title: Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies.
abstract: 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. These findings reveal a new insight into the role of phosphorylation in regulating NPM targeting during mitosis. However, caution should be used when using commercially available phospho-MKK1/MKK2 antibodies to examine the regulation of MKK1/MKK2 during mitotic transitions, owing to their cross-reactivity with phosphorylated NPM at this time of the cell cycle.
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.
authors.forename: Hyukjin
authors.initials: H
authors.lastname: Cha
authors.forename: Chad
authors.initials: C
authors.lastname: Hancock
authors.forename: Surabhi
authors.initials: S
authors.lastname: Dangi
authors.forename: Dony
authors.initials: D
authors.lastname: Maiguel
authors.forename: France
authors.initials: F
authors.lastname: Carrier
authors.forename: Paul
authors.initials: P
authors.lastname: Shapiro
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle; D002460:Cell Line; D018385:Centrosome / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D003429:Cross Reactions; D006801:Humans; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000276:immunology* / Q000378:metabolism; D008938:Mitosis; D009687:Nuclear Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D000090243:Nucleophosmin; D010766:Phosphorylation; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism
chemical_list: C000716967:NPM1 protein, human; C000715570:Npm1 protein, mouse; D009687:Nuclear Proteins; D000090243:Nucleophosmin; C482128:MAP2K2 protein, human; D011505:Protein-Tyrosine Kinases; D016203:CDC2 Protein Kinase; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases