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Nucleophosmin (NPM), an oligomeric phosphoprotein and nucleolar target of the ARF tumor suppressor, contributes to several critical cellular processes. Previous studies have shown that the human NPM’s phosphorylation by cyclin E–cyclin-dependent kinase 2 (cdk2) on threonine (Thr) 199 regulates its translocation from the centrosome during cell cycle progression. Given our previous finding that ARF directly binds NPM, impeding its transit to the cytoplasm and arresting cells before S-phase entry, we hypothesized that ARF might also inhibit NPM phosphorylation. However, ARF induction did not impair phosphorylation of the cdk2 target residue in murine NPM, Thr198. Furthermore, phosphorylation of Thr198 occurred throughout the cell cycle and was concomitant with increases in overall NPM expression. To investigate the cell’s presumed requirement for NPM-Thr198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference. Here, we show that the T198A mutant is fully capable of executing NPM’s described roles in nucleocytoplasmic shuttling, ribosome export and cell cycle progression. Moreover, the proliferative defects observed with stable NPM knockdown were restored by mutant NPM-T198A expression. Thus, we demonstrate that the reduction in NPM protein expression blocks cellular growth and proliferation, whereas phosphorylation of NPM-Thr198 is not essential for NPM’s capacity to drive cell cycle progression and proliferation.
A highly abundant and evolutionarily conserved nucleolar phosphoprotein, nucleophosmin/B23 (NPM), exhibits a dynamic subcellular localization throughout the cell cycle and has been reported to interact with RNA and a diverse suite of proteins, including p19/p14ARF, p53, nucleolin, ribosomal protein L5, GADD45a and a host of viral proteins (Li, 1997; Liu and Yung, 1999; Colombo et al., 2002; Brady et al., 2004; Gao et al., 2005; Yu et al., 2006). Consequently, NPM has been described as a key player in a number of cellular processes, such as the genotoxic stress response, ribosome biogenesis and centrosome duplication (Spector et al., 1984; Okuda, 2002; Yang et al., 2002; Maggi et al., 2008). Although a proteomic analysis of isolated centrosomes failed to corroborate previous reports of NPM’s direct association with the centrosome, several studies in cell culture systems and mouse models have indicated that NPM is a critical regulator of genomic stability and centrosome duplication, be it through a direct or indirect mechanism (Tokuyama et al., 2001; Grisendi et al., 2005).
To ensure the transmission of an intact, diploid genome from one generation to the next, mitotic cells must temporally coordinate the processes of centrosome duplication, DNA replication and cell cycle progression (Winey, 1999). Fibroblasts derived from Npm1−/− embryos rapidly display centrosomal amplification and chromosomal instability in the culture, leading to activation of p53, induction of p21-mediated growth arrest and premature expression of senescence markers (Grisendi et al., 2005). Previous studies have shown that human NPM was bound to single, unreplicated centrosomes in late G1 and underwent phosphorylation by cyclin E–cyclin-dependent kinase 2 (cdk2) at threonine 199 (Thr199; Thr198 in murine NPM), prompting NPM’s dissociation from the centrosome and its subsequent duplication (Okuda et al., 2000; Tokuyama et al., 2001). Other groups have observed NPM’s interaction with duplicated centrosomes in mitotic cells (Zatsepina et al., 1999), yet independent groups failed to detect NPM in preparations of purified centrosomes (Andersen et al., 2003; Cha et al., 2004). Consequently, NPM’s physical association with the centrosome and its purported role as a direct catalyst of centrosome duplication continue to be subjects of discussion and debate in the field.
