Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018193.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018195.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018195.mzid_QEplus018195.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018193.mzid.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018193.mzid_QEplus018193.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018195.mzid.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018195.mzid_QEplus018195.MGFftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018193.mzid_QEplus018193.MGFftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/UniProt_ProteomeSet_Mouse_Isoform.fastaftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018195.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018193.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018193.datftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/06/PXD008875/QEplus018195.datprimaryOK200bettina.warscheid@biologie.uni-freiburg.deFriedel DrepperMass SpectrometryGel-based experimentNot availableC2c12 myotubesFilamin cFilip1http://www.ebi.ac.uk/pride/archive/projects/PXD008875Skeletal Muscle CellSkeletal Muscle Cell LineHis6-tagged FILIP1-2 CT was used to pull-down putative binding partners from extracts prepared from C2C12 cells. Differentiated C2C12 myotubes were scraped in PBS from 4 their 10 cm culture dishes, pelleted and frozen until use. Cell pellets were lysed in 3 ml lysis buffer (50 mM Na2HPO4, 300 mM NaCl, 20 mM Imidazol, 0.5 % (w/v) Tween-20, pH 8.0), and extensively sonified (UP50H, Hielscher, Teltow, Germany). Insoluble material was removed by centrifugation (30 min, 4,000 x g). 1 ml of the cleared lysates was incubated while rotating at 4°C for 1 h with 150 µl empty Ni2+-NTA-agarose beads or beads with bound FILIP1-2 CT (aa 776-1177). As a control, beads with bound FILIP1-2 CT were incubated with lysis buffer only. After extensive washing (50 mM Na2HPO4, 300 mM NaCl, 40 mM imidazole, pH 8.0), FILIP1-2 CT with associated proteins was eluted from the beads by addition of 120 µl elution buffer (50 mM Na2HPO4, 300 mM NaCl, 200 mM imidazole, pH 8.0). The eluate was mixed with 30 l 5x Laemmli sample buffer and heated for 10 min at 95°C. Eluted proteins (30 µl ) were analyzed by SDS-PAGE. A band observed only in the sample obtained from FILIP1-2 CT-coated beads incubated with cell extract was cut from the gel and analyzed by LC-MS.PrideNot availablePhosphoOxidationQExactive Plus raw files were converted into mascot generic files with the ProteomeDiscoverer 1.4. Resulting mgf files were searched against the Uniprot ProteomeSet Mouse Isoform database (release 02_2016, 58,761 entries), enabeling trypsin as protease with a maximum number of 2 missed cleavages, carbamidomethyl as fixed modification, oxidation (M), phospho (ST) and phospho (Y) as variable modifiation. Peptide mass tolerance was set to 5 ppm and fragment mass tolerance to 0.1 Da.ProteomicsBettina WarscheidQ Exactive PlusDepartment of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg im Breisgau, GermanyCOMPLETEMus Musculus (mouse)friedel.drepper@biologie.uni-freiburg.de32444788 Reimann L, Schwäble AN, Fricke AL, Mühlhäuser WWD, Leber Y, Lohanadan K, Puchinger MG, Schäuble S, Faessler E, Wiese H, Reichenbach C, Knapp B, Peikert CD, Drepper F, Hahn U, Kreutz C, van der Ven PFM, Radziwill G, Djinović-Carugo K, Fürst DO, Warscheid B. Phosphoproteomics identifies dual-site phosphorylation in an extended basophilic motif regulating FILIP1-mediated degradation of filamin-C. Commun Biol. 2020 3(1):253 10.1038/s42003-020-0982-5BiologicalAG WarscheidBiologie IIAlbert-Ludwigs-Universität FreiburgSchänzlestr. 