{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063470/checksum.txt"],"Fasta":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063470/PDZ-DEP.fasta"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063470/Ion-Outputs.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063470/211208_HDX_sample.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063470/220210_PDZ.zip"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["Cherine.bechara@umontpellier.fr"],"submitter":["Functional Proteomics Platform FPP"],"technology_type":["HDMSE","Mass Spectrometry"],"software":["Not available"],"submitter_keywords":["Phosphorylation","Hdx-ms","Dishevelled"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD063470"],"tissue":["Cell Suspension Culture"],"sample_protocol":["DVL3 PDZ-DEP was produced by using E. coli as a host organism. To obtain the phosphorylated sample, PDZ-DEP was co-expressed with CK1ε. PDZ-DEP was purified using IMAC (His tag), anion exchange chromatography and SEC. HDX-MS experiments were performed using a Synapt G2-Si HDMS coupled to nanoAQUITY UPLC with HDX Automation technology (Waters Corporation). Dvl3 was concentrated up to 34µM and optimization of the sequence coverage was performed on undeuterated controls. Analysis of phosphorylated and non-phosphorylated PDZ-DEP were performed as follows: 3 µL of sample were diluted in 57 µL of undeuterated for the reference or deuterated equilibration buffer (10 mM TrisCl pH 7.5, 100 mM NaCl). The final percentage of deuterium in the deuterated buffer was 95%. Deuteration was performed at 20°C for 0.5, 5, 10, 20, 40, 60, 120 and 240 min. Next, 50 µL of reaction sample was quenched in 50 µL of quench buffer (KH2PO4 50 mM, K2HPO4 50mM, pH 2.3) at 0°C. 80 µL of quenched sample was loaded onto a 50 µL loop and injected on an online EnzymateTM pepsine column (Waters) maintained at 20°C, with 0.2% formic acid at a flowrate of 100 µL/min. The peptides were then trapped at 0°C on a Vanguard column (ACQUITY UPLC BEH C18 VanGuard Pre-column, 130Å, 1.7 µm, 2.1 mm X 5 mm, Waters) for 3 min, before being loaded at 40 µL/min onto an Acquity UPLC column (ACQUITY UPLC BEH C18 Column, 1.7 µm, 1 mm X 100 mm, Waters) kept at 0°C. Peptides were subsequently eluted with a linear gradient (0.2% formic acid in acetonitrile solvent at 5% up to 35% during the first 6 min, then up to 40% and 95% over 1 min each) and ionized directly by electrospray on a Synapt G2-Si mass spectrometer (Waters). HDMSE data were obtained by 20-30 V trap collision energy ramp. Lock mass accuracy correction was made using a mixture of Leucine enkephalin and GFP. Deuteration timepoints were performed in triplicates for each condition.  Peptide identification was performed from undeuterated data using ProteinLynx global Server (PLGS, version 3.0.3, Waters). Peptides were filtered by DynamX (version 3.0, Waters) using the following parameters: minimum intensity of 1000, minimum product per amino acid of 0.2, maximum error for threshold of 10 ppm. All peptides identified in the linker region were discarded and we only kept peptides peptides present in both protein states, phosphorylated and non-phosphorylated, for comparison. All peptides were manually checked, and data was curated using DynamX."],"repository":["Pride"],"modification":[""],"quantification_method":[""],"data_protocol":["DVL3 PDZ-DEP was produced by using E. coli as a host organism. To obtain the phosphorylated sample, PDZ-DEP was co-expressed with CK1ε. PDZ-DEP was purified using IMAC (His tag), anion exchange chromatography and SEC. HDX-MS experiments were performed using a Synapt G2-Si HDMS coupled to nanoAQUITY UPLC with HDX Automation technology (Waters Corporation). Dvl3 was concentrated up to 34µM and optimization of the sequence coverage was performed on undeuterated controls. Analysis of phosphorylated