NetworksofBiological

SignalingPathways

信號傳遞網(wǎng)絡康海岐高方遠馬欣榮一、生物體內(nèi)的信號傳遞1.Thesenseofsignaltransduction：intercelluarinformationexchange,regulationofmetobolism,onbodylevel2.Typeofsignals：

neuroregulation:neurotransmitter(乙酰膽堿,胺類氨基酸,調節(jié)肽類等),neuroregulator

chemicalsignals：cAMP,Ca2+,hormone，3.Mechanisms:3.1pr.←→pr.,3.2Ereaction(±p)3.3Eactivity3.4pr.degradation3.5intracelluarmessager3.6secondermessager

Ecell一、生物體內(nèi)的信號傳遞4.Signalingpathways：4.1Ca2+

4.2cAMP4.3tyrosinekinase:EGFR,insulinR4.4otherpr.kinasecascade:PKC,PKA,PKG4.5intracelluarproteasecascadeSignaltransmissionoccur:i.Pr.—pr.Interactionii.Enzymaticreaction:±piii.Pr.Degradationiiii.ProductionofintracellularmessagerPeptideSignalinginPlants

PNAS,Nov.6,2001,vol.98no.23Inplants,onlyafewpeptidehavebeenidentified

thatactassignalingmolecules.

Incontrast,signalingpeptidesaremajorplayers

inallaspectsofthelifecycleinanimalsandyeast.suggeststhatsignalingmechanismsacrossthe

eukaryotickingdomarefundamentallydifferent.目前有關植物中信號肽的研究主要基于以下5種：番茄systeminPSKENOD40CLV3SCR18aa10-13

aa72-75aa53-55aa

2.最近分離到另外3種活性信號肽：RALF:rapidalkalinizationfactor,5kd;Tobaccosystemin:TobsysI,TobsysII1）tomatosystemin:由食草動物損傷后引起的系統(tǒng)損傷反應(asystemicwoundingresponse)

在懸浮培養(yǎng)細胞中可以激活促細胞分裂蛋白激酶[mitogen-activatedprotein(MAP)kinase]

并誘導培養(yǎng)基地堿化(alkalinization)

誘導蛋白酶抑制蛋白編碼基因的表達(induceexpressionofproteinase-inhibitorprotein-encodinggenes)3.功能：Fromthefollowingssupporttheideathat

peptideandnonpeptidehormone-activatedsignalingcascadesarelinkedinplantsastheyareinanimals:

植物生長素類似5－羥色胺，乙烯類似一氧化碳，油菜素類固醇是類固醇，茉莉酮酸與前列腺素相關；

Systemin-inducedwoundresponseis

regulatedthroughtheoctadecanoidpathway,

involvingjasmonicacid;4.信號調控網(wǎng)絡

PSK-inducedcellproliferationrequiresthehormonesauxinorcytokinin;SomeofthedevelopmentaldistortionsinrootsinducedonadditionofRALFarereminiscentofimpairednonpeptide

hormone-controlledprocesses.因此，揭開兩種信號cascades之間關系，將是非常有趣的事。二、海馬趾CA1神經(jīng)元區(qū)室化模型

中的15個信號途徑A:EGF,SOSB:GEF,RasC:cAMP,AC1,AC2D:GE:AA,PLA2F:PLC,PLC

G:DAG,IP3H:MAPKCascadeI:CaMKIIJ:PKAK:PKCL:Ca,IP3M:CaMN:CaNO:PP1

ReactionA:EGF,SOS

ReactionB:GEF,RasReactionD:GReactionE:AA,PLA2ReactionsF,G:PLC,PLC,DAG,IP3ThevariousphosphorylationstatesofCaMKIIhavedifferentenzymekinetics,andeachofthesewereexplicitlymodeled.Forsimplicitytheautophosphorylationstepsarerepresentedbyasingleenzymearrowinthisfigure,withCaMKII_aasthecombinedactivityofthevariousphosphorylationstates.Theindividualkinetictermsusedinthemodelareindicatedbythemultipleratereferencesonthearrows.ReactionI:CaMKIIReactionJ:PKAReactionK:PKC

ReactionM:CaMReactionN:CaN2.Materialsandmethord(1).HippocampalCA1neuron(inGENSIS),(2).NMDAR[ondendriticspine(樹突棘)onthemodel](3).Synapticinput(3tetanicburstsat100HZ,1seach)→LTP→Ca2+waveforms3.ComputationformulationGenesisformulation:

S+E<--k2---k1-->SE---k3--->P+E

Vmax=maxvelocity=k3.

Substrateissaturating,soallofEisinSEform.

SoVmax.[Etot]=[SE].k3==[Etot].k3Km=(k3+k2)/k1

k2=k3*4

Kd=Kb/KfIf[A]*[Bhalf]*Kf=[Chalf=Bhalf]*Kbthen[A]=Kb/Kf=KdKa=Kf/Kb=1/Kd4.verification

(i).

Model

simplekineticschemes

thatcouldbecalculatedanalytically,compare

simulatedresults

with

analyticalresults.

(ii).

