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]