179104 FreiburgGermanyGermany10.6019/PXD008875The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig-like domain 20 of FLNc, is doubly phosphorylated. The protein kinases responsible for this dual-site phosphorylation are Akt and PKCα. Proximity proteomics and interaction analysis identified filamin A-interacting protein 1 (FILIP1) as direct FLNc binding partner. FILIP1 binding induces filamin degradation, thereby negatively regulating its function. Here, dual-site phosphorylation of FLNc not only reduces FILIP1 binding, providing a mechanism to shield FLNc from FILIP1-mediated degradation, but also enables fast dynamics of FLNc necessary for its function as signaling adaptor in cross-striated muscle cells.Phosphoproteomics identifies dual-site phosphorylation in an extended basophilic motif regulating FILIP1-mediated degradation of filamin-C.Reimann Lena L, Schwäble Anja N AN, Fricke Anna L AL, Mühlhäuser Wignand W D WWD, Leber Yvonne Y, Lohanadan Keerthika K, Puchinger Martin G MG, Schäuble Sascha S, Faessler Erik E, Wiese Heike H, Reichenbach Christa C, Knapp Bettina B, Peikert Christian D CD, Drepper Friedel F, Hahn Udo U, Kreutz Clemens C, van der Ven Peter F M PFM, Radziwill Gerald G, Djinović-Carugo Kristina K, Fürst Dieter O DO, Warscheid Bettina BBPBS, imidazole acetate, F10B6_15, Beliefs, d230, l(1)VE614, sarcoma of the breast, Sepharose C1 4B, Gene, dTAFII250, SDH, lysis, EfW1, extracted material, CG7826, F10B6.15, Buffer, dmTAF[[II]]230, SDS, Cut, Background, halite, dmTAF1, l(1)7Ba, Taf230, l(1)7Bb, FILIP, Cultural, Q disc, CG7835, imidazole monohydrochloride, Gene Products, CG42273, imidazole citrate, C2C12, Min, H2im(+), DmelCG42273, Backgrounds, TAF250, insoluble, Sepharose, ethnicity, Taf200, dTAF[[II]]250, sarcoma of breast, TFIID TAF250, reference sample, cel, cell, ligand, chlorure de sodium, min, 4733401P19Rik, Cultural Relativisms, Taf1p, mAPC, IBP, pk18, proteins, kf, MBR, AI047805, cloruro sodico, dTAF250, salt, C1 4B, DmelCG11387, SOLO DANCERS, 6-anhydro-alpha-L-galactopyranosyl-(1->3)-beta-D-galactopyranan, sample, Agarose, Kochsalz, PTBR, Customs, CT, Cu-imidazole, autolysin activity, necrosis, culture, Bladder pain syndrome, associated, TAF, imidazole sodium, 6-An-alpha-L-Galp-(1->3)-beta-D-Galp-(1->]n, Al-imidazole, Controlled, imidazole monophosphonate, DYRK1, Controlling, dTAF[[II]]230, TAF[[II]]250, rock salt, BZRP, DBI, eluted material, 4B, Proteins, Ct, TAF200, Dmel_CG7826, AI836084, l(3)84Ab, BG:DS00004.13, Cultural Backgrounds, TAFII-250, breast sarcoma, TAF250/230, buffer, Cell, PBS, Belief, dTAF230, PBR, Cultural Background, TAFII250, common salt, imidazole conjugate monoacid, PKBS, Cultures, Natriumchlorid, Dm1, p230, [4)-3, Protein, Sepharose C1, TAF[[II]]250/230, CG11387., TFIID, bacteriocin activity, table salt, RCB0987, Dyrk1, 1H-imidazol-3-ium, BcDNA:GH10590, NaCl, Dmel_CG7835, Mnb, MNB, Taf[[II]]250, AU020952, Painful Bladder Syndrome, TAF[[II]]230, holin, A disc, CC1, SWDS, TAF[II]250, AI843021, imidazolium, anisotropic disc, AW124434, (1->4)-3, CGI-97, mDRC, CG17603, natrii chloridum, Filip, TAF[[II]], sample population, ME-IV, Protein Gene Products, incised, Gene Proteins, DmelCG17603, lysin activity, imidazolium chloride, Painful bladder syndrome, Taf250, SR3-5, Sepharose 4B, bacteriolytic toxin activity, Relativisms, imidazolium ion, transverse disc, 5730485H21Rik, Relativism, Cultural Relativism, IMIDAZOLE, TAF230, Bladder Pain Syndrome, TAF1, sodium chlorideFLN-C, multicellular organismal catabolic process, single-organism catabolic process, gamma-filamin, ABPL, positional, ABP-280-like protein, breakdown, filamin-2, positional polypeptide feature, degradation, FLN2, region or site annotation, catabolism, INSDC_feature:misc_binding, Phosphorylations, AI843021, Filip, FLNC, phosphorylation, binding site, binding_or_interaction_site, FILIP, site, actin-binding-like protein., 5730485H21Rik, FLNc, region, ABP-LBru, IPP2A2, Clo, Raw, AW549739, Slf, SLF, Somatomedin-C, number, FBN, FPH2, contrasted, PHAPII, 5730420M11Rik, peptide, Polypeptides, Drug Tolerance, ClvPrd, peptido, template-activating factor I, ECTOL1, Del(8)44H, WMS, C79691, Svc, SET, peptides, TAF-I, phosphatase 2A inhibitor I2PP2A, ipp2a2, beta-Trypsin, somatomedin-C, SF, 2pp2a, Kitl, number