Specific phosphorylation of nucleophosmin on Thr(199) by cyclin-dependent kinase 2-cyclin E and its role in centrosome duplication.
The kinase activity of cyclin-dependent kinase 2 (CDK2)-cyclin E is required for centrosomes to initiate duplication. We have recently found that nucleophosmin (NPM/B23), a phosphoprotein primarily found in nucleolus, associates with unduplicated centrosomes and is a direct substrate of CDK2-cyclin E in centrosome duplication. Upon phosphorylation by CDK2-cyclin E, NPM/B23 dissociates from centrosomes, which is a prerequisite step for centrosomes to initiate duplication. Here, we identified that threonine 199 (Thr(199)) of NPM/B23 is the major phosphorylation target site of CDK2-cyclin E in vitro, and the same site is phosphorylated in vivo. NPM/T199A, a nonphosphorylatable NPM/B23 substitution mutant (Thr(199) --> Ala) acts as dominant negative when expressed in cells, resulting in specific inhibition of centrosome duplication. As expected, NPM/T199A remains associated with the centrosomes. These observations provide direct evidence that the CDK2-cyclin E-mediated phosphorylation on Thr(199) determines association and dissociation of NPM/B23 to the centrosomes, which is a critical control for the centrosome to initiate duplication.
Tokuyama
Y
Y
Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521, USA.
Horn
H F
HF
Kawamura
K
K
Tarapore
P
P
Fukasawa
K
K
D016475:3T3 Cells; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002467:Cell Nucleus / Q000378:metabolism; D018385:Centrosome / Q000502:physiology*; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000090243:Nucleophosmin; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine; D014162:Transfection
D019927:Cyclin E; C000716967:NPM1 protein, human; C000715570:Npm1 protein, mouse; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000090243:Nucleophosmin; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases