PINK 1 autophosphorylation is required for ubiquitin recognition
2018; Springer Nature; Volume: 19; Issue: 4 Linguagem: Inglês
10.15252/embr.201744981
ISSN1469-3178
AutoresShafqat Rasool, Naoto Soya, Luc Truong, Nathalie Croteau, Gergely L. Lukács, Jean‐François Trempe,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle23 February 2018free access Transparent process PINK1 autophosphorylation is required for ubiquitin recognition Shafqat Rasool Shafqat Rasool Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Department of Biochemistry, McGill University, Montréal, QC, Canada Search for more papers by this author Naoto Soya Naoto Soya orcid.org/0000-0003-0086-4389 Department of Physiology and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Luc Truong Luc Truong Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Nathalie Croteau Nathalie Croteau Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Gergely L Lukacs Gergely L Lukacs Department of Biochemistry, McGill University, Montréal, QC, Canada Department of Physiology and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Jean-François Trempe Corresponding Author Jean-François Trempe [email protected] orcid.org/0000-0002-6543-3371 Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Shafqat Rasool Shafqat Rasool Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Department of Biochemistry, McGill University, Montréal, QC, Canada Search for more papers by this author Naoto Soya Naoto Soya orcid.org/0000-0003-0086-4389 Department of Physiology and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Luc Truong Luc Truong Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Nathalie Croteau Nathalie Croteau Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Gergely L Lukacs Gergely L Lukacs Department of Biochemistry, McGill University, Montréal, QC, Canada Department of Physiology and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Jean-François Trempe Corresponding Author Jean-François Trempe [email protected] orcid.org/0000-0002-6543-3371 Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada Search for more papers by this author Author Information Shafqat Rasool1,2, Naoto Soya3,‡, Luc Truong1,‡, Nathalie Croteau1, Gergely L Lukacs2,3 and Jean-François Trempe *,1 1Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada 2Department of Biochemistry, McGill University, Montréal, QC, Canada 3Department of Physiology and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +1 514 398 6833; E-mail: [email protected] EMBO Reports (2018)19:e44981https://doi.org/10.15252/embr.201744981 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Small-angle X-ray scattering and hydrogen–deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin. Synopsis The mitochondrial kinase PINK1 autophosphorylates rapidly in trans at a single conserved serine. This phosphorylation step is required for PINK1 to interact with its substrates ubiquitin and Parkin. PINK1 has a higher affinity for the Parkin ubiquitin-like (Ubl) domain, which it binds via a surface conserved with ubiquitin. Insect PINK1 autophosphorylates in trans at Ser205 (Ser228 in human PINK1). Mutation of that serine strongly reduces ubiquitin and Parkin Ubl phosphorylation. Structural analysis of insect PINK1 in solution reveals that Ser205 phosphorylation induces significant changes in the dynamics of the protein near the active site. Introduction Mutations in PINK1 (PARK6 gene) and Parkin (PARK2 gene) cause autosomal recessive early-onset forms of Parkinson's disease 12. PINK1 is a mitochondrially targeted serine–threonine kinase 13, and Parkin is a RING-between-RING (RBR)-type E3 ubiquitin ligase, and they are both involved in a mitochondrial quality control pathway 4. The pathway comes into play when mitochondria undergo damage, which triggers the stabilization of PINK1 on the outer mitochondrial membrane (OMM) as an active kinase 5. There, it phosphorylates ubiquitin (Ub) 678 and cytosolic Parkin on its N-terminal ubiquitin-like domain (Ubl) specifically at Ser65 9—a residue conserved in both Ub and Ubl. The phosphorylation of Parkin and its binding to phospho-Ub results in the activation of its E3 ligase activity and localization to the mitochondria, allowing it to ubiquitinate OMM proteins 1011. Build-up of ubiquitin chains on mitochondrial proteins recruits cargo receptors, including optineurin and NDP52, and subsequently autophagy machinery to the mitochondria to initiate mitophagy 1213. In healthy mitochondria, PINK1 is imported, cleaved by the