Identification of DDX6 ubiquitylated lysines in cAMP-stimulated cells. stimulation

DDX6 is an RNA helicase mostly located in the P-bodies, membrane-less organelles that play a fundamental role in the post-transcriptional regulation of gene expression. We found that stimulation of the G protein-coupled receptor (GPCR)-cAMP pathway induces a non-proteolytic polyubiquitylation of DDX6 mediated by the RING E3 ligase Praja2. To identify potential lysine residues of DDX6 accepting ubiquitin moieties mediated by Praja2, we performed a proteomic analysis on DDX6 purified from total lysates from cells treated with Forskolin, a diterpene that raises cAMP levels. The analysis identified K26 and K286 as ubiquitylated sites upon cAMP stimulation.