In addition to NPM’s phosphorylation by cyclin E–cdk2, its nuclear export by the Ran–Crm1 complex has also been implicated in NPM’s induction of centrosome duplication. Overexpression of NPM nuclear export signal mutants or treatment with leptomycin B, an inhibitor of Crm1-mediated nuclear export, effectively impedes NPM export, resulting in NPM’s accumulation in the nucleus and its dissociation from the centrosome (Shinmura et al., 2005; Wang et al., 2005). In addition, human cells treated with leptomycin B or small interfering RNAs (siRNAs) targeting NPM display centrosome amplification, indicating that Crm1-mediated NPM nuclear export suppresses repeated centrosome duplication cycles, presumably through NPM’s observed localization to the centrosome (Shinmura et al., 2005; Wang et al., 2005). Using similar methods in primary mouse embryonic fibroblasts (MEFs), we have previously demonstrated that NPM expression and nucleocytoplasmic shuttling are required for cell cycle progression (Brady et al., 2004; Yu et al., 2006). The integration of our findings with previously published reports (Tokuyama et al., 2001; Shinmura et al., 2005; Wang et al., 2005) suggests that NPM may use its robust expression, nuclear export and phosphorylation at Thr198 to temporally coordinate the processes of centrosome duplication and cellular proliferation.
To date, phosphorylation of NPM-Thr198 has not definitively been shown to be essential for cell growth and proliferation. Nonetheless, centrosomes and their duplication are believed to play a crucial role in cell cycle progression, although recent studies have challenged this view (Hinchcliffe et al., 2001; Khodjakov and Rieder, 2001; Uetake et al., 2007). Recalling that an alanine substitution mutant (T199A) of human NPM failed to dissociate from the centrosome and initiate duplication (Tokuyama et al., 2001), we reasoned that parallel mutation of Thr198 in the murine NPM ortholog would severely compromise the proliferation of primary MEFs. Also, given our previous finding that the ARF tumor suppressor effectively blocked NPM nuclear export (Brady et al., 2004), a critical factor in NPM’s promotion of centrosome duplication and cellular proliferation, we hypothesized that ARF might also inhibit NPM-Thr198 phosphorylation. Here, we report that ARF cannot attenuate the phosphorylation of NPM. Moreover, we demonstrate that NPM expression levels, and not Thr198 phosphorylation, define the cell’s capacity to synthesize and export ribosomes, progress through the cell cycle and proliferate.
To further investigate NPM’s contribution to cell proliferation and transformation, the impact of NPM overexpression in immortal Arf−/− or p53−/− MEFs was tested. Similar to transduction with oncogenic RasV12, exogenous expression of NPM induced a significant increase in Arf−/− cell size, as evidenced by flow cytometric measurements of forward and side scatter (Figure 1a). In agreement with previous findings in immortalized rodent cells (Kondo et al., 1997), overexpression of NPM significantly increased the size of p53−/−-transformed cell colonies that grew in soft agar, although not to the extent of RasV12 (Figure 1b).
To further address the putative role of NPM in promoting cell proliferation and transformation, 60 tissue samples from breast, prostate and colon carcinomas, were analyzed using NPM immunohistochemistry. Approximately 10–18% of Ki-67-positive tumor samples exhibited negative staining for NPM (Figure 1c, top panels), indicating that a subset of highly proliferative tumors does not upregulate NPM expression to drive proliferation. However, the remaining 82–90% of Ki-67-positive tumors did show positive staining for NPM, and nearly 50% of these samples displayed a strong nuclear/nucleolar NPM expression pattern, regardless of tumor type (Figure 1c, bottom panels).
Arf−/− MEFs, although immortal, remain diploid (Kamijo et al., 1997) and retain normal numbers of centrosomes when passaged in culture (Figure 1d, right panels). Genetic ablation of Npm1 results in centrosome amplification and genomic instability in MEFs (Grisendi et al., 2005), suggesting that NPM plays a critical regulatory role maintaining proper centrosome duplication. Given this and other corroborating reports (Okuda et al., 2000; Tokuyama et al., 2001; Wang et al., 2005), the influence of exogenous NPM expression on the ploidy and centrosome amplification in Arf−/− MEFs was examined. As shown in Figure 1d, NPM overexpression did not impact the overall chromosome number in Arf−/− MEFs, nor did it alter the number of centrosomes in these cells. Taken together, these findings demonstrate that the pro-growth and transforming properties of NPM are not coupled to the regulation of DNA ploidy changes or centrosome number.
