文字方塊: Mkk1 



 Phosphorylation specific antibodies provide a powerful tool for analyzing the regulation and activity of proteins in the mitogen activated protein (MAP) kinase and other signaling pathways. Using synchronized cells, phosphorylation specific antibodies developed against the active form of MAP kinase kinase-1 and 2 (MKK1/2) reacted with a protein that was approximately 35kDa during G2/M-phase of the cell cycle. Failure of the 35 kDa protein to react with phosphorylation-independent MKK1/2 antibodies suggested that this protein was not related to MKK1 or 2. Thus, the 35kDa protein was isolated by immunoprecipitation with the phospho-MKK1/2 antibody and identified by mass spectrometry. Peptide sequence analysis revealed matches with nucleophosmin/B23 (NPM), a phosphoprotein involved in nucleolar assembly, centrosome duplication, and ribosome assembly and transport. Biochemical and immunocytochemistry analyses verified that the phospho-MKK1/2 antibodies cross-reacted with NPM that was phosphorylated on threonine residues 234 and 237 (T234/237) during G2/M-phase, which are the same sites that are targeted by the mitotic Cdc2 kinase. Using phosphorylation site mutants, we show that phosphorylation of T234/237 is required for NPM immunoreactivity with the phospho-MKK1/2 antibody. Moreover, T234/237 phosphorylation was demonstrated to regulate NPM localization to the centrosome after nuclear envelope breakdown in mitotic cells. These findings reveal new insight into the role of phosphorylation in regulating NPM targeting during mitosis. However, caution should be used when using commercially available phospho-MKK1/2 antibodies to examine regulation of MKK1/2 during mitotic transitions due to their cross-reactivity with phosphorylated NPM at this time of the cell cycle.

PMID: 14670079 [PubMed - as supplied by publisher]