Molecular Biology of the Cell
Vol. 19, 2870–2875, July 2008
Nucleophosmin Is a Binding Partner of Nucleostemin in
Human Osteosarcoma Cells
Hanhui Ma and Thoru Pederson
Program in Cell Dynamics, Department of Biochemistry and Molecular Pharmacology, University of
Massachusetts Medical School, Worcester, MA 01605
Submitted February 7, 2008; Revised March 19, 2008; Accepted April 23, 2008
Monitoring Editor: Wendy Bickmore
Nucleostemin (NS) is expressed in the nucleoli of adult and embryonic stem cells and in many tumors and tumor-derived
cell lines. In coimmunoprecipitation experiments, nucleostemin is recovered with the tumor suppressor p53, and more
recently we have demonstrated that nucleostemin exerts its role in cell cycle progression via a p53-dependent pathway.
Here, we report that in human osteosarcoma cells, nucleostemin interacts with nucleophosmin, a nucleolar protein
believed to possess oncogenic potential. Nucleostemin (NS) and nucleophosmin (NPM) displayed an extremely high
degree of colocalization in the granular component of the nucleolus during interphase, and both proteins associated with
prenucleolar bodies in late mitosis before the reformation of nucleoli. Coimmunoprecipitation experiments revealed that
NS and NPM co-reside in complexes, and yeast two-hybrid experiments confirmed that they are interactive proteins,
revealing the NPM-interactive region to be the 46-amino acid N-terminal domain of NS. In bimolecular fluorescence
complementation studies, bright nucleolar signals were observed, indicating that these two proteins directly interact in
the nucleolus in vivo. These results support the notion that cell cycle regulatory proteins congress and interact in the
nucleolus, adding to the emerging concept that this nuclear domain has functions beyond ribosome production.
tion of the nucleolus is its role in ribosome biosynthesis, various
nonribosomal proteins began to be observed in the nucleolus a
decade ago (Pederson, 1998b). Subsequently, several cell cycle
regulatory proteins have been observed in the nucleolus by pro-
teomics analysis of purified nucleoli (Andersen et al., 2002; Scherl
et al., 2002) and by in situ localization methods (Pederson, 1998a;
Boisvert et al., 2007). Meanwhile, and possibly related, we have
consists of both RNA-containing complexes and proteinaceous
particles that lack detectable RNA (Politz et al., 2005). We have
therefore begun to consider the possibility that the nucleolus is a
staging site for the assembly of cell cycle regulatory machinery,
either to function within the nucleolus by regulating ribosome
or to shuttle to the nucleoplasm to impact DNA replication or
other key steps in cell cycle progression.
Nucleostemin (NS) is a nucleolar protein required for
embryogenesis and cell cycle progression (Beekman et al.,
2006; Zhu et al., 2006), but its mode of action is unclear. NS
shuttles between the nucleolus and surrounding nucleo-
plasm based on its state of guanosine triphosphate (GTP)
binding (Tsai and McKay, 2005). A likely key to understand-
ing the action of NS would be to define the proteins with
which it interacts. An interaction between N-terminal basic
region of NS and the tumor suppressor p53 was initially
observed by pull-down and coimmunoprecipitation experi-
ments (Tsai and McKay, 2002), and subsequently we dem-
onstrated a role of p53 in the arrest of cell cycle progression
in NS-depleted cells (Ma and Pederson, 2007). Moreover, the
regulatory subunit B of human protein phosphatase-2
(PPP2R5A) has been identified as an NS-interactive protein
by yeast two-hybrid experiments (Yang et al., 2005). In ad-
dition, the protein RSL1D1, which contains a ribosomal pro-
tein-homologous element, was found to interact with both
the N-terminal basic domain and the GTP binding domain
of NS and also was found to be important for the nucleolar
location of NS (Meng et al., 2006). Finally, NS has also been
found to interact with telomeric repeat-binding factor 1
(TRF1) and to negatively regulate the stability of TRF1 via
ubiquitination (Zhu et al., 2006).
