Polo-Like Kinase 2-Dependent Phosphorylation of NPM/B23 on Serine 4 Triggers Centriole Duplication

Duplication of the centrosome is well controlled during faithful cell division while deregulation of this process leads to supernumary centrosomes, chromosome missegregation and aneuploidy, a hallmark of many cancer cells. We previously reported that Polo-like kinase 2 (Plk2) is activated near the G1/S phase transition, and regulates the reproduction of centrosomes. In search for Plk2 interacting proteins we have identified NPM/B23 (Nucleophosmin) as a novel Plk2 binding partner. We find that Plk2 and NPM/B23 interact in vitro in a Polo-box dependent manner. An association between both proteins was also observed in vivo. Moreover, we show that Plk2 phosphorylates NPM/B23 on serine 4 in vivo in S-phase. Notably, expression of a non-phosphorylatable NPM/B23 S4A mutant interferes with centriole reduplication in S-phase arrested cells and leads to a dilution of centriole numbers in unperturbed U2OS cells. The corresponding phospho-mimicking mutants have the opposite effect and their expression leads to the accumulation of centrioles. These findings suggest that NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication and that phosphorylation on serine 4 can trigger this process

Phosphorylation of NPM/B23 serine 4 is cell cycle regulated.

<p><b>A</b> Cell extracts from HeLa cells, asynchronously (exp) grown or synchronized in S-phase (S) by 4 mM hydroxyurea for 40 h or in M-phase (M) by 100 ng/ml nocodazole for 15 h, were examined by Western blotting using the indicated antibodies. GAPDH, loading control. <b>B</b> Plk1 or Plk2 were immunoprecipitated from HeLa extracts, either exponentially grown or arrested in M-phase or in S-phase as described in (A), and subjected to <i>in vitro</i> kinase assays using α-Casein as a substrate in the presence of [γ³²P]-ATP. For experimental details see in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009849#pone-0009849-g003" target="_blank">Fig. 3A</a>. HC, IgG heavy chain. <b>C</b> Plk1 and Plk2 were immunoprecipitated from HeLa extracts as described in (B) and subjected to <i>in vitro</i> kinase assays with His-tagged NPM/B23 as substrate. Western blotting was performed by NPM/B23 pS4 antibodies. Equal His-NPM/B23 levels are shown by Ponceau S staining. <b>D </b><i>In vitro</i> kinase assays using GST-tagged Plk1 and His-NPM/B23 were performed in the presence of 100 mM BI2536 or DMSO. Phosphorylation of His-NPM/B23 was detected by Western blot with anti-pS4 NPM/B23 antibodies. Loading control, NPM/B23; unt., untreated. <b>E </b><i>In vitro</i> kinase assays using immunoprecipitated Flag-Plk2 WT from 293T cells and His-NPM/B23 as substrate. The experiment was performed as described in (D). <b>F</b> HeLa cells were incubated with 100 nM BI2536 or DMSO for 2 h. Cell extracts were used for immunoprecipitations with anti-Plk1 or anti-Plk2 antibodies and subjected to <i>in vitro</i> kinase assays using α-Casein as substrate, supplemented with [γ³²P]-ATP. Kinase assays were analyzed as described in (B). <b>G</b> Asynchronous HeLa or U2OS cells or S-phase arrested HeLa cells as in (A) were treated with 100 nM BI2536 for 2 h. Cell extracts were prepared for Western blotting with anti-NPM/B23 and anti-pS4 NPM/B23 antibodies. Loading control, α-Tubulin. Quantifications of kinase activities or protein levels in this figure were generated by ImageJ (NIH).</p

NPM/B23 serine 4 phosphorylation in S-phase is mediated by Plk2.

