Mechanism of phospho-ubiquitin-induced PARKIN activation

Wauer, Tobias; Šimíček, Michal; Schubert, Alexander F; Komander, David

doi:10.1038/nature14879

Cited by 388 publications

(504 citation statements)

References 37 publications

(98 reference statements)

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“…NMR dynamics experiments show significant mobility for the IBR domain and segments on either side of a short helix within the tether region where poor electron density is frequently observed in crystal data (see fig S4 in Kumar et al , 2015). Details of the partial E3 ligase activation of parkin have been shown in complexes with phosphorylated ubiquitin (pUb; Kumar et al , 2015, 2017; Wauer et al , 2015), where pUb binds to a broad crevasse between the RING0 and RING1 domains (Fig 1A). The association of pUb causes rearrangement of the IBR domain and results in the formation of a large gap between the IBR and RING1 domains.…”

Section: Resultsmentioning

confidence: 96%

“…These regions undergo multiple rearrangements in structure and orientation upon pUb and Ubl binding (Kumar et al , 2015; Wauer et al , 2015; Kumar et al , 2017). The increase in HDX could indicate further rearrangement occurs upon UbcH7‐Ub binding leading to a more extended structure.…”

Section: Resultsmentioning

confidence: 99%

“…One exception is the C‐terminus of the pUbl domain (D62‐M80) that is significantly more protected. This region contains the phosphorylated S65 residue suggested to interact with three basic residues (K161, R163 and K211) in the RING0 domain that show attenuated parkin autoubiquitination when substituted (Wauer et al , 2015). Two of these sites are locations of ARJP substitutions (K161N, K211R, K211N).…”

Section: Resultsmentioning

confidence: 99%

“…Under oxidative stress conditions, PINK1 is activated and phosphorylates ubiquitin (pUb) near the outer mitochondrial membrane. This in turn helps recruit parkin to the membrane through binding of pUb to the RING1–IBR region of the E3 ligase (Sauvé et al , 2015; Wauer et al , 2015; Kumar et al , 2017) and subsequent phosphorylation of parkin's Ubl (pUbl) domain (Ordureau et al , 2014). These two events greatly stimulate ubiquitination activity (Kondapalli et al , 2012; Shiba‐Fukushima et al , 2012; Kane et al , 2014; Kazlauskaite et al , 2014; Koyano et al , 2014) through an allosteric displacement mechanism of the pUbl domain from parkin (Kumar et al , 2015; Sauvé et al , 2015).…”

Section: Introductionmentioning

confidence: 99%

“…What is less clear is how parkin positions the E2~Ub conjugate to enable transfer of the Ub molecule to the RING2(Rcat) domain as a necessary step for catalysis. Current crystal structures of parkin show the proposed E2 binding site on the RING1 domain is > 50 Å from the catalytic site (C431) in the RING2(Rcat) domain suggesting a significant conformational rearrangement is needed (Riley et al , 2013; Trempe et al , 2013; Wauer & Komander, 2013; Kumar et al , 2015, 2017; Sauvé et al , 2015; Wauer et al , 2015). A similar dilemma arises from recent structures of HHARI in complex with UbcH7‐Ub that show the ubiquitin molecule is 47–53 Å from the catalytic site (Dove et al , 2017; Yuan et al , 2017).…”

Section: Introductionmentioning

confidence: 99%

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Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

et al. 2018

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The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early‐onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N‐terminal ubiquitin‐like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho‐ubiquitin‐loaded C‐terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re‐modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

et al. 2018

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Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

et al. 2016

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Edited by Michael IbbaThe activity of the Parkinson's disease-linked E3 ligase parkin is stimulated by phosphorylation at ubiquitin Ser65 (pUb S65 ). The role of other ubiquitin phospho-sites and their kinases are unknown. We produced pUb variants (pS7, pS12, pS20, pS57, pS65) by genetically encoding phosphoserine with the UAG codon. In release factor-deficient Escherichia coli (DRF1), intended to enhance UAG read-through, we discovered ubiquitin variants lacking the UAG-encoded residue, demonstrating previously undocumented +3 frame shifting. We successfully purified each pUb variant from mistranslated products. While pUb S20 failed to stimulate parkin, parkin was partially active with pUb S12 . We observed significant ubiquitination when pUb S65 was the sole substrate.

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Programmed ubiquitin acetylation using genetic code expansion reveals altered ubiquitination patterns

2019

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Edited by Michael IbbaUbiquitination is a post-translational modification (PTM) capable of being regulated by other PTMs, including acetylation. However, the biological consequences of acetylated ubiquitin (acUb) variants are poorly understood, due to their transient nature in vivo and poor characterization in vitro. Since Ub is known to be acetylated in human cells, we produced all possible acUb variants using genetic code expansion. We also developed a protocol that optimizes acetyl-lysine addition to minimize mistranslated proteins and maximize site-specific acUb protein production. Purified acUb proteins were used in pilot ubiquitination assays and found to be competent with IpaH3CT and RNF8 E3 ligases. Overall, this work provides an optimized method to express and purify all acetyl-lysine variants for ubiquitin and shows these proteins can be used to identify potential unique ubiquitination patterns.Covalent attachment of ubiquitin (Ub) to a substrate serves as a signaling event that determines the fate of the target protein. A series of E1, E2, and E3 enzymes specify the position of ubiquitination and the types of linkage between ubiquitin molecules in the case of polyubiquitinated substrates. In this latter case, K48-linked polyubiquitin chains target the substrate for proteasomal degradation [1], while other linkages are signals for DNA damage repair [2], cell cycle progression [3], or downstream cascades such as the MEK-ERK pathway [4]. In addition to ubiquitination, protein function can also be attenuated by a variety of other post-translational modifications (PTMs) such as phosphorylation and acetylation, and there is often cooperativity between these PTMs. For example, current knowledge holds that phosphorylation of Ub at Ser65 by PINK1 is required for the activation of the E3 ligase parkin [5]. Ser65 is just one of the eleven residues in Ub that can be phosphorylated and at least one other phosphorylation site has been identified in vivo [6] and can exhibit similar activation levels for parkin [7]. Additionally, acetylation could occur at any of the seven Lys residues with unknown downstream effects.With the recent advances in enrichment methods and improvements to mass spectrometry, there has been a significant shift in modified proteome analysis. As a result, the acetylome has been mapped in great detail with various acetyl-proteome datasets demonstrating ubiquitin acetylation. Many of these datasets demonstrate that fluctuations in acetylated peptides can be dependent on different cell stimuli such as deacetylase (HDAC, SIRT) inhibition [8-10] and/or knockout [11], or cell stress. For example, ionizing radiation [12] or induction of autophagy [13] results in

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Mechanism of phospho-ubiquitin-induced PARKIN activation

Cited by 388 publications

References 37 publications

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

Programmed ubiquitin acetylation using genetic code expansion reveals altered ubiquitination patterns

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