Nucleophosmin (NPM, also known as B23 protein) is an
abundant and multifunctional nucleolar phosphoprotein
that has been implicated in rRNA processing, ribosome as-
sembly, centrosome duplication, cell proliferation, and ma-
lignancy (Grisendi et al., 2006; Naoe et al., 2006). NPM has
been variously reported to have either oncogenic or tumor
suppressor-like activities, and these difference are thought to
be attributable, at least in part, to the p53 expression status
of the cell (e.g., Colombo et al., 2002). In the present inves-
tigation, we have found that NPM directly interacts with NS
in the nucleoli of living human tumor cells.
MATERIALS AND METHODS
Cell Culture, Transfection, and Establishment of Stable
U2OS (human osteosarcoma cells) were cultured at 37°C in DMEM supple-
mented with 10% fetal bovine serum (FBS). The kDa.1 derivative of U2OS was
kindly provided by Dawn E. Quelle (College of Medicine, University of Iowa;
This article was published online ahead of print in MBC in Press
on April 30, 2008.
Address correspondence to: Hanhui Ma (hanhui.ma@umassmed.
edu) or Thoru Pederson (firstname.lastname@example.org).
Abbreviations used: BiFC, bimolecular fluorescence complementa-
tion; NPM, nucleophosmin; NS, nucleostemin; PNB, prenucleolar
2870© 2008 by The American Society for Cell Biology
Meng et al., 2006), and it was maintained in DMEM containing 10% FBS and
500 ?g/ml geneticin. A stably transformed, green fluorescent protein (GFP)-
NS–inducible cell line, U2OSiNS-GFP, was constructed by cotransfection of
U2OS cells at 40–60% confluence with pcDNA4-rNS-GFP-TO (described
under Plasmids) and pcDNA6/TR (Invitrogen, Carlsbad, CA) (1:6 ratio) by
using Lipofectamine 2000, followed by selection with 500 ?g/ml hygromycin
B and 5 ?g/ml blasticidin S. After 2 wk in the selection medium, single cells
were cultured in 96-well plates. After another 2 wk, each clone was subcul-
tured and examined for expression of NS-GFP after induction by 0.5 ?g/ml
doxycycline for 24–48 h.
The plasmid encoding red fluorescent human nucleostemin (pmRFP-hNS-C1)
was described previously (Ma and Pederson, 2007). pEGFP-B23-C1 was
kindly provided by Sui Huang (Feinberg School of Medicine, Northwestern
University; Chen and Huang, 2001). pEGFP-rNS was kindly provided by
Robert Tsai (Texas A&M Health Science Center, Houston, TX; Tsai and
McKay, 2002). The nucleostemin (rNS) coding region was cloned into pEGFP-
N1, and the resulting rNS-GFP coding region was subcloned into the
pcDNA4/TO vector to generate pcDNA4-rNS-GFP-TO for construction of the
NS-GFP–inducible stable cell line (see above). The yeast two-hybrid plasmids
were kindly provided by Peter Pryciak (University of Massachusetts Medical
School). DNA fragments encoding human NPM, wild-type human NS, hu-
man NS mutants consisting of amino acids 1–46 (NSB), amino acids 1–267
(NSBG), amino acids 47-549 (NSdB), or amino acids 268–549 (NSdBG) were
inserted into the vector containing the activation domain to obtain pAD-
NPM, pAD-NS, pAD-NSB, pAD-NSBG, pAD-NSdB, and pAD-NSdBG. NPM
was inserted into the vector containing the DNA binding domain and re-
porter genes to obtain pBD-NPM. Yeast transformation and filter ?-galacto-
sidase assays were performed as described previously (Winters and Pryciak,
2005). Parental BiFC plasmids were kindly provided by Tom Kerppola (Hu et
al., 2002) and modified by insertion of cyan fluorescent protein (CFP) and
monomeric red fluorescent protein (mRFP) into the YN and YC vectors,
respectively, to obtain pBiFC-CFP-YN and pBiFC-mRFP-YC. DNA fragments
encoding wild-type NS, the N-terminal 46 amino acids (NSB), or NS lacking
the 46-amino acid N-terminal domain (i.e., amino acids 47-549) (NSdB) were
inserted into pBiFC-CFP-YN to obtain pBiFC-NS-CFP-YN, pBiFC-NSB-CFP-
YN, and pBiFC-NSdB-CFP-YN. The DNA fragment encoding NPM was in-
serted into pBiFC-mRFP-YC to obtain pBiFC-NPM-mRFP-YC.
