ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-ribosylation

Cell Communication and Signaling

Research

Elisabeth Kremmer¹  Karla LH Feijs²  Patricia Verheugd²  Nicolas Herzog²  Bernhard Lüscher²  Alexandra H Forst²  Anne Braczynski³  Ulrike Linzen⁴  Henning Kleine⁵ 

[1] Helmholtz Zentrum München, Institute for Molecular Immunology, Marchioninistr. 25, 81377, München, Germany;Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany;Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany;Department of Neurology, Medical School, RWTH Aachen University, 52074, Aachen, Germany (AB);Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany;Home office, Schoppershofstraße 65, 90489, Nürnberg, Germany (AB);Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany;Present addresses: Abbott GmbH & Co. KG, Max-Planck-Ring 2a, 65205, Wiesbaden, Germany (HK);

关键词: ARTD;    Posttranslational modification;    ADP-ribosyltransferase;    PARP10;    PARP14;    ARTD8;    NAD;    Kinase activity;    Regulation;   

DOI  :  10.1186/1478-811X-11-5

 received in 2012-10-31, accepted in 2012-12-20,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundAlthough ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification.ResultsWe have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3β revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro. Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3β activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling.ConclusionsOur findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro. By studying one of the ARTD10 substrates more closely, the kinase GSK3β, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.

【 授权许可】

Unknown   
© Feijs et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.