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

Abstract

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.

Small-Molecule Chemical Probe Rescues Cells from Mono-ADP-Ribosyltransferase ARTD10/PARP10-Induced Apoptosis and Sensitizes Cancer Cells to DNA Damage

“…OUL35 will aid further studies to assess the roles of ARTD10 in different processes. In vitro screening identified 78 target proteins for MARylation by ARTD10 of which the majority were kinases (Feijs et al, 2013a), highlighting the need to understand its roles in various physiological and stress/disease related conditions.…”

“…While some ARTDs modify substrates by transferring iteratively multiple ADP-ribose units resulting in poly-ADP-ribosylation (PARylation), most ARTDs mono-ADP-ribosylate (MARylate) their substrates (Kleine et al, 2008). ARTDs function in DNA damage repair (Malanga and Althaus, 2005;Nicolae et al, 2014Nicolae et al, , 2015, the unfolded protein response (Jwa and Chang, 2012), apoptosis Koh et al, 2005), heat shock (Petesch and Lis, 2008), cellular signaling (Feijs et al, 2013a;, cell division (Chang et al, 2004(Chang et al, , 2005(Chang et al, , 2009Ha et al, 2012), as well as transcription and chromatin regulation Schreiber et al, 2006). PARylating ARTDs (pARTDs; ARTD1-6), most prominently ARTD1, have been the focus of cancer related research during the past two decades.…”

“…ARTD10 has a conserved C-terminal ART domain responsible for its enzymatic activity (Hottiger et al, 2010;Kleine et al, 2008;Otto et al, 2005;Yu et al, 2005). In vitro screening of more than 8000 proteins identified 78 substrates for ARTD10 (Feijs et al, 2013a), the majority of these being kinases. ADP-ribosylation of GSK3-β by ARTD10 negatively regulates the kinase activity of GSK3-β in vitro and knockdown of ARTD10 increased the kinase activity of GSK3-β in cells (Feijs et al, 2013a).…”

Regulation of NF-κB signalling by the mono-ADP-ribosyltransferase ARTD10

“…Mutants were generated using standard mutagenesis procedures and were verified by sequencing. Cloning of pSUPER-ARTD10_1 and pSUPER-ARTD10_6 is described elsewhere 34 . Plasmids expressing FLAG-TRAF6, HA-TAK1, FLAG-NEMO, His-ubiquitin K63only and 3xNF-kB luciferase reporter constructs are described elsewhere [35][36][37][38][39] .…”

The ADP-ribosyltransferase PARP10/ARTD10 Interacts with Proliferating Cell Nuclear Antigen (PCNA) and Is Required for DNA Damage Tolerance

“…Similar to other PARPs, PARP10 has auto-catalytic activity (30). Recent work has uncovered a number of other substrates targeted for MARylation by PARP10: GSK3␤ MARylation was shown to inhibit its kinase activity (34), while MARylation of NEMO was shown to block the activation of NF-B pathway by extracellular signals (35). A protein microarray screen identified over 70 substrates for PARP10-catalyzed MARylation (34), but the extent to which most of these substrates are modified in vivo is unknown.…”