Cells grown on coverslips were fixed for 12 min in phosphate-buffered saline
(PBS) containing 4% formaldehyde, followed by permeabilization with 0.5%
Triton X-100 for 5 min. Coverslips were then incubated with primary anti-
bodies in PBS, 1% bovine serum albumin for 1–2 h before washing and
incubation with the appropriate secondary antibodies. All these steps were
carried out at room temperature. Coverslips were mounted in Prolong Anti-
fade (Invitrogen), and two- or three-dimensional images were captured and in
some cases subjected to deconvolution as described previously (Ma and
Pederson, 2007). The primary antibodies and dilutions were as follows: rabbit
anti-human NS polyclonal antibody (1:200; Millipore Bioscience Research
Reagents, Temecula, CA) and mouse anti-human NPM monoclonal antibody
(1:500; Santa Cruz Biotechnology, Santa Cruz, CA).
Immunoprecipitation and Immunoblotting
U2OS cells were lysed on ice in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM
EDTA, and 0.5% NP-40, containing 1 mM phenylmethylsulfonyl fluoride and
a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) as specified
by the manufacturer. Protein concentration was determined using the bicin-
choninic acid assay (Pierce Chemical, Rockford, IL). Samples were incubated
with antibody for human NPM (see below) or with nonimmune human
immunoglobulin G (IgG) (2 ?g/each) at 4°C for overnight, and then protein
A- or protein G-agarose beads were added for an additional 2 h. The beads
were washed and the eluted proteins were separated by SDS-polyacrylamide
gel electrophoresis followed by transfer to Immobilon-P membranes (Milli-
pore, Billerica, MA) which were then incubated with specific primary anti-
bodies followed by their detection with appropriate horseradish peroxidase-
conjugated secondary antibodies and enhanced chemiluminescence substrate
(Pierce Chemical). The primary antibodies and dilutions used for immuno-
blotting were rabbit anti-human NS (1:5000; Millipore Bioscience Research
Reagents) and mouse anti-human NPM (1:5000; Santa Cruz Biotechnology).
Time-Lapse Fluorescence Microscopy of Mitotic Cells
The U2OSiNS-GFPcell line was grown on Lab-Tek chambered coverglasses
(Nalge Nunc Intlernational, Rochester, NY) and transfected with pBiFC-
NPM-mRFP-YC by using standard procedures. The microscope and chamber
were kept in 37°C and 5% CO2during observation and imaging. A Leica
DM-IRB microscope, equipped with a 100? objective (numerical aperture
1.4), a Quantix 57 charge-coupled device camera (Photometrics, Tucson, AZ),
the appropriate filter sets, and MetaMorph acquisition software (Molecular
Devices, Sunnyvale, CA) were used, as detailed previously (Jacobson and
Pederson, 1997; Politz et al., 2007).
Bimolecular Fluorescence Complementation (BiFC)
Imaging in Living Cells
U2OS cells grown on a Lab-Tek chambered coverglasses were cotransfected
with plasmids encoding the desired fusion proteins. Twenty-four to 48 h after
transfection, cells were washed with PBS, and then they were incubated at
30°C (5% CO2) for 2–6 h. Fluorescence was observed in living cells as
described above using a Leica DM-IRB microscope equipped with the appro-
priate filter sets: CFP (436/20, BP480/40), mRFP (546/14, LP580), and yellow
fluorescent protein (YFP0 (510/20, BP560/40). BiFC signal was evaluated only
in cells exhibiting similar levels of CFP and mRFP expression. Controls
established that there was no spectral cross-talk between channels at the
exposure times used in these experiments.