mitochondrial processing peptidase (MPP), presenilin-associated rhomboid-like (PARL) protease, and AFG3L2 1415, and then degraded through the ubiquitin–proteasome pathway 16. Hence, PINK1 is kept at low levels under basal condition, and its accumulation on mitochondria is a specific cellular response to mitochondrial damage. In recent years, the role of phosphorylation of Ub and Ubl at Ser65 by PINK1 has been discovered in terms of the molecular mechanism for the activation of Parkin 1718192021. While some proteomics studies in yeast have found multiple sites of phosphorylation on cellular Ub including Ser57, phosphorylation at Ser65 is so far the best characterized 22. PINK1 is also the only known ubiquitin kinase to date. Despite this well-characterized phosphorylation of Ub and Ubl at Ser65 by PINK1 and its consequences for Parkin activation and localization on mitochondria, the molecular mechanisms governing the recognition and phosphorylation of Ub and Ubl by PINK1 upstream of Parkin activation are not well understood. Human PINK1 (hPINK1) is a 581 amino acid protein with an N-terminal mitochondrial targeting sequence (MTS) followed by a putative transmembrane helix, a linker region (here on referred to as NT linker), a kinase domain, and a C-terminal segment (Fig EV1A). The kinase domain is reported to be a canonical bilobular domain weakly homologous (~20–25% sequence identity) to the calmodulin-dependent kinase (CamK) and DMPK families 23. The N-lobe of the kinase domain of hPINK1 harbors three insertions relative to homologous Ser/Thr kinases. The NT linker and C-terminal segment do not have homology to known proteins and hence are unique features of PINK1 as well 24. Some studies suggest that the C-terminal segment might play a role in regulating the kinase activity of PINK1 25. The exact functions of these unique features remain to be understood. Owing to the poor expression of hPINK1 in bacterial expression systems and low levels of activity in vitro, most recent studies have made use of PINK1 orthologs from insect species Tribolium castaneum (red flour beetle) and Pediculus humanus corporis (louse) for in vitro functional assays 6782627. PINK1 displays a high degree of conservation across species, with about 40–45% sequence identity in the cytosolic domain between human and insect orthologs (Fig EV1B). Click here to expand this figure. Figure EV1. Domain structure and sequence alignment of PINK1 Domain structure of human Parkin and PINK1. Selected PD mutations are shown on top, and residue numbers are shown below. The phosphorylation site Ser228 is highlighted in red. Multiple sequence alignment of PINK1 orthologs; Tribolium castaneum (Tc, red beetle), Homo sapiens (human), Mus musculus (mouse), Gallus gallus (chicken), Danio rerio (zebra fish), Drosophila melanogaster (fruit fly), and Aplysia californica (sea slug). The alignment shows residues conserved across all species (blue), sites of autophosphorylation found in purified TcPINK1 are indicated with *. Ser205 is colored in red, and active site residues Lys196 and Asp337 are indicated. The alignment was performed using the MUSCLE server (https://www.ebi.ac.uk/Tools/msa/muscle/) for the segment corresponding to TcPINK1121–570. The N-terminal segments (corresponding to TcPINK11–120) have low conservation and were not aligned for better visual display of their lengths. Download figure Download PowerPoint Multiple studies have indicated that PINK1 is autophosphorylated in its activated form on the mitochondria 392829. Three different phosphorylation sites have been found in different studies: Ser228 (located just upstream of the putative regulatory helix in the N-lobe), Thr257 (located in the second insertion of the N-lobe), and Ser402 (located in the putative kinase activation segment). Thr257 was not found to be required for PINK1 activity in terms of Parkin recruitment 9. Ser228 and Ser402 were found to be required for Parkin recruitment as serine-to-alanine mutations at these positions abolished Parkin recruitment to mitochondria under conditions of CCCP-induced damage 29. Later, it was shown in another study that S402A rendered PINK1 temperature sensitive and phosphorylation at this position was not required for PINK1 activity 30. Nonetheless, the precise role of autophosphorylation at other sites on PINK1's kinase activity or substrate recognition is not understood. Herein, we present our findings about the recognition and phosphorylation of Ub and Ubl by PINK1 using the Tribolium castaneum