Nucleostemin stabilizes ARF by inhibiting the ubiquitin ligase ULF.
Upregulated expression of nucleolar GTPase nucleostemin (NS) has been associated with increased cellular proliferation potential and tumor malignancy during cancer development. Recent reports attribute the growth regulatory effects of NS protein to its role in facilitating ribosome production. However, the oncogenic potential of NS remains unclear, as imbalanced levels of NS have been reported to exert growth inhibitory effect by modulating p53 tumor-suppressor activity. It also remains in questions if aberrant NS levels might have a p53-independent role in regulation of cell proliferation and growth. In this study, we performed affinity purification and mass spectrometry analysis to explore protein-protein interactions influencing NS growth regulatory properties independently of p53 tumor suppressor. We identified the alternative reading frame (ARF) protein as a key protein associating with NS and further verified the interaction through in vitro and in vivo assays. We demonstrated that NS is able to regulate cell cycle progression by regulating the stability of the ARF tumor suppressor. Furthermore, overexpression of NS suppressed ARF polyubiquitination by its E3 ligase Ubiquitin Ligase for ARF and elongated its half-life, whereas knockdown of NS led to the decrease of ARF levels. Also, we found that NS can enhance NPM stabilization of ARF. Thus, we propose that in the absence of p53, ARF can be stabilized by NS and nucleophosmin to serve as an alternative tumor-suppressor surveillance, preventing potential cellular transformation resulting from the growth-inducing effects of NS overexpression.
Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, LA, USA.
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Lo
Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, LA, USA.
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Zhang
Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA.
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MS
Dai
Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA.
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Sun
Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, LA, USA.
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Zeng
Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, LA, USA.
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Lu
D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D045744:Cell Line, Tumor; D059585:G1 Phase Cell Cycle Checkpoints; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000502:physiology; D025803:Tumor Suppressor Protein p14ARF / Q000737:chemistry / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors*
D002352:Carrier Proteins; C469949:GNL3 protein, human; D009687:Nuclear Proteins; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; C530813:TRIP12 protein, human; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases; D019204:GTP-Binding Proteins