IndraLab

"NPM phosphorylation at Ser4 and Thr199 may play a role in maintaining or generating the structure of the nucleolus."

"Thr199 and Ser4 of NPM are phosphorylated by G2/M checkpoint kinase CDK1 xref and polo-like kinase PLK1 xref , respectively."

"An in vitro kinase assay showed that phosphorylated-NPM at Thr199 by CDK1 enhanced NPM phosphorylation at Ser4 by PLK1 as revealed by Western blotting with the phospho-specific NPM antibodies ( xref )."

"Other antibodies used include: Mouse monoclonal anti-p53 (DO-1) and either a rabbit or goat polyclonal anti-HA (HA-probe (Y-11); Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-actin (Ab-1; Calbiochem, La Jolla, CA, USA); mouse monoclonal anti-NPM (mouse anti-Nucleophosmin (FC-61991); Invitrogen, Carlsbad, CA, USA); rabbit monoclonal anti-pNPM(S4) (Phospho-NPM (Ser4) (D19C1) XP® Rabbit mAb), rabbit polyclonal anti-pNPM(T199) (Phospho-NPM (Thr199) Antibody), rabbit monoclonal anti-pCDC25C(S216) (Phospho-CDC25C (Ser216)(63F9) Rabbit mAb) and rabbit monoclonal anti-CDC25C (cdc25C (5H9) rabbit mAb; all from Cell Signaling Technology, Beverly, MA, USA); and rat monoclonal anti-HA (Anti-HA affinity clone3F10; Roche Applied Science, Indianapolis, IN, USA)."

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"These results suggest that phosphorylation of NPM at Ser4 and Thr199 plays a role in determination of nucleolar number."

"Furthermore, we show for the first time that sequential phosphorylation of NPM at Thr199 and Ser4 is important for regulating nucleolar formation."

"These results suggest that phosphorylation of NPM at Ser4 and Thr199 plays a role in determination of nucleolar number."

"B23/nucleophosmin serine 4 phosphorylation mediates mitotic functions of polo-like kinase 1."

"(f) These results suggest that sequential phosphorylation of NPM at Thr199 and Ser4 by a novel signalling cascade, PPM1D-CDC25C-CDK1-PLK1, influences nucleolar formation."

"Polo-like kinase 2-dependent phosphorylation of NPM/B23 on serine 4 triggers centriole duplication."

"(d) Decrease of phosphorylated-NPM at Thr199 and Ser4 by CDC25 inhibitor in MCF-7."

"Furthermore, CDC25 inhibitor decreased the phosphorylation level of NPM at Thr199 and Ser4 (Fig. 5d)."

"Phosphoprotein profiling by Kinetworks trade mark analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue. Consistent with the resemblance of the Ser-4 phosphorylation site to the Polo-like kinase 1 (Plk1) consensus recognition sequence, inhibition of Plk1 by a kinase-defective mutation (K82M) abrogated B23 Ser-4 phosphorylation, whereas activation of Plk1 by a constitutively active mutation (T210D) enhanced its phosphorylation following in vivo transfection and in vitro phosphorylation assays."

"Based on phosphopeptide profiling, we identified three additional phosphorylation sites on NPM (Ser4, Ser70, and Ser125; Supplemental Table S2 and Supplemental Figure S3E)."

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"We also demonstrate for the first time that sequential phosphorylation of NPM on Thr199 and Ser4 by a novel PPM1D-CDC25C-CDK1-PLK1 pathway regulates nucleolar formation."

"(e) PLK1 binds to Thr199-phosphorylated NPM through its PBD and induces phosphorylation of Ser4 on the same NPM molecule. It is suggested that phosphorylation of Thr199 of NPM by CDK1 enhances phosphorylation of Ser4 of NPM by PLK1."

"Phosphoprotein profiling by Kinetworks trade mark analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue. Consistent with the resemblance of the Ser-4 phosphorylation site to the Polo-like kinase 1 (Plk1) consensus recognition sequence, inhibition of Plk1 by a kinase-defective mutation (K82M) abrogated B23 Ser-4 phosphorylation, whereas activation of Plk1 by a constitutively active mutation (T210D) enhanced its phosphorylation following in vivo transfection and in vitro phosphorylation assays. Depletion of endogenous Plk1 by RNA interference abolished B23 Ser-4 phosphorylation."

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"Phosphoprotein profiling by Kinetworks trade mark analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue. Consistent with the resemblance of the Ser-4 phosphorylation site to the Polo-like kinase 1 (Plk1) consensus recognition sequence, inhibition of Plk1 by a kinase-defective mutation (K82M) abrogated B23 Ser-4 phosphorylation, whereas activation of Plk1 by a constitutively active mutation (T210D) enhanced its phosphorylation following in vivo transfection and in vitro phosphorylation assays. Depletion of endogenous Plk1 by RNA interference abolished B23 Ser-4 phosphorylation. The physical interaction of Plk1 and B23 was further demonstrated by their co-immunoprecipitation and glutathione S-transferase fusion protein pull-down assays. Interference of Ser-4 phosphorylation of B23 induced multiple mitotic defects in HeLa cells, including aberrant numbers of centrosomes, elongation and fragmentation of nuclei, and incomplete cytokinesis."

"NPM phosphorylation at Ser4 and Thr199 may play a role in maintaining or generating the structure of the nucleolus."

"NPM phosphorylation at Ser4 and Thr199 may play a role in maintaining or generating the structure of the nucleolus. To investigate the effect of the phosphorylation status at these NPM sites on the nucleolar number, we expressed HA-tagged NPM mutants in MCF-7 subsequent to endo-NPM knockdown and co-stained with NPM antibody and HA antibody (Fig. S3). All mutants localized to the nucleolus (Fig. S3). The cells with HA-NPM(WT) had an average of 4.5 nucleoli in one cell (Fig. 4, Table 4). The cells transfected with S4A or T199A mutants showed a decreased nucleolar number of 4.0. The cells transfected with NPM double mutant (S4A/T199A) exhibited a similar number of nucleoli with NPM(S4A) and NPM(T199A). The cells transfected with S4D or T199E phosphor-mimic mutants showed same number of nucleoli as WT. These results suggest that phosphorylation of NPM at Ser4 and Thr199 plays a role in determination of nucleolar number."

"Phosphoprotein profiling by Kinetworks trade mark analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue."

"PPM1D knockdown caused a decrease of phosphorylated-NPM at Thr199 and a significant decrease of phosphorylated-NPM at Ser4 (Fig. 3)."

"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."

"We also demonstrated for the first time that PPM1D up-regulated phosphorylation of Thr199 and Ser4 of NPM sequentially via the CDC25C-CDK1-PLK1 signalling cascade thereby modulating nucleolar formation (Fig. 6)."

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