Diverse Effects of Pathogenic Mutations of Parkin That Catalyze Multiple Monoubiquitylation in Vitro

Abstract

Mutational dysfunction of PARKIN gene, which encodes a double RING finger protein and has ubiquitin ligase E3 activity, is the major cause of autosomal recessive juvenile Parkinsonism. Although many studies explored the functions of Parkin, its biochemical character is poorly understood. To address this issue, we established an E3 assay system using maltose-binding protein-fused Parkin purified from Escherichia coli. Using this recombinant Parkin, we found that not the front but the rear RING finger motif is responsible for the E3 activity of Parkin, and it catalyzes multiple monoubiquitylation. Intriguingly, for autosomal recessive juvenile Parkinsonism-causing mutations of Parkin, whereas there was loss of E3 activity in the rear RING domain, other pathogenic mutants still exhibited E3 activity equivalent to that of the wild-type Parkin. The evidence presented allows us to reconsider the function of Parkin-catalyzed ubiquitylation and to conclude that autosomal recessive juvenile Parkinsonism is not solely attributable to catalytic impairment of the E3 activity of Parkin.