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

Abstract

Although p73α 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 p73α as bait. Among the clones that showed a specific interaction with p73α was AMP-activated protein kinase α (AMPKα). Additional yeast two-hybrid assays indicated that the βγ-binding domain of AMPKα is critical for the interaction with p73α. The interaction was further confirmed in vitro by glutathione S-transferase pull-down, and in vivo by immunoprecipitation and immunofluorescence microscopy. Transient coexpression of AMPKα resulted in downregulation of the effect of p73α, but not of p53, on various p53-responsive promoters. Chromatin immunoprecipitation indicated p73α-dependent recruitment of AMPKα to the p21WAF1 promoter. Treatment with 5-aminoimidazole-4-carboxamide ribonucleotide, an agonist of AMPKα, and expression of dominant-negative versions of AMPKα revealed that the repression of p73α was independent of AMPKα kinase activity. In addition, cisplatin-induced growth repression was impaired when AMPKα was overexpressed. Upon the knock down of AMPKα by siRNA, the induction of p21WAF1 by p73α was significantly increased. Taken together, these data indicate that AMPKα specifically regulates p73α by a direct interaction without affecting its phosphorylation status. From these data, we speculate that AMPKα may provide a molecular clue to understand the repressive role of the C-terminus of p73α in transcription and DNA damage response.

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Abbreviations

AMPK:

AMP-activated protein kinase

AICAR:

5-aminoimidazole-4-carboxamide ribonucleotide

DBD:

DNA-binding domain

GST:

glutathione S-transferase

ChIP:

chromatin immunoprecipitation

GFP:

green fluorescent protein

β-gal:

β-galactosidase

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium) bromide

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Acknowledgements

This study was supported by a grant of the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (0420030-3). All authors were supported by BK21 project from Ministry of Education and Human Resources Development.

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Correspondence to E-J Kim or S-J Um.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Lee, YG., Lee, SW., Sin, HS. et al. Kinase activity-independent suppression of p73α by AMP-activated kinase α (AMPKα). Oncogene 28, 1040–1052 (2009). https://doi.org/10.1038/onc.2008.452

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