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Digestion was quenched by 0.1% TFA  and resulting peptides were desalted using C18 stage-tips. LC-MS/MS was performed by  coupling an UltiMate 3000 RSLCnano LC system to a Q Exactive Plus mass spectrometer  (Thermo Fisher Scientific). Digested peptides were loaded into a trap column (Acclaim™ PepMap™ 100 C18 LC Columns 5 µm, 20 mm length) for 3 min at a flow rate of 10 µL/min in  0.1% formic acid. Then, peptides were transferred to an EASY-Spray PepMap RSLC C18  column (Thermo) (2 µm, 75 µm x 50 cm) operated at 45 °C and separated using a 60 min  effective gradient (buffer A: 0.1% FA; buffer B: 100% ACN, 0.1% FA) at a flow rate of 250  nL/min. The gradient used was, from 4% to 6% B in 2 min, from 6% to 33% B in 58 min, plus  10 additional min at 98% B. Peptides were sprayed at 1.5 kV into the mass spectrometer via  the EASY-Spray source. The capillary temperature was set to 300 °C. The mass spectrometer  was operated in a data-dependent mode, with an automatic switch between MS and MS/MS  scans using a top 15 method (intensity threshold ≥ 4.5 x 104 , dynamic exclusion of 5 or 10 sec  and excluding charges unassigned, +1 and > +6). MS spectra were acquired from 350 to 1500  m/z with a resolution of 70,000 FWHM (200 m/z). Ion peptides were isolated using a 2.0 Th  window and fragmented using higher-energy collisional dissociation (HCD) with a normalized  collision energy of 27. MS/MS spectra resolution was set to 35,000 (200 m/z). The ion target  values were 3 x 106 for MS (maximum IT of 25 ms) and 1x105 for MS/MS (maximum IT of 110  msec). Crosslinking:Both, phosphorylated (1 mM ATP, 2 mM MgCl2, 30 min) and un-phosphorylated CCDC6-RET (4-6 µM) were crosslinked with 200-fold molar excess of disuccinimidyl dibutyric urea (DSBU,  Thermo scientific) in 20 mM HEPES pH 7.5, 500 mM NaCl, 1mM TCEP. All the crosslinking  reactions were performed at RT for 50 min and then quenched with 20 nM NH4HCO3. The  cross-linked samples were vacuum-dried and resuspended in 4% SDS in 50 mM Tris/HCl pH  8.0. Reduction and alkylation were performed with 15 mM TCEP and 25 mM chloroacetamide  30 min at 45 ºC, respectively. Samples were then trypsinized (16 hours, 37 °C) at an enzymeto-substrate ratio of 1:50 following an automated SP3 protocol (Hughes et al., 2019). Digested  samples were adjusted to 20 mM NH4OH and fractionated in four fractions by micro reverse  phase C18 high pH chromatography, using homemade EmporeC18 Stage-Tips. Fractions  were vacuum-dried and resuspended in 1% (v/v) formic acid, 0.5% DMSO for LC-MS/MS  analysis. Peptides were analysed by LC-MS/MS in an Exploris480 mass spectrometer  (Thermo) coupled to an UltiMate 3000 RSLCnano LC system (Thermo). Peptides were loaded  into a trap column (Acclaim™ PepMap™ 100 C18 LC Columns 5 µm, 20 mm length) for 3 min  at a flow rate of 10 µl/min in 0.1% formic acid. Then, peptides were transferred to an EASYSpray PepMap RSLC C18 column (Thermo) (2 µm, 75 µm x 50 cm) operated at 45 °C and  separated using a 60 min effective gradient (Solvent A: 0.1% FA; solvent B: 100% ACN, 0.1%  FA) at a flow rate of 250 nL/min. The gradient used was, from 4% to 6% B in 2 min, from 6%  to 33% B in 58 min, plus 10 additional min at 98% B. The mass spectrometer was operated in  a data-dependent mode, with an automatic switch between MS and MS/MS scans using a top  12 method. (Intensity threshold ≥ 7.5 x 104 , dynamic exclusion of 30 sec and excluding  charges (+1 and ≥ +8). MS spectra were acquired from 400 to 1500 m/z with a resolution of  60,000 FWHM (200 m/z). Ion peptides were isolated using a 0.7 Th window and fragmented  using higher-energy collisional dissociation (HCD) with a stepped normalized collision energy  14 of 29, 31 and 33. MS/MS spectra resolution was set to 60,000 (200 m/z). The ion target values  were 3 x 106 for MS (maximum IT of 25 msec) and 1 x 105 for MS/MS (maximum IT set to  auto, according to the transient length)"],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Raw files were processed with Maxquant (v 1.6 and higher) using the standard settings against  the corresponding sequences of the expressed recombinant proteins. Carbamidomethylation  of cysteines was set as a fixed modification whereas oxidation of methionines, acetylation and  phosphorylation of serines, threonines and tyrosines were set as variable modifications.  