Specific Phosphorylation of Nucleophosmin on Thr¹⁹⁹ 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 pre-requisite step for centrosomes to initiate duplication. Here, we identified that threonine 199 (Thr¹⁹⁹) 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 (Thr¹⁹⁹ → 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 Thr¹⁹⁹ determines association and dissociation of NPM/B23 to the centrosomes, which is a critical control for the centrosome to initiate duplication.