NPM/B23:THE EFFECTOR OF CDK2 IN THE CONTROL OF CENTROSOME DUPLICATION AND mRNA PROCESSING
Nucleophosmin (NPM/B23) is a phosphoprotein predominantly localized in the nucleolus, and is believed to function in ribosome assembly, cytonuclear shuttling, and molecular chaperoning. Here we report novel functions of NPM/B23 in initiation of centrosome duplication and mRNA processing. The centrosome, a major microtubule organizing center of cells, directs the formation of bipolar mitotic spindles, which is essential for proper chromosome segregation to daughter cells. Cancer cells frequently contain abnormal numbers of centrosomes suggesting that centrosome amplification is the major contributing factor for chromosome instability. It has been shown that cyclin-dependent kinase 2 (CDK2) activity is required for initiation of centrosome duplication. NPM/B23 is a substrate of CDK2/Cyclin E in initiation of centrosome duplication. Here we identified that threonine 199 (Thr199) of NPM/B23 is the major phosphorylation site of CDK2/Cyclin E. NPM/B23 associates with single centrosomes and upon phosphorylation by CDK2/Cyclin E, NPM/B23 is lost from the centrosomes resulting in initiation of centrosome duplication. A non-phosphorylatable mutant of NPM/B23 (NPM/T199A) acts as a dominant negative, resulting in inhibition of centrosome duplication. Thus, NPM/B23 is a key regulator in initiation of centrosome duplication. We further examined the role of CDK2/Cyclin E phosphorylation of NPM/B23 on Thr199 (phospho-Thr199 NPM). We found that phospho-Thr199 NPM specifically localizes to nuclear structures known as nuclear speckles, which consist of pre-mRNA splicing factors. We found that phosphorylation on Thr199 of NPM/B23 enhances NPM/B23’s RNA binding activity. Also, non-phosphorylated NPM/B23 binds with specific splicing factors, whereas phosphorylation of NPM/B23 decreases these interactions. Moreover, we found that phosphorylation of NPM/B23 on Thr199 represses pre-mRNA splicing. These findings suggest an interesting connection between cell cycle progression and mRNA processing. We also found NPM/B23 to be ubiquitinated. NPM/B23 is maximally ubiquitinated at the G1/S transition of the cell cycle, when CDK2/Cyclin E is also maximally active. Furthermore, phosphorylation on Thr199 targets NPM/B23 for ubiquitination. Studies have shown that ubiquitin ligases such as SCF and BRCA1-BARD1, localize to centrosomes, and ubiquitin-mediated proteolysis plays an important role in centrosome duplication. Thus, it is possible that ubiquitin modification of NPM/B23 is a key event in the centrosome duplication cycle.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:cdk2 centrosome duplication genomic instability pre mrna splicing nuclear speckles
Date of Publication:01/01/2004