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DrepperNot availableSkeletal Muscle MyoblastMyoblast Cell LineMouse C2 cells transiently expressing BirA* FLNc d18-21 were treated for with 10 µM MK-2206 (Selleck) or 10 µM Gö6976 (Merck) for 1 h or with 300 nM PMA (Sigma Aldrich) for 15 min. In experiments using both, inhibitor and activator, cells were first treated with the respective inhibitor (MK-2206, Gö6976) for 1 h and PMA was added for the last 15 min of the treatment. Cells were lysed on ice with 500 µl modified RIPA buffer. Enrichment of the Myc-tagged FLNc fragment was performed with 10 µl Myc-Ddynabeads (Invitrogen) according to the manufacturer’s protocol. On-bead tryptic digestion was performed in 200 µl ammonium bicarbonate solution (50 mM) for 3.5 h at 42°C and 800 rpm on a thermoshaker using sequencing grade trypsin (Promega) in a 1:50 (w/w) ratio. Phosphopeptides were enriched as described above and mixed with 47.5-95 fmol phosphopeptides of a phosphopeptide standard comprising 181 phosphopeptides (Intavis). For targeted MS analysis on a QExactive Plus instrument, an inclusion list comprising 16 precursors with 96 transitions was generated with Skyline 2.6.0.6709 and is available in the PanoramaWeb interface. One scan cycle consisted of a full MS1 scan at a resolution of 70,000, an automatic gain control (AGC) target of 3e6 ions, a max. ion time of 20 ms and a scan range from m/z 400 to 1600, followed by 10 PRM scans. Each PRM scan targeted one precursor of the inclusion list at a resolving power of 17,500, an AGC target of 2e5, a max. ion time of 49 ms and an isolation window of 2 m/z. Raw files were analyzed with Skyline and intensities of hFLNc phosphopeptides were normalized to the summed intensities of eight phosphopeptides (SEGpSPVLPHEPAK, TGMGSGpSAGKEGGPFK, pSTVASMMHR, VIEDNEpYTAR, LIEDNEpYTAR, pSFNGSLKNVAVDELSR, pSGGQRHSPLSQR, AYpTHQVVTR) from the standard under control conditions.PrideBettina WarscheidQ ExactiveDepartment 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, GermanyCOMPLETE32444788 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/s42003-020-0982-5AG WarscheidBiologie IIAlbert-Ludwigs-Universität FreiburgSchänzlestr. 179104 FreiburgGermanybettina.warscheid@biologie.uni-freiburg.deSRM/MRMMass SpectrometryFilamin cPkcAktMyocyteshttp://www.ebi.ac.uk/pride/archive/projects/PXD009228Not availablePhosphoOxidationRaw files from PRM analyses were pre-processed into mascot generic files using the ProteomeDiscoverer and searched against Uniprot Proteomeset database (release 11.2016, 92,933 protein entries) including the sequence of the recombinantly expressed FLNc d18-21 and its mutants as background for FDR calculation. Precursor and fragment mass tolerances were set to 5 ppm and 0.1 Da, respectively. Search parameters were as follows: proteolytic enzyme: trypsin, max. number of missed cleavages: 1, variable modifications: methionine oxidation and phosphorylation of serine, threonine and tyrosine. Mascot .dat files, all raw files and the FLNc d18-21 sequence were imported into Skyline 4.1.0. MS1 intensities were calculated as described by (Schilling et al, 2012) using the MS1 filtering tutorial provided by the software developers. Skyline peptide settings were as follows: tryptic peptides with 1 missed cleavage, a