and non-phosphorylated PDZ-DEP were performed as follows: 3 µL of sample were diluted in 57 µL of undeuterated for the reference or deuterated equilibration buffer (10 mM TrisCl pH 7.5, 100 mM NaCl). The final percentage of deuterium in the deuterated buffer was 95%. Deuteration was performed at 20°C for 0.5, 5, 10, 20, 40, 60, 120 and 240 min. Next, 50 µL of reaction sample was quenched in 50 µL of quench buffer (KH2PO4 50 mM, K2HPO4 50mM, pH 2.3) at 0°C. 80 µL of quenched sample was loaded onto a 50 µL loop and injected on an online EnzymateTM pepsine column (Waters) maintained at 20°C, with 0.2% formic acid at a flowrate of 100 µL/min. The peptides were then trapped at 0°C on a Vanguard column (ACQUITY UPLC BEH C18 VanGuard Pre-column, 130Å, 1.7 µm, 2.1 mm X 5 mm, Waters) for 3 min, before being loaded at 40 µL/min onto an Acquity UPLC column (ACQUITY UPLC BEH C18 Column, 1.7 µm, 1 mm X 100 mm, Waters) kept at 0°C. Peptides were subsequently eluted with a linear gradient (0.2% formic acid in acetonitrile solvent at 5% up to 35% during the first 6 min, then up to 40% and 95% over 1 min each) and ionized directly by electrospray on a Synapt G2-Si mass spectrometer (Waters). HDMSE data were obtained by 20-30 V trap collision energy ramp. Lock mass accuracy correction was made using a mixture of Leucine enkephalin and GFP. Deuteration timepoints were performed in triplicates for each condition.  Peptide identification was performed from undeuterated data using ProteinLynx global Server (PLGS, version 3.0.3, Waters). Peptides were filtered by DynamX (version 3.0, Waters) using the following parameters: minimum intensity of 1000, minimum product per amino acid of 0.2, maximum error for threshold of 10 ppm. All peptides identified in the linker region were discarded and we only kept peptides peptides present in both protein states, phosphorylated and non-phosphorylated, for comparison. All peptides were manually checked, and data was curated using DynamX."],"omics_type":["Proteomics"],"labhead":["Cherine Bechara"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["University of Montpellier, France"],"species":["Homo Sapiens (human)","Escherichia Coli"],"publication":["Not available"],"submitter_mail":["contact@fpp.cnrs.fr"],"submitter_affiliation":["FPP CNRS"],"submitter_country":["France"],"additional_accession":[]},"is_claimable":false,"name":"Multisite phosphorylation of intrinsically disordered region of Dishevelled","description":"When WNT binds to its receptor Frizzled (FZD) to activate the signaling, Dishevelled (DVL), the central component of Wnt signaling, is phosphorylated by redundant Casein kinase 1 (CK1) isoforms ε or δ, to transduce the signal downstream. Although the basic principles of Wnt signal transduction are known, the mechanistic aspects of DVL phosphorylation remain elusive. Here we identify Wnt-dependent multisite phosphorylation of a large and conserved serine/threonine (S/T) cluster within intrinsically disordered region (IDR), located between the PDZ and DEP domains. The detailed analysis of the phosphorylation and its effect on the flanking domains was performed with the construct containing both domains PDZ and DEP flanking the IDR (hDVL3 PDZ-DEP, aa 243-496).","dates":{"publication":"2026-04-07","submission":"2025-04-30"},"accession":"PXD063470","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:3555","NEWT:241368","NEWT:2","NEWT:157546","NEWT:190802","NEWT:35554","NEWT:9778","NEWT:150475","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216979","NEWT:307972","NEWT:32046","NEWT:544496","NEWT:5180","NEWT:256737","NEWT:2042546","NEWT:115104","NEWT:1081927","NEWT:67825","NEWT:185579","NEWT:43179","NEWT:13076","NEWT:1249668","NEWT:317","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:1211601","NEWT:415540","NEWT:876138","NEWT:4081","NEWT:554","