Specific phosphorylation of nucleophosmin on Thr(199) by cyclin-dependent kinase 2-cyclin E and its role in centrosome duplication.

The kinase activity of cyclin-dependent kinase 2 (CDK2)-cyclin E is required for centrosomes to initiate duplication. We have recently found that nucleophosmin (NPM/B23), a phosphoprotein primarily found in nucleolus, associates with unduplicated centrosomes and is a direct substrate of CDK2-cyclin E in centrosome duplication. Upon phosphorylation by CDK2-cyclin E, NPM/B23 ...
dissociates from centrosomes, which is a prerequisite step for centrosomes to initiate duplication. Here, we identified that threonine 199 (Thr(199)) of NPM/B23 is the major phosphorylation target site of CDK2-cyclin E in vitro, and the same site is phosphorylated in vivo. NPM/T199A, a nonphosphorylatable NPM/B23 substitution mutant (Thr(199) --> Ala) acts as dominant negative when expressed in cells, resulting in specific inhibition of centrosome duplication. As expected, NPM/T199A remains associated with the centrosomes. These observations provide direct evidence that the CDK2-cyclin E-mediated phosphorylation on Thr(199) determines association and dissociation of NPM/B23 to the centrosomes, which is a critical control for the centrosome to initiate duplication.
Mesh Terms:
3T3 Cells, Animals, CDC2-CDC28 Kinases, Cell Nucleus, Centrosome, Cyclin E, Cyclin-Dependent Kinase 2, Cyclin-Dependent Kinases, HeLa Cells, Humans, Kinetics, Mice, Nuclear Proteins, Phosphorylation, Protein-Serine-Threonine Kinases, Recombinant Proteins, Substrate Specificity, Threonine, Transfection
J. Biol. Chem.
Date: Jun. 15, 2001
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