IndraLab
Statements
rlimsp
"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."
rlimsp
"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)."
rlimsp
"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."
rlimsp
"This revealed that NPM1 phosphorylation at threonine-199 and threonine-234/237 was more prominent in the cytoplasm than in the nucleus in both cell lines. By contrast, the phosphorylation at serine-125 was more pronounced in the nucleus. The analysis of the phosphorylation of serine-4 delivered inconclusive results: it was less prominent in the cytoplasm of A549 cells compared to the nucleus while being nearly equally phosphorylated in nucleus and cytoplasm in HeLa cells."
rlimsp
"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."
rlimsp
"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."
rlimsp
"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."