In response to hyper-proliferative cues, such as oncogenic signals emanating from Myc, E1A and Ras, ARF is induced, and antagonizes Mdm2, to promote p53-dependent pathways of growth arrest (Sherr and Weber, 2000). We have previously shown that ARF uses a common domain at its N terminus to bind both Mdm2 and NPM, resulting in the nucleolar sequestration of each protein independent of the other (Brady et al., 2004). ARF not only delocalizes Mdm2 to the nucleolus, away from active pools of nucleoplasmic p53, but also impairs Mdm2’s E3 ubiquitin ligase activity, thereby negatively regulating Mdm2 through two distinct mechanisms (Honda and Yasuda, 1999; Tao and Levine, 1999; Weber et al., 1999). Thus, ARF might employ a similar two-pronged approach attenuating NPM’s growth-promoting functions. As phosphorylation of human NPM by cyclin E–cdk2 was reported to be essential for the initiation of centrosome duplication in late G1 (Tokuyama et al., 2001), we considered that ARF might inhibit NPM phosphorylation in addition to retaining it in the nucleolus to arrest cell growth before S-phase entry (Weber et al., 2000).
Alignment of human and mouse NPM amino acid sequences revealed 94% identity and 97% similarity. As shown in Figure 2a (lower panel), Thr199 in human NPM corresponds to Thr198 in murine NPM. A polyclonal antibody raised against a phosphopeptide surrounding Thr198 in murine NPM was generated to specifically detect phospohoThr198 in NPM (Figure 2a, underlined sequence). The phosphospecific NPM-Thr198 antibody (NPM-pT198) reacted with a protein band migrating at approximately 38 kDa in whole cell lysates from asynchronously growing triple knockout (TKO) MEFs (Arf−/− p53−/− Mdm2−/−) (Figure 2a, lane 1), but failed to detect the corresponding band in lysates from contact-inhibited TKO MEFs (Figure 2a, lane 2) or in purified recombinant NPM proteins expressed in Escherichia coli (Figure 2a, lane 3). Re-probing of this membrane with a monoclonal antibody recognizing NPM showed that a 38 kDa protein band was present in all three lanes, indicating that the polyclonal antibody reacts specifically with NPM phospho-Thr198 proteins, but does not cross-react with non-phosphorylated NPM. In addition, TKO MEFs infected with siRNAs targeting the 3′-UTR of endogenous NPM were used to show specificity of the antibody to Thr198. Phosphorylation of Thr198 was reduced at a level consistent with reduction in total NPM protein after siNPM infection (Figure 2a, right panel). Rescue of NPM knockdown with an ectopic RNA interference-resistant NPM-GFP (green fluorescent protein) protein resulted in a restoration of NPM phosphorylation at Thr198 (Figure 2a, right panel, lane 3 arrow), whereas rescue with an NPM T198A-GFP mutant resulted in a non-observable phosphorylation with the phospho-T198 antibody (Figure 2a, right panel, lane 4). This demonstrates that our NPM phospho-T198 antibody is specific for Thr198.
