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 require d for centrosomes to initiate duplication. We have recently found that nucl eophosmin (NPM/B23), a phosphoprotein primarily found in nucleolus, associa tes 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 vi tro, and the same site is phosphorylated in vivo. NPM/T199A, a nonphosphory latable NPM/B23 substitution mutant (Thr(199) --> Ala) acts as dominant neg ative when expressed in cells, resulting in specific inhibition of centroso me duplication. As expected, NPM/T199A remains associated with the centroso mes. These observations provide direct evidence that the CDK2 cyclin E-medi ated 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.