NEWT:98334","NEWT:426428","NEWT:237561","NEWT:6928","NEWT:10036","NEWT:7574","NEWT:1351","NEWT:7215","NEWT:29204","NEWT:272563","NEWT:507601","NCBITaxon:79857","NCBITaxon:6157","NEWT:95648","NEWT:3885","NEWT:746360","NEWT:6239","NEWT:3888","NEWT:1589","NEWT:470150","NEWT:135622","NEWT:216257","NEWT:6915","NEWT:9986","NEWT:101510","NEWT:4054","NEWT:3880","NEWT:272559","NEWT:226186","NEWT:3641","NEWT:383379","NEWT:8782","NEWT:1263854","NEWT:1000589","NEWT:435590","NEWT:1902","NEWT:85962","NEWT:160488","NEWT:28104","NEWT:317447","NEWT:7955","NCBITaxon:2","NEWT:985076","NEWT:7959","NEWT:2261","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:216595","NEWT:243230","NEWT:8355","NEWT:1283","NEWT:931281","NEWT:4550","NEWT:1000561","NEWT:7029","NEWT:1283300","NEWT:6183","NEWT:6063","NEWT:334747","NEWT:61235","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:436486","NEWT:6287","NEWT:2242","NEWT:300641","NEWT:4784","NEWT:727","NEWT:9796","NEWT:725","NEWT:360106","NEWT:260707","NEWT:287","NEWT:10117","NEWT:10239","NCBITaxon:6191","NEWT:10116","NEWT:1280","NEWT:1836","NEWT:1735272","NEWT:83334","NEWT:185431","NEWT:83332","NEWT:29760","NEWT:260704","NEWT:703612","NEWT:260705","NEWT:80863","NEWT:44685","NEWT:2697049","NEWT:1148","NEWT:11676","NEWT:55571","NEWT:100226","NCBITaxon:6073","NEWT:4530","NEWT:4896","NEWT:6279","NEWT:1123869","NEWT:7370","NEWT:75058","NEWT:83906","NEWT:607699","NEWT:6282","NEWT:1094343","NEWT:208964","NEWT:1134506","NEWT:575584","NEWT:296543","NEWT:1773","NEWT:38783","NEWT:8727","NEWT:1895","NEWT:4006","NEWT:1182590","NEWT:8726","NEWT:6669","NEWT:10090","NEWT:935293","NEWT:64152","NEWT:749200","NEWT:4120","NEWT:51515","NEWT:5693","NEWT:8724","NEWT:51511","NEWT:92867","NEWT:8723","NEWT:990346","NEWT:5334","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:230741","NEWT:284812","NCBITaxon:10359","NCBITaxon:1313","NEWT:43330","NEWT:242619","NEWT:44544","NEWT:632957","NEWT:373995","NEWT:645463","NEWT:544404","NEWT:3702","NEWT:129249","NEWT:9925","NEWT:8839","NEWT:4232","NEWT:990119","NEWT:2758385","NEWT:4113","NEWT:837","NEWT:11298","NEWT:171101","NEWT:421932","NEWT:196627","NEWT:408172","NEWT:5691","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:627025","NEWT:400772","NEWT:1097677","NEWT:3708","NEWT:128161","NEWT:106592","NEWT:536231","NEWT:61674","NEWT:1117957","NEWT:9913","NEWT:1432138","NEWT:10312","NEWT:1424507","NEWT:4100","NEWT:1076","NEWT:6763","NEWT:803","NEWT:8030","NEWT:29722","NEWT:380394","NEWT:1692259","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:180066","NEWT:67767","NEWT:46835","NEWT:135588","NEWT:1843183","NEWT:95486","NEWT:58002","NEWT:9103","NEWT:4577","NEWT:1416333","NEWT:5664","NEWT:2157","NEWT:146479","NEWT:10306","NCBITaxon:2759","NEWT:1911079","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:3635","NEWT:5811","NEWT:1480154","NEWT:235443","NEWT:1274414","NEWT:27606","NEWT:59202","NEWT:9975","NEWT:3197","NEWT:9615","NEWT:10299","NEWT:860688","NEWT:411483","NEWT:884019","NEWT:411490","NEWT:169963","NEWT:36329","NEWT:1147787","NCBITaxon:3044782","NEWT:72407","NEWT:476272","NEWT:349741","NEWT:9606","NEWT:367830","NEWT:157295","NEWT:641501","NEWT:178616","NEWT:410289","NEWT:373153","NEWT:915099","NEWT:74940","NEWT:9721","NEWT:1450511","NEWT:360094","NEWT:470","NEWT:411901","NEWT:1313","NEWT:411469","NEWT:84023","NEWT:9838","NCBITaxon:9615","NCBITaxon:5811","NEWT:58334","NEWT:411464","NEWT:1193501","NEWT:3055","NEWT:6326","NEWT:6689","NEWT:411460","NEWT:2762","NEWT:5476","NEWT:1174673","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NEWT:1274432","NEWT:1274426","NEWT:1423","NEWT:4932","NEWT:70448","NEWT:9825","NEWT:1274423","NEWT:3603","NEWT:698936","NEWT:2759","NEWT:3847","NEWT:39946","NEWT:9823","NEWT:178876","NEWT:9940","NEWT:327160","NEWT:573","NEWT:521001","NEWT:9031","NEWT:1274420","NEWT:7091","NEWT:578458"]}}