Figure 2.

PINK1 phosphorylation of Parkin at Ser⁶⁵ mediates activation of Parkin E3 ligase activity. Wild-type (a) but not kinase-inactive (b) PINK1 activates wild-type Parkin, but does not affect the activity of Ser⁶⁵Ala (S65A) mutant Parkin (c). Two micrograms of wild-type or S65A Parkin were incubated with indicated amounts of wild-type or kinase-inactive (D359A) MBP-TcPINK in a kinase reaction (50 mM Tris-HCl (pH 7.5), 0.1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM MgCl₂, 1% 2-mercaptoethanol and 0.1 mM [γ-³²P] ATP (approx. 500 cpm pmol⁻¹) (in parallel to confirm the phosphorylation) for 60 min. The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris-HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl₂, 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli, 0.05 mM Flag-Ubiquitin (MW approx. 9.5 kDa) (Boston Biochem) and 2 mM ATP). Reactions were terminated after 60 min by addition of SDS-PAGE loading buffer and resolved by SDS-PAGE. Ubiquitin, Parkin and PINK1 were detected using anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Incorporation of [γ-³²P] ATP was detected by autoradiography (lower panel). Ubiquitin attached to the E1 (Ub-Ube1) and ubiquitin dimer (Ub₂) formation occurred in the assay in all conditions (a–c). Ubiquitylation of PINK1 (Ub-PINK1) is indicated (a). Formation of polyubiquitin chains (poly-Ub) upon Parkin activation (a) is indicated. As mentioned in §4, further work is required to establish the nature of these chains and whether they are linked to UbcH7. Representative of five independent experiments.