<p><b>A</b> Flag-Plk2 WT, KD or Flag-alone were overexpressed in 293T cells. 8 h after transfections cells were arrested with 4 mM hydroxyurea (HU) for 40 h. Exponentially growing cells were harvested 48 h after transfections. Cell extracts were analyzed by SDS-PAGE and Western blot using anti-Flag, anti-NPM/B23, anti-pS4 NPM/B23 and anti-α-Tubulin (loading control) antibodies. <b>B</b> RNAi of Plk2 was mediated by overexpression of pSuper-RNAi-Plk2 and pSuper-RNAi-Luciferase as control in 293T cells. Cell cycle synchronization and analyses were carried out as in (A). To detect Plk2 downregulation in Western blot, membranes were probed with anti-Plk2 antibodies. <b>C</b> RNAi of Plk1 in HeLa cells was mediated by Lipofectamine2000-transfections of Plk1 specific siRNAs. GL2 siRNAs were used as control. Cell extracts were analyzed by Western blotting with the indicated antibodies. Loading control, α-Tubulin. <b>D</b> Plk4 was downregulated in U2OS cells by Lipofectamine2000 mediated siRNA transfections, as control GL2 was used. S-phase arrest was achieved as described in (A). Downregulation of Plk4 and pS4 NPM/B23 levels were examined by Western blot analysis with the indicated antibodies. Loading control, α-Tubulin.</p

Treatment with BI2536, a Polo-kinase inhibitor, interferes with centriole reduplication.

<p><b>A</b> U2OS GFP-Centrin1 cells were treated with 1.9 µg/ml aphidicolin to induce centriole reduplication for 75 h. In parallel, treatment with 100 nM BI2536 or DMSO as control was performed. Cell extracts were resolved by SDS-PAGE and analyzed on Western blot level with anti-NPM/B23 and anti-pS4 NPM/B23 antibodies. α-Tubulin, loading control. <b>B</b> Indirect immunofluorescence analysis from the experiment described in (A) was carried out specific for γ-Tubulin after methanol fixation. Insets show enlargement of centrioles. GFP-Centrin1, green; γ-Tubulin, red; DNA, blue. Scale bar, 10 µm. <b>C</b> For statistics, the experiment in (B) was also scored for centriole reduplication (>4 GFP-Centrin1 dots/centrioles or >2 γ-Tubulin dots/centrosomes) by microscopy. For quantification, more than 200 cells were analyzed for centriole reduplication in three independent experiments. Black bars, GFP-Centrin1 signal; grey bars, γ-Tubulin staining; unt, untreated; aph, aphidicolin.</p

Overexpression of the phospho-mimicking NPM/B23 S4E mutant rescues inhibition of centriole reduplication induced by Plk2 RNAi.

<p><b>A</b> U2OS GFP-Centrin1 cells were subjected to siRNA transfections (siPlk2 or GL2 as control) using Lipofectamine2000, transfected with Flag-NPM/B23 constructs for 8 h and then subjected to a centrosome duplication assay in the presence of 1.9 µg/ml aphidicolin for 75 h. Western blot analysis demonstrated the downregulation of Plk2 by specific antibodies. loading control, α-Tubulin <b>B</b> After fixing cells from experiment in (A) with methanol indirect immunofluorescence analyses were carried out with anti-Flag antibodies. Insets show enlargements of centrioles. GFP-Centrin1, green; Flag, red; DNA, blue. Scale bar, 10 µm. <b>C</b> For statistics, U2OS GFP-Centrin1 cells from indirect immunofluorescence analyses in (B) were scored for centriole reduplication (>4 GFP-Centrin1 dots/centrioles). In case of transfections, only Flag-positive cells were analyzed. In total, 200 cells were counted each in 3 independent experiments to calculate means +/− standard deviation. unt, untreated, aph, aphidicolin.</p

Plk2 interacts with NPM/B23 <i>in vivo</i> and <i>in vitro</i>.