ortholog (TcPINK1). Using enzyme kinetic assays, we show that Ubl is a more favored phosphorylation substrate for PINK1 compared to Ub. Using two-dimensional NMR and phosphorylation assays, we establish the binding site of PINK1 on Ubl. Furthermore, we show that TcPINK1 autophosphorylates in trans specifically at Ser205 (Ser228 in hPINK1) and that phospho-Ser205 is required for substrate recognition and phosphorylation. Finally, hydrogen–deuterium exchange (HDX) mass spectrometry reveals that phosphorylation of Ser205 changes the conformation and dynamics of the C-helix region and activation loop in the kinase domain. Results Parkin Ubl is the preferred substrate of PINK1 While multiple studies have examined the phosphorylation of Ub and Ubl by PINK1, the relative affinities of both substrates to PINK1 and preference as phosphorylation targets are poorly characterized. Despite the high protein fold conservation, and the conservation of many key residues and features such as Ser65 and the Ile44 hydrophobic patch, Ub and Parkin Ubl only share 32% sequence identity 31, which could result in a difference in affinity and phosphorylation kinetics. In order to achieve a better understanding of Ubl and Ub phosphorylation, we performed kinetic analyses of the phosphorylation of both substrates by TcPINK1. As opposed to its human ortholog, the kinase domain from TcPINK1 is soluble, can be expressed in an active form in E. coli 26, and can phosphorylate mammalian Parkin Ubl and Ub (Appendix Fig S1A). Moreover, we found that PD-like mutations in TcPINK1, for the most part, abolish the Ub kinase activity (Appendix Fig S1C), as observed in hPINK1 for Parkin 32. Phosphorylated Ubl and Ub were loaded on phos-tag gels to perform densitometry analysis (Appendix Fig S1A and B). The results show that Ub phosphorylation has a 10-fold higher Km than Ubl phosphorylation (Fig 1A). This indicates that the Ubl may have a higher affinity for TcPINK1, assuming that kcat is greater than koff. While the kcat for Ub is twofold higher than for the Ubl, the overall catalytic efficiency (kcat/Km ratio) for Ubl is more than fourfold higher than Ub (0.22 versus 0.05). This marked preference for Ubl was also maintained in full-length TcPINK1, suggesting that residues upstream of the kinase domain do not contribute significantly to substrate selectivity (Appendix Fig S1D). Overall, the results of the kinetics indicate that the Ubl domain of Parkin is a preferred substrate for TcPINK1 compared to Ub. Figure 1. Parkin Ubl is the preferred substrate for PINK1 Enzyme kinetics of Ub and Ubl phosphorylation by TcPINK1. 5-min phosphorylation assays were conducted with different concentrations of Ub or Ubl with GST-TcPINK1(143–570), visualized on phos-tag gels and modeled to the Michaelis–Menten equation. The given graphs represent global fits to data collected from two sets of reactions for both Ub and Ubl performed independently. Bars represent the mean ± SD (n = 2). 1H-15N TROSY-HSQC NMR spectra of 2H,15N-labeled Ubl or Ub alone (blue), or titrated with different concentrations of GST-TcPINK1 (red, green, and black). The spectra are shifted on the y-axis to better visualize the decrease in the peak intensity (there is no chemical shift displacement). Structure of Parkin Ubl (PDB 4ZYN) and Ub (PDB 1UBQ) showing regions (pink) with backbone amides experiencing greatest loss of signal upon addition of 25 μM and 150 μM GST-TcPINK1, respectively. Fractional levels of phosphorylation of WT and point mutants of Ubl. The phosphorylation assays were performed with 30 μM GST-Ubl and 2 μM GST-TcPINK1 and loaded on phos-tag gels followed by densitometry (n = 1). Original gels and similar experiments performed under different conditions can be found in Fig EV3. Phosphorylation time course of Ub or Ub2 (K6-, K48-, or K63-linked). Experiments were performed with 30 μM substrate and 2.5 μM GST-TcPINK1 and analyzed by intact mass spectrometry (n = 1). Original data shown in Appendix Fig S3. Download figure Download PowerPoint NMR studies reveal how PINK1 engages its substrates To determine how PINK1 engages its substrates, we titrated 2H,15N-labeled Ub or Ubl with GST-TcPINK1 and recorded 1H-15N TROSY-HSQC spectra. Upon binding, the fully protonated PINK1 should induce line-broadening in the Ubl resonances that are in proximity to PINK1, while other resonances should remain sharp in spite of the decreased rotational tumbling via TROSY selection. We indeed observe a selective loss of signal with increasing GST-TcPINK1 