Journal: Science Advances

Article Title: RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway

doi: 10.1126/sciadv.aav0443

Figure Lengend Snippet: ( A and B ) HFT PARKIN WT cells treated with doxycycline (DOX) were depolarized with AO for the indicated periods of time before analysis of whole-cell extracts (WCE) by SDS–polyacrylamide gel electrophoresis (PAGE) using the indicated antibodies. ( C ) Schematic for the creation of HFT-PARKIN WT cells (with or without previous deletion of PINK1) expressing GFP-TBK1 using CRISPR-Cas9. ( D ) HFT-PARKIN WT cells with or without either PINK1 or GFP-TBK1 were depolarized with AO for 1 hour before immunoblotting (IB) of whole-cell extracts with the indicated antibodies. ( E and F ) Quantitative analysis of pS172 in TBK1 in the presence and absence of AO (1 hour) was performed using PRM. Error bars represent SEM from biological triplicate measurements. UT, untreated. ( G ) Schematic workflow for TBK1-dependent phosphoproteome discovery. Whole-cell extracts from the indicated cells (duplicates for untreated and 1-hour treatments and a single replicate for 1.5 hours of depolarization) were cleaved with Lys-C and trypsin, phosphopeptides were enriched using TiO 2 , and samples were labeled for 10-plex TMT before analysis using SPS-MS 3 . m/z , mass/charge ratio. ( H ) Peptide and protein quantification from TMT proteomics of experiment outlined in (G). ( I ) Dynamics of pS65-Ub in response to mitochondrial depolarization from the experiment outlined in (G). ( J ) Dynamics of phosphorylation sites in RAB7A, RMD3, VIM, and SQSTM1 from the experiment outlined in (G). ( K ) Structure of RAB7A (Protein Data Bank: 1T91) showing the location of switch I in blue, switch II in magenta, S72 in green, and GTP in red.

Article Snippet: A collection of four heavy labeled TBK1 reference peptides ( 163 ELEDDEQFV S LYGTEEYLHPD M Y ER 187 phosphorylated at Ser 172 and with methionine oxidized and 144 VIGEDGQS V YK 154 , 242 PSGAISG V QK 251 , and 647 YQEYTNELQETL P QK 661 ) was produced at Cell Signaling Technology and quantified by amino acid analysis, and a collection of two heavy labeled RAB7 reference peptides ( 70 FQ S LGVAFY R 79 phosphorylated or not at Ser 172 ) was produced (0.1 mg) at SynPeptide Co. Ltd (underlined residue is phosphorylated, and bold amino acid is heavy).

Techniques: Polyacrylamide Gel Electrophoresis, Expressing, CRISPR, Western Blot, Labeling, Phospho-proteomics

Journal: Science Advances

Article Title: RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway

doi: 10.1126/sciadv.aav0443

Figure Lengend Snippet: ( A ) Schematic for experiments examining the phosphorylation of S72 in RAB7A by TBK1. The indicated cells were subjected to depolarization, and cell extracts were subjected to α-FLAG immunoprecipitation (IP) before either Phostag-PAGE or PRM proteomics. ( B ) Immunoblotting of whole-cell extracts from the indicated cells using α-RAB7A or α-actin as a loading control. ( C ) α-FLAG immunoprecipitates from the indicated cells were separated by Phostag-PAGE followed by immunoblotting with the indicated antibodies. ( D and E ) Quantitative analysis of pS72 in RAB7A in the presence and absence of AO (1 hour) was performed using PRM. Error bars represent SEM from triplicate measurements. ( F ) TBK1 requirement for RAB7A S72 phosphorylation in response to AO (1 hour). The indicated cells were subjected to depolarization, and cell extracts were subjected to α-FLAG immunoprecipitation before Phostag-PAGE and immunoblotting with α-RAB7A antibodies. ( G ) HFT-PARKIN WT or S65A cells with or without PINK1 were depolarized for the indicated times and purified mitochondria enriched for phosphopeptides using immobilized metal anion chromatography (IMAC) before TMT-based proteomics. RP, reversed-phase. ( H ) Volcano plot of −log( P value) versus the log 2 FC (fold change) for quantified phosphopeptides for PARKIN WT cells with or without AO treatment for 1 hour. Phosphorylation site and number of peptides quantified are shown in parenthesis. ( I ) Relative abundance of pS72 RAB7A normalized to RAB7A abundance also measured by TMT. Error bars represent SEM from biological triplicate measurements. n.s., not significant.