of, Mast cell growth factor, CG10574, Lccp, OCTD, DmelCG4299, mKIAA0989, Con, set, IGAAD, 2PP2A, 10^[-6], DmelCG10574, taf-ibeta, IGF1, ppm, has or lacks parts of type, Mechano growth factor, dSET, dSet, blz, peptidos, Soluble KIT ligand, house mouse, Sl, GPHYSD2, drug tolerance, SGS, phapii, IGF-I, steel factor, immune system tolerance, hematopoietic growth factor KL, mouse, igaad, somatomedin, StF-IT-1, Immunological Tolerance., extra or missing physical or functional parts, IBP1, Peptide, sKITLG, ACMICD, mereological quality, polypeptide, Immune Tolerance, Mus, I-2PP2A, mechano growth factor, Dm I-2, I2PP2A, Immunologic Tolerance, MFS1, Igf-1, Data Base, WMS2, SHEP7, Col4a-1, HLA-DR-associated protein II, mast cell growth factor, DI-2, mice, I-2Dm, Stem cell factor, Tripcellim, MASCOT, MASS, CG4299, inhibitor of granzyme A-activated DNase, STAT5, beta Trypsin, I-2PP1, Trypure, stem cell factor, Kitlg, dSET/TAF-Ibeta, 2610030F17Rik, KL-1, Self Tolerance, TAF-IBETA, Tolerance, MGF, Mgf, c-Kit ligand, cardinality, SSKS, KITLG, Peptid, Mouse, TAF-Ibeta, Polypeptide, SCF, variable, AA407739, i2pp2a, Gb, Immunological ToleranceFUSED, F10B6_15, measuring, knobbly, l(1)VE614, Extract, protein complex, Ct, Fused, AI836084, SDH, protein, Spectrum Analyses, protein-containing complex, Spectrum Analysis, Cell, B930045J24, F10B6.15, Spectroscopy, Axin, SDS, any method, Cut, protein polypeptide chains, Experiment, native protein, natural protein, polypeptide chain, l(1)7Ba, kinky, l(1)7Bb, FILIP, Protein, Human Skeletal Muscle Cells, Mass, Extracts, Analysis, BcDNA:GH10590, protein aggregate, END, Mass Spectrum Analyses, Mass Spectroscopy, Mass Spectrum Analysis, Mass Spectrum, Yeast, Analyses, FU, Spectrometry, 4733401P19Rik, SWDS, proteins, AI843021, CGI-97, merged with, Filip, study assay, kf, CG11387, incised, ligand., Cell Extract, DmelCG11387, SOLO DANCERS, scientific observation, joined with, AI316800, Fu, CT, fused to, mKIAA1278, fused, Kb, 5730485H21Rik, ORW1, 1700112N14Rik, coalesced, HHT1, KiFLN-C, multicellular organismal catabolic process, single-organism catabolic process, Striated Muscles, PRKBA, Akt/PKB, dP60, CG5373, ATVPS34, Vps34, determination, DAKT1/PKB, postnatal development, Myocyte, primitive node, PI3K, Dp110, BcDNA:LD15217, growth and development, protein, AKT1, VPS34, 11621, AKT signalling cascade, phosphorylation, binding site, PI-3 kinase, p110-alpha, protein polypeptide chains, PHOSPHATIDYLINOSITOL 3-KINASE, DmelCG2699, F8A5.4, dorsal marginal zone, node, Filamin-2, ABP280A, Kinase, phosphatidylinositol 3-kinase activity, protein aggregate, Muscle Cell, Dp60, Pi3K92D, CG4141, PI3K_59F, catabolism, catalyst activity, 1110055E19Rik, Pi3k, PI3-kinase activity, proteins, AKT signaling cascade, Striated, PI[[3]]K, PI3Kgamma, PKB|Akt, Fast, RacPK, PKB signaling cascade, MCAP, PKB signalling cascade, p55alpha, Muscles, Mature Muscle Cell, Hensen node, signaling process, PI3K 68D, Hensen's node, Pi3K, PI3k, protein kinase B signal transduction, MCMTC, embryo organizer, FAST, ATP - 1-phosphatidyl-1D-myo-inositol 3-phosphotransferase activity, single organism signaling, PKB alpha, dVps34, gamma-filamin, PI3K68D, AKT signal transduction, dakt, P110DELTA, phosphatidylinositol 3-kinase signaling/protein kinase B signal transduction, PtdIns-3-kinase activity, rea, p50alpha, FASTK, RAC-PK-alpha, l(3)89Bq, CG2699, vacuolar protein sorting 34, DmelCG5373, dPIK, 1-phosphatidylinositol 3-kinase activity, AKT1 kinase, PKB/Akt, PKB/AKT, AKT/PKB, protein kinase B alpha, C530050K14, dAkt, dAKT, type-1 PI3K, p-Akt, protein kinase B signaling, Henson's node, DAkt, region, l(3)04226, CG11621, DPKB, MFM5, breakdown, positional polypeptide feature, pAkt, p85alpha, growth pattern, FLN2, Fln2, non-developmental growth, actin-binding-like protein, proto-oncogene c-Akt, AI843021, PI3K 68_D, Mature, dAKT/dPKB, PKB/dAKT, FLNC, Filip, DmelCG4141, F8A5_4, protein kinase B, APDS, p110D, Dakt, PI3K/Akt signal