To determine whether or not phosphorylation of murine NPM-Thr198 is a cyclin E–cdk2-specific event within the context of cell cycle progression, TKO MEFs were serum-starved and synchronized in G0, evidenced by the cells’ low expression levels of cyclin D1 protein (Figure 2b, lane 2). After release into serum, phospho-Thr198 NPM expression increased, achieving maximal levels at 24-h post-serum addition (Figure 2b). Notably, the observed increase in phospho-Thr198 NPM levels coincided with the increased expression of total NPM protein (Figure 2b). Quantitative comparison of protein band intensities confirmed that phospho-Thr198 NPM protein levels increased in parallel with total NPM protein expression. Given that cyclin D1 protein expression levels were maximal at approximately 8 h after the cells’ release into serum, yet abundant levels of phospho-T198 NPM were already evident by 4-h post-stimulation, this result suggests that cyclin E–cdk2 is not the sole kinase which phosphorylates NPM-Thr198 within the cell (Figure 2b). These data instead indicate that NPM-Thr198 seems to be constitutively phosphorylated throughout the cell cycle rising only when overall protein levels of NPM increase, and likely undergoes phosphorylation at Thr198 by one or more kinases, with overall NPM abundance being the limiting substrate. To further explore this possibility, cells were growth arrested at various points of the cell cycle. Aphidicolin-induced G1/S-phase arrest did not alter phospho-T198 compared with dimethyl sulfoxide controls (Figure 2c, lane 2). We did observe a modest increase in Thr198 phosphorylation (1.4-fold) with nocodazole treatment, consistent with an overall increase in NPM abundance (Figure 2b). Inhibition of cdk2 with roscovitine resulted in no change in Thr198 phosphorylation (Figure 2c, lane 4), suggesting that kinases other than cdk2 are quite capable of phosphorylating this residue throughout the cell cycle.
Given that ARF’s interaction with NPM represents one of its p53-independent functions, ARF’s impact on NPM-Thr198 phosphorylation in TKO MEFs was examined. Retroviral-mediated transduction of p19ARF into TKO MEFs failed to produce an appreciable change in phospho-Thr198 NPM protein levels (Figure 2d, lanes 1 and 3). TKO MEFs that were transduced to express p19ARFΔ1 – 14, a mutant lacking the NPM-binding domain (Brady et al., 2004), showed a very subtle increase in phospho-Thr198 NPM levels (Figure 2d, lane 2). In combination with the earlier result showing that NPM-Thr198 is constitutively phosphorylated, these data indicate that this particular NPM phosphorylation site is not subject to either positive (that is, cdk-mediated) or negative (that is, ARF-directed) regulation throughout the cell cycle, but is instead constantly being phosphorylated as total levels of NPM rise in the cell.
Although this current study demonstrates that ARF induction does not influence NPM-Thr198 phosphorylation (Figure 2c), our previously published findings have shown that ARF effectively blocks NPM nucleocytoplasmic shuttling, a critical function of NPM that is essential for cellular growth and proliferation (Brady et al., 2004; Yu et al., 2006). Thus, the requirement of phosphorylation of Thr198 for NPM’s nucleocytoplasmic shuttling was examined using a non-phosphorylatable alanine substitution mutant, T198A.
Given NPM’s well-documented capacity to form homo-oligomers (Liu and Chan, 1991; Yung and Chan, 1987; Namboodiri et al., 2004), the ability of ectopically-expressed T198A to hetero-oligomerize with endogenous NPM was examined. We have previously shown that NPM functional mutants often form hetero-oligomers with wild-type NPM and act as dominant-negative NPM molecules, inhibiting the function of wild-type NPM (Yu et al., 2006). Immunoprecipitation of retrovirally transduced His-tagged wild-type NPM or mutant T198A proteins from TKO MEFs, followed by NPM western blot analysis revealed that T198A formed complexes with endogenous NPM proteins, similar to ectopic wild-type NPM (Figure 3a). If NPM-T198A mutants were non-functional, we expected that they would act as dominant-negative mutants, preventing the function of endogenous wild-type NPM. Heterokaryon shuttling assays using constructs encoding either wild-type NPM or mutant T198A were then performed to answer this biological question. This experimental system assesses NPM’s capacity to shuttle between the nucleus and cytoplasm, a property that defines NPM’s role in promoting cell growth and proliferation (Yu et al., 2006; Maggi et al., 2008), demonstrated by the transit of the visibly-tagged protein of interest from a transfected human donor cell into an untransfected murine recipient cell (Tao and Levine, 1999; Yu et al., 2006). Similar to wild-type NPM (24/24, 100% shuttling), mutant NPM-T198A shuttled from the nuclei/nucleoli of transiently transfected human HeLa cells into the nuclei/nucleoli of fused, untransfected mouse NIH3T3 cells, demonstrating that NPM’s nucleocytoplasmic shuttling is not dependent on its phosphorylation at Thr198 and is not inhibited by mutant NPM-T198A molecules (Figure 3b).
