E3 ubiquitin-protein ligase activity of Parkin is dependent on cooperative interaction of RING finger (TRIAD) elements

The parkin gene codes for a 465-amino acid protein which, when mutated, results in autosomal recessive juvenile parkinsonism (AR-JP). Symptoms of AR-JP are similar to those of idiopathic Parkinson's disease, with the notable exception being the early onset of AR-JP. We have cloned and expressed human Parkin in Escherichia coli and have examined Parkin-mediated ubiquitination in an in vitro ubiquitination assay using purified recombinant proteins. We found that Parkin has E3 ubiquitin ligase activity in this system, demonstrating for the first time that the E3 activity is an intrinsic function of the Parkin protein and does not require posttranslational modification or association with cellular proteins other than an E2 (human Ubc4 E2 was utilized in this ubiquitination assay). Mutagenesis of individual elements of the conserved RING TRIAD domain indicated that at least two elements were required for ubiquitin ligase activity and suggested a functional cooperation between the RING finger elements. Since the activity assays were conducted with recombinant proteins purified from E. coli, this is the first time TRIAD element interaction has been demonstrated as an intrinsic feature of Parkin E3 activity. Copyright (C) 2001 National Science Council, ROC and S. Karger AG, Basel.