concentrations (Figs 1B and EV2A) and not with free GST (Appendix Fig S2A). Consistent with our observations that Ubl is a preferred substrate of PINK1 compared to Ub, loss of signals is observed at a TcPINK1/Ubl concentration ratio of 1:3 whereas signal loss is registered at much higher TcPINK1/Ub ratio, indicating a weaker binding to Ub. Click here to expand this figure. Figure EV2. TROSY NMR analysis of TcPINK1 binding to Ubl and Ub Full 1H-15N TROSY NMR spectra of 2H,15N-labeled Ubl or Ub with different concentrations of GST-TcPINK1121–570 (corresponding to Fig 1B). Bar graph of peak height ratios between the 75 μM Ubl + 25 μM GST TcPINK1121–570 titration point and 75 μM Ubl for all the backbone amides from the H1-N15 TROSY experiment shown in (A). Amides with the lowest value of peak height ratio are marked by red boxes. Bar graph of peak height ratios between the 37.5 μM Ub + 150 μM GST-TcPINK1121–570 and 37.5 μM Ub for all the backbone amides from the H1-N15 TROSY experiment shown in (A). Amides with the lowest value of peak height ratio are marked by red boxes. Download figure Download PowerPoint Site-specific analysis of signal loss for the titrations with Ubl revealed that the fastest signal loss occurs at the backbone amides in four distinct regions: residues 7–8, 45–49, 62–66, and 69–72 (Figs 1C and EV2B). This implies that the binding site of PINK1 is located on the β-sheet face of Ubl and involves the solvent-exposed side chains of Asn8, Ile44, Ser65, His68, Val70, and Arg72. With the exception of Asn8—a leucine in Ub—these residues are conserved between Ub and Ubl. We observed a more widespread broadening in the titration with 2H,15N-labeled Ub (Fig EV2C). We attribute this primarily to relaxation broadening from residual 1H in Ub, combined with the slower tumbling induced by oligomerization that takes place in TcPINK1 at high concentrations (required to saturate the weaker binding), or to chemical exchange broadening arising from widespread structural changes in Ub in the PINK1-bound form [33; see Discussion]. Nevertheless, we could unambiguously identify the amides of residues 46–49 in Ub as undergoing the fastest signal loss upon TcPINK1 binding (Fig 1C), which also show selective broadening in the Ubl. To confirm the binding site derived from the NMR experiments, we mutated residues in these regions of the Ubl to alanine and found that they display decreased phosphorylation by both GST-TcPINK1121–570 and GST-TcPINK1143–570 compared to WT Ubl (Figs 1D and EV3A and B). In particular, I44A and H68A displayed the most robust reduction in phosphorylation, followed by R72A, R6A, and K48A. These mutations did not unfold the Ubl, since 1H NMR spectra of all the mutants show a characteristic peak at −0.2 ppm, corresponding to the side-chain methyl Cδ2 of Leu61 (Fig EV3C). This residue is in the hydrophobic core of the Ubl domain and located “above” the aromatic ring of Phe45, conferring a shielded chemical shift 34. To investigate the determinants of a more favorable binding for Ubl compared to Ub, we mutated residues in the Ubl-binding site corresponding to different residues in Ub and performed phosphorylation assays. While only a minor decrease in phosphorylation was seen for the I66T Ubl, N8L underwent a robust decrease in phosphorylation compared to WT Ubl (Fig 1D). To confirm the role of Asn8 in increasing its interaction with PINK1, we mutated ubiquitin to the corresponding residues in the Ubl of Parkin. Phosphorylation assays show that the L8N mutation modestly increases activity compared to Ub WT (Fig EV3D). Moreover, mutations K63Q and E64Q, adjacent to the phosphorylation site, also increased phosphorylation. Click here to expand this figure. Figure EV3. Phosphorylation assays with Ubl and Ub mutants confirm the PINK1 binding site (Left) Phos-tag gels of phosphorylation assays shown in Fig 1D. (Right) Fractional levels of phosphorylation of WT and point mutants of Ubl (left). The phosphorylation assays were performed with 30 μM of GST-Ubl and 2 μM GST-TcPINK1143–570 and loaded on phos-tag gels followed by densitometry (n = 1). (Top) Phos-tag gels of phosphorylation assays and (bottom) fractional levels of phosphorylation of WT and point mutants of Ubl. The phosphorylation assays were performed with 12.5 μM of GST-Ubl and 2 μM GST-TcPINK1121–570 and loaded on phos-tag gels followed by densitometry (n = 2). Error bars represent mean ± range of two independent phosphorylation experiments, loaded on the same gel. 1D proton NMR spectra of 30 μM