The Pyridoxal 5 '-Phosphate (PLP)-Dependent Enzyme Serine Palmitoyltransferase (SPT):Effects of the Small Subunits and Insights from Bacterial Mimics of Human hLCB2a HSAN1 Mutations

The pyridoxal 5′-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) catalyses the first step of de novo sphingolipid biosynthesis. The core human enzyme is a membrane-bound heterodimer composed of two subunits (hLCB1 and hLCB2a/b), and mutations in both hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) have been identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Here we measure the activities of the hLCB2a mutants in the presence of ssSPTa and ssSPTb and find that all decrease enzyme activity. High resolution structural data of the homodimeric SPT enzyme from the bacterium Sphingomonas paucimobilis (Sp SPT) provides a model to understand the impact of the hLCB2a mutations on the mechanism of SPT. The three human hLCB2a HSAN1 mutations map onto Sp SPT (V246M, G268V, and G385F), and these mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

This is the author accepted manuscript. The final version is available from EMBO Press via the DOI in this recordMutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65).Wellcome Trust Senior Research Fellowship in Clinical Science101022/Z/13/Z; Medical Research Council; Wellcome Trust; Parkinson's UK; Michael J. Fox Foundation for Parkinson's Disease Research; Tenovus Scotland; Wellcome/MRC; UCL Institute of Neurology; University of Sheffield; MRC‐PPU of University of Dundee; Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer‐Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Serine⁶⁵

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser(65) within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser(65) that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser(65) in ubiquitin lies in a similar motif to Ser(65) in the Ubl domain of Parkin. Remarkably, PlNK1 directly phosphorylates Ser(65) of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser(65) (Delta Ubl-Parkin) is robustly activated by ubiquitin(Phospho-Ser65), but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser(65) (Ubl(phospho-Ser65)) can also activate Delta Ubl-Parkin similarly to ubiquitin(PhosPh-Ser65). Thirdly, we establish that ubiquitin(PhosPh-Ser65), but not non-phosphorylated ubiquitin or Ubl(PhosPh-Ser65) activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser(65) and ubiquitin at Ser(65), since only mutation of both proteins at Ser(65) completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser(65), but also by phosphorylating ubiquitin at Ser(65). We propose that phosphorylation of Parkin at Ser(65) serves to prime the E3 ligase enzyme for activation by ubiquitin(PhosPh-Ser65), suggesting that small molecules that mimic ubiquitin(PhosPh-Ser65) could hold promise as novel therapies for Parkinson's disease

PINK1 and Parkin - mitochondrial interplay between phosphorylation and ubiquitylation in Parkinson's disease

The discovery of mutations in genes encoding protein kinase PTEN-induced kinase 1 (PINK1) and E3 ubiquitin ligase Parkin in familial Parkinson's disease and their association with mitochondria provides compelling evidence that mitochondrial dysfunction is a major contributor to neurodegeneration in Parkinson's disease. In recent years, tremendous progress has been made in the understanding of how PINK1 and Parkin enzymes are regulated and how they influence downstream mitochondrial signalling processes. We provide a critical overview of the key advances in the field and also discuss the outstanding questions, including novel ways in which this knowledge could be exploited to develop therapies against Parkinson's disease

Phosphorylation of Parkin at Serine65 is essential for activation:elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser(65)) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser(65) phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser(65) by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser(65) phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin's ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser(65) is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson's disease

PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD

Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog

Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access--including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop