(Patho‐)physiological relevance of
            <scp>PINK</scp>
            1‐dependent ubiquitin phosphorylation

Fiesel, Fabienne C.; Ando, Maya; Hudec, Roman; Hill, Anneliese R.; Castanedes‐Casey, Monica; Caulfield, Thomas R.; Moussaud-Lamodière, Elisabeth L.; Stankowski, Jeannette N.; Bauer, Peter; Lorenzo‐Betancor, Oswaldo; Ferrer, Isidre; Arbelo, José Matías; Siuda, Joanna; Chen, Li; Dawson, Valina L.; Dawson, Ted M.; Wszołek, Zbigniew K.; Ross, Owen A.; Dickson, Dennis W.; Springer, Wolfdieter

doi:10.15252/embr.201540514

Cited by 155 publications

(176 citation statements)

References 42 publications

Supporting

Mentioning

167

Contrasting

Order By: Relevance

“…3A). As pH decreases, the integral for the upfield peak increases and reaches ∼80% of the total pUb; as pH increases, the integral for the downfield 31 P increases and reaches ∼60% of the total pUb (Fig. 3B) (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…Thus, the peak intensities measured from 2D To understand what triggers the interconversion between the two states, we collected a series of 1D 31 P spectra over a broad range of pH from 4 to 9. Unlike 2D 1 H- 15 N HSQC spectra, we could easily integrate the volume for each 31 P peak, which corresponds to the exact population of the associated conformational state. At pH 7.4, two 31 P peaks can be observed, and the integral for the downfield peak is slightly larger than the integral for the upfield peak (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, phosphorylated polyUb can be either recruited or generated onsite. Upon oxidative stress, neurodegeneration, and aging, Ub phosphorylation level drastically increases (4,8,31), whereas mitophagy, apoptosis, ischemia, inflammation, aging, and many pathophysiological conditions are all associated with changes in cellular pH (32)(33)(34)(35). Altogether, it is likely that certain functions of pUb are only turned on in response to changes in both Ub phosphorylation level and environmental pH.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Gong

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last β-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pK a value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pHsensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals., a 76-residue signaling protein, is found ubiquitously in cells. Two or more Ub molecules can be covalently linked to form a diubiquitin (diUb) and then a polyubiquitin (polyUb), as an isopeptide bond is formed between the carboxylate group of one Ub (called the distal Ub) and the amine group of another Ub (called the proximal Ub). Owing to the characteristic quaternary structures of polyUb and specific interactions between polyUb and its target proteins (1, 2), a polyUb with a specific linkage can be involved in a distinctive set of cellular functions (3).The heterogeneity of Ub also arises from other types of covalent modifications. Proteomics studies have indicated that Ub is phosphorylated at multiple sites (4, 5). However, PINK1 is the only Ub kinase known to date, which specifically phosphorylates Ub at S65 (6, 7). Under normal conditions, only a fraction of Ub is S65-phosphorylated. However, upon oxidative stress, neurodegeneration, or aging, the level of S65 phosphorylation increases significantly (4, 8). S65-phosphorylated Ub (pUb) in turn can activate PARKIN, a ubiquitin ligase, and induce mitophagy (9-12). However, no other pUb-specific targets have been clearly identified, and how phosphorylation affects Ub signaling in general remains unclear.Previously, Komander and coworkers showed that pUb gives two distinct sets of NMR peaks, which correspond to the two conformational states of pUb exchanging at a slow timescale (13). Based on NMR long-r...

show abstract

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Gong

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Recent data show that pUb S65 is not uniformly distributed in the cell. Rather, pUb S65 was shown by immuno-detection to colocalize with parkin and the mitochondrial membrane [38]. The abundance of pUb S65 increases with oxidative stress in cell-based studies and with age and PD status in patient tissue samples [39].…”

Section: Codon Skippingmentioning

confidence: 96%

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

et al. 2016

View full text Add to dashboard Cite

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.

show abstract

“…The following primary antibodies were used for Western blot (WB) and/or immunofluorescence (IF): anti-CDC37 (WB, 1:1,000, 4793, Cell Signaling), anti-HSP90 (WB, 1:2,000, 610419, BD), mouse anti-GAPDH (WB, 1: 150, 000, H86504M, Meridian Life science), mouse anti-Mitofusin 1 (WB, 1:5,000, ab57602, Abcam), rabbit anti-PINK1 (WB, 1:2,000, BC100-494, Novus Biologicals), rabbit anti-PINK1 (WB, 1: 2, 000, IF, 1: 100, #6946, Cell Signaling), mouse anti-TOM20 (IF, 1: 250, sc-17764, Santa Cruz Biotechnology), anti-TOM40 (WB, 1: 1,000, 18409-1-AP, ProteinTech Group), mouse anti-Ub (WB, 1: 1, 000, MAB1510, ubi-1, Millipore), rabbit anti-pS65-Ub (WB, 1:5,000-1:15,000, IF, 1:250, in house 18) ), mouse anti-V5 (WB, 1: 5, 000, R960-25, Invitrogen), rabbit anti-V5 (WB, 1: 5, 000, ab9115, Abcam), Streptavidin HRP (WB, 1: 150, 0000, Pierce).…”

Section: Antibodiesmentioning

confidence: 99%