Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies

被引:41
作者
Cha, H
Hancock, C
Dangi, S
Maiguel, D
Carrier, F
Shapiro, P [1 ]
机构
[1] Univ Maryland, Sch Pharm, Dept Pharmaceut Sci, Baltimore, MD 21201 USA
[2] Univ Maryland, Sch Med, Dept Biochem, Baltimore, MD 21201 USA
关键词
cell cycle; MAP kinase (mitogen-activated protein kinase kinase); MKK (MAP kinase kinase); mitosis; nucleophosmin; phosphorylation;
D O I
10.1042/BJ20031173
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 G(2)/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 Thr(234) and Thr(231) during G(2)/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 Tbr(234) and Thr(237) is required for NPM immunoreactivity with the phospho-MKK1/MKK2 antibody. Moreover, phosphorylation of Thr(234) and Thr(211) 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.
引用
收藏
页码:857 / 865
页数:9
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