time window of 3 min, min. and max. peptide length 8 and 30 aa, respectively, exclusion of cysteine-containing peptides, phosphorylation of serine, threonine and tyrosine and oxidation of methionine as variable modifications, and max. number of variable modifications and neutral losses 3 and 1, respectively. Orbitrap default parameters were used for transition settings. Extracted ion chromatograms of the imported peptides were manually inspected for correct peak picking and peak integration was manually adjusted, if necessary. Total MS1 areas for all peptides with ≥6 MS/MS spectra, a mass error of ≤ 3 ppm were exported into a pivot table and processed using Excel 2010 and Origin 9.1. Mean and standard error of the mean (SEM) were first calculated for the three biological replicates and subsequently for the technical replicates. Intensities of all phosphopeptides were summed and were normalized by the respective calculated summed intensity.ProteomicsMus Musculus (mouse)friedel.drepper@biologie.uni-freiburg.deBiologicalGermany10.6019/PXD009228The 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 Bprojections, FLN-C, Soluble, PRKBA, Akt/PKB, dP60, Vps34, DAKT1/PKB, alpha-Spe, BcDNA:LD15217, insuline humaine, growth and development, AKT1, VPS34, 11621, alpha-Spc, CG31654, AKT signalling cascade, phosphorylation, Calcium Phospholipid-Dependent Protein Kinase, 2-Amino-3-hydroxypropionic acid, PI-3 kinase, 5730420M11Rik, p110-alpha, protein polypeptide chains, Employee Workloads, Protein Kinase M, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, Employee, Kinase, ABP280A, SET, Dp60, PI3K_59F, phosphatase 2A inhibitor I2PP2A, PI3-kinase activity, proteins, AKT signaling cascade, PI3Kgamma, RacPK, PKB signaling cascade, DmelCG4299, MCAP, 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, PKB signalling cascade, set, xigf1, PI3K 68D, s, protein kinase B signal transduction, Sgk, SGK, ATP Phosphotransferases, Work Load, ATP - 1-phosphatidyl-1D-myo-inositol 3-phosphotransferase activity, single organism signaling, Calcium Activated Phospholipid Dependent Kinase, insulinum humanum, dVps34, Regular, anatomical protrusion, phosphatidylinositol 3-kinase signaling/protein kinase B signal transduction, rea, p50alpha, somatomedin, RAC-PK-alpha, IBP1, igf1-A, CG2699, DmelCG5373, mice C57BL/6xCBA/CaJ hybrid, ada, Regular Insulin, Phospholipid Sensitive Calcium Dependent Protein Kinase, AKT/PKB, insulin (recombinant), D-Ada, C530050K14, type-1 PI3K, shelf, p-Akt, dAP-2a, Calcium-Activated, Soluble Insulin, l(2)06694, CG11621, MFM5, Serine, HLA-DR-associated protein II, DI-2, FLN2, Fln2, I-2Dm, insulina humana, LY 294002, alpha, inhibitor of granzyme A-activated DNase, AP-2alpha, projection, dAKT/dPKB, PKB/dAKT, FLNC, ridge, DmelCG4141, F8A5_4, DmelCG4260, APDS, I-2PP1, TAF-IBETA, PI3KBETA, Mouse, TAF-Ibeta, RAC-ALPHA, DmelCG4006, anon-EST:fe2E2, PI3K/PKB signal transduction, lamellae, Mining, alpha-Spect, number, 2-(morpholin-4-yl)-8-phenyl-4H-chromen-4-one, PKB signal transduction, protein-containing complex, process of organ, fs(3)neo61, lamella, CG10422, PIK3, dVps34/PI3K59F, Pi3Kp60, PI3K-dp110, Insulin, dPI3K, anon-92Ed, FLNc, class I, Chain, Employee Work Load, ABP-280-like protein, dre3, 2pp2a, Pi3Kp110, Alpha-Adaptin, Employee Workload, CG10574, P110BETA, PI3K21B, 