ARTD10 substrate identification on protein microarrays: regulation of GSK3beta by mono-ADP-ribosylation

We have optimized a novel screening method employing protein microarrays, ProtoArrays(R), 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 GSK3beta 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 GSK3beta 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 GSK3beta, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.