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.
Negi
Sandeep S
SS
Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Olson
Mark O J
MO
D000595:Amino Acid Sequence; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism; D002466:Cell Nucleolus / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000090243:Nucleophosmin; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D000090243:Nucleophosmin; D047390:Casein Kinase II; D018844:Cyclin-Dependent Kinases