Identification Of The NIRF-interacting Domain With The P53in NIRF-mediated-P3Ubiquitination

ObjectiveNp95/ICBP90-like RING finger protein (NIRF) is a newly nuclearprotein identified in2002by Prof. Kochi of Fukushima Medical University.It possesses802amino acid residues, contained UBL, PHD, YDG/SRA andRING finger domains. Since the NIRF was identified, research on NIRF hasmade its debut in regulation of cellular proliferation, protein degradation ofpolyubiquitination, controlling cellular cancerization on certain conditionsand probably more hitherto unknown directions. Previous reports show thatNIRF physically interacts with Cdk2and let-7a, and over expression ofNIRF induced an increase in G1phase cells. In addition, NIRF functions asan E3ubiquitin ligase and has auto-ubiquitination activity, suggesting thatNIRF manifests an intrinsic capacity to mediate the degradation ofprotein.The tumour suppressor protein p53is a stress-activated transcriptionfactor which has vital significance in cancerous derivatives. It is believedthat p53acts in response to stresses and abnormalities in cell physiology bymobilizing the repair processes or by leading to cell cycle arrest for senescence and programmed cell death for removing the diseased cells. Ithas been reported that NIRF can interacts with p53, and NIRF itself is ahighly regulated protein which was thought to be the E3ligase responsiblefor p53degradation under normal physiologic conditions. However, theNIRF-interacting domain within the p53is not clear now. Here, we reportthat NIRF could bind to p53and identify the NIRF-interacting domain withthe p53in NIRF-mediated p53ubiquintination in order to provideexperimental basis for the further ubiquintination research and exploretherapy of tumors.Methods（1） HEK293cells were cultured in DMEM nutrient, supplementedwith10%fetal calf serum, penicillin and streptomycin.（2） Cells were transiently transfected with the indicated expressionconstructs for48h with additional treatment of presence or absenceof MG-132for10h.（3） The total proteins from HEK293cells were collected RIPAbuffer.（4） NIRF deletion mutants were expressed in HEK293cells.HEK293cells transfected with pCMV-3×FLAG,pCMV-FLAG-NIRF and NIRF deletion mutants tagged with theFlag, were lysed for analysis by SDS-PAGE and Western blottingwith anti-NIRF, anti-FLAG and anti-β-Actin (loading control) antibodies.（5） Co-immunoprecipitation and Western blot were performed inidentity the NIRF-interacting domain with the p53. HEK293cellsco-transfected with pcDNA3-P53, pCMV-FLAG-NIRF and NIRFdeletion mutant plasmids were lysed, immunoprecipitated withanti-p53antibody and then analyzed by Western blotting withanti-p53or anti-FLAG antibody. WT NIRF, which binds stronglywith p53, was used as a positive control.ResultThe results show that all the deletion mutants can be expressed in cellsand the conditions indentified by two antibodies were accordant with eachother. While the NIRF mutants which carried a deletion of UBL, YDG orRING finger domain were still able to pull-down the p53protein, a deletionof PHD domain completely abolished the interaction between the twoproteins.ConclusionTogether, these results suggest that NIRF binds to p53through its PHDdomain to form complexes, which may be involved in important cell eventssuch as protein sorting and degradation, DNA repair and apoptosis, andconstitute a novel signaling pathway with some relation to cell proliferation,which may play roles in the development and progression of cancers.