The T198A mutant’s ability to hetero-oligomerize with endogenous NPM and shuttle to the cytoplasm could potentially mask this mutant’s true phenotype. More specifically, the T198A mutant does not display dominant-negative behavior within the cell, unlike our previously described NPMdL mutant, which blocks NPMdL-NPM hetero-oligomers from shuttling (Yu et al., 2006). To address this possibility, an NPM knockdown-rescue lentiviral construct was engineered encoding both a short hairpin RNA targeting the 3′-UTR of murine NPM (siNPM) and an siRNA-resistant cDNA corresponding to either wild type (siNPM + NPM-GFP) or mutant (siNPM + T198A-GFP) murine NPM. This strategy allowed the simultaneous reduction of endogenous NPM protein levels and ectopic expression of GFP-tagged NPM rescue proteins with high efficiency in TKO MEFs, as confirmed by NPM western blot analysis (Figure 4a).
Knockdown of endogenous NPM in TKO MEFs resulted in an increase in the number of cells containing a single centrosome and a concomitant decrease in the number of cells exhibiting two centrosomes (Figure 4b, black bars). A slight, but reproducible, increase in the number of cells displaying more than two centrosomes was observed, which is consistent with another group’s findings in Npm1−/− MEFs (Figure 4b, black bars) (Grisendi et al., 2005). Ectopic expression of wild-type NPM and T198A reversed some of the centrosome defects observed upon NPM loss (cells with two centrosomes), but neither was capable of limiting cells with centrosome numbers greater than two (Figure 4b, gray and hatched bars). In addition, colocalization of ectopic wild type or T198A NPM with centrosomes was not observed, although cells displaying NPM-GFP-positive nucleoli adjacent to tubulin-positive centrosomes were observed (Figure 4b, arrows).
We have previously shown that NPM nucleocytoplasmic shuttling is essential for nuclear export and for the formation of cytosolic ribosomes (Yu et al., 2006; Maggi et al., 2008). Having confirmed that the T198A mutant efficiently shuttles from the nucleolus/nucleus to the cytoplasm (Figure 3b), we next aimed to determine whether NPM-Thr198 phosphorylation is necessary for NPM’s established role in the assembly and transport of translationally competent ribosomes. We observed that knockdown of NPM in TKO MEFs produced a striking reduction in the populations of 40S, 60S and 80S cytosolic ribosomal subunits, as well as a significant attenuation in the levels of actively translating polysomes (Figure 4c). Importantly, expression of either wild-type NPM or the T198A mutant was sufficient to rescue the siNPM-induced ribosomal defect, restoring all cytosolic ribosomal populations to levels present in control siLuc-infected cells (Figure 4c). Consistent with our findings from nuclear export assays, this result demonstrates that NPM plays a critical role in ribosome biogenesis that is not dependent on its phosphorylation at Thr198.
A previous study has suggested that phosphorylation of human NPM at Thr199 is necessary for proper S-phase entry and cellular proliferation (Tokuyama et al., 2001). Given that the T198A mutant was fully competent in executing NPM’s described roles in shuttling, centrosome duplication and ribosome biogenesis (Figures 3 and and4),4), the influence of the T198A mutant on cellular proliferation was examined. Stable knockdown of endogenous NPM in TKO MEFs severely compromised the cells’ ability to enter S-phase, as evidenced by decreased cyclin A expression (Figure 5a) and bromodeoxyuridine (BrdU) incorporation into replicating DNA (Figure 5c). Ectopic expression of either wild-type NPM or T198A-mutant siRNA-resistant proteins was sufficient to fully rescue incorporation of BrdU into the DNA of NPM knockdown cells (Figure 5c). In addition, knockdown of NPM in diploid Arf−/− MEFs resulted in a substantial increase in G1 cells (Figure 5b), suggesting that loss of NPM imposes a block before S-phase entry. To further investigate the potential long-term effects of NPM loss on cell proliferation, foci formation assays were conducted in parallel. Stable knockdown of NPM significantly inhibited foci formation by TKO MEFs, a proliferative defect that was fully reversed upon rescue with either wild-type NPM or T198A-mutant siRNA-resistant proteins (Figure 5d). Thus, these data demonstrate that phosphorylation of NPM on Thr198 is dispensable for cell cycle progression and cellular proliferation, whereas adequate NPM protein expression is essential.
