ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-ribosylation
1 Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany
2 Helmholtz Zentrum München, Institute for Molecular Immunology, Marchioninistr. 25, 81377, München, Germany
3 Present addresses: Abbott GmbH & Co. KG, Max-Planck-Ring 2a, 65205, Wiesbaden, Germany (HK
4 Department of Neurology, Medical School, RWTH Aachen University, 52074, Aachen, Germany (AB
5 Home office, Schoppershofstraße 65, 90489, Nürnberg, Germany (AB
Cell Communication and Signaling 2013, 11:5 doi:10.1186/1478-811X-11-5Published: 19 January 2013
Although 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.
We 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.
Our 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.