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Nucleophosmin protein expression level, but not threonine 198 phosphorylation, is essential in growth and proliferation

Oncogene volume 28, pages 3209–3220 (2009)Cite this article

Abstract

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.

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Acknowledgements

We are indebted to Sheila Stewart, Gregory Longmore, J Alan Diehl, Martine Roussel, Charles Sherr and Gerard Zambetti for gifts of plasmid constructs, antibodies and primary TKO MEFs. In addition, we would like to thank Sheila Stewart, Helen Piwnica-Worms, Michael Tomasson and John Majors for insightful discussions throughout the course of this study.

SNB was supported by the Cancer Biology Pathway. CLP was a trainee in the Lucille P Markey Special Emphasis Pathway in Human Pathobiology. JDW was funded through the National Institutes of Health and Department of Defense Era of Hope Scholar Award in Breast Cancer Research.

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  1. S N Brady and L B Maggi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine, Division of Molecular Oncology, Siteman Cancer Center, St Louis, MO, USA

    S N Brady, L B Maggi Jr, C L Winkeler, E A Toso, A S Gwinn, C L Pelletier & J D Weber

  2. Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, USA

    J D Weber

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  1. S N Brady
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Correspondence to J D Weber.

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Brady, S., Maggi, L., Winkeler, C. et al. Nucleophosmin protein expression level, but not threonine 198 phosphorylation, is essential in growth and proliferation. Oncogene 28, 3209–3220 (2009). https://doi.org/10.1038/onc.2009.178

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