A multifunctional and dynamic nucleolar phosphoprotein, NPM, has been described as a critical mediator and regulator of numerous processes within the cell, including protein chaperoning, ribosome biogenesis, centrosome duplication and genomic stability (Okuwaki et al., 2001; Okuda, 2002; Okuwaki et al., 2002; Colombo et al., 2005; Maggi et al., 2008). Given this list of disparate, but basic, cellular functions that require NPM, it is not surprising that NPM also plays essential roles in embryonic development (Grisendi et al., 2005) and cell cycle progression (Brady et al., 2004).
In support of this hypothesis, ectopic expression of NPM in immortalized fibroblasts not only increased cell size but also supplied the cell with signals that are necessary for enhanced proliferation and anchorage-independent growth. On the basis of our data and that of other groups, we propose that upregulation of NPM can promote transformation. In agreement with this idea, a subset of adult leukemias carries an NPM mutation, which encodes a second nuclear export signal at NPM’s extreme carboxy terminus (Falini et al., 2005). Further study of this mutant revealed that it dictates increased nucleocytoplasmic shuttling of NPM (Colombo et al., 2006), and our laboratory has previously shown that proper cell cycle progression requires NPM nuclear export (Brady et al., 2004). In addition, numerous laboratories (Itahana et al., 2003; Bertwistle et al., 2004; Brady et al., 2004) have demonstrated that NPM is a functional target of the nucleolar ARF tumor suppressor, implying that the transformation properties of NPM can be antagonized by the ARF tumor suppressor. The fact that we have shown NPM to be oncogenic in the absence of p53 and Arf suggests that NPM’s role in promoting transformation is not to simply antagonize these two tumor suppressors.
Previous studies have demonstrated that human NPM undergoes phosphorylation at Thr199 (Thr198 in mouse), and that cyclin E–cdk2 targets this Thr residue to relieve NPM-mediated repression of centrosome duplication and cell cycle progression (Okuda et al., 2000; Tokuyama et al., 2001). In considering this argument, one would predict that centrosome duplication would be repressed under conditions of increased NPM expression or nuclear export. However, this has not been observed in acute myelogenous leukemia patients who carry NPMc+ mutants (Falini et al., 2005) or in our current study of the cellular effects of NPM overexpression. Although intriguing, the existing model concerning the role of NPM and its phosphorylation at Thr199 in the process of centrosome duplication does not account for the mounting evidence which links NPM overexpression and nuclear export to increased cell growth and proliferation. We have provided evidence that induction of NPM protein expression is the critical limiting factor in NPM’s ability to promote cell growth and proliferation.