transduction, PI3KBETA, dPKB, DMZ, p120-PI3K, 5730485H21Rik, Protein Kinase, droPIK57, RAC-ALPHA, DRAC-PK85, skeletal muscle system., DmelCG4006, biological signaling, PI3K/PKB signal transduction, Myocytes, region or site annotation, Peptidomics, PI3K92E, number, STK10, Pi3K_59F, Gene, PKB signal transduction, type I phosphatidylinositol kinase activity, PKB signaling, protein-containing complex, skeletal muscle, presence, phosphatidylinositol 3-kinase, Dpkb, PIK3, Pi3Kp60, dVps34/PI3K59F, polypeptide chain, p60, FILIP, PI3K-dp110, Gene Products, dPI3K, PHOSPATIDYLINOSITOL 3-KINASE, anon-92Ed, FLNc, Dp110/PI3K, PI3K-68D/E, class I, shield, Gamma-filamin, positional, ABP-280-like protein, nodus primitivus, ligand, Spemann's organizer, PI3'K, Pi3Kp110, Muscle Cells, ATP-protein phosphotransferase, P110BETA, PI3K-68D, PI3K21B, Striated Muscle, AKT, ATP:Fas-activated serine/threonine protein phosphotransferase activity, Akt, DRAC-PK, site, MCM, organizer, MPD4, Cpk, striated muscle, dP110, protein kinase B signalling cascade, AKT signaling, filamin-2, AA414921, degradation, akt, p120, class II, protein complex, type III phosphoinositide 3-kinase activity, Proteins, Phosphorylations, p110, Actin-binding-like protein, Vps34p, DAkt1, DAKT1, function, embryonic organizer, PI(3)K, cpk, PI3K-PKB/Akt pathway, PI3K-92E/Dp110, development, Mature Muscle Cells, PIK3C1, somatic muscle, count in organism, ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase activity, PKB, CWS6, native protein, CWS5, vps34, natural protein, Protein, chemical analysis, PKB-ALPHA, Spemann Mangold organizer, dp110, D-Akt, CG4006, PI3CG, protein kinase B signaling cascade, ABP-280, PI-3-K, Dakt1, serine-threonine kinase Akt, ABPL, PI3K-59F, PI3K-Dp110, akt1, dAkt1, ABPA, Kinases, INSDC_feature:misc_binding, DRAC-PK66, postnatal growth, class III, DmelCG11621, p110gamma, signalling, Protein Gene Products, Gene Proteins, dAkt/PKB, dakt1, CLOVE, binding_or_interaction_site, signalling process, DmVps34, IMD14, PI3K_68D, PI3K-92D, PKBalpha, embryonic shield, Dmp110, dAKT1, Rac, RAC, assay, quantitative, growth, ABP-L, thymoma viral proto-oncogene, skeletal muscle system, presence or absence in organismFILIP., AI843021, 5730485H21Rik, FilipfalseFILIP1 CT pull-downFILIP1 was identified as potential new filamin C interaction partner by a SILAC-based BioID experiment. Interaction of the two protein via the carboxy-terminal end of FILIP and domain 18-21 of filamin C was prooven by yeast-2-hybrid sreens and dot-blot assays, respectively. To verify this interaction by mass spectrometry, recombinantly expressed FILIP1 carboxy-term was fused via its His6-tag to Ni2+-NTA beads. Subsequently. beads were incubated with cell extracts from human skeletal muscle cells. Bands observed in SDS-PAGE from the pull-down assay were cut and analysed by LC-MS. As a result Filamin C could be identified as FILIP1 binding partner.2022-06-062018-02-06PXD0088754466449917558258038804333029535851114512067811080612461962461972842188090320637522995811363350906836351524260275838538791187947398007186801NCBITaxon:1280572307111430118726355557146835145262364915084727467073641135461388580358232903138886915216778688953997844751870435112329412624696928123977133103430436548135833346079251981836592198131432059655819476214936329572637371922226938124781866132838828139558434513586722294914081691804541619341390363453058611204162326446071121041170612744321085956149636746941283166694369455972945281132081003923751463308793694478447517420654220896431744712704459741194669400667145458793299352932010198126469041066114575512165994048339251216597216595160621213312831282128033952321832172920435315227794428112454349825741948147871720309498019752555536231439841356742951053948824261914548140479NCBITaxon:21575515343989903712285775975320596876231935163098005888455818178081736309431796989361604885661409859566416242515059071187021187053204963063863235554320466322261866331299456586068845351216129567171360441001602