title: Kinase activity-independent suppression of p73alpha by AMP-activated kinase alpha (AMPKalpha).
abstract: Although p73alpha induces many of the same cellular events as p53, it is structurally distinct from p53 in that it possesses a unique COOH-terminal domain. To dissect the function of this domain, we performed yeast two-hybrid screening of a HeLa cDNA library using residues 552-636 of p73alpha as bait. Among the clones that showed a specific interaction with p73alpha was AMP-activated protein kinase alpha (AMPKalpha). Additional yeast two-hybrid assays indicated that the betagamma-binding domain of AMPKalpha is critical for the interaction with p73alpha. The interaction was further confirmed in vitro by glutathione S-transferase pull-down, and in vivo by immunoprecipitation and immunofluorescence microscopy. Transient coexpression of AMPKalpha resulted in downregulation of the effect of p73alpha, but not of p53, on various p53-responsive promoters. Chromatin immunoprecipitation indicated p73alpha-dependent recruitment of AMPKalpha to the p21WAF1 promoter. Treatment with 5-aminoimidazole-4-carboxamide ribonucleotide, an agonist of AMPKalpha, and expression of dominant-negative versions of AMPKalpha revealed that the repression of p73alpha was independent of AMPKalpha kinase activity. In addition, cisplatin-induced growth repression was impaired when AMPKalpha was overexpressed. Upon the knock down of AMPKalpha by siRNA, the induction of p21WAF1 by p73alpha was significantly increased. Taken together, these data indicate that AMPKalpha specifically regulates p73alpha by a direct interaction without affecting its phosphorylation status. From these data, we speculate that AMPKalpha may provide a molecular clue to understand the repressive role of the C-terminus of p73alpha in transcription and DNA damage response.
authors.affiliation: Department of Bioscience & Biotechnology/Institute of Bioscience, Sejong University, Kwangjin-Gu, Seoul, Korea.
authors.forename: Y-G
authors.initials: YG
authors.lastname: Lee
authors.forename: S-W
authors.initials: SW
authors.lastname: Lee
authors.forename: H-S
authors.initials: HS
authors.lastname: Sin
authors.forename: E-J
authors.initials: EJ
authors.lastname: Kim
authors.forename: S-J
authors.initials: SJ
authors.lastname: Um
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D047369:Chromatin Immunoprecipitation; D002945:Cisplatin / Q000494:pharmacology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000502:physiology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000494:pharmacology; D012263:Ribonucleosides / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D000970:Antineoplastic Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D012263:Ribonucleosides; C495901:TP53 protein, human; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D000620:Aminoimidazole Carboxamide; C011651:acadesine; D008156:Luciferases; D005982:Glutathione Transferase; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; D002945:Cisplatin