<p><b>A</b> After overexpression of Myc-Plk2 KD and Flag-NPM/B23 in 293T cells, cell extracts were subjected to immunoprecipitations using anti-Flag antibodies. Co-precipitation of Myc-Plk2 KD by Flag-NPM/B23 was detected by Western blotting using anti-Plk2 and anti-NPM/B23 antibodies. Asterisks (*), endogenous NPM/B23. <b>B</b> Myc-Plk2 KD was overexpressed in 293T cells and immunoprecipitated with anti-Myc antibodies. Complex formation of Myc-Plk2 KD and endogenous NPM/B23 was analyzed by Western blot with anti-Plk2 and anti-NPM/B23 antibodies. <b>C</b> Endogenous Plk2 was immunoprecipitated from HeLa lysates by specific antibodies. The interaction was verified by Western blotting using anti-NPM/B23 and anti-Plk2 antibodies. IgG, random antibody control. <b>D</b> Far Western blot analysis. Recombinant His-tagged NPM/B23 was resolved by SDS-PAGE, blotted onto nitrocellulose and incubated with GST-Plk2 PBD, -PBD mutant or GST (control) to allow interaction. Protein bound to NPM/B23 was detected with anti-GST antibodies. Equal amounts of GST-PBD, -PBD mutant and GST used for ligand binding are demonstrated by Coomassie blue staining. <b>E</b> Scheme of Plk2 truncated versions. <b>F</b> Flag-Plk2 truncated versions (E) were overexpressed in 293T cells, immunoprecipitated by anti-Flag antibodies and incubated with recombinant His-NPM/B23 in <i>in vitro</i> binding assays. Complexes were separated by SDS-PAGE and analyzed by Western blot using anti-NPM/B23 and anti-Flag antibodies. <b>G</b> Scheme of NPM/B23 truncated versions. <b>H</b> Glutathione sepharose-bound GST-NPM/B23 truncated versions (G) or GST (control) were incubated with <i>in vitro</i> translated, [³⁵S]-methionine-labeled HA-Plk2 in pull down assays. Complexes were resolved by SDS-PAGE. After Coomassie blue staining, interactions were detected by autoradiography. <b>I</b> NPM/B23 was downregulated by siRNA in U2OS cells as demonstrated in a Western blot analysis (left panel). Indirect immunofluorescence analysis was performed for Plk2 (red) and centriolar GT335 (green) (middle panel). Localization of centriolar Plk2 was quantified in 3 independent experiments with >150 analyzed cells each (right panel). GL2, siRNA control; loading control, α-Tubulin.</p

Plk2 phosphorylates NPM/B23 on serine 4.

<p><b>A</b> Flag-NPM/B23 was overexpressed in 293T cells. After lysis, immunoprecipitations with anti-Flag antibodies or random IgGs as control were performed. Immunoprecipitated Flag-NPM/B23 and controls were subjected to <i>in vitro</i> kinase assays with Zz-Plk2 WT or KD, purified from bacteria, in the presence of [γ³²P]-ATP. To assess Zz-Plk2 WT and KD kinase activity, <i>in vitro</i> kinase assays, supplemented with [γ³²P]-ATP, were performed using α-Casein as substrate. Kinase assays were resolved by SDS-PAGE. After Coomassie staining of the gel, phosphorylation was detected by autoradiography. <b>B</b> To identify the Plk2 phosphorylation site on NPM/B23, <i>in vitro</i> kinase assays were carried out using Zz-tagged Plk2 WT in combination with GST-tagged NPM/B23 fragments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009849#pone-0009849-g002" target="_blank">Fig. 2G</a>, as control GST alone, in the presence of [γ³²P]-ATP. After SDS-PAGE, followed by Coomassie blue staining of the gel, analysis was done by autoradiography. <b>C</b> To verify the NPM/B23 phosphorylation site, NPM/B23 serine 4 and threonine 95 were mutated to alanine (A). GST-NPM/B23 (aa 1–120) WT, S4A and T95A were incubated with Zz-Plk2 in an <i>in vitro</i> kinase assay and analyzed as in (B). <b>D </b><i>In vitro</i> kinase assay using immunoprecipitated Flag-NPM/B23 S125A mutant from 293T cells together with Zz-Plk2 WT and Zz-Plk2 KD from bacteria. Analysis was done as in (A). <b>E </b><i>In vitro</i> kinase assay, assembled and analyzed as in (A) besides using immunoprecipitated Flag-NPM/B23 WT, S4A or T199A as substrates. <b>F </b><i>In vitro</i> kinase assay using immunoprecipitated Flag-Plk2 WT or KD from 293T cells together with His-tagged NPM/B23 WT or S4A from bacteria. Reactions were subjected to Western blot analysis using anti-pS4 NPM/B23 and anti-NPM/B23 antibodies. <b>G </b><i>In vitro</i> kinase assay, assembled and analyzed as described in (F) besides using GST-tagged Plk1 WT or KD.</p

Serine 4 phosphorylated NPM/B23 promotes centrosome duplication.

