Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy
Figure 7
Phosphorylated α-synuclein is not a Parkin substrate.
Analysis of a representative GST-pull-down assay performed by incubating GST-Parkin [26] or GST with the TBS-soluble protein fraction from a symptomatic hA30Pα-syn mouse. Antibodies against human α-synuclein, PS129-α-synuclein or α-tubulin were used for western blotting. GST-Parkin interacts specifically with α-tubulin [54], but not with α-synuclein or PS129-α-synuclein. The asterisk indicates the GST-moiety resulting from the degradation of the proteolytically unstable recombinant GST-Parkin protein. (B) Results of an in vitro ubiquitylation assay performed by incubating GST-Parkin, or the enzymatically inactive GST-ParkinC418R variant, with ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, UbcH7, FLAG-tagged ubiquitin and ATP, in the presence or absence of in vitro-phosphorylated α-synuclein (PS87,129-α-syn). Once the reaction was completed, GST-Parkin was immobilized on glutathione-sepharose beads; the beads and the supernatant fraction were analyzed by western blotting with anti-FLAG, PS129-α-synuclein, or anti-Parkin antibodies. GST-Parkin (but not GST-ParkinC418R) promoted its own ubiquitylation in a dose-dependent manner, as demonstrated by the amounts of ubiquitylated species in the bead fraction being proportional to the amounts of GST-Parkin used for the ubiquitylation reactions. In addition, GST-Parkin (but not GST-ParkinC418R) promoted the formation of ubiquitin chains, as indicated by the presence of a ladder of regularly spaced ubiquitin-immunoreactive protein bands in the supernatant fraction. However, GST-Parkin did not promote the ubiquitylation of PS129-α-synuclein: PS129-α-synuclein-immunoreactive proteins with higher apparent molecular mass than the monomer were not detected in the supernatant fraction at any of the GST-Parkin concentrations used in the assay (PS129-α-syn); moreover, we did not observe any ubiquitin-immunoreactive bands specific for PS129-α-synuclein in the supernatant fraction (FLAG). The asterisk indicates a FLAG immunoreactive band most likely corresponding to a thioester adduct formed between the E2 enzyme UbcH7 and FLAG-tagged ubiquitin in the presence of the E1 enzyme.