Minimal peptide length was set to 7 amino acids and a maximum of two tryptic missedcleavages were allowed. Results were filtered at 0.01 FDR (peptide and protein level). Raw  data were imported into Skyline. Label free quantification of identified phosphopeptides was  performed using the extracted ion chromatogram of the isotopic distribution. Only peaks  13 without interference were used for quantification. Phosphopeptides intensities were  normalized by the intensity of non-modified peptides from the target protein Crosslink data:For data analysis, Xcalibur raw files were converted  into the MGF format through MSConvert (Proteowizard) and used directly as input files for  MeroX (Iacobucci et al., 2018). Searches were performed against an ad-hoc protein database  containing the sequences of the complexes and a set of randomized decoy sequences  generated by the software. The following parameters were set for the searches: maximum  number of missed cleavages 3; targeted residues K, S, Y and T; minimum peptide length five  amino acids; variable modifications: carbamidomethyl-Cys (mass shift 57.02146 Da), Metoxidation (mass shift 15.99491 Da); BuUrBu modification fragments: 85.05276 Da and  111.03203 (precision: 10 ppm MS1 and 20 ppm MS2); false discovery rate cut-off: 5%. Finally,  each fragmentation spectra were manually inspected and validated."],"omics_type":["Proteomics"],"labhead":["Marta Isasa"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["CNIO"],"species":["Homo Sapiens (human)"],"publication":["41792117 Martín-Hurtado A, Contreras J, Sánchez-Wandelmer J, Zarzuela E, García F, Le Coq J, Boskovic J, Muñoz IG, Gago F, Muñoz J, Isasa M, Plaza-Menacho I. The oncogenic CCDC6-RET fusion protein is a dual ATP- and ADP-dependent kinase. Nat Commun. 2026 17(1):3595 10.1038/s41467-026-69833-y"],"submitter_mail":["eduzarfer@gmail.com"],"submitter_affiliation":["Centro Nacional de Investigaciones Oncológicas"],"submitter_country":["Spain"],"pubmed_abstract":["Gene fusion products involving protein kinases are known drivers in human cancers and actionable targets for personalized therapy, yet the structural and molecular determinants that control their function are largely unexplored. Here we show that a CCDC6-RET fusion protein, a driver and therapeutic target in lung and thyroid cancers, is a highly active dimeric kinase. Time-resolved mass spectrometry together with a robust biochemical and biophysical characterization reveal that CCDC6-RET functions as a dual ATP- and ADP-dependent kinase able to bind both nucleotides and to use them as phosphoryl donors. We also identify a crosstalk between the C-terminal and the activation segments controlling both the processing and the catalytic activity of the fusion protein. Furthermore, a 3D-structural assembly of a CCDC6-RET homodimer was generated combining single particle electron microscopy, small-angle X-ray scattering and in silico molecular dynamics simulations. Our structural model together with cross-linking mass spectrometry data demonstrated that CCDC6-RET in the inactive state forms a face-to-face dimer characterized by intermolecular-crosslinked activation segments. Upon nucleotide binding the catalytic domains swing apart and fast activation loop phosphorylation could be driven by a mechanism in cis. Our work uncovers the molecular and structural determinants that control the mechanism of CCDC6-RET autoactivation."],"pubmed_title":["The oncogenic CCDC6-RET fusion protein is a dual ATP- and ADP-dependent kinase."],"pubmed_authors":["Martín-Hurtado Ana A, Contreras Julia J, Sánchez-Wandelmer Jana J, Zarzuela Eduardo E, García Fernando F, Le Coq Johanne J, Boskovic Jasminka J, Muñoz Inés G IG, Gago Federico F, Muñoz Javier J, Isasa Marta M, Plaza-Menacho Iván I"],"additional_accession":[]},"is_claimable":false,"name":"Phospho-proteomic characterization of CCDC6-RET","description":"Here we show that a CCDC6-RET fusion product, a driver and therapeutic target in lung and thyroid cancers, is a highly active dimeric kinase in solution. Time-resolved mass spectrometry analysis together with a robust biochemical and biophysical characterization reveal the autophosphorylation kinetics and nucleotide dependency. Structural analyses together with cross-linking mass spectrometry data  provided clear insights into the mechanism of