RESULTS AND DISCUSSION
Nucleolar Colocalization of NS and NPM during
We previously demonstrated that NS resides in the granular
component of the nucleolus at sites that lack 28S rRNA
(Politz et al., 2005). NPM had been shown previously to be
localized in the granular component of the nucleolus (Goessens,
1984), and we were therefore interested to know the degree
to which these two proteins are colocalized. As shown in
Figure 1, top, when mRFP-tagged human NS and GFP-
tagged human NPM were coexpressed in U2OS cells, both
mRFP-NS and GFP-NPM were concentrated in nucleoli. Us-
ing high-resolution digital imaging and deconvolution anal-
ysis, the intensity distribution of mRFP-nucleostemin was
found to spatially overlap very extensively with GFP-NPM
in subnucleolar regions, indicated by yellow in the merged
image and the quantitative linescans (Figure 1). To assess
whether these results might be due to overexpression of the
two fluorescent proteins, we also determined the degree of
intranucleolar colocalization of endogenous NS and NPM
by immunostaining. Because resolving the intranucleolar
distribution of NPM can be difficult due to its high concen-
tration and issues of antibody accessibility, we used a U2OS-
derived stably transformed cell line in which the level of
NPM expression is depressed ?50% due to small interfering
RNA knockdown (Korgaonkar et al., 2005). The intranucleo-
lar localizations of endogenous NS and NPM were again
found to be extensively overlapping (Figure 1, bottom).
Colocalization of NS and NPM in Anaphase and during
Given the high degree of colocalization of NS and NPM in
the nucleoli of interphase cells, we examined the temporal
and spatial features of their association as nucleoli reform
during the late stages of mitosis. We constructed a stably
transformed U2OS cell line that conditionally expresses
NS-GFP (Supplemental Figure S1) and tracked this protein
along with transiently expressed NPM-mRFP in live mitotic
cells. NPM is known to be released as nucleoli disassemble
during prophase and to then associate with prenucleolar
bodies (PNBs) during telophase, followed by the coalescence
of PNBs into nucleoli once active rRNA polymerase I and
early rRNA processing factors have been recruited to the
nucleolar organizer regions (Dundr et al., 2000; Savino et al.,
2001; Angelier et al., 2005). We found NS and NPM to be
extensively colocalized in the immediate periphery of the
chromosomes in anaphase (0.00 min; Figure 2) and to remain
colocalized because PNBs occurred in early telophase (15
min) and after complete nucleolar reformation had occurred
in late telophase (45 min) and early G1 (90 min). To verify
that the mitotic association of NS and NPM is not due to the
GFP tag on the NS, we carried out double antibody staining
Vol. 19, July 20082871
experiments with the parental U2OS cells of the stable cell
line (Figure 3), which confirmed that endogenous, non-GFP–
tagged NS displays a high degree of colocalization with
NPM during telophase. These results (Figures 2 and 3) par-
allel the previous description of NPM during anaphase and
telophase (Dundr et al., 2000; Savino et al., 2001; Angelier et
al., 2005), and they demonstrate that the colocalization of
NPM with NS seen in interphase is reflected in the earliest
stages of nucleolar reformation during late mitosis.
A 46-Amino Acid N-Terminal Domain of NS Is Essential
for Interaction with NPM
Notwithstanding the very high degree of colocalization of
NS and NPM in nucleoli and during mitosis, these results do
not address actual molecular complexing. As shown in Fig-
ure 4A, NS was detected in complexes precipitated by NPM
antibody but not by nonimmune IgG. Moreover, an interac-
tion between NS and NPM was observed in yeast-two hy-
brid experiments (Figure 4B, top right), as was a ho-
modimerization of NPM (Figure 4B, bottom left) in
confirmation of the well-known self-association of this pro-
tein as determined in biochemical studies (Yung and Chan,
1987; Chan and Chan, 1995; Herrera et al., 1996). To identify
the region(s) within NS required for interaction with NPM,
mutants were constructed containing only a 46-amino acid
N-terminal basic region (Figure 4C, NSB) as well as mutants
lacking this N-terminal basic region (Figure 4C, NSdB), con-
taining the N-terminal basic and GTP binding region do-
mains (Figure 4C, NSBG), or containing only the C-terminal
half of NS (Figure 4C, NSdBG). Yeast two-hybrid analyses
with these mutants demonstrated that the 46-amino acid
N-terminal domain of NS is both necessary (Figure 4D, top
live imaging was performed 24 h after transfection. Bottom row, endogenous NS and NPM in nontransfected U2OS-derived kDa.1 cells
determined by immunostaining. Far right, images were subjected to deconvolution and the intensity of the two colors at each pixel along red
lines drawn in the merged panels was quantified and plotted. Bar, 5 ?m.
