von-Hippel Lindau protein (pVHL) suppresses tumorigenesis in the kidney, in part through regulation of hypoxiainducible factor α (HIFα). However, HIF has been proposed to be necessary but insufficient for renal tumorigenesis. p53 was implicated as a transcription factor that is regulated by pVHL, but the molecular mechanism by which pVHL regulates p53 on DNA damage is unknown. We demonstrated that checkpoint kinase-2 (Chk2) binds to the β-domain of pVHL and phosphorylates Ser 111 on DNA damage. Notably, this modification enhances pVHL-mediated transactivation of p53 by recruiting p300 and Tip60 to the chromatin of p53 target gene. Further, the naturally occurring pVHL mutants pVHL-S111R and pVHL-S111C showed diminished binding to coactivators, ultimately retarding p53-mediated growth arrest and apoptosis. In this study, we determined the molecular mechanism by which pVHL transactivates p53 on DNA damage and demonstrated that p53-related pVHL subtype mutants regulate tumorigenecity in VHL diseases.
Bibliographical noteFunding Information:
Chromatin immunoprecipitation assay (ChIP). ChIP assays We thank Dr. V.M. Vogt (Cornell University, USA), Dr. S.H. were performed using suitable antibodies as described in refer-Baek (Seoul National University), and Dr. R.H. Seong (Seoul ence 42. National University) for invaluable materials. This work was sup-Neutral comet assay. Neutral comet assays were performed ported by grants from the Mid-Career Research Program, the using Comet Assay kit (Trevigen, 4250-050-K) according to National Research Foundation (NRF-2010-0018896 and 2010-the manufacturer’s instruction with modification. Briefly, cells 0007646 to H.D.Y.), the National R&D Program for Cancer were treated with adriamycin (0.8 μg/ml) and harvested (~105 Control, Ministry of Health and Welfare (0720460), the Korea per pellet). Harvested cells were counted, and then 15 μl of 105 Healthcare Technology R&D Project (A090281) and the World cells/ml single cell suspension were mixed with 85 μl of melted Class University Program of the MEST and the NRF (R31-LMAgarose. A 50 μl of mixed cells then layered onto Comet 2008-000-10103-0) to H.D.Y. J.S.R. was supported by NRF Slide and maintained in the dark at 4°C for 1 h. Slides were (NRF-2011-355-E00015). submerged in pre-chilled lysis solution for 1.5 h, washed with Tris buffer and incubated for 1x TBE buffer for 30 min at room temperature. After electrophoresis (7 mA, 30 min), immerse slides in distilled water for 10 min then washed with 70%
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Developmental Biology
- Cell Biology