autoactivation.","dates":{"publication":"2026-04-27","submission":"2024-07-12"},"accession":"PXD053907","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:3555","NEWT:2","NEWT:157546","NEWT:35554","NEWT:38942","NEWT:307972","NEWT:32046","NEWT:544496","NEWT:2042546","NEWT:43179","NEWT:4513","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:1211601","NEWT:876138","NEWT:237561","NEWT:6928","NEWT:10036","NEWT:1351","NEWT:1438992","NEWT:2649997","NEWT:272563","NEWT:224326","NCBITaxon:79857","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:1589","NEWT:135622","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:3880","NEWT:272559","NEWT:3641","NEWT:383379","NEWT:10029","NEWT:1000589","NEWT:85962","NEWT:317447","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:1283","NEWT:931281","NEWT:4550","NEWT:1000561","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:1270","NEWT:2242","NEWT:4784","NEWT:11320","NEWT:360106","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:44685","NEWT:1148","NEWT:580240","NEWT:11676","NEWT:55571","NEWT:100226","NEWT:4530","NEWT:4896","NEWT:75058","NEWT:13616","NEWT:1094343","NEWT:296543","NEWT:1773","NEWT:1895","NEWT:1182590","NEWT:935293","NEWT:64152","NEWT:4924","NEWT:749200","NEWT:990346","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:263","NEWT:230741","NEWT:284812","NCBITaxon:1313","NEWT:43330","NEWT:44544","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:990119","NEWT:408172","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:257313","NEWT:400772","NEWT:3708","NEWT:128161","NEWT:332648","NEWT:106592","NEWT:536231","NEWT:1432138","NEWT:10312","NEWT:1424507","NCBITaxon:1773","NEWT:9598","NEWT:8030","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:67767","NEWT:46835","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:10306","NCBITaxon:2759","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:240906","NEWT:593117","NEWT:3635","NEWT:5811","NEWT:235443","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:169963","NEWT:499175","NEWT:476272","NEWT:367830","NEWT:178616","NEWT:410289","NEWT:373153","NEWT:360094","NEWT:470","NEWT:1313","NEWT:411469","NEWT:84023","NCBITaxon:5811","NEWT:411464","NEWT:411460","NEWT:2762","NEWT:1174673","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:28038","NEWT:1423","NEWT:4932","NEWT:3603","NEWT:2759","NEWT:3847","NEWT:178876","NEWT:327160","NEWT:573","NEWT:9031","NEWT:7091","NEWT:241368","NEWT:42528","NEWT:190802","NEWT:9778","NEWT:150475","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216979","NEWT:5180","NEWT:256737","NEWT:115104","NEWT:1081927","NEWT:1238993","NEWT:67825","NEWT:185579","NEWT:13076","NEWT:1249668","NEWT:317","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:9402","NEWT:415540","NEWT:4081","NEWT:554","NEWT:98334","NEWT:426428","NEWT:7574","NEWT:7215","NEWT:575412","NEWT:29204","NEWT:507601","NCBITaxon:6157","NEWT:746360","NEWT:6239","NEWT:470150","NEWT:216257","NEWT:9986","NEWT:4054","NEWT:226186","NEWT:1268063","NEWT:8782","NEWT:1263854","NEWT:435590","NEWT:1902","NEWT:160488","NEWT:28104","NCBITaxon:2","NEWT:985076","NEWT:1215323","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:2829","NEWT:216599","NEWT:216595","NEWT:243230","NEWT:8355","NEWT:9685","NEWT:7029","NEWT:1283300","NEWT:6183","NEWT:6063","NEWT:334747","NEWT:61235","NEWT:6289","NEWT:436486","NEWT:6287","NEWT:300641","NEWT:727","NEWT:9796","NEWT:725","NEWT:260707","NCBITaxon:6191","NEWT:1836","NEWT:185431","NEWT:29760","NEWT:260704","NEWT:703612","NEWT:260705","NEWT:80863","NEWT:2697049","NCBITaxon:6073","NEWT:884204","NEWT:6279","NEWT:1123869","NEWT:9544","NEWT:7370","NEWT:83906","NEWT:607699","NEWT:6282","NEWT:208964","NEWT:1134506","NEWT:575584","NEWT:38783","NEWT:8727","NEWT:4006","NEWT:8726","NEWT:6426","NEWT:6669","NEWT:10090","NEWT:4120","NEWT:51515","NEWT:5693","NEWT:8724","NEWT:51511","NEWT:92867","NEWT:8723","NEWT:5334","NCBITaxon:10359","NEWT:242619","NEWT:632957","NEWT:373995","NEWT:544404","NEWT:9925","NEWT:8839","NEWT:4232","NEWT:2758385","NEWT:4113","NEWT:837","NEWT:11298","NEWT:171101","NEWT:714","NEWT:421932","NEWT:196627","NEWT:5691","NEWT:627025","NEWT:1097677","NEWT:61674","NEWT:1117957","NEWT:9913","NEWT:4100","NEWT:1076","NEWT:6763","NEWT:803","NEWT:29722","NEWT:380394","NEWT:1692259","NEWT:180066","NEWT:135588","NEWT:1843183","NEWT:58002","NEWT:4577","NEWT:1416333","NEWT:5664","NEWT:2157","NEWT:418985","NEWT:146479","NEWT:1911079","NEWT:1480154","NEWT:1274414","NEWT:27606","NEWT:59202","NEWT:9975","NEWT:9612","NEWT:38865","NEWT:51953","NEWT:3197","NEWT:9615","NEWT:10299","NEWT:860688","NEWT:36329","NEWT:1147787","NCBITaxon:3044782","NEWT:72407","NEWT:349741","NEWT:9605","NEWT:9606","NEWT:157295","NEWT:641501","NEWT:7668","NEWT:91509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