Our studies have revealed that ARF’s binding to NPM cannot block phosphorylation of NPM at Thr198. In addition, a non-phosphorylatable mutant of NPM, T198A, does not block cell cycle progression, centrosome duplication, nuclear export or cytosolic ribosome accumulation in the absence of endogenous wild-type NPM. Moreover, we observed that NPM-Thr198 is constitutively phosphorylated throughout the cell cycle, and any increase in Thr198 phosphorylation parallels the increase in total NPM protein expression. Although our data indicates that phosphorylation of NPM-Thr198 does not influence NPM function, we do not discount the importance of NPM in centrosome duplication. In agreement with others’ published findings from NPM knockout mice (Grisendi et al., 2005) and cell lines (Okuda et al., 2000; Tokuyama et al., 2001), we have shown that loss of NPM deregulates centrosome duplication. However, we propose that this might be a downstream effect, which may not be directly mediated by NPM. In cells undergoing acute NPM loss, we observed a decrease in the number of actively translating ribosomes at time points (48 h) preceding the observed defects in centrosome duplication and S-phase entry (96–120 h). Therefore, our data supports a model in which NPM’s direct command over ribosome biogenesis and protein translation could result in indirect changes in a downstream target that plays a critical role in the process of centrosome duplication. Thus, translational targets of the ribosome might in turn also promote cellular proliferation and transformation.
The Arf−/− MEFs, Arf−/−/p53−/−/Mdm2−/− MEFs (TKO MEFs, provided by Gerard Zambetti, St Jude Children’s Research Hospital), NIH3T3 and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM non-essential amino acids and 100 U each of penicillin and streptomycin. TKO MEFs were synchronized into quiescence by culturing at sub-confluency in medium supplemented with 0.1% fetal bovine serum for 48 h.
The pSRα-MSV-tkneo retroviral expression vectors encoding p19ARF, p19ARFΔ1 14 and full-length murine NPM were used as described previously (Brady et al., 2004). The His-T198A NPM mutant was amplified from pET28a-NPM using the following mutagenic primers: 5′-ATCTGTACGAGATGCA CCAGCCAAAAATGC-3′ (sense) and 5′-GTGCATTTTTGG CTGGTGCATCTCGTACAG-3′ (antisense). The resultant His-T198A cDNA was sub-cloned into pcDNA3.1 using EcoRI and BamHI, and into pSRα-MSV-tkneo using EcoRI; pcDNA3.1-Myc-NPC-M9 was gift from J Alan Diehl (University of Pennsylvania, USA). The pFLRu-GFP-siLuc and pFLRu-GFP-siNPM vectors were provided by Gregory Longmore (Washington University, USA) (Pelletier et al., 2007). To generate the pFLRu-siNPM-NPM-GFP and pFLRu-siNPM-T198A-GFP rescue constructs, murine cDNAs encoding wild type or T198A-mutant NPM were sub-cloned into the EcoRI and BamHI sites of the pFLRu-GFP-siNPM vector. The lentiviral envelope and packaging vectors, pHCMV.G and CMVΔR8.2, were gifts from Sheila Stewart (Washington University).
Retroviral production and infection using pBabe-H-RasV12 and SRα-MSV-tkneo vectors were carried out according to methods described previously (Brady et al., 2004; Roussel et al., 1995). Lentiviruses encoded by the pFLRu-GFP vectors were packaged in 293T cells after cotransfection of the pHCMV.G, CMVΔR8.2 and pFLRu-GFP lentiviral vectors using Fugene 6 (Roche, Indianapolis, IN, USA). Primary MEFs were infected for 4 h with freshly harvested lentiviral supernatants in the presence of 8 μg/ml protamine sulfate, and at 24-h post-infection, puromycin (2 μg/ml) was added to the cells for a selection period of 48 h where appropriate.
The Arf−/− MEFs were infected with retroviruses encoding the control vector, His-NPM or RasV12, and were harvested at 72 h. Cells were fixed and resuspended in 1X phosphate-buffered saline/1% fetal bovine serum with or without propidium iodide before analysis using a FACSCalibur (Becton Dickson, Rockville, MD, USA).
Mouse embryonic fibroblasts were infected with lentiviral expression supernatants and were seeded (2 × 103) onto 100 mm dishes. Cells were grown for 14 days in complete medium, fixed in 100% methanol and stained for 30 min with 50% Giemsa.
The p53−/− MEFs were infected with control vector, His-NPM, His-NPM-T198A or RasV12 retroviruses, and were seeded (1 × 103) in triplicates onto 60 mm dishes. Colonies were allowed to grow for 14 days in complete medium supplemented with fetal bovine serum and Noble Agar.