2PP2A, Insulin recombinant, AKT, Text, Akt, dSET, dSet, MCM, MPD4, laminae, l(3)dre3, striated muscle, Cpk, serine, AKT signaling, alpha-Spectrin, AA414921, akt, class II, anatomical process, type III phosphoinositide 3-kinase activity, Phosphorylations, Actin-binding-like protein, DAkt1, DAKT1, PI(3)K, cpk, PI3K-PKB/Akt pathway, PI3K-92E/Dp110, PIK3C1, Serin, PKB, CWS6, native protein, CWS5, PKC, I-2PP2A, chemical analysis, Dm I-2, PKB-ALPHA, Insulin A Chain, MENE(2L)-A, CG4006, Sodium, phosphorylation enrichment, Novolin, alphaSp, protein kinase B signaling cascade, organ process, Dakt1, ABPL, mating_type_alpha, ABPA, postnatal growth, DmelCG11621, PKC Serine-Threonine Kinase, signalling, processes, AP2, dAkt/PKB, CLOVE, signalling process, DmVps34, IMD14, Bam-C, PI3K_68D, PI3K-92D, MGF, alpha mating type (yeast), Dmp110, quantitative, ABP-L, thymoma viral proto-oncogene, Insulin B Chain, IPP2A2, Staff, BamF, CG5373, ATVPS34, determination, BamC, AP-2, Blood, postnatal development, sgk-A, PI3K, Dp110, protein, Serum, Serine-Threonine Kinase, l(3)04276, PHOSPHATIDYLINOSITOL 3-KINASE, DmelCG2699, F8A5.4, human insulin, alpha-adaptin, Filamin-2, phosphatidylinositol 3-kinase activity, DmelCG10422, protein aggregate, alpha-spec, insulin, CG4260, Pi3K92D, CG4141, TAF-I, sgk1, catalyst activity, 1110055E19Rik, somatomedin-C, Pi3k, PI[[3]]K, PKB|Akt, alpha-spectrin, Work Loads, IGAAD, insulin human, p55alpha, 2-amino-3-hydroxypropanoic acid, DmelCG10574, 3-Hydroxyalanine., IGF1, papilla, signaling process, Workload, C730016P09Rik, Pi3K, PI3k, house mouse, MCMTC, ATP, PKB alpha, phapii, IGF-I, gamma-filamin, PI3K68D, AKT signal transduction, dakt, P110DELTA, PtdIns-3-kinase activity, lamina, mouse, Igf-I, flanges, StF-IT-1, Phospholipid-Sensitive Calcium-Dependent Protein Kinase, ham, l(3)89Bq, Blood Serum, Workloads, vacuolar protein sorting 34, dPIK, 1-phosphatidylinositol 3-kinase activity, AKT1 kinase, Literatures, PKB/Akt, PKB/AKT, alphaSpec, protein kinase B alpha, dAkt, dAKT, 3-Hydroxyalanine, mechano growth factor, cg4260, protein kinase B signaling, DAkt, Igf-1, spectrin, l(3)04226, MENE (2L)-A, Transphosphorylases, DPKB, pAkt, p85alpha, growth pattern, mice, non-developmental growth, shelves, actin-binding-like protein, proto-oncogene c-Akt, CG4299, Staff Workloads, PI3K 68_D, Calcium Phospholipid Dependent Protein Kinase, protein kinase B, immunoglobulin, p110D, Dakt, PI3K/Akt signal transduction, spine, Text Mining, dPKB, L Serine, p120-PI3K, L-Serine, i2pp2a, droPIK57, DRAC-PK85, alpha-Ada, biological signaling, l(3)alpha-Spec, PI3K92E, Pi3K_59F, Insulin B, type I phosphatidylinositol kinase activity, PKB signaling, skeletal muscle, presence, l(2)SH2 0460, PHAPII, phosphatidylinositol 3-kinase, protrusion, Dpkb, antibody, alpha-ada, template-activating factor I, polypeptide chain, p60, klo, PHOSPATIDYLINOSITOL 3-KINASE, alpha-Sp, CG1977, Phosphotransferase, Dp110/PI3K, PI3K-68D/E, Gamma-filamin, Employee Work Loads, ipp2a2, ridges, PI3'K, Transphosphorylase, igf-1, PI3K-68D, Iletin, taf-ibeta, Staff Work Load, DRAC-PK, Staff Workload, PKC Serine Threonine Kinase, dP110, protein kinase B signalling cascade, filamin-2, 3A9, p120, protein complex, DmelCG1977, igaad, p110, Vps34p, Phosphotransferases, Mus muscaris, development, somatic