358700264203694569410167634102107612773511316582150480227933428146100293578617463313252911187002157641809699745774449156666754760445246767561917265181301941119294396336641136336721751117645102398709NCBITaxon:40559325823590927883965264982174982163472565691569387224982116544872056971666905108944835525663661410NCBITaxon:49328715568910245980875741710005894526808732287535311561263730069303911415542193255521872687238724872987271235987555295476242231414654773055180066556484198822445299767446982510216998231174673897641041051155744689444474153NCBITaxon:50557960167767446851000364960065349118226370824854801299114578458359741276473194439137086326547876569835566369838192011164034624136810893161001428099612NCBITaxon:548681NCBITaxon:58205464143077479464098609958652283196627290587668960596061572951372218759446708756227086171101335483309034974129829387834306154319474703342494729859610896158865597464998703920211107287827215407821190857075298352061894804189853502471587796275630411902528674636046147964696443759391462472746128746011345062534526591972219638191552638588596NCBITaxon:615750521105172375059576131302409263419610526419407722774694163481123869NCBITaxon:27036125061747679274596153840052573131368626979686662990346941713443463603475153632964404210867310185109760635586331496161286287966927294351338968583551865368595968912801726071018568729411009118696141633316780781186941436183NCBITaxon:6191118698840231719118691443906NCBITaxon:102982100071257118387279694836419080218787870384766455347095145122578656870861822607046183260705260707NCBITaxon:780NCBITaxon:1502451516658457702921158612184520928574707856100713370565295076192126385454195NCBITaxon:30555044573915383379913998192875NCBITaxon:10239NCBITaxon:714282912680631309570448300641751585923810555241590841228233446900815025754122810451930174830085210977970671356225833136185509924NCBITaxon:5693826883064052779617946995833424090615101515NCBITaxon:631240674482428130767070876138370828167728503515819901195195337024924053608881756261583558333760212671928532177357267119350184645177299808616787917743737111049056371215348112153239576769014726647091317047571256274579031133355085962859634792491308126387121203511082332648373153214693NCBITaxon:961538323318586260799141262200298131449376083334111497019281239655833321018413856959111006581237561275045426662551519788749200375036009412934971530094114903524725175014012573983649908515151438992NCBITaxon:11552374539883747445446415259311792866432359839062887051580119459932716011497861603293338922853422999810894535936457428412754121536024271831627713512251179948875058NCBITaxon:1040724271550277916797181673871349130627415895521010306502780291595412629158515849327159570210022633889128768913558893094514647920312421641332803828035192843436909398580894621080348179392118259028788929502745609906901749342725632273218903829286757221247190400727NCBITaxon:5796537457114311233521140206411981087135547714245072295337969835552222129167141262161154293157546139622421392139077020114319338293550833292307411292211484896160106772526499975744727580240550717629968335366581214053576229412816078395763206672400540296543282311843183224659620575549922957575759284593936053307972551811354970667143673353502611595564442143213818492231124060721054211109697622611117957529818184319219496340161433479866265281046603815181044214547448500796267825356709747731751351240565050NCBITaxon:1313607699389424688795588012014887519795962141140822414082234219728909101412285316435186763185431520767737677764940894144242327742NCBITaxon:17732140922088931157781154756208895929793244704207559101177499067499073129899136645112273231663170187367451011610358991513284262658721036098334139269611307981336795358893164071034015303337330158191306413992530688391033721233681994044586449811535765261111471612249111313166343053120175549935129938505779983176669642688451979401151345842157535326787286238621115104160791298315392227468911983511255228562532326604691361651056121491676336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