B23 (or nucleophosmin, NPM) is a multifunctional protein involved in ribosome biogenesis, control of centrosome duplication and in sensing cellular stress. It is phosphorylated during interphase by casein kinase 2 (CK2) and during mitosis by cyclin-dependent kinase (CDK). In this study we have addressed the role of these phosphorylation events in the dynamics and location of protein B23. Mutation of the CK2 phosphorylation site to alanine results in slower recovery of the mutant compared with the wild-type protein as measured by fluorescence recovery after photobleaching (FRAP). Immunofluorescence studies using an antibody against phosphorylated Thr199 revealed that B23 is phosphorylated at this CDK1 site at the start of mitosis and is dephosphorylated during anaphase. The CDK1-type phosphorylation sites are in the nucleic acid binding region of B23 and may contribute to its dissociation from the nucleolus during mitosis. A Thr to Glu mutant of the CDK1-type sites as well as other members of the nucleoplasmin family that lack the C-terminal nucleic-acid-binding region showed a greater mobility and/or faster recovery than wild-type B23.1, the longer variant. These results provide evidence that phosphorylation at these sites reduces the affinity of B23 for nucleolar components and might be a factor in regulating its location during the cell cycle.

Ribosome biogenesis is a fundamental cellular process and is elevated in cancer cells. As one of the most energy consuming cellular processes, it is highly regulated by signaling pathways in response to changing cellular conditions. Many of the regulators of this process are aberrantly activated in various cancers. Recently two novel rRNA synthesis inhibitors, CX-5461 and BMH-21, have been shown to selectively kill cancer cells while sparing normal cells. Here, we tested the effectiveness of pre-rRNA synthesis inhibitor CX-5461 on acute lymphoblastic leukemia cells with different cytogenetic abnormalities. Acute lymphoblastic leukemia cells are more sensitive to rRNA synthesis inhibition compared to normal bone marrow cells. CX-5461 treated cells undergo caspase-dependent apoptosis independent of their p53 status. More-over, CX5461, activates checkpoint kinases and arrests cells in G2 phase of cell cycle. Finally, overcoming this G2 arrest by inhibiting ATR kinase leads to robust cell killing. These results show that CX-5461 can be even more potent in combination with ATR inhibitors.

Protein B23/nucleophosmin is a multifunctional protein that plays roles in ribosome biogenesis, control of centrosome duplication, and regulation of p53 expression. A yeast two-hybrid screen was performed in a search for interaction partners of B23. The complementary DNA for a highly acidic protein, nucleoplasmin 3 (NPM3), was found in multiple positive clones. Protein NPM3 and its interaction with B23 were further characterized. Endogenous B23 was able to be co-immunoprecipitated with NPM3, and this complex was resistant to ribonuclease treatment and high concentrations of salt. The N-terminal 35-90 amino acids of B23 were found to be required for their interaction. Separate co-immunoprecipitation studies of B23 and NPM3 suggested the existence of two different complexes, one containing B23 and 28 S ribosomal RNA (rRNA) and another composed of B23, NPM3, and other proteins, but no RNA. NPM3 was localized in the nucleolus, and its nucleolar localization depended on active rRNA transcription. In the cells overexpressing NPM3, there were decreased rates of pre-rRNA synthesis and processing. Overexpression of a mutant of NPM3 that did not interact with B23 did not alter pre-rRNA synthesis and processing, suggesting that the interaction of NPM3 with B23 plays a role in the ribosome biogenesis.Ribosome biogenesis in eukaryotic cells is a multistep process that takes place primarily in the nucleolus where the individual stages of assembly correlate with specific subclasses of ultrastructures (1-4). The process begins with transcription of the ribosomal DNA at the border between the fibrillar center and the dense fibrillar components of the nucleolus. The product of transcription, 47 S pre-ribosomal RNA (pre-rRNA) 1 in mammals, is processed into smaller pre-rRNA intermediates, which finally become 28, 5.8, and 18 S rRNA. The nascent pre-ribosomal particles of the dense fibrillar components eventually mature into granular components, with ribosomal proteins added at various steps in the process. Numerous nonribosomal proteins and small nucleolar RNA participate in these steps.Protein B23 (NPM1, nucleophosmin) is an abundant nucleolar non-ribosomal protein whose locations and multiple activities suggest it plays a role in ribosome biogenesis. This protein is primarily localized to the granular component region with lesser amounts in the dense fibrillar components of the nucleolus (5-8). The nucleolar localization of B23 is dependent on the presence of active rDNA transcription (9), and it is found in association with maturing pre-ribosomal RNP particles (10, 11). In vitro experiments indicate B23 has nucleic acid binding activity and ribonuclease activities (12-17). The nucleic acid binding activity has been mapped to its C-terminal end (17,18) and is believed to be important in its nucleolar localization (15).In support of this, a splicing variant, B23.2, in which the C-terminal 35-amino acid sequence is absent, exists both in the nucleoplasm and cytoplasm (15,17). More recent studies provide evidence for a direct role ...