Dunkerley, K. M., Rintala-Dempsey, A. C., Salzano, G. , Tadayon, R., Hadi, D., Barber, K. R., Walden, H. and Shaw, G. S. (2022) Distinct phosphorylation signals drive acceptor versus free ubiquitin chain targeting by Parkin. Biochemical Journal, 479(6), pp. 751-766. (doi: 10.1042/bcj20210741) (PMID:35262643) (PMCID:PMC9022993)
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Abstract
The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of the Ubl domain in parkin, both mediated by the OMM kinase, PINK1. How an OMM protein is selected for ubiquitination is unclear. Parkin targeted OMM proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we used chimeric proteins, tagged with ubiquitin (Ub), to evaluate parkin ubiquitination of mitochondrial substrates. We find that pUb tethered to the mitochondrial target proteins, Miro1 or CISD1, is necessary for parkin recruitment and essential for target protein ubiquitination. Surprisingly, phosphorylation of parkin is not necessary for the ubiquitination of either Miro1 or CISD1. Thus, parkin lacking its Ubl domain efficiently ubiquitinates a substrate tethered to pUb. Instead, phosphorylated parkin appears to stimulate free Ub-chain formation. We also demonstrate that parkin ubiquitination of pUb-tethered substrates occurs on the substrate, rather than the pUb modification. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb on the OMM protein and subsequent parkin phosphorylation triggers free Ub chain formation. This finding accounts for the broad spectrum of OMM proteins ubiquitinated by parkin and has implications on target design for therapeutics.
Item Type: | Articles |
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Additional Information: | This research was supported by a grant (PJT 166019) from the Canadian Institutes of Health Research (G.S.S.) and a grant (209347/Z/17/Z) from Wellcome Trust U.K. (H.W.). K.M.D. was the recipient of a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship |
Keywords: | Cell Biology, Molecular Biology, Biochemistry |
Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Salzano, Dr Giulia and Walden, Professor Helen |
Authors: | Dunkerley, K. M., Rintala-Dempsey, A. C., Salzano, G., Tadayon, R., Hadi, D., Barber, K. R., Walden, H., and Shaw, G. S. |
College/School: | College of Medical Veterinary and Life Sciences > School of Molecular Biosciences |
Journal Name: | Biochemical Journal |
Publisher: | Portland Press |
ISSN: | 0264-6021 |
ISSN (Online): | 1470-8728 |
Published Online: | 09 March 2022 |
Copyright Holders: | Copyright © The Author(s) 2022 |
First Published: | First published in Biochemical Journal 479(6): 751-766 |
Publisher Policy: | Reproduced under a Creative Commons licence |
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