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
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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