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Title: AMP-activated protein kinase is a positive regulator of poly(ADP-ribose) polymerase

Abstract

AMPK acts as a cellular fuel gauge and responds to decreased cellular energy status by inhibiting ATP-consuming pathways and increasing ATP-synthesis. The aim of this study was to examine the role of AMPK in modulating poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in maintaining chromatin structure and DNA repair. HT-29 cells infected with constitutively active AMPK demonstrated increased PARP automodification and an increase in bioNAD incorporation. AMPK and PARP co-immunoprecipitated under basal conditions and in response to H{sub 2}O{sub 2}, suggesting a physical interaction under both resting and stress-induced conditions. Incubation of PARP with purified AMPK resulted in the phosphorylation of PARP; and the inclusion of AMP as an AMPK activator potentiated PARP phosphorylation. Using immobilized PARP, the incorporation of bioNAD by PARP was dramatically increased following the addition of AMPK. These data suggest a novel role for AMPK in regulating PARP activity through a direct interaction involving phosphorylation.

Authors:
 [1];  [1];  [2]
  1. Division of Gastroenterology, University of Alberta, Edmonton, Alberta (Canada)
  2. Division of Gastroenterology, University of Alberta, Edmonton, Alberta (Canada). E-mail: Karen.madsen@ualberta.ca
Publication Date:
OSTI Identifier:
20798877
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 342; Journal Issue: 1; Other Information: DOI: 10.1016/j.bbrc.2006.01.145; PII: S0006-291X(06)00242-7; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ADP; AMP; ATP; BIOLOGICAL STRESS; BIOSYNTHESIS; CHROMATIN; DNA; DNA REPAIR; ENZYMES; FUEL GAGES; HYDROGEN PEROXIDE; INTESTINES; PHOSPHORYLATION; RIBOSE

Citation Formats

Walker, John W., Jijon, Humberto B., and Madsen, Karen L. AMP-activated protein kinase is a positive regulator of poly(ADP-ribose) polymerase. United States: N. p., 2006. Web. doi:10.1016/j.bbrc.2006.01.145.
Walker, John W., Jijon, Humberto B., & Madsen, Karen L. AMP-activated protein kinase is a positive regulator of poly(ADP-ribose) polymerase. United States. doi:10.1016/j.bbrc.2006.01.145.
Walker, John W., Jijon, Humberto B., and Madsen, Karen L. Fri . "AMP-activated protein kinase is a positive regulator of poly(ADP-ribose) polymerase". United States. doi:10.1016/j.bbrc.2006.01.145.
@article{osti_20798877,
title = {AMP-activated protein kinase is a positive regulator of poly(ADP-ribose) polymerase},
author = {Walker, John W. and Jijon, Humberto B. and Madsen, Karen L.},
abstractNote = {AMPK acts as a cellular fuel gauge and responds to decreased cellular energy status by inhibiting ATP-consuming pathways and increasing ATP-synthesis. The aim of this study was to examine the role of AMPK in modulating poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in maintaining chromatin structure and DNA repair. HT-29 cells infected with constitutively active AMPK demonstrated increased PARP automodification and an increase in bioNAD incorporation. AMPK and PARP co-immunoprecipitated under basal conditions and in response to H{sub 2}O{sub 2}, suggesting a physical interaction under both resting and stress-induced conditions. Incubation of PARP with purified AMPK resulted in the phosphorylation of PARP; and the inclusion of AMP as an AMPK activator potentiated PARP phosphorylation. Using immobilized PARP, the incorporation of bioNAD by PARP was dramatically increased following the addition of AMPK. These data suggest a novel role for AMPK in regulating PARP activity through a direct interaction involving phosphorylation.},
doi = {10.1016/j.bbrc.2006.01.145},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 342,
place = {United States},
year = {Fri Mar 31 00:00:00 EST 2006},
month = {Fri Mar 31 00:00:00 EST 2006}
}
  • Purpose: Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR). Methods and Materials: The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kineticsmore » assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini. Results: DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K{sub dapp} = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang. Conclusion: DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.« less
  • The cDNA of human poly(ADP-ribose) polymerase (pADPRP), encoding the entire protein, was subcloned into the Escherichia coli expression plasmid pYUb. In this expression system, the carboxyl terminus of ubiquitin is fused to the amino terminus of a target protein, in this case pADPRP, stabilizing the accumulation of the cloned gene product. Following induction of the transformed cells, the sonicated extract contained a unique protein immunoreactive with both pADPRP and ubiquitin antibodies and corresponding to the predicted mobility of the fusion protein in SDS-PAGE. Fusion of ubiquitin to pADPRP increased the yield of pADPRP approximately 10-fold compared to that of themore » unfused enzyme. The resulting recombinant fusion protein had catalytic properties which were nearly identical to those of native pADPRP obtained from mammalian tissues. An initial analysis by deletion mutagenesis of pADPRP's functional domains revealed that deletions in the NAD binding domain eliminated all activity; however, partial polymerase activity resulted from deletion in the DNA binding or automodification domains. The activities were not enhanced by breaks in DNA. The authors further report a colony filter screening procedure designed to identify functional polymerase molecules which will facilitate structure/function studies of the polymerase.« less
  • Recently the authors described a full-length cDNA for the human nuclear enzyme poly(ADP-ribose) polymerase. Here, they report the chromosomal localization and partial map of the human gene for this enzyme as well as the complete coding sequence for this protein. The nucleotide sequence reveals a single 3042-base open reading frame encoding a protein with a predicted M/sub r/ of 113,135. A comparison of this deduced amino acid sequence with the amino acid sequence of three peptides derived from human poly(ADP-ribose) polymerase revealed a match of 27 amino acid residues. A computer-derived structural analysis of the enzyme and a search formore » similarities with other proteins confirmed that the polymerase belongs to a subfamily of DNA/NAD-binding proteins and DNA-repair proteins. Possible Zn/sup 2 +/-binding fingers, a nucleotide-binding fold, and a nuclear transport signal were noted. Additionally, chromosomal mapping has identified polymerase-hybridizing sequences on human chromosomes 1 (the active gene), 13, and 14 (processed pseudogenes). Using the polymerase cDNA as a probe, they also have detected several DNA restriction fragment length polymorphisms in normal humans.« less
  • Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs,more » and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination.« less
  • SV40-3T3 cells were exposed in monolayer cultures to 5{times}10{sup {minus}7} M methotrexate (MTX), that inhibited thymidylate synthetase, arrested cell growth without cell killing in 24 h and did not induce single- (ss) or double-strand (ds) breaks in DNA. Following 24, up to 72 h, the poly(ADP-ribose) polymerase content of attached cells was induced by 5{times}10{sup {minus}7} MTX and the augmentation of the enzyme increased with the time of exposure to the drug. Inhibition of protein or RNA synthesis abolished augmentation of enzymatic activity; so too did the initiation of maximal cell growth by thymidine + hypoxanthine, by-passing the inhibitory sitemore » of MTX. Isolation of the ADP-ribosylated enzyme protein by gel electrophoresis identified poly(ADP-ribose) polymerase protein as the molecule that was induced by 5{times}10{sup {minus}7} M MTX. Under identical conditions, the poly(ADP-ribose) polymerase induction in 3T3 cells could not be demonstrated. A possible cell-cycle dependent biosynthesis of the enzyme protein is proposed in SV40 3T3 cells.« less