muscle, count in organism, Phospholipid-Dependent Kinase, ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase activity, Phosphopeptide, Sodium Insulin, vps34, natural protein, Mus, Staff Work Loads, Protein, I2PP2A, dp110, D-Akt, PI3CG, flange, ABP-280, PI-3-K, opsonin activity, serine-threonine kinase Akt, PI3K-59F, Exubera, D-alphaAda, PI3K-Dp110, akt1, dAkt1, Kinases, DRAC-PK66, INS, class III, FCP-B, p110gamma, process, dSET/TAF-Ibeta, dakt1, 2610030F17Rik, Data, l(3)62Bd, PKBalpha, BAM, Bam, B cell receptor activity, l(2)SH0460, processus, alfa-Spec, dAKT1, RAC, Rac, assay, AA407739, growth, Calcium-Activated Phospholipid-Dependent Kinase, Serums, Spec, skeletal muscle system, presence or absence in organismFLN-C, Bru, IL1BC, dmMOF, ion, Raw, determination, fond, CASP-1, instrument configuration, CG7788, crice, myc-B, DmelCG10798, MYC, Myc, 7b-dihydroxy-3-(hydroxymethyl)-1, CycEI, 4]benzo[1, Long Term, B430311C09Rik, DmelCG9648, 1bS, c-Myc, c-MYC, Ccne, tetradecanoylphorbol acetate, EG:BACN5I9.1, dMOF, 1, ARC, Min, 4, Filamin-2, ABP280A, 5, 6, myc, 8, 9, Il1bc, Effect, DmelCG42273, Svc, c-myc, reference sample, dm/dMyc, dMax, weight-weight percentage, 1110055E19Rik, 1b, min, HardwareType, mAPC, purification, T6G21.3, SCAN, IL-1BC, AI047805, isolation and purification, AHA1, ICE, OPEN STOMATA 2, 9a-decahydro-1H-cyclopropa[3, house mouse, Long-Term Effects, ratio, gamma-filamin, Stars, Caspase-1 subunit p10, 7aS, ice, iCE, dmyc1, AU016757, drice, H(+)-ATPase 1, mouse, Dmel_CG7826, CG9648, Longterm Effect, Th, 8-tetramethyl-5-oxo-1a, Cyc E, br37, mice C57BL/6xCBA/CaJ hybrid, DmelCG7788, bHLHd7, bHLHd6, bHLHd9, bHLHd8, bHLHd5, bHLHd4, 7bS, BG:DS07108.3, Dm1, Dm, Ionen, drIce, drICE, 4a, NOP, dmax, 9aS)-9a-acetoxy-4a, mMyc, l(2)05206, Dmel_CG7835, phorbol 12-tetradecanoate 13-acetate, Mnb, MNB, parent ion, l(1)G0354, MFM5, (1aR, FLN2, instrument, Fln2, mice, l(1)G0359, actin-binding-like protein, P45, proportionality, fmol, rate, AW124434, d-myc, study protocol, FLNC, beta Trypsin, D-Myc, w/w, Myc2, Niard, MOF, Mof, 12-tetradecanoylphorbol 13-acetate, l35Dd, phorbol-12-myristate-13-acetate, myc2, precursor ion, Drice, p45, Mouse, dMyc1, DRICE, MRTL, STARS, time, CG10798, 12-O-tetradecanoylphorbol 13-acetate, cycline, 7a, dm/myc, 7b, Effects, DrIce, DrICE, l(1)G0139, DmcyclinE, cycE, precursor, 8R, thomson, dMyc, dMYC, l(2)br37, CG7826, hardware, CYCLE, Buffer, mass-to-charge ratio, cdi7, period, method, anon-WO03040301.171, DmelCG3025, MYCC, cyclinE, Ms1, isolation, method used in an experiment, CG7835, c-myc II, Cdi7, CDI7, CG42273, Nop30, bHLHe57, Del(8)44H, 9R, FLNc, Pro-Mega, Gamma-filamin, TPA, proportion, 4aR, MAX, ABP-280-like protein, CYCE, F19F24_16, 2-e]azulen-9-yl tetradecanoate, MS1, Longterm, DmelCG3938, iones, CyclE, PCE-2, beta-Trypsin, inhibiteur, Dmyc, DMYc, 3938, Long-Term, DmcycE, ions, Solution, l(2)k05007, dm-cycE, max, F19F24.16, OST2, inhibidor, RNCMYC, Nird, Caspase-1 subunit p20, male sterility 1, Long-Term Effect, MPD4, Controlled., bHLHe39, dmyc, Controlled, inhibitors, DYRK1, Controlling, mass percentage, phorbol 12-myristate 13-acetate, filamin-2, bead, caspase 3, CG3025, inhibitor, Actin-binding-like protein, CE-2, buffer, Cell, Mus muscaris, AI875693, Phosphopeptide, l(2)k02514, DmCycE, Mus, chemical analysis, Long