GST-Ubl WT or mutants, or untagged Ubl showing the characteristic Leu61 peak at −0.2 ppm (*) observed in all the mutants shown. (Top) Phos-tag gels of phosphorylation assays and (bottom) fractional levels of phosphorylation of mutants of point of Ub made by mutating residues in Ub to corresponding residues in Ubl. The assays were conducted with 30 μM of GST-Ubl and 0.5 μM GST-TcPINK1121–570 for 30 min (n = 1). Download figure Download PowerPoint We also noticed that three lysine acceptor sites for polyubiquitin chains are located at the PINK1-binding site (Lys6, Lys48, and Lys63). We thus performed a phosphorylation time course with K6-, K48-, and K63-linked diubiquitin (Ub2). All Ub2 chains were singly phosphorylated at rates slightly faster than Ub (Fig 1E and Appendix Fig S3). However, the chains differed in their ability to be doubly phosphorylated. Notably, only a single phosphorylation site could be observed in K6-Ub2, suggesting K6 linkage interferes with PINK1 binding, as well as K48 to a lesser degree. This is consistent with both Arg6 and Lys48 in the Ubl being important for PINK1 binding. In conclusion, our results show that PINK1 engages its substrates through a common interface that comprises Ile44, Lys48, His68, and Arg72, while Asn8, Gln63, and Gln64 in the Ubl mediate interactions that make it a preferred substrate for PINK1. PINK1 competes with Parkin RING1 and SH3 for Ubl binding In the structure of full-length Parkin, the Ubl domain binds to the RING1 domain via the Ile44-centered site 1835. The Parkin Ubl also interacts with the SH3 domain of endophilin-A1 (referred to as SH3 from here onwards) via the same Ile44 surface 34. Comparing our Parkin Ubl NMR and mutagenesis results to these previous studies, we find that the Ubl:RING1 and Ubl:SH3 interactions involve the same residues implicated in binding PINK1 (Fig 2A). Hence, we hypothesized that Parkin RING1 and SH3 would compete with PINK1 for binding Ubl. To test this hypothesis for Parkin, we conducted enzyme kinetics assays with full-length WT or L266K Parkin. We have previously shown that mutation of Leu266 disrupts the interface of RING1 for Ubl at the Ile44 patch 18. Consistent with this, our enzyme kinetics experiments reveal that L266K Parkin is phosphorylated by TcPINK1 with a Km of 31 ± 7 μM, similar to the value obtained for the Ubl domain alone (Fig 2B). Phosphorylation of WT Parkin was slower than L266K, and the concentration range did not enable a reliable estimation of Km. This result reinforces our previous observation that the Ubl is not accessible to PINK1 due to its engagement with RING1 18. Figure 2. PINK1 shares binding site on Ubl with Parkin RING1 and endophilin-A1 SH3 Binding interface of Parkin Ubl (green) and RING1 domain (black; PDB 4ZYN), and Parkin Ubl (green) and endophilin-A1 SH3 (magenta; PDB 1KNB). Enzyme kinetics of the phosphorylation of full-length Parkin WT or L266K. 5-min assays were performed with different concentrations of Parkin WT or L266K with GST-TcPINK1 (143-570) and visualized using phos-tag gels. 1H-15N HSQC NMR spectra of competition assays between 15N-labeled endophilin-A1 SH3 domain and GST-TcPINK1 for Ubl binding. The peaks represent backbone amide signals from the spectra of 62 μM SH3 alone (red), following the addition of 48 μM Ubl (black), 200 μM GST-TcPINK1 WT (green), or 48 μM Ubl and 200 μM GST-TcPINK1 WT (blue). Chemical shift differences for different 1H-15N SH3 cross peaks plotted as a function of Ubl concentrations (12, 24, 48 μM), with and without 200 μM GST-TcPINK1. Data from 10 peaks were fitted to an exact competition model, with the affinity constants displayed in the boxed area (average ± SD). Download figure Download PowerPoint The competition hypothesis with SH3 was tested by performing NMR titrations of 15N-labeled SH3 domain with Ubl and TcPINK1 as a competitor (Fig 2C). As reported previously, addition of Ubl to 15N-SH3 induces chemical shifts perturbations that reflect complex formation 34. Addition of WT TcPINK1 to 15N-SH3 and Ubl caused a decrease in the peak shift indicating that it competes with SH3 for Ubl binding. The chemical shift changes from these titrations points were fitted to an exact binding competition model 36, to calculate a Kd of 15 ± 4 μM for the Ubl–SH3 interaction and 43 ± 11 μM for the Ubl–PINK1 interaction (Fig 2D). The Kd for the Ubl–SH3 interaction is similar to the one reported previously 34. Moreover, the equilibrium dissociation constant of the Ubl–PINK1 interaction is similar to the Km