Article Snippet: A collection of four heavy labeled TBK1 reference peptides ( 163 ELEDDEQFV S LYGTEEYLHPD M Y ER 187 phosphorylated at Ser 172 and with methionine oxidized and 144 VIGEDGQS V YK 154 , 242 PSGAISG V QK 251 , and 647 YQEYTNELQETL P QK 661 ) was produced at Cell Signaling Technology and quantified by amino acid analysis, and a collection of two heavy labeled RAB7 reference peptides ( 70 FQ S LGVAFY R 79 phosphorylated or not at Ser 172 ) was produced (0.1 mg) at SynPeptide Co. Ltd (underlined residue is phosphorylated, and bold amino acid is heavy).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Control, Purification, Chromatography

Journal: Science Advances

Article Title: RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway

doi: 10.1126/sciadv.aav0443

Figure Lengend Snippet: ( A ) Coomassie blue PAGE analysis of purified GST-RAB7A WT and RAB7A S72A after purification from E. coli . ( B ) The indicated GST-RAB7A proteins were incubated with recombinant TcPINK1 ( E. coli ) or GST-TBK1 (insect cells) (1 hour at 30°C, with or without ATP), and reaction products were separated by Phostag-PAGE before immunoblotting with α-RAB7A antibody. ( C ) Alignment of the amino acid sequence around S72 in RAB7A (red asterisk) and selected RABs. ( D ) Coomassie blue SDS-PAGE analysis of purified GST-RAB1A, GST-RAB1B, and GST-RAB7L (with or without mutation of the S72 equivalent to Ala) after purification from E. coli . ( E ) The indicated GST-RABs were incubated with or without GST-TBK1 (1 hour at 30°C, with ATP), and reaction products were separated by Phostag-PAGE and immunoblotted with α-GST antibody. ( F ) Coomassie blue SDS-PAGE analysis of purified GST-RAB8A WT , RAB8A S72A , and RAB8A S111A after purification from E. coli . ( G ) The indicated GST-RAB8A proteins were incubated with or without GST-TBK1 and analyzed as in (E).

Article Snippet: A collection of four heavy labeled TBK1 reference peptides ( 163 ELEDDEQFV S LYGTEEYLHPD M Y ER 187 phosphorylated at Ser 172 and with methionine oxidized and 144 VIGEDGQS V YK 154 , 242 PSGAISG V QK 251 , and 647 YQEYTNELQETL P QK 661 ) was produced at Cell Signaling Technology and quantified by amino acid analysis, and a collection of two heavy labeled RAB7 reference peptides ( 70 FQ S LGVAFY R 79 phosphorylated or not at Ser 172 ) was produced (0.1 mg) at SynPeptide Co. Ltd (underlined residue is phosphorylated, and bold amino acid is heavy).

Techniques: Purification, Incubation, Recombinant, Western Blot, Sequencing, SDS Page, Mutagenesis

Journal: Science Advances

Article Title: RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway

doi: 10.1126/sciadv.aav0443

Figure Lengend Snippet: ( A ) Triton X-100 (1%) lysates from HFT PARKIN WT ;RAB7A −/− cells reconstituted with FLAG-HA-RAB7A WT or the S72A or S72E mutants (see ) were subjected to α-FLAG immunoprecipitation-MS in triplicate, and interacting proteins were quantified by TMT-based proteomics. Volcano plots of −qlog( P value) versus the log 2 FC of the indicated pairs of proteins for triplicate measurements are shown. ( B ) As in (A) but depolarized for 1 hour with AO. ( C ) Histogram of the relative abundance of selected proteins found in association with RAB7A WT and either S72A or S72E mutants. Error bars represent SEM from biological triplicate measurements. ( D ) The indicated cell lines were left untreated or depolarized for 1 hour with AO, and α-FLAG immunoprecipitates were subjected to immunoblotting with the indicated antibodies. ( E ) GST-RAB7A and pS72-GST-RAB7A were made in E. coli . For pS72-GST-RAB7A, protein was made using the amber codon suppressor system in the presence of a tRNA system for activation of phospho-Ser . Both proteins were purified using glutathione (GSH)–Sepharose and separated by SDS-PAGE before Coomassie staining. ( F ) The stoichiometry of GST-RAB7A phosphorylation on S72 was measured using PRM proteomics and found to be ~70%. ( G ) Schematic for use of immobilized pS72-GST-RAB7A for binding to FLCN in cell extracts. ( H ) Immobilized GST-RAB7A, pS72-GST-RAB7A, or GSH-resin was incubated with cell extracts (4 hours at 4°C), and washed beads were subjected to SDS-PAGE and immunoblotting with either α-FLCN or α-RAB7A antibodies. Extract (2% of input) was run as a loading control. ( I ) HA-FLCN/FLAG-FNIP1 complexes (6 μg, purified from mammalian cells and immobilized on α-FLAG resin) were incubated with 20 μg of GST-RAB7A WT or S72A, S72E, or T22N mutants (3 hours at 4°C). Washed resin was subjected to SDS-PAGE and immunoblotting with the indicated antibodies. Input proteins were similarly analyzed in parallel.

Article Snippet: A collection of four heavy labeled TBK1 reference peptides ( 163 ELEDDEQFV S LYGTEEYLHPD M Y ER 187 phosphorylated at Ser 172 and with methionine oxidized and 144 VIGEDGQS V YK 154 , 242 PSGAISG V QK 251 , and 647 YQEYTNELQETL P QK 661 ) was produced at Cell Signaling Technology and quantified by amino acid analysis, and a collection of two heavy labeled RAB7 reference peptides ( 70 FQ S LGVAFY R 79 phosphorylated or not at Ser 172 ) was produced (0.1 mg) at SynPeptide Co. Ltd (underlined residue is phosphorylated, and bold amino acid is heavy).

