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...

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The E2F family of transcription factors is critical for the control of cell cycle progression. We now show that the specific inactivation of E2F3 in mouse embryo fibroblasts (MEFs) results in a disruption of the centrosome duplication cycle. Loss of E2F3, but not E2F1, E2F2, E2F4, or E2F5 results in unregulated cyclin E-dependent kinase activity, defects in nucleophosmin B association with centrosomes, and premature centriole separation and duplication. Consequently, this defect leads to centrosome amplification, mitotic spindle defects, and aneuploidy. Our findings implicate the E2F3 transcription factor as an important link that orchestrates DNA and centrosome duplication cycles, ensuring the faithful transmission of genetic material to daughter cells.

The serotonin neurons of the dorsal and medial raphe nuclei project to all areas of the forebrain and play a key role in mood disorders. Hence, any loss or degeneration of serotonin neurons could have profound ramifications. In a monkey model of surgical menopause with hormone replacement and no neural injury, E and P decreased gene expression in the dorsal raphe nucleus of c-jun n-terminal kinase (JNK1) and kynurenin mono-oxygenase (KMO) that promote cell death. In concert, E and P increased gene expression of superoxide dismutase (SOD1), VEGF, and caspase inhibitory proteins that promote cellular resilience in the dorsal raphe nucleus. Subsequently, we showed that ovarian steroids inhibit pivotal genes in the caspase-dependent and caspase-independent pathways in laser captured serotonin neurons including apoptosis activating factor (Apaf1), apoptosis inducing factor (AIF) and second mitochondria-derived activator of caspases (Smac/Diablo). SOD1 was also increased specifically in laser captured serotonin neurons. Examination of protein expression in the dorsal raphe block revealed that JNK1, phosphoJNK1, AIF and the translocation of AIF from the mitochondria to the nucleus decreased with hormone therapy, whereas pivotal execution proteins in the caspase pathway were unchanged. In addition, cyclins A, B, D1 and E were inhibited, which would prevent re-entry into the cell cycle and catastrophic death. These data indicated that in the absence of gross injury to the midbrain, ovarian steroids inhibit the caspase-independent pathway and cell cycle initiation in serotonin neurons. To determine if these molecular actions prevented cellular vulnerability or death, we examined DNA fragmentation in the dorsal raphe nucleus with the TUNEL assay (terminal deoxynucleotidyl transferase nick end labeling). Ovarian steroids significantly decreased the number of TUNEL positive cells in the dorsal raphe. Moreover, TUNEL staining prominently colocalized with TPH immunostaining, a marker for serotonin neurons.In summary, ovarian steroids increase the cellular resilience of serotonin neurons and may prevent serotonin neuron death in women facing decades of life after menopause. The survival of serotonin neurons would support cognition and mental health.