Nucleophosmin protein expression level, but not threonine 198 phosphorylation, is essential in growth and proliferation.
Nucleophosmin (NPM), an oligomeric phosphoprotein and nucleolar target of the ARF tumor suppressor, contributes to several critical cellular processes. Previous studies have shown that the human NPM's phosphorylation by cyclin E-cyclin-dependent kinase 2 (cdk2) on threonine (Thr) 199 regulates its translocation from the centrosome during cell cycle progression. Given our previous finding that ARF directly binds NPM, impeding its transit to the cytoplasm and arresting cells before S-phase entry, we hypothesized that ARF might also inhibit NPM phosphorylation. However, ARF induction did not impair phosphorylation of the cdk2 target residue in murine NPM, Thr198. Furthermore, phosphorylation of Thr198 occurred throughout the cell cycle and was concomitant with increases in overall NPM expression. To investigate the cell's presumed requirement for NPM-Thr198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference. Here, we show that the T198A mutant is fully capable of executing NPM's described roles in nucleocytoplasmic shuttling, ribosome export and cell cycle progression. Moreover, the proliferative defects observed with stable NPM knockdown were restored by mutant NPM-T198A expression. Thus, we demonstrate that the reduction in NPM protein expression blocks cellular growth and proliferation, whereas phosphorylation of NPM-Thr198 is not essential for NPM's capacity to drive cell cycle progression and proliferation.
Brady
S N
SN
Department of Internal Medicine, Division of Molecular Oncology, Siteman Cancer Center, St Louis, MO, USA.
Maggi
L B
LB
Winkeler
C L
CL
Toso
E A
EA
Gwinn
A S
AS
Pelletier
C L
CL
Weber
J D
JD
D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000090243:Nucleophosmin; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D000090243:Nucleophosmin; D049452:Green Fluorescent Proteins; D013912:Threonine; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2