IndraLab

"Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies. Phosphorylation-specific antibodies provide a powerful tool for analysing the regulation and activity of proteins in the MAP (mitogen-activated protein) kinase and other signalling pathways. Using synchronized cells, it was observed that phosphorylation-specific antibodies developed against the active form of MKK1/MKK2 (MAP kinase kinase-1 and -2) reacted with a protein that was approx. 35 kDa during G2/M-phase of the cell cycle. Failure of the 35 kDa protein to react with phosphorylation-independent MKK1/MKK2 antibodies suggested that this protein was not related to MKK1 or MKK2. Thus the 35 kDa protein was isolated by immunoprecipitation with the phospho-MKK1/MKK2 antibody and identified by MS. Peptide sequence analysis revealed matches with NPM (nucleophosmin/B23), a phosphoprotein involved in nucleolar assembly, centrosome duplication and ribosome assembly and transport. Biochemical and immunocytochemistry analyses verified that the phospho-MKK1/MKK2 antibodies cross-reacted with NPM that was phosphorylated at Thr234 and Thr237 during G2/M-phase, which are the same sites that are targeted by Cdc2 (cell division cycle protein-2) during mitosis. Using phosphorylation site mutants, we show that phosphorylation of Thr234 and Thr237 is required for NPM immunoreactivity with the phospho-MKK1/MKK2 antibody. Moreover, phosphorylation of Thr234 and Thr237 was demonstrated to regulate NPM localization to the centrosome after nuclear envelope breakdown in mitotic cells."

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"Nucleoplasmic NPM1 displays the particularity to be phosphorylated [33], we next asked if the two phosphorylated forms of NPM1, Thr199 and Thr234/237, could colocalize with AR."

"CDK-1 mediated phosphorylation of NPM1 at T199, T219, T234 and T237 has been shown to play a critical role in mitosis [42] ."

"Biochemical and immunocytochemistry analyses verified that the phospho-MKK1/MKK2 antibodies cross-reacted with NPM that was phosphorylated at Thr234 and Thr237 during G2/M-phase, which are the same sites that are targeted by Cdc2 (cell division cycle protein-2) during mitosis."

"E. Quantification of cells displaying strong staining of phosphorylated Thr199 or Thr234/237 NPM1."

"Altogether, these results suggest that N6L inhibits NPM1 phosphorylations on Thr 199 and Thr 234/237 and AR activity."

"Using phalloidin staining, we found that stress fiber formation was enhanced in NPM stable transfectants, while its effect was abolished in its phosphorylation site mutant (Thr234/237A) (Figure 5F)."

"Phosphorylation of nucleophosmin at threonine 234/237 is associated with HCC metastasis."

"By overexpressing Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) into low p-NPM-Thr234/237 expressing cells (Hep3B and Huh7) using a lentiviral based approach, we demonstrated that p-NPM-Thr234/237 is critical in invasion and migration of HCC cells, and this effect was mediated by cyclin-dependent kinase 1 (CDK1)."

"HeLa cells depleted of either PP1 isoform as well as control were UV irradiated (Supplemental Figure S3A), and the level of phosphorylation of NPM on Thr199 or Thr234/237 was measured at 0, 3, and 6 h after treatment."

"Our findings reveal the role of Thr199 and Thr234/237 phosphorylated NPM1 in PCa progression and define N6L as a new drug candidate for PCa therapy."

"Upon analysis, we found phosphorylated level of nucleophosmin (NPM) at Threonine 234/237 (p-NPM-Thr234/237) had remarkably high level in metastatic HCC cells (PLC-LM) than the corresponding primary HCC cell line (PLC-PT). Similar observation was observed in another match primary and their metastatic counterparts (MHCC-97L and MHCC-97H). By immunohistochemical staining, p-NPM-Thr234/237 was consistently found to be preferentially expressed in metastatic HCCs when compared with primary HCC in 28 HCC cases (p < 0.0001). By overexpressing Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) into low p-NPM-Thr234/237 expressing cells (Hep3B and Huh7) using a lentiviral based approach, we demonstrated that p-NPM-Thr234/237 is critical in invasion and migration of HCC cells, and this effect was mediated by cyclin-dependent kinase 1 (CDK1). Wild-type NPM was found to physically interact with a metastatic gene, ROCK2, and defective in Thr234/237 phosphorylation decreased its binding affinity, resulting in decrease in ROCK2 mediated signaling pathway."

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"Using CelluSpot™ Serine/Threonine kinase peptide array analysis, we compared the phosphorylation profiling of the two matched HCC cell lines and found phosphorylated level of nucleophosmin (NPM) at Threonine 234/237 (p-NPM-Thr234/237) had remarkably high level in metastatic HCC cells (PLC-LM) than the corresponding primary HCC cell line (PLC-PT)."

"Accompanied with the reduction in CDK1 expression, p-NPM-Thr234/237 was consistently suppressed in two shCDK1 clones when compared to NTC control, which suggesting that CDK1 is upstream kinase leading to NPM phosphorylation at Thr234 and Thr237 (Figure 4B)."

"Phosphorylation of Nucleophosmin at Threonine 234/237 is associated with HCC metastasis."

"To further investigate whether NPM phosphorylation regulates cell motility and cancer invasion through modulation of actin stress fiber, we examined stress fiber formation and polymerized actin in EV, Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) of Hep3B cells."

"Accompanied with the reduction in CDK1 expression, p-NPM-Thr 234/237 was consistently suppressed in two shCDK1 clones when compared to NTC control, which suggesting that CDK1 is upstream kinase leading to NPM phosphorylation at Thr234 and Thr237 (Figure xref )."

"Since NPM is phosphorylated on Thr234 and Thr237 by cyclin-dependent kinase (CDK) 1/cyclinB [ xref ], we tested whether knockdown of CDK1 had a role in the regulation of p-NPM-Thr 234/237 mediated HCC metastasis."