The TARP4 tissue array was purchased from NCI Tissue Array Research Project. The tissues used to construct arrays were obtained from the Cooperative Human Tissue Network (CHTN). Each tissue array slide contained 600 samples. De-paraffinized tissue sections were first treated with 3% H2O2 for 30 min followed by antigen retrieval by heating in citra plus solution (BioGenex, San Ramon, CA, USA). After subjecting to avidin block, biotin block and power block for 15 min, the sections were incubated with mouse anti-NPM antibody (Zymed, San Francisco, CA, USA) for 1 h. After further incubation with biotinylated multi-link antibody for 45 min and peroxidase-labeled streptavidin for 30 min, the staining was developed by reaction with 3,3′-diaminobenzidine tetrahydrochloride substrate–chromogen solution.
The Arf−/− MEFs were infected with control vector or His-NPM retroviruses, and at 72-h post-infection were treated with colcemid (10 μg/ml) for 16 h. Cells were harvested in 75 mM KCl for 6 min at 37 °C. Cells were fixed in methanol:acetic acid (3:1) and washed. The cells were resuspended in 2 ml fixative and one drop was allowed to fall onto frosted glass slide. DNA was stained with DAPI (4′,6-diamidino-2-phenylindole) and fluorescent signals were detected.
Immunoprecipitation of cell lysates was performed as previously described (Brady et al., 2004). Antibodies recognizing γ-tubulin, cyclin D1, His (Santa Cruz, Santa Cruz, CA, USA), p19ARF (Abcam, Cambridge, MA, USA), NPM (Zymed) and NPM (custom rabbit polyclonal, Sigma, St Louis, MO, USA) were used in western blot analyses. The custom phosphospecific polyclonal antibody recognizing phospho-NPM (Thr198) was generated commercially (Zymed) and raised against the following phosphopeptide: CSVRDpTPAKN (Tufts University Peptide Core).
The HeLa cells (2 × 105) were seeded onto glass cover slips in six-well dishes and transfected with constructs encoding either His-tagged wild type or T198A-mutant NPM in combination with a Myc-tagged NPC-M9 plasmid (a gift from J Alan Diehl, University of Pennsylvania). Heterokaryon assays were performed as previously described (Yu et al., 2006).
The Arf−/− or TKO MEFs were infected with SRα-MSV-tkneo retroviruses or pFLRu-GFP lentiviruses as indicated, and seeded onto glass cover slips. Cells were washed with phosphate-buffered saline, fixed at room temperature using 10% formalin/10% methanol, followed by 1% NP-40 in phosphate-buffered saline for 5 min at room temperature. Cells were stained with an antibody recognizing γ-tubulin (Sigma), followed by FITC or rhodamine X-conjugated immunoglobulins. Nuclei were counterstained with DAPI.
The Arf−/− or TKO MEFs were infected with SRα-MSV-tkneo retroviruses or pFLRu-GFP lentiviruses as indicated. Cells were seeded onto glass cover slips and subjected to BrdU incorporation analysis (Brady et al., 2004).
At 4 days post-infection with pFLRu-GFP lentiviruses, TKO MEFs were subjected to ribosome fractionation analysis (Maggi et al., 2008).
We are indebted to Sheila Stewart, Gregory Longmore, J Alan Diehl, Martine Roussel, Charles Sherr and Gerard Zambetti for gifts of plasmid constructs, antibodies and primary TKO MEFs. In addition, we would like to thank Sheila Stewart, Helen Piwnica-Worms, Michael Tomasson and John Majors for insightful discussions throughout the course of this study. SNB was supported by the Cancer Biology Pathway. CLP was a trainee in the Lucille P Markey Special Emphasis Pathway in Human Pathobiology. JDW was funded through the National Institutes of Health and Department of Defense Era of Hope Scholar Award in Breast Cancer Research.
Conflict of interest
The authors declare no conflict of interest.