Term Effects, Dyrk1, CES2A1, CyeE, ABP-280, PLASMA MEMBRANE PROTON ATPASE, AU020952, ABPL, l(2)k02602, Col4a-1, MALE STERILITY 1 PROTEIN, ABPA, CC1, l(2)35Dd, Striated muscle activator of Rho-dependent signaling, Tripcellim, IL-1 beta-converting enzyme, Interleukin-1 beta-converting enzyme, Longterm Effects, ME-IV, Interleukin-1 beta convertase, plan specification, Trypure, D-CycE, PMA, Ion, AA960152, 3.4.22.36, quotient, assay, CG3938, ABP-LFLN-C, multicellular organismal catabolic process, single-organism catabolic process, Striated Muscles, PRKBA, Akt/PKB, CG5373, dP60, Vps34, ATVPS34, 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, DmelCG5373, vacuolar protein sorting 34, 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, 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, dVps34/PI3K59F, Pi3Kp60, 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, phosphorylation enrichment, 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 organismFLN-C, Gamma-filamin, ABPL, gamma-filamin, MFM5, ABP-280-like protein, filamin-2, FLN2, determination, Fln2, ABPA, actin-binding-like protein, 1110055E19Rik, chemical analysis., Filamin-2, Actin-binding-like protein, ABP280A, assay, MPD4, FLNc, FLNC, ABP-280, ABP-LFLN-C, CPD photolyase activity, para Tyrosine, DmErk, dmMOF, Raw, PhrB photolyase activity, ion, phosphorylation, Long Term, 2-Amino-3-hydroxypropionic acid, 5730420M11Rik, DmelCG9648, Polypeptides, protein polypeptide chains, dMOF, 3, NUP96, ABP280A, Software Engineering, outer pigmented layer of retina, DA transporter, epithelium, SEM, Sem, WMS, Threonin, C79691, MAP-k, SET, C, M, phosphatase 2A inhibitor I2PP2A, 3-(p-Hydroxyphenyl)alanine, 2-amino-3-(4-hydroxyphenyl)propanoic acid, Dsor2, proteins, T6G21.3, number of, Y, sem, SUPPRESSOR OF AUXIN RESISTANCE 3, Lccp, DmelCG4299, AI047805, set, pigment epithelium of retina, Solute carrier family 6 member 3, pigmented retina epithelium, Half Cystine, SGS, ERK-A, Stars, dpERK, dpErk, Zinc Cysteinate, SPENCD, Longterm Effect, Roifman Immunoskeletal syndrome, DmMAPK, para-Tyrosine, dp-ERK, Software Tools, ACMICD, bHLHd7, bHLHd6, bHLHd9, bHLHd8, Computer Applications, bHLHd5, bHLHd4, Dm1, Computer Applications Software, dipyrimidine photolyase (photosensitive), pMAPK, pMapK, Methionin, methionine, Hmet, (2R)-2-amino-3-sulfanylpropanoic acid, MFM5, Serine, Software Applications, DmERKA, HLA-DR-associated protein II, rl/MAPK, FLN2, DI-2, Fln2, Source Software, I-2Dm, MASCOT, l(2)41Ac, inhibitor of granzyme A-activated DNase, FLNC, beta Trypsin, L isomer, I-2PP1, Applications, CT34260, TAF-IBETA, dpERk, TAF-Ibeta, L-Tyrosine, STARS, Liquimeth, Computer Software Applications, 2-amino-4-(methylsulfanyl)butanoic acid, region or site annotation, Effects, number, L-Zystein, 12559, precursor, protein-containing complex, deoxyribodipyrimidine photolyase activity, CG7826, stratum pigmentosum retinae, EK2-1, period, DmelCG3025, CG7835, CG42273, Half-Cystine, FLNc, Application, Erk, ERK, Open Source Softwares, positional, MAX, ABP-280-like protein, Longterm, Software Application, L Cysteine, E-920, beta-Trypsin, F23A5.3, Open Source Software, 2pp2a, Long-Term, ions, CG10574, Computer Software Application, erk, mKIAA0989, max, 2PP2A, 2-amino-4-(methylthio)butanoic acid, Tools, CYSTEINE, dSET, dSet, FREE