<p><b>A</b> After transfections with Flag-tagged NPM/B23 WT and non-phosphorylatable mutants (S4A, T199A, S4A/T199A) or phospho-mimicking mutants (S4D, S4E), centriole reduplication was performed in U2OS GFP-Centrin1 cells with 1.9 µg/ml aphidicolin for 75 h. Afterwards, cells were fixed for immunofluorescence and cells lysates were prepared for Western blot analysis using anti-Flag antibodies to show comparable expression levels of the transfected constructs. Loading control, GAPDH. <b>B</b> Quantification of the centriole reduplication phenotype of experiments described in (A). Cells were analyzed by immunofluorescence using anti-Flag antibodies to detect transfected cells. Flag-positive cells were analyzed for centriole reduplication (>4 GFP-Centrin1 dots/centrioles). For quantification, >150 transfected cells were counted in three independent experiments. Insets show representative centriole reduplication phenotypes. GFP-Centrin1, green. <b>C</b> Non-phosphorylatable or phospho-mimicking Flag-tagged NPM/B23 mutants described in (A) were overexpressed in U2OS GFP-Centrin1 cells for 48 h. After immunofluorescence using anti-Flag antibodies, Flag-positive cells were scored for centriole dilution (<2 GFP-Centrin1 dots/centrioles) or centriole amplification (4 or >4 GFP-Centrin1 dots/centrioles). Quantifications are means +/− standard deviation from three independent experiments, >150 transfected cells were counted for each experiment.</p

Combination of antiangiogenic therapy using the mTOR-inhibitor everolimus and low-dose chemotherapy for locally advanced and/or metastatic pancreatic cancer: a dose-finding study

Pancreatic adenocarcinomas are associated with a poor survival prognosis. Besides curative surgical resection, only limited therapies with modest impact are available. New evidence suggests that the mammalian target of rapamycin pathway may be involved in the pathogenesis of neuroendocrine tumors, and breast and renal cell cancer. The phase I study described here was therefore designed to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of escalating doses of the mammalian target of rapamycin inhibitor everolimus in combination with gemcitabine in patients with advanced pancreatic cancer. Eligible patients had histologically confirmed locally advanced and/or metastatic pancreatic carcinoma and were administered 5 mg everolimus every second day (cohort 1, 2, 3) or 5 mg daily (cohort 4, 5) in combination with escalating low-dose gemcitabine. It was found that if two patients showed DLTs, MTD was reached and gemcitabine dose escalation was stopped at this level. Twenty-seven patients were enrolled in the study (cohort 1: n=3; cohort 2: n=4; cohort 3: n=6; cohort 4: n=7; cohort 5: n=7) and received a maximum 600 mg gemcitabine/week. In cohort 5, two of the six patients experienced DLTs (grade 3 liver toxicity lasting for>7 days). MTD was measured as 400 mg/m2/week gemcitabine plus 5 mg/day everolimus. The MTD of a low-dose gemcitabine treatment in combination with everolimus was determined and no new safety concerns were identified in patients with advanced pancreatic cancer

Early and late effects of radiochemotherapy on cerebral blood flow in glioblastoma patients measured with non-invasive perfusion MRI

Background and purpose To provide a systematic measure of changes of brain perfusion in healthy tissue following a fractionated radiotherapy of brain tumors. Materials and methods Perfusion was assessed before and after radiochemotherapy using arterial spin labeling in a group of 24 patients (mean age 54.3±14.1 years) with glioblastoma multiforme. Mean relative perfusion change in gray matter in the hemisphere contralateral to the tumor was obtained for the whole hemisphere and also for six regions created by thresholding the individual dose maps at 10 Gy steps. Results A significant decrease of perfusion of -9.8 ± 20.9% (p = 0.032) compared to the pre-treatment baseline was observed 3 months after the end of radiotherapy. The decrease was more pronounced for high-dose regions above 50 Gy (-16.8 ± 21.0%, p = 0.0014) than for low-dose regions below 10 Gy (-2.3 ± 20.0%, p = 0.54). No further significant decrease compared to the post-treatment baseline was observed 6 months (-0.4 ± 18.4%, p = 0.94) and 9 months (2.0 ± 15.4%, p = 0.74) after the end of radiotherapy. Conclusions Perfusion decreased significantly during the course of radiochemotherapy. The decrease was higher in regions receiving a higher dose of radiation. This suggests that the perfusion decrease is at least partly caused by radiotherapy. Our results suggest that the detrimental effects of radiochemotherapy on perfusion occur early rather than later