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Nucleophosmin: A Ran/Crm1-associated Licensing Factor That Regulates Centrosome Duplication
The cellular components that regulate nucleocytoplasmic transport are also independently implicated in spindle assembly (Weis K. Cell 112: 441–51, 2003). This process involves import (importins α and β) and export (Crm1) receptors that bind to nuclear localization signals (NLS) or nuclear export signals (NES), respectively. Ran, a small GTPase, controls the interaction of these receptors with their substrates. A fraction of Ran, Crm1, and RanBP1 (a major regulator of Ran that promotes Crm1 dissociation from Ran) is found on centrosomes. Crm1 inactivation, either by a Crm1-specific inhibitor, leptomycin B (LMB), or hepatitis B virus (HBV) HBx protein interaction (via its NES motif), results in supernumerary centrosomes and multipolar spindles (Forgues M et al. Mol Cell Biol 23: 5282–92, 2003; Forgues M et al. J Biol Chem 276: 22797–803, 2001). This implies that Crm1 may function to prevent unscheduled centriole splitting during mitosis, thereby ensuring the formation of a bipolar spindle. Aneuploidy can be detected in livers chronically infected with HBV, a preneoplastic condition predisposing individuals to hepatocellular carcinoma. Multipolar spindles and mitotic abnormalities are also a consequence of Ran mutations or overexpression of RanBP1. Since Ran/Crm1 acts as a receptor to shuttle cellular proteins and interacts with many known cell cycle regulators, components of the Ran/Crm1 pathway likely function as licensing factors to ensure appropriate centrosome duplication during different stages of the cell cycle. Thus, one attractive model is that the Ran/Crm1 complex, through its NES binding activity, may ensure that proteins that regulate centrosome duplication are present at the correct location and time to safeguard the fidelity of this process. Nucleophosmin (NPM or B23) is a ubiquitously expressed phosphoprotein that mainly localizes in the nucleolus and shuttles between the nucleus and the cytoplasm during the cell cycle. NPM associates with unduplicated centrosomes and dissociates from centrosomes upon phosphorylation by CDK2/cyclin E, which coincides with the initiation of centrosome duplication and DNA replication. During mitosis, NPM reassociates with centrosomes. Since NPM has been implicated as a regulator of centrosome synthesis, we thought it plausible that NPM is a substrate for Ran/Crm1 to regulate centrosome duplication. This hypothesis was first tested by a heterokaryon assay to evaluate cytoplasmic-to-nuclear transport of NPM or an NPM NES mutant. In our study, the NES motif of NPM was shown to be functional and its local trafficking was mediated by a Ran/Crm1-dependent process. Since Crm1 and NPM could associate with centrosomes, we determined whether their localization required a functional NES motif. We demonstrated the association of NPM and Crm1 by immunofluorescence-based co-localization on centrosomes and co-fractionation from sucrose gradients to isolate centrosomes. Mutation of NPM NES or disruption of Crm1 function by LMB, RanBP1, or HBx led to NPM dissociation from centrosomes and initiation of premature centrosome duplication. Therefore, this process was mediated by the Ran/Crm1 pathway and required a functional NES motif. To examine the role of NPM in regulating centrosome duplication, we knocked down the expression of endogenous NPM with NPM siRNA. Consistently, NPM siRNA resulted in supernumerary centrosomes that could be nucleated in mitotic cells to form mitotic spindles, an activity that could be effectively inhibited by coexpression of NPM. Thus, loss of NPM is associated with centrosome reduplication. Furthermore, a novel proline-dependent kinase (PDK) phosphorylation site was identified at threonine residue (T95) within the NES motif of NPM. We tested whether phosphorylation at T95 could alter the NES property, thereby preventing NPM binding to Crm1 and docking on centrosomes. A mutant mimicking T95 phosphorylation displayed decreased centrosome binding and supernumerary centrosomes. Since this phosphorylation site is within the NPM NES, it may regulate the binding of NPM to Crm1. Taken together, these results suggest that proper centrosome duplication is mediated by NPM binding to centrosomes through the interaction of its NES motif with Crm1. These findings suggest that the Crm1/Ran complex may act as a “loading dock,” to spatially and temporally coordinate various checkpoint factors to regulate the fidelity of centrosome duplication during cell cycle progression. We have used genetic and biochemical approaches to demonstrate that NPM localization is mediated by a Ran/Crm1-dependent process and, through its NES-based interaction with Crm1, is a potential licensing factor for centrosome duplication (Figure 1). The disruption of such processes may lead to genomic instability and oncogenic acceleration. Figure 1. Ran/Crm1 functions as a loading dock to coordinate “licensing factors” that regulate centrosome duplication. The small GTPase, Ran, switches between an inactive GDP and an active GTP-bound state through interaction with RanBP1 and RCC1, respectively. During early G1 or mitosis, nucleophosmin (NPM) associates with centrosomes through its nuclear export signal (NES) interaction with the Ran/Crm1 complex, thus preventing centrosome reduplication. NPM is then phosphorylated and dissociates from the G1 centrosome upon activation of CDK2/cyclin E, or other kinases, to initiate centrosome duplication. NPM reassociates with mitotic centrosomes upon dephosphorylation. Thus, the Ran/Crm1 network serves as a loading lock to spatially and temporally coordinate NES-containing “licensing factors” that ensure the fidelity of the centrosome duplication process. |
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mammalian cells, centrosome duplication is a critical event that must be completed
before mitosis, the culminating stage of the cell cycle, during which a bipolar
mother cell partitions into two genetically equivalent daughter cells. Centrosomes
are the major microtubule organizing centers that direct bipolar spindle assembly
and balance mitotic chromosome segregation, controlling microtubule number,
nucleation, polarity, and orientation. This process, which begins at G