199D K263R mutant at two different concentrations with vimentin as a substrate
(first panel). Aliquots of the kinase reaction mixtures were immunoblotted with antivimentin, anti-NPM/B23, and anti-GST antibodies. We also tested
GST–full-length ROCK II, and we obtained similar results (data not shown). (C) NIH 3T3 cells prearrested by Aph treatment for 24 h were cotransfected with
GFP-CAT plus FLAG-tagged wild-type NPM/B23, GFP-CAT plus the T199D mutant, GFP-CAT plus the K263R mutant, or GFP-CAT plus the T199D/
K263R mutant. For controls, cells were cotransfected with the GFP vector (GFP-vec) plus the FLAG vector (FLAG-vec), the GFP vector plus FLAG-tagged
wild-type NPM/B23, or GFP-CAT plus the FLAG vector. After transfection, cells were incubated in the presence of Aph for 18 h, and the centrosome profiles
of the GFP-positive cells were determined (?200 cells). The results shown are averages ? standard errors from three independent experiments.
VOL. 26, 2006 CENTROSOME DUPLICATION CONTROL BY ROCK II AND NPM/B23 9031
We then tested the K263R mutant for its ability to aid
ROCK II to promote centrosome duplication by centrosome
reduplication assay. Since we also tested the T199D K263R
double mutant, the modified centrosome reduplication assay
(18 h of Aph exposure after transfection) described for Fig. 8A
was used. We cotransfected GFP-CAT with FLAG-tagged
wild-type NPM/B23 and the T199D, K263R, or T199D/K263R
mutant into NIH 3T3 cells prearrested with Aph. After trans-
fection, cells were further exposed to Aph for 18 h, and the
centrosome profiles of the GFP-positive cells were determined
(Fig. 10C). As shown in Fig. 8A, wild-type NPM/B23 aug-
mented the ROCK II-mediated promotion of centrosome re-
duplication (?60%), and the T199D phospho-mimetic mutant
did so more efficiently (?90%). However, both the K263R and
T199D/K263R mutants failed to augment the activity of GFP-
CAT to promote centrosome reduplication; the frequency of
centrosome amplification was similar to that of the vector-
transfected control cells (?30%). Thus, NPM/B23 requires
centrosome localization to augment ROCK II to promote cen-
trosome duplication, implying that NPM/B23 and ROCK II
need to interact with each other at centrosomes to promote
In this study, we identified ROCK II as a centrosomal pro-
tein that interacts with NPM/B23 with a high affinity. We
further found that ROCK II promotes centrosome duplication
in its kinase and centrosome localization-dependent manner.
In cells in which ROCK II expression is silenced, the initiation
of centrosome duplication, which normally occurs at late G1/
early S phase, was significantly delayed, although centrosomes
were eventually duplicated prior to mitosis. Thus, ROCK II
may be dispensable for centrosome duplication per se but
required for the timely initiation of centrosome duplication,
and thus the initiation of centrosome duplication and S-phase
entry are coupled. We further found that NPM/B23 superac-
tivates ROCK II by physical interaction, and this interaction is
enhanced by the Thr199phosphorylation of NPM/B23. For
instance, NPM/B23 with a phospho-mimetic mutation at
Thr199shows a significantly higher binding affinity for ROCK
II than unphosphorylated NPM/B23. Moreover, the phospho-
mimetic NPM/B23 mutant superactivates ROCK II with a min-
imal concentration. Since the quantities of specific proteins are
limited in cells, the acquisition of a higher affinity of binding to
the partner proteins by posttranslational modifications (e.g.,
phosphorylation) becomes a critical event. Indeed, the com-
plex formation of NPM/B23 and ROCK II is cell cycle depen-
dent and occurs in association with the activation of CDK2/
cyclin E and the emergence of phospho-Thr199NPM/B23.
