ABSTRACT
Retinoblastoma protein (RB) plays critical roles in tumor suppression and is degraded through the proteasomal pathway. However, E3 ubiquitin ligases responsible for proteasome-mediated degradation of RB are largely unknown. Here we characterize a novel RB E3 ubiquitin ligase (NRBE3) that binds RB and promotes RB degradation. NRBE3 contains an LXCXE motif and bound RB in vitro. NRBE3 interacted with RB in cells when proteasome activity was inhibited. NRBE3 promoted RB ubiquitination and degradation via the ubiquitin-proteasome pathway. Importantly, purified NRBE3 ubiquitinated recombinant RB in vitro, and a U-box was identified as essential for its E3 activity. Surprisingly, NRBE3 was transcriptionally activated by E2F1/DP1. Consequently, NRBE3 affected the cell cycle by promoting G1/S transition. Moreover, NRBE3 was up-regulated in breast cancer tissues. Taken together, we identified NRBE3 as a novel ubiquitin E3 ligase for RB that might play a role as a potential oncoprotein in human cancers.
Subject(s)
E2F1 Transcription Factor/physiology , Gene Expression Regulation/physiology , Retinoblastoma Protein/metabolism , Transcription, Genetic/physiology , Ubiquitin-Protein Ligases/physiology , Amino Acid Sequence , Base Sequence , Breast Neoplasms/physiopathology , Cell Line, Tumor , DNA , Female , Humans , Molecular Sequence Data , Promoter Regions, Genetic , Proteolysis , Retinoblastoma Binding Proteins , Sequence Homology, Amino Acid , Ubiquitin-Protein Ligases/chemistry , Up-RegulationABSTRACT
Ribosome biogenesis is required for normal cell function, and aberrant ribosome biogenesis can lead to p53 activation. However, how p53 is activated by defects of ribosome biogenesis remains to be determined. Here, we identified human UTP14a as an SSU processome component by showing that hUTP14a is nucleolar, associated with U3 snoRNA and involved in 18 S rRNA processing. Interestingly, ectopic expression of hUTP14a resulted in a decrease and knockdown of hUTP14a led to an increase of p53 protein levels. We showed that hUTP14a physically interacts with p53 and functionally promotes p53 turn-over, and that hUTP14a promotion of p53 destabilization is sensitive to a proteasome inhibitor but independent of ubiquitination. Significantly, knockdown of hUTP14a led to cell cycle arrest and apoptosis. Our data identified a novel pathway for p53 activation through a defect in rRNA processing and suggest that a ribosome biogenesis factor itself could act as a sensor for nucleolar stress to regulate p53.
Subject(s)
Apoptosis/physiology , Cell Cycle/physiology , Proteasome Endopeptidase Complex/metabolism , Ribonucleoproteins, Small Nucleolar/metabolism , Tumor Suppressor Protein p53/metabolism , Gene Knockdown Techniques , HeLa Cells , Humans , Proteasome Endopeptidase Complex/genetics , Protein Stability , RNA Processing, Post-Transcriptional/physiology , RNA, Ribosomal, 18S/biosynthesis , RNA, Ribosomal, 18S/genetics , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/metabolism , Ribonucleoproteins, Small Nucleolar/genetics , Ribosomes/genetics , Ribosomes/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
OBJECTIVE: To prepare and characterize the polyclonal antibody against KIAA0649 and identify the localization and the functional motif of KIAA0649. METHODS: Three polypeptides were synthesized based on the bioinformatics analysis of KIAA0649 protein. New Zealand rabbits were immunized with the mixture of the three KIAA0649 peptides coupled with keyhole limpet hemocyanin (KLH). The titer of the antisera was detected with ELISA. The antisera were purified with immuno-affinity chromatography when the titer reached 1:10(5). Western blot was performed with the purified antisera on the cell lysates of U2OS cells transfected with either Flag-KIAA0649 or KIAA0649-targeting siRNA. Immunofluorescence was performed with the purified antisera and anti-Flag antibody on the cells transfected with Flag-KIAA0649. A series of Flag-KIAA0649 deletion mutants was constructed by PCR cloning. The cellular compartmentation of full-length Flag-KIAA0649 and its deletion mutants were analyzed with immunofluorescence. RESULTS: The results of Western blot and immunofluorescence demonstrated that the antisera from the KIAA0649 polypeptides-immunized rabbits specifically recognized endogenous and exogenous KIAA0649. The full-length Flag-KIAA0649 displayed specific nuclear foci. The Flag-KIAA0649 deletion mutant containing PENF motif showed the same nuclear foci as the full length of Flag-KIAA0649, suggesting that the PENF motif could be the minimum functional motif of KIAA0649. CONCLUSION: We have obtained anti-KIAA0649 polyclonal antibody which will be useful for further investigation. The PENF motif could be the minimum functional domain of KIAA0649.