value from the kinetics experiment reported above (Fig 1A). Thus, the interaction of the Parkin Ubl with endophilin-A1 SH3 domain and PINK1 are mutually exclusive, and the competitive nature of these interactions allows us to estimate the Kd for the Ubl:TcPINK1 interaction. TcPINK1 autophosphorylates in trans at Ser205 The ability of kinases to interact with their substrates is often modulated by autophosphorylation 37, and thus, we sought to better characterize PINK1 autophosphorylation. Multiple sites of phosphorylation for PINK1 have indeed been reported in the literature 2629. Mass spectrometry analysis of intact WT TcPINK1 revealed multiple peaks of different intensities greater than the theoretical mass of WT TcPINK1 and 80 Da apart from each other, implying that there were at least eight different phosphorylation sites (Fig EV4A). No autophosphorylation was detected for the TcPINK1 D337N kinase-dead mutant, confirming that the observed phosphorylation results from autocatalytic reactions. LC-MS/MS analysis of a tryptic digest from WT TcPINK1 revealed major phosphorylation sites (with high identification scores) at S-1 (linker), Ser154, Thr186, Ser205, Thr218, Ser267, and Thr530, as well as a number of other minor sites (Fig 3A and Table EV1). Click here to expand this figure. Figure EV4. Mass spectrometry analysis of TcPINK1 autophosphorylation Intact mass spectra of TcPINK1121–570 WT or D337N expressed for different times in E. coli. The double-edged arrows on the spectra are used to indicate a difference of 80 Da (1 phosphorylation) between peaks. “0p” indicates the theoretical mass of the unphosphorylated protein. MS/MS spectrum for the peptide 197-212 of TcPINK1121–570 D337N, phosphorylated by GST-TcPINK1121–570 WT (see Fig 3C). The b and y series indicate that phosphorylation takes place at Ser205. The y* series (green) show neutral loss of a phosphate group (H3PO4, −97.7 amu). Intact mass spectra of the time course phosphorylation assay of TcPINK1 D337N with CIP-treated 15N-TcPINK1 WT (corresponding to the plot in Fig 3D). MS/MS spectrum for the peptide 197–212 of TcPINK1121–570 14N-D337N and 15N-WT, after 2-min phosphorylation (see Fig 3E). The b and y series indicate that phosphorylation takes place at Ser205 for the 14N and 15N-labeled peptides eluting at 36.5 min, and Ser207 for the 15N-labeled peptide eluting earlier at 35.8 min. The y* series (green) show neutral loss of a phosphate group (H3PO4, −97.7 amu). The different phosphorylation sites give rise to y(6) and y(7) ions with different masses. Download figure Download PowerPoint Figure 3. TcPINK1 autophosphorylates in trans at Ser205 Schematic diagram of TcPINK1 WT (121–570) showing the location of different autophosphorylation sites in the protein expressed and purified in E. coli. “S-1” refers to the phosphorylation at the remnant serine from the 3C cleavage of the N-terminal GST tag. Intact mass spectra of TcPINK1 D337N before and after phosphorylation with GST-tagged WT, S205N or K196A. The assays were performed with 25 μM substrate and 0.5 μM enzyme for 5 min. Extracted ion chromatograms (top) and precursor ion spectrum (bottom) of a.a. 197–212 from TcPINK1 D337N, before (blue) and after (red) phosphorylation by GST-TcPINK1 WT. The two peaks correspond to the elution of the non-phosphorylated and Ser205-phosphorylated peptide (see MS/MS, Fig EV4B). Time course of a 2-min phosphorylation assay with 1 μM TcPINK1-D337N and 1 μM CIP-treated 15N-labeled TcPINK1 WT. Peak intensity from intact mass spectra of different phosphorylated species (0, 1, or 2 sites) is plotted as a function of time. The diagram above displays how WT PINK1 can theoretically phosphorylate in cis or in trans, whereas kinase-dead D337N can only be phosphorylated in trans. The rate of WT phosphorylation is the sum of cis and trans phosphorylation. LC-MS/MS analysis of the digests of the 0 and 2 min time points from the time course shown in (D), demonstrating phosphorylation at Ser205. The phosphorylation sites were identified by MS/MS (see Fig EV4D). Download figure Download PowerPoint Ser205 (Ser228 in human PINK1) is located at the base of the putative regulatory αC-helix in the N-lobe of the kinase domain and is the only phosphorylation site that is conserved across all PINK1 orthologs (Fig EV1B). Moreover, we found that a shorter induction time for protein expression in E. coli leads to a reduced number of phosphorylation sites (Fig EV4A). We thus hypothesized t
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