Colocalization of NS and NPM within the nucleolus. Top row, mRFP-NS and GFP-NPM were cotransfected into U2OS cells and
S1) by addition of doxycycline together with transient transfection with NPM-mRFP. Twenty-four hours later NS-GFP and NPM-mRFP were
imaged in the same focal plane in dividing cells. Top row, NS-GFP in a cell entering anaphase (0.00 min) and proceeding through telophase
and into G1. Middle row, NPM-mRFP in the same cell. Bottom row, merged NS-GFP and NPM-mRRF. Bar, 10 ?m.
Localization of NS and NPM during anaphase and telophase. NS-GFP was induced in U2OSiNS-GFPcells (see Supplemental Figure
H. Ma and T. Pederson
Molecular Biology of the Cell 2872
right) and indeed sufficient (Figure 4D, top center) for NPM
Molecular Interaction of NS and NPM in Living
We next investigated the occurrence and subcellular local-
ization of NS and NPM interaction in living U2OS cells by
using BiFC. BiFC is based on the generation of a fluorescent
signal when the two halves of YFP combine and refold, only
when in sufficient proximity to one another by virtue of their
attachment to two other dimerizing proteins (Hu et al., 2002).
For our experiments, we used a modified system initially
reported by Wolff et al. (2006) that enabled the levels of the
two expressed proteins to be assessed in a given cell by also
inserting cyan or red fluorescent protein coding sequences
into each plasmid, as diagrammed in Figure 5A. When cells
were transfected with the two plasmids lacking NS and
NPM, the expressed proteins were predominantly cytoplas-
mic (Figure 5B, top row, CFP and RFP) and little interaction
was observed, reflected by the very low intensity of yellow
signal (Figure 5B, top row, YFP). In contrast, when NS and
NPM were present on the plasmids, the NS and NPM fusion
proteins were directed to nucleoli (Figure 5, second row,
CFP and RFP, respectively) showing that the presence of the
cyan or red fluorescent protein elements and the hemi-YFP
did not interfere with the ability of NS and NPM to properly
Shown is a typical telophase cell. The images were subjected to deconvolution and then merged (right). Bar, 5 ?m.
Double immunostaining of NS and NPM in telophase. Parental U2OS cells were immunostained for NS (left) and NPM (middle).
NPM. Proteins captured from U2OS cell extracts by NPM antibody (middle lane) or nonimmune IgG (left lane) were subjected to
immunoblotting for NS or NPM. Right lane, immunoblot of total cell protein. The two regions of the blot containing the NS and NPM
antibody-reactive bands are juxtaposed in this composite figure. (B) Yeast two-hybrid analysis of NS-NPM interaction. Shown are the
?-galactosidase signals for strains carrying pBD-NPM and pAD (activation domain) vector alone (top left), pBD-NPM and pAD-NS (top
right), and pBD-NPM and pAD-NPM (bottom left). (C) Mutants of NS. “Basic” denotes the N-terminal domain previously implicated in
nucleolar localization, and “G1” and “G4” indicate GTP binding domains. The ? and ? signs at the right indicate the interaction of each
mutant with NPM, as determined by two-hybrid analysis (D). (D) Yeast two-hybrid analysis of NPM interaction with mutant forms of NS.
