Kinase activity-independent suppression of p73α by AMP-activated kinase α (AMPKα)

Although p73 alpha 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 p73a as bait. Among the clones that showed a specific interaction with p73a was AMP-activated protein kinase alpha (AMPK alpha). Additional yeast two-hybrid assays indicated that the beta gamma-binding domain of AMPKa is critical for the interaction with p73 alpha. The interaction was further confirmed in vitro by glutathione S-transferase pull-down, and in vivo by immunoprecipitation and immunofluorescence microscopy. Transient coexpression of AMPKa resulted in downregulation of the effect of p73 alpha, but not of p53, on various p53-responsive promoters. Chromatin immunoprecipitation indicated p73 alpha-dependent recruitment of AMPKa to the p21WAF1 promoter. Treatment with 5-aminoimidazole-4-carboxamide ribonucleotide, an agonist of AMPK alpha, and expression of dominant-negative versions of AMPKa revealed that the repression of p73 alpha was independent of AMPK alpha kinase activity. In addition, cisplatin-induced growth repression was impaired when AMPK alpha was overexpressed. Upon the knock down of AMPKa by siRNA, the induction of p21WAF1 by p73 alpha was significantly increased. Taken together, these data indicate that AMPKa specifically regulates p73 alpha by a direct interaction without affecting its phosphorylation status. From these data, we speculate that AMPKa may provide a molecular clue to understand the repressive role of the C-terminus of p73 alpha in transcription and DNA damage response.