Fox Foundation for Parkinson’s Research Langston Award to Dario R Alessi

Fox Foundation for Parkinson’s Research Langston Award to Dario R Alessi. RH1 Medical Research Council MC_UU_12016/2 to Dario R RH1 Alessi. National Institutes of Health DK37332 to Suzanne R Pfeffer. Additional information Competing interests No competing interests declared. Author contributions Conceptualization, Data curation, Validation, Investigation, Visualization, Methodology, Designed and executed experiments in Figures 2, 3, 5, 6, 7, 8, 9 with PL and was involved in discussing and interpreting the data. Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Methodology, Writingoriginal draft, Writingreview and editing, Designed and executed experiments in Figures 2, 3, 5, 6, 7, 8, 9 with KB and was involved in discussing and interpreting the data. Data curation, Formal analysis, Validation, Investigation, Visualization, Designed and executed Figure 11, Designed and executed Figures 4 and 10 with PSW and was involved in discussing and interpreting the data. Data curation, Investigation, Visualization, Methodology, Designed and executed Figures 4 and 10 with WMY and was involved in discussing and interpreting the data. Data curation, Formal analysis, Investigation, Visualization, Designed and executed mass spectrometry experiments for Figures 6B, 6D, 9B, Figure 8Figure supplement 2, Figure supplement 5 and expression analysis of PPM1H and PPM1M in Figure 12Figure Supplements 1 and 2 and was involved in discussing and interpreting the data. Data curation, Investigation, Methodology, Undertook most of the cloning required for this study. Data curation, Investigation, Methodology, Generated expression constructs for CRISPR/CAS9 gene editing studies. Investigation, Methodology, Developed the expression and purification system to produce active recombinant PPM1H, PPM1M and PPM1J phosphatases, and MST3 kinase. Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Expressed, purified and phosphorylated Rab8A for experiments shown in Figure 8, Involved in discussing and interpreting the data. Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Designed and executed experiments in Figure 1, Involved in discussing and interpreting the data. Conceptualization, Data curation, Supervision, Funding acquisition, Visualization, Writingoriginal draft, Project administration, Writingreview and editing, Supervised the project with DRA and wrote the manuscript. Conceptualization, Formal analysis, Supervision, Funding acquisition, Writingoriginal draft, Project administration, Writingreview and editing, Supervised the project with SRP and wrote the manuscript. Additional files Supplementary file 1.Numerical data of the pRab10/Total Rab10 ratios relative to scrambled siRNA control from Screens 1, 2 and 3 (experiments shown in Figure 2). addition to the quantitation of the pRab10/Total Rab10 ratios in Supplementary file 1. The file also contains all RNA sequences of siRNA library. All Plasmids, antibodies and proteins (including datasheets and sequence information) that we have generated for this study can be requested and information downloaded from MRC PPU Reagents and Services (https://mrcppureagents.dundee.ac.uk/). The following dataset was generated: Kerryn Berndsen, Pawel Lis, Raja S Nirujogi. 2019. PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins. ProteomeXchange. PXD014794 Abstract Mutations that activate LRRK2 protein kinase cause Parkinsons disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A substrate-trapping PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H RH1 acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinsons disease. DH5 and purified using a Hi\Speed Plasmid Maxi Kit (Qiagen). siRNA G-CSF screens The siRNA screen was performed using a human siRNA library (Dharmacon) designed to target 322 phosphatases. The list of siRNA targets and the sequences of all siRNA oligonucleotides used are provided in Supplementary File 1. A549 cells were seeded in 6-well plates at 150,000 cells/well. After 24 h cells were transfected using 2 l Lipofectamine RNAi Max and 20 pmol of siRNA per well. Cells were then cultured for a further 72 h. In Screen 1 and 2, cells were directly lysed without further treatment, whereas in Screen 3, cells were treated for 5 min with 100 nM MLi-2 prior to lysing. Lysates were centrifuged at 20,800 g for 15 min at 4C, quantified by Bradford assay (Thermo Scientific) and subjected to immunoblot analysis. Heavy synthetic peptides Heavy phosphorylated either 13C615N4 (Arg*) or 13C615N2 (Lys*) containing pRab1(FRpTITSSYYR*), pRab3 (YRpTITTAYYR*), pRab8(FRpTITTAYYR*), pRab10(FHpTITTSYYR*), total Rab10 (NIDEHANEDVER*, AFLTLAEDILR*) non-phosphorylated Thr73 pRab10(FHTITTSYYR*), pRab35(FRpTITSTYYR*) and pRab43(FRpTITQSYYR*) peptides were synthesized from JPT innovative peptide technologies (https://www.jpt.com/). All of the synthesized peptides are of >95% isotopic purity and an independent verification for the absolute amounts were determined by Amino acid analysis (AAA analysis), HPLC and LC-MS/MS analysis. Generation of PPM1H CRISPR/Cas9 knockout CRISPR was performed using a paired nickase approach to minimize off-target effects (Ran et al., 2013a). Analysis of the locus (ENSG00000111110) showed the expression of a single verified transcript (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_020700.2″,”term_id”:”1519315875″,”term_text”:”NM_020700.2″NM_020700.2, ENST00000228705.7) and exon one specific guide pairs with low combined off-targeting scores were subsequently identified using the Sanger Institute CRISPR webtool (http://www.sanger.ac.uk/htgt/wge/find_crisprs). Complementary oligos for the optimal guide pair A (G1 and G2 transformed with either Plasmid DU62835 (expresses His-SUMO-PPM1H[wild-type]), Plasmid DU68104 (His-SUMO-PPM1H[H153D]), or Plasmid DU68087 (His-SUMO-PPM1H[D288A]). RH1 Bacteria were cultured at 37C until OD600 0.4C0.6. The temperature was reduced to 15C and protein expression was induced by addition of isopropyl -D-1-thiogalactopyranoside to 50 M in addition to MnCl2 to 2 mM as PPM family of phosphatases require Mn or Mg for stability (Das et al., 1996). Cells were cultured for 16 hr before harvesting by centrifugation at 4200 x g for 20 min at 4C. The pellet was resuspended in 200 ml of ice cold lysis buffer (50 mM Tris/HCl pH7.5, 150 RH1 mM NaCl, 1% (by vol) Triton, 2 mM MnCl2, 0.5 mM TCEP (tris(2-carboxyethyl)phosphine)), 1 mM Pefabloc (4-(2-aminoethyl)-benzene-sulfonyl fluoride) and 1 mM benzamidine. Cells were lysed using a cell disruptor (passing sample through twice) and extracts clarified by centrifugation at 30,000 x g for 20 min at 4C. Lysates were incubated in 2 ml of Cobalt-Agarose (Amintra Cobalt NTA Affinity Resin, Expedeon) that was equilibrated in lysis buffer and incubated on a roller at 4C for 90 min. The resin was loaded onto a column and washed with 20 column volumes of High Salt Wash Buffer (50 mM Tris/HCl pH7.5, 500 mM NaCl, 2.