Chemical synthesis of phosphorylated ubiquitin and diubiquitin exposes positional sensitivities of E1-E2 enzymes and deubiquitinases

Abstract Modification of ubiquitin by phosphorylation extends the signaling possibilities of this dynamic signal, as it could affect the activity of ligases and the processing of ubiquitin chains by deubiquitinases. The first chemical synthesis of phosphorylated ubiquitin and of Lys63-linked diubiquitin at the proximal, distal or both ubiquitins is reported. This enabled the examination of how such a modification alters E1-E2 activities of the ubiquitination machinery. It is found that E1 charging was not affected, while the assembly of phosphorylated ubiquitin chains was differentially inhibited with E2 enzymes tested. Moreover, this study shows that phosphorylation interferes with the recognition of linkage specific antibodies and the activities of several deubiquitinases. Notably, phosphorylation in the proximal or distal ubiquitin unit has differential effects on specific deubiquitinases. These results support a unique role of phosphorylation in the dynamics of the ubiquitin signal. Modifying the modifier: Phosphorylation of ubiquitin has emerged very recently as a new mode of regulation of the ubiquitin signal. Here, the first chemical synthesis of phosphorylated ubiquitin and diubiquitin (see figure) is reported and how such a modification affects the E1-E2 of the ubiquitination machinery and deubiquitinases, the enzymes which oppose ubiquitination, is examined.