CYSTEINE, peptidos, rll, Tyrosin, MPD4, (2R)-2-amino-3-mercaptopropanoic acid, metionina, serine, spondylometaphyseal dysplasia with enchondromatous changes, CG3025, Phosphorylations, Actin-binding-like protein, L-Isomer, SPENCDI, Tool, polypeptide, Serin, native protein, I-2PP2A, tirosina, Dm I-2, Long Term Effects, MFS1, phosphorylation enrichment, background, Met, AU020952, ABPL, RPE, Col4a-1, CG12559, ABPA, dpERK1, Striated muscle activator of Rho-dependent signaling, (R)-2-amino-3-mercaptopropanoic acid, retinal pigment epithelium, primary structure of sequence macromolecule, Longterm Effects, p. pigmentosa retinae, ME-IV, Computer Programs, Applications Softwares, dpMAPK, E920, deoxyribonucleate pyrimidine dimer lyase (photosensitive), threonine, ABP-L, Bru, IPP2A2, E 920, PKDYS, cleavage, L-Threonine, protein, L-cysteine, pigmented epithelium, SR2-1, peptide, L-Isomer Methionine, Methionine, peptido, ClvPrd, Min, Filamin-2, protein aggregate, Effect, Computer Program, DmelCG42273, Svc, Erk/Map kinase, peptides, TAF-I, dMax, Open, 1110055E19Rik, pigmented retina, DERK-A, Computer Programs and Programming, min, mAPC, E(sina)7, Rl, Spondyloenchondrodysplasia with immune dysregulation, DNA cyclobutane dipyrimidine photolyase activity, IGAAD, reaction, DERK, 2-amino-3-hydroxypropanoic acid, DmelCG10574, ppm, Tyr, spondyloenchondromatosis, dERK, GPHYSD2, Racemethionine, Long-Term Effects, phapii, spondyloenchondrodysplasia with immune dysregulation, PRE, gamma-filamin, Mapk, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, 2-Amino-4-(methylthio)butyric acid, 2-Amino-3-(p-hydroxyphenyl)propionic acid, CG9648, Dmel_CG7826, Erk1, StF-IT-1, extra or missing physical or functional parts, pigmented retinal epithelium, Cys, Source Softwares, Tyrosine, Programs, Program, retinal pigment, 3-Hydroxyalanine, Ionen, MapK, MAPK, combined immunodeficiency with autoimmunity and spondylometaphyseal dysplasia, dmax, Computer Applications Softwares, Softwares, retinal pigment layer, tyrosine, region, Dmel_CG7835, mapk, parent ion, Mnb, MNB, ERKa, positional polypeptide feature, BcDNA:RE08694, l(2R)EMS45-39, retinal pigmented epithelium, actin-binding-like protein, L-isomer, DmelCG12559, L-Cysteine, MASS, CG4299, AW124434, L-Cystein, DpErk, DpERK, ErkA, ERKA, phr A photolyase activity, MOF, Mof, DNA-photoreactivating enzyme, precursor ion, L Serine, Polypeptide, L-Serine, i2pp2a, time, pERK, Cysteine Hydrochloride, GroupII, AW549739, L Isomer, FBN, Computer, photoreactivating enzyme activity, Phosphopeptide., PHAPII, spondyloenchondrodysplasia, polypeptide chain, template-activating factor I, Ms1, ECTOL1, L-Methionine, stratum pigmentosa retinae, Del(8)44H, Pedameth, Gamma-filamin, MOS3, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, PRECOCIOUS, MS1, iones, ipp2a2, L Threonine, spondylometaphyseal dysplasia with combined immunodeficiency, CG18732, OCTD, Table, 10^[-6], L Tyrosine, taf-ibeta, male sterility 1, has or lacks parts of type, site, DmERK-A, Long-Term Effect, DAT, F23A5_3, Applications Software, Open Source, DYRK1, DAT1, filamin-2, Computer Software, DL-Methionine, protein complex, igaad, CT39192, Peptide, AI875693, mereological quality, stratum pigmentosum (retina), MODIFIER OF SNC1, Software Tool, natural protein, Protein, deoxyribonucleic