What is new in the nucleolus?: workshop on the nucleolus: new perspectives.
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Animals↗
Biomedical subjects
Mark O J Olson
Publications and source records attributed to Mark O J Olson.
Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23.
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.
Amino Acid Sequence↗
Protein NPM3 interacts with the multifunctional nucleolar protein B23/nucleophosmin and inhibits ribosome biogenesis.
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.
Amino Acid Sequence↗
Sensing cellular stress: another new function for the nucleolus?
Recent research suggests that the nucleolus communicates with the p53 regulatory system and acts as a stress sensor. Cellular stress causes nucleolar disruption, triggering the release of regulatory factors to the nucleoplasm. These factors, especially ARF (alternative reading frame), inhibit the degradation of p53, thereby facilitating its intracellular accumulation.
Animals↗
Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23.
Protein B23 is a multifunctional nucleolar protein whose molecular chaperone activity is proposed to play role in ribosome assembly. Previous studies (Szebeni, A., and Olson, M. O. J. (1999) Protein Sci. 8, 905-912) showed that protein B23 has several characteristics typical of molecular chaperones, including anti-aggregation activity, promoting the renaturation of denatured proteins, and preferential binding to denatured substrates. However, until now there has been no proposed mechanism for release of a bound substrate. Protein B23 can be phosphorylated by protein kinase CK2 (CK2) in a segment required for chaperone activity. The presence of bound substrate enhanced the rate of CK2 phosphorylation of protein B23 by 2-3-fold, and this enhancement was dependent on a nonpolar region in its N-terminal end. Formation of a complex between B23 and chaperone test substrates (rhodanese or citrate synthase) was inhibited by CK2 phosphorylation. Furthermore, CK2 phosphorylation of a previously formed B23-substrate complex promoted its dissociation. The dissociation of complexes between B23 and the human immunodeficiency virus-Rev protein required both CK2 phosphorylation and competition with a Rev nuclear localization signal peptide, suggesting that Rev binds B23 at two separate sites. These studies suggest that unlike many molecular chaperones, which directly hydrolyze ATP, substrate release by protein B23 is dependent on its phosphorylation by CK2.
Animals↗
Conventional and nonconventional roles of the nucleolus.
As the most prominent of subnuclear structures, the nucleolus has a well-established role in ribosomal subunit assembly. Additional nucleolar functions, not related to ribosome biogenesis, have been discovered within the last decade. Built around multiple copies of the genes for preribosomal RNA (rDNA), nucleolar structure is largely dependent on the process of ribosome assembly. The nucleolus is disassembled during mitosis at which time preribosomal RNA transcription and processing are suppressed; it is reassembled at the end of mitosis in part from components preserved from the previous cell cycle. Expression of preribosomal RNA (pre-rRNA) is regulated by the silencing of individual rDNA genes via alterations in chromatin structure or by controlling RNA polymerase I initiation complex formation. Preribosomal RNA processing and posttranscriptional modifications are guided by a multitude of small nucleolar RNAs. Nearly completed ribosomal subunits are exported to the cytoplasm by an established nuclear export system with the aid of specialized adapter molecules. Some preribosomal and nucleolar components are transiently localized in Cajal bodies, presumably for modification or assembly. The nonconventional functions of nucleolus include roles in viral infections, nuclear export, sequestration of regulatory molecules, modification of small RNAs, RNP assembly, and control of aging, although some of these functions are not well established. Additional progress in defining the mechanisms of each step in ribosome biogenesis as well as clarification of the precise role of the nucleolus in nonconventional activities is expected in the next decade.
Active Transport, Cell Nucleus↗