Interaction of NS and NPM by both coimmunoprecipitation and yeast two-hybrid analysis. (A) Coimmunoprecipitation of NS and
Vol. 19, July 20082873
localize in nucleoli. Significantly, bright yellow BIFC signals
were observed in the nucleoli (Figure 5B, second row, YFP)
indicating a direct molecular complexing of the two pro-
teins. When the NS BiFC plasmid carried only the 46-amino
acid N-terminal region of NS, this protein displayed strong
nucleolar localization (Figure 5B, third row, CFP), and as
shown in the YFP panel (Figure 5B, third row, YFP), a BiFC
signal was observed comparable with the intensity seen with
wild-type NS. These results strongly confirm the yeast two-
hybrid results and indicate that the N-terminal domain of
NS is sufficient for the heterodimerization of NS and NPM in
the nucleoli of living human cells. Finally, as shown in the
bottom row, a NS mutant lacking this N-terminal domain
failed to localize in nucleoli, or even the nucleus, and no
BiFC was observed despite the demonstrable nucleolar pres-
ence of NPM (Figure 5B, bottom row).
These results establish that NPM is a NS-interactive pro-
tein within the nucleoli of human osteosarcoma cells and
identify the NPM-interactive region of NS as its 46-amino
acid N-terminal domain, both by yeast two-hybrid and BiFC
experiments. Although NPM has previously been impli-
cated as a RNA-binding protein involved in rRNA process-
ing (Wang et al., 1994; Savkur and Olson, 1998), not all of the
NPM in the nucleolus seems to be RNA-associated based on
the observation that a fraction of NPM is resistant to release
by extensive RNase digestion (unpublished data). The fact
that NPM and NS are demonstrably heterodimerized sug-
gests that they may contribute to the RNA-deficient protein-
aceous particles we previously observed in the granular
component at the electron microscopic level (Politz et al.,
2005). This idea is also compatible with the known homo-
oligomerization (Yung and Chan, 1987; Chan and Chan,
1995; Herrera et al., 1996) and protein chaperone (Szebeni
and Olson, 1999) activities of NPM, which might be expected
to result in large complexes. Indeed, NS and NPM have
recently been reported to co-reside in large (?670-kDa) com-
plexes with the Myb-binding protein 1a in HeLa cells (Yamauchi
et al., 2008). Further studies are now in order to potentially
extend the list of NS- and NPM-interactive proteins in the
nucleolus, by using the same approaches as taken in the
The present study does not address whether NS and NPM
function in a dimeric/multimeric form to control the cell
cycle, nor whether the initially reported interactivity of NS
with p53, in coimmunoprecipitation and pull-down assays
(Tsai and McKay, 2005), means there are heterotrimeric com-
plexes of NS, NPM, and p53. Beyond the obvious need for
further studies on these protein–protein interactions, there is
the vexing problem of the conflicting reports on the role of
NPM in the cell cycle, with some studies suggesting an
oncoprotein-like function and others a tumor suppressor-
like property. It has been suggested that these disparate
findings on NPM reflect differences in the levels of p53
expression in the cell types investigated (Colombo et al.,
2002), but it is also plausible that variations in the expression
levels of other NPM-interactive proteins, including proteins
yet to be discovered, may modulate its cell cycle progression
activity, and, of course, the same point applies to NS-inter-
active proteins. The conceptual landscape should now envi-
sion these two nucleolar proteins as potential binding part-
cyan and red fluorescent protein, respectively; YN, N-terminal domain of YFP; YC, C-terminal domain of YFP; YFP, reconstituted yellow
fluorescent protein. (B) Expression and BiFC in U2OS cells expressing the pairs of plasmids indicated on the left. Images were acquired 36 h
after transfection. The images in each column were scaled the same. Bar, 5 ?m.
BiFC of NS and NPM in U2OS cells. (A) Schematic representation of modified BiFC. X, NS or mutant NS; Y, NPM; CFP and RFP,
H. Ma and T. Pederson
Molecular Biology of the Cell 2874
ners in any cell cycle control scenarios investigated, with the
possibility of a multitude of additional proteins co-residing
with them in complex machines.
We thank Joan Ritland Politz and Fan Zhang for constructive advice and
comments on the manuscript, and we gratefully acknowledge the investiga-
tors, identified in the text, for kindly providing materials. This investigation
was supported by National Science Foundation grant MCB-0445841 (to T.P.).
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