Nucleophosmin Protein Dephosphorylation by DUSP3 Is a Fine-Tuning Regulator of p53 Signaling to Maintain Genomic Stability.

The dual-specificity phosphatase 3 (DUSP3), an atypical protein tyrosine phosphatase (PTP), regulates cell cycle checkpoints and DNA repair pathways under conditions of genotoxic stress. DUSP3 interacts with the nucleophosmin protein (NPM) in the cell nucleus after UV-radiation, implying a potential role for this interaction in mechanisms of genomic stability. Here, we show a high-affinity binding between DUSP3-NPM and NPM tyrosine phosphorylation after UV stress, which is increased in DUSP3 knockdown cells. Specific antibodies designed to the four phosphorylated NPM's tyrosines revealed that DUSP3 dephosphorylates Y29, Y67, and Y271 after UV-radiation. DUSP3 knockdown causes early nucleolus exit of NPM and ARF proteins allowing them to disrupt the HDM2-p53 interaction in the nucleoplasm after UV-stress. The anticipated p53 release from proteasome degradation increased p53-Ser15 phosphorylation, prolonged p53 half-life, and enhanced p53 transcriptional activity. The regular dephosphorylation of NPM's tyrosines by DUSP3 balances the p53 functioning and favors the repair of UV-promoted DNA lesions needed for the maintenance of genomic stability.

Loss of DUSP3 activity radiosensitizes human tumor cell lines via attenuation of DNA repair pathways.

BACKGROUND: Radiotherapy causes the regression of many human tumors by increasing DNA damage, and the novel molecular mechanisms underlying the genomic instability leading to cancer progression and metastasis must be elucidated. Atypical dual-specificity phosphatase 3 (DUSP3) has been shown to down-regulate mitogen-activated protein kinases (MAPKs) to control the proliferation and apoptosis of human cancer cells. We have recently identified novel molecular targets of DUSP3 that function in DNA damage response and repair; however, whether DUSP3 affects these processes remains unknown. METHODS: Tumor cell lines in which DUSP3 activity was suppressed by pharmacological inhibitors or a targeted siRNA were exposed to gamma radiation, and proliferation, survival, DNA strand breaks and recombination repair pathways were sequentially analyzed. RESULTS: The combination of reduced DUSP3 activity and gamma irradiation resulted in decreased cellular proliferation and survival and increased cellular senescence compared with the effects of radiation exposure alone. Gamma radiation-induced DNA damage was increased by the loss of DUSP3 activity and correlated with increased levels of phospho-H2AX protein and numbers of ionizing radiation-induced γ-H2AX foci, which were reflected in diminished efficiencies of homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. Similar results were obtained in ATM-deficient cells, in which reduced DUSP3 activity increased radiosensitivity, independent of increased MAPK phosphorylation. CONCLUSION: The loss of DUSP3 activity markedly increases gamma radiation-induced DNA strand breaks, suggesting a potential novel role for DUSP3 in DNA repair. GENERAL SIGNIFICANCE: The radioresistance of tumor cells is effectively reduced by a combination of approaches through the inhibition of DUSPs.