photolyase activity, I2PP2A, sequence, Dyrk1, Dat1, Software, Data Base, ABP-280, WMS2, photolyase activity, MALE STERILITY 1 PROTEIN, Su(Raf)2B, CC1, Engineering, Tripcellim, L-2-Amino-3-mercaptopropionic acid, EY2-2, introduction, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, D-ERK, Ion, SSKS, AA960152, cardinality, alpha-amino-gamma-methylmercaptobutyric acid, Peptid, variable, AA407739FLN-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, phosphorylation enrichment, actin-binding-like protein., 5730485H21Rik, FLNc, region, ABP-LfalseFLNc S2233 S2236 PRM analysisSkeletal muscle is known to adapt dynamically to changes in workload by regulatory processes of the phosphatidylinositide 3-kinase (PI3K)/Akt pathway. We performed a global quantitative phosphoproteomics analysis of contracting mouse C2 myotubes treated with insulin growth factor 1 (IGF-1) or LY294002 to activate or inhibit PI3K/Akt signaling, respectively. Among the significantly regulated phosphopeptides we identified the novel extended basophilic motif RxRxxp[S/T]xxp[S] to be enriched in the set of down-regulated phosphopeptides following inhibition of PI3K/Akt signaling. Using literature-based text mining we identified the kinases Akt, serum and glucocorticoid-regulated kinase 1 (SGK1) and p70S6 kinase to be potentially involved in the phosphorylation of the first serine in the RxRxxp[S/T]xxp[S] motif, whereas no kinase targeting the serine in the +3 position was revealed. In the signaling adapter protein filamin c (FLNc) we found this novel motif in immunoglobulin (Ig)-like domain 20 which is involved in various protein interactions. Through in vitro and in cellulo kinase assays we identified Akt and protein kinase C alpha as the responsible kinases phosphorylating FLNc in this motif at the first and the second serine, respectively.2020-06-302018-03-16PXD009228646099180498511145422492156843544071284218327042363336351198343006636362758385696363121766611143015557114526314526236493461312695736664923916193641364510665688951136364762723661112329442229555832681304232312258636543460792519365910632143205971293865581912502321195774818665867225680761755437359253668586274081691804542787751201089367414795641003923694204943344597458641NCBITaxon:776436715675936438677160540483131528269304562716391624292042779443696121810032962922113763948840479NCBITaxon:215713152731051067205968720165163684071736309698936148364320429294101061320491381693320464535111103217459216129288671288676410069456941016763410210761277351131658213584301417298174633675412873567568403013506218130194111929439367110411364459139498432025301270320221498751104759412047886134749515372110971652998279051355513980317585439801260866398081375755518320196447111415542193255521138343911931819813981810269701069955529NCBITaxon:47519833547641465477982515727398238976484074186287054824153NCBITaxon:505579844675251000364548021035974137081154665478983532215998381067710348198523487804225314884142228634794615718712593991599191572952929211752719860148840633424951869123423523815233598589859886559746499870392021110723635272159874287434182350241451139692931902593907190174636012374746361722625360013197221191585310588596989511051989972379897263419610264266722758858156899628737562690724420237172019998570724074767927459615333554319311671833554184005136861505