Thus, the ROCK II binding of NPM/B23 and consequential
superactivation of ROCK II in vivo is likely controlled by
phosphorylation on Thr199mediated by CDK2/cyclin E. Since
the centrosome-binding mutant NPM/B23 can no longer aug-
ment the activity of ROCK II to promote centrosome dupli-
cation, interaction between ROCK II and NPM/B23 must oc-
cur at centrosomes to promote centrosome duplication. All
these findings converge to model the molecular events associ-
ated with the initiation of centrosome duplication (Fig. 11). At
late G1, CDK2/cyclin E is activated by the temporal expression
of cyclin E, which phosphorylates NPM/B23 on Thr199at cen-
trosomes. Thr199phosphorylation triggers the majority of
NPM/B23 proteins to dissociate from centrosomes, but some
NPM/B23 proteins remain at centrosomes. Indeed, we could
readily detect NPM/B23 phosphorylated on Thr199in the cen-
trosomes isolated from cells exposed to Aph for 36 h (?80% of
cells after 36 h of Aph exposure contain duplicated centro-
somes), and NPM/B23 phosphorylated on Thr199could be
detected immunocytochemically on one of the duplicated cen-
trosomes, presumably the one with the mother centriole of the
FIG. 11. Model of the ROCK II-NPM/B23-mediated regulation of
centrosome duplication. During the early to mid-G1phase of the cell
cycle, NPM/B23 localizes between the paired centrioles of undupli-
cated centrosomes, likely functioning in pairing the centrioles. In late
G1, CDK2/cyclin E becomes activated and phosphorylates NPM/B23
on Thr199. Upon Thr199phosphorylation, the majority of NPM/B23
proteins dissociate from centrosomes, but some remain at centrosomes
and translocate toward the mother centriole of the pair. These Thr199-
phosphorylated NPM/B23 proteins have high binding affinities to
ROCK II and bind to ROCK II present at centrosomes. ROCK II is
superactivated by NPM/B23 binding and rapidly targets the protein
(shown as “X”) which plays a key role in the initiation of centrosome
9032 MA ET AL.MOL. CELL. BIOL.
original pair, based on our previous findings (38; Z. Ma, un-
published observation). Upon Thr199phosphorylation, NPM/
B23 acquires a high binding affinity for ROCK II and interacts
with ROCK II, which in turn superactivates ROCK II. ROCK
II then rapidly and efficiently targets the key centrosomal pro-
tein(s) for initiation of centrosome duplication. Although the
identification of such a target protein(s) is under way in our
laboratory, we predict that it likely functions in the splitting of
the paired centrioles, an initial event of centrosome duplica-
tion, for the following reasons. Within the unduplicated cen-
trosome, NPM/B23 localizes between the paired centrioles
(38). In late G1, the majority of NPM/B23 proteins dissociate
from centrosomes upon CDK2/cyclin E-mediated phosphory-
lation on Thr199. However, some NPM/B23 proteins remain at
centrosomes, and they move toward the mother centriole of
the pair prior to centriole splitting (38). Thus, after centriole
splitting, only the mother centriole is bound by NPM/B23,
which makes it highly unlikely that the ROCK II-NPM/B23
complex functions in procentriole formation. Procentrioles
form on both centrioles. Therefore, it is more likely that the
ROCK II-NPM/B23 complex formed at mother centrioles
upon the phosphorylation of NPM/B23 by CDK2/cyclin E
functions in the process of splitting of the paired centrioles.
Such a function of the ROCK II-NPM/B23 complex may be
critical for the timely initiation of centrosome duplication and
the coupling of centrosome duplication and DNA replication.
It has been shown that the majority of ROCK II-null em-
bryos die early in development, yet a small percentage of mice
develop normally (45), suggesting that ROCK II deficiency can
be inefficiently compensated for by other proteins, perhaps
ROCK I, another member of the ROCK kinase family. With
respect to the duplication of centrosomes, ROCK II expres-
sion-silenced cells eventually duplicate centrosomes despite
the delayed initiation of centrosome duplication. At present,
we do not know whether centriole splitting occurs eventually
without the activity of ROCK II or whether the ROCK II
activity is compensated for (inefficiently) by other kinases in
cells whose ROCK II expression is silenced. If the latter is the
case, ROCK I is the most likely candidate. It has been shown
that ROCK I also localizes to centrosomes and is involved in
the proper pairing and positioning of centrioles: the down-
regulation of ROCK I results in the erratic motion of mother
centrioles during G1and premature centriole migration in the
midbody during mitosis (8). ROCK I and ROCK II have been
shown to share many substrates, although they target the sub-
strate with different efficiencies (50). Although NPM/B23 does
not bind to ROCK I, ROCK I, like ROCK II, is already active
by binding to Rho but not superactivated. Thus, ROCK I may
be able to inefficiently compensate for ROCK II, resulting in
the eventual initiation of centrosome duplication after a sig-
NPM/B23 is involved in diverse biological events and path-
ways and often simultaneously participates in two functionally
opposing events/pathways. For instance, NPM/B23 influences
cellular proliferation and transformation both positively (on-
cogenically) and negatively (antioncogenically). Thus, it is not
surprising that NPM/B23 is involved in the regulation of cen-
trosome duplication through multiple functionally opposing
pathways. We have previously shown that the unphosphorylat-
able NPM/B23 T199A mutant acts as a dominant negative
when expressed in cells, resulting in the suppression of centro-
some duplication (46). NPM/B23 localizes between paired cen-
trioles within the unduplicated centrosome, likely functioning
as a glue-like protein for centriole pairing. Upon phosphory-
lation on Thr199, NPM/B23 either dissociates from centro-
somes or shifts its subcentrosomal localization. The T199A
mutant can neither dissociate from centrosomes nor shift its
subcentrosomal localization even in the presence of active
CDK2/cyclin E, resulting in a failure of the paired centrioles to
undergo physical separation; hence, centrosome duplication is
suppressed. In this context, NPM/B23 negatively controls cen-
trosome duplication, which is released by Thr199phosphoryla-
tion by CDK2/cyclin E. Here, we found that NPM/B23 upon
phosphorylation on Thr199acquires a high affinity for binding
to ROCK II, physically associating with and superactivating
ROCK II at centrosomes, which in turn promotes centrosome
duplication. Why then do the cells derived from mice whose
NPM/B23 expression is depleted to 10 to 30% of the normal
level suffer centrosome amplification (15)? If the protein is
involved in the regulation of one biological event through
multiple pathways, when the expression of the protein is either
lost or reduced in mice, it is difficult to predict which pathway
will be more affected than the others by loss/depletion of the
protein and emerge as a predominant phenotype. Among its
multiple functions, the functions which are more readily com-
pensated for by other proteins or mutations will more likely be
masked and will not result in abnormal phenotypes. In the case
of NPM/B23, with respect to the regulation of centrosome
duplication, the positive regulatory function of ROCK II may
be more readily compensated for by other proteins or muta-
tions than its negative regulatory function exerted on centriole
pairing. Thus, the loss of the negative regulatory function may
emerge as a predominant phenotype in mice with reduced
We thank K. George for technical assistance.
This research is supported by a grant from the National Institutes of
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