Ubiquitin phosphorylated at Ser57 hyper-activates parkin
2017
George, Susanna | Wang, Sabrina M. | Bi, Yumin | Treidlinger, Margot | Barber, Kathryn R. | Shaw, Gary S. | O'Donoghue, Patrick
Malfunction of the ubiquitin (Ub) E3 ligase, parkin, leads to defects in mitophagy and protein quality control linked to Parkinson's disease. Parkin activity is stimulated by phosphorylation of Ub at Ser65 (pUbS⁶⁵). Since the upstream kinase is only known for Ser65 (PINK1), the biochemical function of other phosphorylation sites on Ub remain largely unknown. We used fluorescently labelled and site-specifically phosphorylated Ub substrates to quantitatively relate the position and stoichiometry of Ub phosphorylation to parkin activation. Fluorescence measurements show that pUbS⁶⁵-stimulated parkin is 5-fold more active than auto-inhibited and un-stimulated parkin, which catalyzes a basal level of auto-ubiquitination. We consistently observed a low but detectable level of parkin activity with pUbS¹². Strikingly, pUbS⁵⁷ hyper-activates parkin, and our data demonstrate that parkin is able to selectively synthesize poly-pUbS⁵⁷ chains, even when 90% of the Ub in the reaction is un-phosphorylated. We further found that parkin ubiquitinates its physiological substrate Miro-1 with chains solely composed of pUbS⁶⁵ and more efficiently with pUbS⁵⁷ chains. Parkin hyper-activation by pUbS⁵⁷ demonstrates the first PINK1-independent route to active parkin, revealing the roles of multiple ubiquitin phosphorylation sites in governing parkin stimulation and catalytic activity. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O’Donoghue.
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