Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy
Significance PINK1 protein kinase and PARKIN UB ligase are mutated in inherited forms of Parkinson’s disease and several cancers. Thus, it is of great significance to understand normal functions that could be disrupted in disease. A role for PARKIN and PINK1 is in mediating autophagy of damaged mito...
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Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 21; pp. 6637 - 6642 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
National Academy of Sciences
26.05.2015
National Acad Sciences |
Subjects | |
Online Access | Get full text |
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Summary: | Significance PINK1 protein kinase and PARKIN UB ligase are mutated in inherited forms of Parkinson’s disease and several cancers. Thus, it is of great significance to understand normal functions that could be disrupted in disease. A role for PARKIN and PINK1 is in mediating autophagy of damaged mitochondria (mitophagy) through polyubiquitylation of numerous mitochondrial outer membrane proteins in a reaction that involves phosphorylation of both PARKIN and ubiquitin (UB) by PINK1. The mechanism remains unclear, however, due to challenges in defining individual steps in the pathway. Here, we use a UB replacement system to elucidate steps in the pathway that require PARKIN and/or UB phosphorylation by PINK1 and provide evidence of a PINK1- and UB-driven feed-forward mechanism important for efficient mitochondrial ubiquitylation and mitophagy.
The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using “UB S⁶⁵ᴬ-replacement,” we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UB S⁶⁵ᴬ-replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains. |
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Bibliography: | http://dx.doi.org/10.1073/pnas.1506593112 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Author contributions: A.O., J.-M.H., D.M.D., B.A.S., and J.W.H. designed research; A.O., J.-M.H., D.M.D., J.A.P., J.L.O., and D.Y. performed research; J.R. contributed new reagents/analytic tools; A.O., J.-M.H., D.M.D., B.A.S., and J.W.H. analyzed data; and A.O., B.A.S., and J.W.H. wrote the paper. Contributed by Brenda A. Schulman, April 6, 2015 (sent for review March 13, 2015) |
ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.1506593112 |