Nucleophosmin is a binding partner of nucleostemin in human osteosarcoma cells

Abstract

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.

Figure 1.

Colocalization of NS and NPM within the nucleolus. Top row, mRFP-NS and GFP-NPM were cotransfected into U2OS cells and 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 μm.

Figure 2.

Localization of NS and NPM during anaphase and telophase. NS-GFP was induced in U2OS^iNS-GFP cells (see Supplemental Figure 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 μm.

Figure 3.

Double immunostaining of NS and NPM in telophase. Parental U2OS cells were immunostained for NS (left) and NPM (middle). Shown is a typical telophase cell. The images were subjected to deconvolution and then merged (right). Bar, 5 μm.

Figure 4.

Interaction of NS and NPM by both coimmunoprecipitation and yeast two-hybrid analysis. (A) Coimmunoprecipitation of NS and 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 β-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.

Figure 5.

BiFC of NS and NPM in U2OS cells. (A) Schematic representation of modified BiFC. X, NS or mutant NS; Y, NPM; CFP and RFP, 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 μm.