In animal cells, duplication of centrosomes and DNA is coordinated. Since CDK2/cyclin E triggers initiation of both events, activation of CDK2/cyclin E is thought to link these two events. We identified nucleophosmin (NPM/B23) as a substrate of CDK2/cyclin E in centrosome duplication. NPM/B23 associates specifically with unduplicated centrosomes, and NPM/B23 dissociates from centrosomes by CDK2/cyclin E-mediated phosphorylation. An anti-NPM/B23 antibody, which blocks this phosphorylation, suppresses the initiation of centrosome duplication in vivo. Moreover, expression of a nonphosphorylatable mutant NPM/ B23 in cells effectively blocks centrosome duplication. Thus, NPM/B23 is a target of CDK2/cyclin E in the initiation of 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 3 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.The centrosome, a major microtubule-organizing center of the animal cells, directs the formation of bipolar mitotic spindles, which is essential for accurate chromosome segregation to daughter cells (for reviews, see Refs. 1-3). Since each daughter cell inherits one centrosome upon cytokinesis, the centrosome must duplicate prior to the next mitosis and do so only once. Thus, centrosome duplication must take place in coordination with other cell cycle events including DNA synthesis. In mammalian cells, the centriole, the core component of the centrosome, initiates duplication at the G 1 /S boundary (reviewed in Refs. 4 -6). Activation of cyclin-dependent kinase 2 (CDK2) 1 -cyclin E has recently been found to be essential for the centrosome to initiate duplication (7,8). The activity of CDK2-cyclin E is regulated by the temporal expression of cyclin E, which normally occurs in late G 1 (9, 10), and it has been known that active CDK2-cyclin E complexes are required for initiation of DNA replication (11, 12). These observations indicate that the late G 1 -specific activation of CDK2-cyclin E plays a key role for the coordinated initiation of centrosome and DNA duplication. Indeed, we have shown that constitutive activation of CDK2-cyclin E by cyclin E overexpression in cultured mammalian cells results in uncoupling of the initiation of centrosome and DNA duplication; in these cells, the centrosomes initiate duplication in early G 1 long before the onset of DNA synthesis (13). Unlike the initiation of DNA synthesis, which can only be triggered by CDK2-cyclin E after completion of a series of necessary events (14, 15), the initiation of centrosome duplication appears to depend primarily on the activation of CDK2-cyclin E. Thus, the late G 1 -specific activation of CDK2-cyclin E may serve as a checkpoint control for timely initiation of centrosome duplication.We have recently identified nucle...

We have previously reported that loss of p53 tumor suppressor protein results in centrosome hyperampli®ca-tion, which leads to aberrant mitosis and chromosome instability. Since p53 is either deleted or mutated in human cancers at a high frequency, we investigated whether human cancers showed centrosome hyperampli®cation. Screening of advanced stage breast ductal carcinomas and squamous cell carcinomas of the head and neck (SCCHN) revealed that centrosome hyperampli®cation is frequent in both tumor types. Moreover, through the analyses of p53 in SCCHN samples by direct sequencing and by loss-of-heterozygosity test, we found that p53 mutations correlated with occurrence of centrosome hyperampli®cation. However, in some cases, we observed centrosome hyperampli®cation in tumors that retained wild-type p53. These tumors contained high levels of Mdm2. Since Mdm2 can inactivate p53 through physical association, we investigated whether Mdm2 overexpression induced centrosome hyperampli®cation. We found that Mdm2 overexpression, like loss of p53, induced centrosome hyperampli®cation and chromosome instability in cultured cells.

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