968666211961748666134751536329NCBITaxon:1763286287348837670580271848186536859598607575527472036105135294296110098402321000734882412336363071047005187878945556449447NCBITaxon:13356265419591399811901781681478281055524NCBITaxon:960613585846900813668481979153519307285930085264114090051184995262254121337591390143811139442370416744312044143708351581990119519532590841370137023943222774862615835583715371633376057267371137121906503714759172073127262981274575727090309031372814243371421044792491108239412NCBITaxon:961565885814780269266100658137352750454135663816475203750644223669502129349763224555253651997649908143899237453747264732376026473128870559553614169152527403271602938511497864794363755293824794372251177529916738719384413765327159100226423444234546700128768959119846704146479293652803428038280351080348179392112621229502711311803738681911265269971028621076015973611432425071140981087135547743658142450722953379698204232124223321803369822347081645733185889716546759928777020141252955081148174488864374526499972830357183085507176299926837041515628876513885639245611520865955516551814321371597495350261432138587817076831949634016142800531646862751104660381518454744850097476974771180512401748418556015554792042742014887110532532972610059412890931643512116049900138943828354232423310829341220585991311227317018799154291312678181871049316407299467303337330485264638301130641399253064236121162065261610337286129940115357652611114716133865431201725323712552329935316317NCBITaxon:11084993841063267872862321933486208621160791392227681196125522814953932132232334774121827532917573129948861814084341033519963139248811533811533933043937556992862655993391120948862415718399758644423748643533244426066533241343909768212717863719798568639998399868655352914865410429863573582576328840198649823801119110286889995866522034163213811938062563188657130344386581144889198466645463NCBITaxon:11072NCBITaxon:110711428006288353743751165971459431165988543311167810778543144080439334NCBITaxon:11082693977381382189931824431935128387145953116885311213278695berry86941003335869724436647015011142869215047514991054842047147223540251208470146497444974311700893062186441930611076323308056603960162179748258539439651321279056881337751298176601711214786105023669465734963205112450189744173941245012885318388919867948853191113791037164735166015294363163119857389693934323795602608712134633432322354431427524143361133456556029169963643680279010560314081883818573370340188127441443755219696971398558408172405598609183827127442340817027598127442012744262354553821176275943282474801199150956481882291690255120455148167238473848190695148498101510693731263753405203860186497405224052141856451804NCBITaxon:813478009152275484793870333849387191722617561094616143512032581205505235259447320549386411257226512138803766861206781122022307432201186705738733486912193879186802398007186801215451468351408186150847120677769183885160861138886915216778672127901008704351231719692812392948677133226126219813411229265716329576938124712461485451328388348392949113903631204162665890771514880036942694169436945187901015080817053942065421055687317447127015329242814111946691169601454586953201019812646905505371408157697392830312383911283128212802811010832825253531522811235315349825741948153623142410145481NCBInr 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