Techniques: Immunoprecipitation, Western Blot, Activation Assay, Purification, SDS Page, Staining, Phospho-proteomics, Binding Assay, Incubation, Control

Journal: Science Advances

Article Title: RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway

doi: 10.1126/sciadv.aav0443

Figure Lengend Snippet: ( A ) The indicated HFT-PARKIN WT cell lines were treated with DOX (16 hours) to induce PARKIN and depolarized with AO (1 hour), and mitochondrial extracts were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of biological triplicate experiments as described in (A). Error bars represent SEM. ( C ) Schematic describing how S72 in RAB7A was replaced by Ala using CRISPR-Cas9. The replacement template used was single-stranded DNA containing S72A. ( D and E ) HFT-PARKIN WT cell lines were treated with DOX (16 hours) to induce PARKIN and depolarized with AO for 1 hour, and either whole-cell lysates (D) or mitochondrial extracts (E) were subjected to SDS-PAGE and immunoblotting using the indicated antibodies. ( F ) Quantification of biological triplicate experiments as described in (B). Error bars represent SEM.

Article Snippet: A collection of four heavy labeled TBK1 reference peptides ( 163 ELEDDEQFV S LYGTEEYLHPD M Y ER 187 phosphorylated at Ser 172 and with methionine oxidized and 144 VIGEDGQS V YK 154 , 242 PSGAISG V QK 251 , and 647 YQEYTNELQETL P QK 661 ) was produced at Cell Signaling Technology and quantified by amino acid analysis, and a collection of two heavy labeled RAB7 reference peptides ( 70 FQ S LGVAFY R 79 phosphorylated or not at Ser 172 ) was produced (0.1 mg) at SynPeptide Co. Ltd (underlined residue is phosphorylated, and bold amino acid is heavy).

Techniques: SDS Page, Western Blot, CRISPR

Journal: Science Advances

Article Title: RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway

doi: 10.1126/sciadv.aav0443

Figure Lengend Snippet: ( A ) Top: Example images of individual HFT-PARKIN WT cells 32 hours after depolarization stained with α-DNA antibodies to detect unaggregated, aggregated, or cleared mitochondrial. Bottom: Histogram of the results of PARKIN-dependent mitophagy assays 32 hours after depolarization using the indicated cells and analyzed as in the top panel of (A). Error bars represent SEM from biological triplicate measurements, with >100 cells analyzed per replicate. Cells were treated with DOX for 2.5 hours to induce PARKIN before depolarization. ( B ) Schematic workflow for analysis of proteins enriched in lysosomes from cells expressing wild-type (WT) or S72A RAB7A. Extracts from the indicated cells were subjected to density centrifugation, and lysosomal fractions were subjected to 10-plex TMT using SPS-MS 3 analysis . ( C ) Volcano plot [−log( P value) versus log 2 FC (RAB7A WT /RAB7A S72A )] of proteins present in the lysosomal fraction 16 hours after depolarization. Proteins in pink or blue represent proteins with P < 0.05 and log 2 FC of >1.5 or <−1.5, respectively. Mitochondrial proteins (based on MitoCarta2.0) are shown in dark red circles, while the location of RAB7A itself, the HOPS complex, and CCZ1-MON1 is shown in yellow, green, and blue circles, respectively. ( D ) Histogram of the relative abundance of selected mitophagy-related proteins in the lysosome based on the data in (C). ( E ) Mitophagy assays as in (A) for cells lacking FLCN with or without rescue with MSCV-FLAG-HA-FLCN. ( F ) The indicated cells were depolarized with AO for 2 hours, and mitochondria were purified before SDS-PAGE and immunoblotting with the indicated antibodies. SA, S72A. ( G ) Working model for TBK1-driven phosphorylation of RAB7A and downstream regulation of mitophagy. See text for details. ER, endoplasmic reticulum; HM, heavy mitochondria; LM, light mitochondria; PM, plasma membrane.

Article Snippet: A collection of four heavy labeled TBK1 reference peptides ( 163 ELEDDEQFV S LYGTEEYLHPD M Y ER 187 phosphorylated at Ser 172 and with methionine oxidized and 144 VIGEDGQS V YK 154 , 242 PSGAISG V QK 251 , and 647 YQEYTNELQETL P QK 661 ) was produced at Cell Signaling Technology and quantified by amino acid analysis, and a collection of two heavy labeled RAB7 reference peptides ( 70 FQ S LGVAFY R 79 phosphorylated or not at Ser 172 ) was produced (0.1 mg) at SynPeptide Co. Ltd (underlined residue is phosphorylated, and bold amino acid is heavy).

Techniques: Staining, Expressing, Centrifugation, Purification, SDS Page, Western Blot, Phospho-proteomics, Clinical Proteomics, Membrane