Mutation-specific and common phosphotyrosine signatures of - Tahir et al. - Unknown - Unknown

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Mutation-specificandcommonphosphotyrosinesignaturesofKRASG12DandG13Dalleles1,234,53,4,53,6RaihaTahir,SantoshRenuse,SavitaUdainiya,AnilK.Madugundu,JevonA.Cutler,RajaSekhar372,32,3*2,3,5,8,*Nirujogi,ChanHyunNa,YaoyuXu,XinyanWu,AkhileshPandey1Biochemistry,CellularandMolecularBiologyGraduateProgram,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD,USA2DepartmentofBiologicalChemistry,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD,USA3McKusick-NathansInstituteofGeneticMedicine,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD,USA4InstituteofBioinformatics,InternationalTechnologyPark,Bangalore,560066India5ManipalAcademyofHigherEducation(MAHE),Manipal576104,Karnataka,India6Pre-DoctoralTrainingPrograminHumanGenetics,McKusick-NathansInstituteofGeneticMedicine,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD,USA7DepartmentofNeurology,InstituteofCellEngineering,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD,USA8DepartmentsofPathologyandOncology,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD,USATABLEOFCONTENTSSupplementalFigure1.DeeperproteomeprofilingofSW48KRASmutantsknockincells.SupplementalFigure2.MutantKRAS-inducedsignaturesinthetotalproteome.SupplementalFigure3.Full-lengthblots/gelscorrespondingtowesternblotsreportedforthisstudy.SupplementalTableS1.AlistofproteinsidentifiedintheglobalproteomeanalysisSupplementalTableS2.AlistofphosphotyrosinesitesidentifiedinthisstudySupplementalTable2A.AlistofphosphotyrosinepeptidesquantifiedinthisstudySupplementalTable2B.Alistof812phosphotyrosinepeptidesquantifiedbyPyQuantSupplementalTable2C.Alistof308phosphotyrosinepeptidesquantifiedinall3replicatesfortheG12DcellsSupplementalTable2D.Alistof308phosphotyrosinepeptidesquantifiedinall3replicatesfortheG13DcellsS-1

1AB1,086pTyrsites(676proteins)SILACQuantification1952,0325,925(PyQuant)812pTyrsitesquantified(523proteins)Quantifiedinall3replicatesHammondetal.2015G12D/ParentalG13D/ParentalCurrentstudy308pTyrsites280pTyrsites(222proteins)(213proteins)CSupplementalFigure1.DeeperproteomeprofilingofSW48KRASmutantsknockincells.(26)(A)Venndiagramcomparingtheproteomesprofiledbypreviouslypublishedstudyandcurrentstudy.(B)Diagramdepictingthequantitativedataprocessforphosphotyrosineprofiling.(C)AlterationsofexpressionandphosphorylationlevelsofproteinsinvolvedincanonicalRASsignalingnetwork.Redstarsrepresentprotein’sexpressionand/orphosphorylationlevelssignificantlychangedinKRASG12D/G13DSW48knockincells.(DerivedfromtheKEGGpathwaydatabase)S-2

2ABSILACRatio(logG13D/Parental)SILACRatio(logG12D/Parental)22CG12DG13DD(134proteins)(182proteins)ProteoglycansincancerCellcycleDNAreplication9044138AxonguidanceRap1signalingOxytocinsignalingDopaminergicsynapseELeukocytetransendothelialmigrationGlutamatergicsynapseParentalG12DG13DReninsecretionWB:anti-CD73GapjunctionGastricacidsecretionWB:anti-KRAPWCLFructoseandmannosemetabolismG13DWB:anti-HSPB1PhenylalaninemetabolismG12DWB:anti-GAPDH02468SupplementalFigure2.MutantKRAS-inducedsignaturesinthetotalproteome.(A-B)Volcanoplotsdepictingrelativedifferencesinintotalproteinabundancesbetweenparentalcellsand(A)G12Dmutatedand(B)G13Dmutatedcell.TheratioofrelativeintensityofaproteininindividualKRASmutantsversusparentalisshownonthex-axis(log2-transformed).They-axisshowsthep-values(log2-transformed).Theverticalred/bluelinesindicate2-foldchangesintheabundanceratiosandthehorizontalgreenlinesignifiesap-valuecutoffof0.05.Proteinsshowingstatisticallysignificant(p-value≤0.05)up-regulationordown-regulationaremarkedinredorblue,respectively.Selectedproteinsarelabeled.(C)VenndiagramshowingthesetofproteinsshowingincreasedabundanceinbothorindividualKRASmutantcellsrelativetoparentalcells.(D)PathwayanalysisusingDAVID,awebgene-annotationenrichmenttool.YellowbarsrepresentthenumberofgenesenrichedinthepathwaysupregulatedinG13DcellsandbluebarrepresentsG12Dcells.(E)Westernblotanalysisofwholecelllysatesfromparentalandmutantcellsusingaprotein-specificantibodydirectedagainst5'-nucleotidaseecto(CD73),KRAS-inducedactin-interactingprotein(KRAP),andheatshockproteinbeta-1(HSPB1).Thebottompanelshowsimmunoblottingwithanti-GAPDHantibodycarriedoutinparallel.Thecorrespondingfull-lengthblots/gelsarepresentedinSupplementaryFig.3F.S-3

3ACDEFGSW48G12DG13DKDaParentalG12DG13DParentalG12DG13DParentalG12DG13DParentalG12DG13DParentalG12DG13DkDasiCONsiPZRsiCONsiPZRsiCONsiPZR140WCLWB:anti-CD7314018511565140115115WB:anti-MET115808080658065656550IP:anti-pTyr505065WCLParentalG12DG13DPZRWCLWB:anti-pY263WCL50WB:anti-ALDH3A140405030(MPZL1)40WB:anti-KRAP3030WB:anti-GAPDH2530253015WCL251525WB:anti-pTyr1550Beta-ac�nB30ParentalG12DG13DParentalG12DG13DParentalG12DG13DParentalG12DG13D140115kDaParentalG12DG13D140WB:anti-LMO7185WCL11514080WB:anti-TNK2115115HWCL6580805080SW48G12DG13D40656565WB:anti-MPZL1IP:anti-pTyr30WCLWCL505050ParentalG12DG13DsiCONsiKRASsiCONsiKRASsiCONsiKRAS4040WB:anti-GAPDH303025K-Ras(F234)18525302515115WB:anti-HSPB1ParentalG12DG13D152515WB:anti-LGALS150Beta-ac�n8014011515306580WCL6550WCL50ParentalG12DG13DkDaParentalG12DG13D4030WB:anti-GAPDH30140140WB:anti-CD442525115WB:anti-FKBP11115WB:anti-TNK2808065WCL6550WCL4050304025140115301525806515WCL5040WB:anti-GAPDH3025SupplementalFigure3.Full-lengthblots/gelscorrespondingtowesternblotsreportedforthisstudy.(A)Full-lengthblot/gelforresultspresentedinFigure1A.(B)Full-lengthblot/gelforresultspresentedinFigure3E.(C)Full-lengthblot/gelforresultspresentedinFigure4C.(D)Full-lengthblot/gelforresultspresentedinFigure4D.(E)Full-lengthblot/gelforresultspresentedinFigure4E.(F)Full-lengthblot/gelforresultspresentedinFigureS2C.(G)Full-lengthblot/gelforresultspresentedinFigure5B.(H)Full-lengthblot/gelforresultspresentedinFigure5C.S-4