Usethelawofmassconservationand

microscopicreversibilityprinciples(微觀可逆性原理)

→test

accuracyincomplexreactionschemes.

(iii).

Run

thesamemodel

atdifferenttimesteps,comparetheresultingsimulatedvalues.5.ProteinKinaseCmodelingexampleSimulationparameters:PKCReactionK:PKC

ReferencesFigure

Reac#

kf

kb

K

1

1

50

K

2

2E-10

0.1

K

3

1.2705

3.5026

K

4

0.000000002

0.1

K

5

1

0.1

K

6

2

0.2

K

7

0.000001

0.5

K

8

1.3333E-08

8.6348

K

9

0.000000001

0.1

K

10

0.00000003

2

ReferencesConcsK:PKC

ReferencesFigure

Name

Conc

K

PKC_inactive

1

1.

Review:Y.Nishizuka,Nature334,661(1988)2.

J.D.SchaechterandL.I.Benowitz,J.Neurosci.13,4361(1993)3.

T.Shinomura,Y.Asaoka,M.Oka,K.Yoshida,Y.Nishizuka,Proc.Natl.Acad.Sci.U.S.A.88,5149(1991)U.Kikkawa,Y.Takai,R.Minakuchi,S.Inohara,Y.Nishizuka,J.Biol.Chem.257,13341(1982).A.BlockdiagramofactivationforPKCpathwaybyCa2+,AAandDAG.builtupsimulationsiteratively:First:matchedAAactivationofPKCatzeroCa.Then:matchedactivationofPKCwithCaatzeroAA,Third:matchedthecurvesinBwith1uMCaandvaryingAA.Four:testthematchforC,withvaryingCaand50uMAA.Last:incorporatedDAGinteractionsintothemodel.B:ActivationofPKCbyAA,with(triangles)orwithout(squares)1mMCa2+.Opensymbolsanddashedlinesrepresentsimulations,solidsymbolsandsolidlinesareexperimentaldata.Shows:Ca2+isnecessaryfortheactivationofPKC.experimentalconcentration-effectcurvesfromtwomainsources:J.D.SchaechterandL.I.Benowitz,J.Neurosci.

13,4361(1993);T.Shinomura,Y.Asaoka,M.Oka,K.Yoshida,Y.Nishizuka,Proc.Natl.Acad.Sci.U.S.A.

88,5149(1991)C:ActivationofPKCbyCa2+,with(triangles)orwithout(squares)50mMAA.Thecurveinthepresenceof50mMAA(triangles)waspredictedfromtheparametersobtainedbymatchingthecurvesinBandthecurvewithoutAA(squares)inC,withoutfurtheradjustment.D:ActivationofPKCbyDAG,with(triangles)orwithout(squares)50mMAA.BothcurvesinDwereobtainedinthepresenceof1mMCa2+.DuetodifferentmethodsforestimatingDAGconcentrationsthelevelsofDAGusedinthemodelarescaled15-foldupwithrespecttotheexperimentalconditionsfromShinomuraetal.四、developethenetworkmodelinstagesFirst:modelindividualpathwaysThen:examinexperimentallydefinedcombinationsoftwoorthreesuchindividualpathwaysandtestthesecombinedmodelsagainstpublisheddata.Third:repeatthisprocessusinglargerassembliesofpathwaysuntiltheentirenetworkmodelofinteractingpathwayswasformed.Pathwayswerelinkedbytwokindsofinteractions:(i)SecondmessengerssuchasAAandDAG,producedbyonepathwaywereusedasinputstootherpathways.(ii)Enzymeswhoseactivationwasregulatedbyonepathwaywerecoupledtosubstratesbelongingtootherpathways.1、one

SignalingpathwaysexampleS

(1).EGF’sstimulationofMAPK1,2Fig.2.EGFreceptorsignalingpathways.(A).Blockdiagramofsignalingpathways.Rectanglesrepresentenzymes,andcirclesrepresentmessengermolecules.ThismodelusedmodulesshowninFig.1,reactionA(EGF),B(Ca2+/CaM),E(AA,PLA2),H(PKC),F(PLCγ,DAG,IP3),H(MAPKascade),K(PKC),I(CaMKII),L(Ca,IP3).

Fig.2BthetimecourseofactivationofMAPKbyEGF(B)Predicted(opentriangle)andexperimental(filledtriangles)timecourseofresponseofMAPKtoasteady

EGFstimulusof100nM.theyaxisrepresentsfractionalactivation.

ThefallintheMAPKactivityaftertheinitialstimulationisduetoacombinationofEGFreceptorinternalizationandMAPKphosphorylationandinactivationofSoS.1、one

SignalingpathwaysexampleS

(2).ActivationofPLCγ

byCa2+

inthepresence(triangles)orabsence(squares)ofEGF.

(C)Concentration-effectcurves.Dashedlinesaremodeldata,andsolidlinesareexperimentaldata.Theyaxisrepresentsactivation.Threestimulusconditions:

10minat5nMEGF(shortbar,circles),100minat2nMEGF(longbar,squares),100minat5nMEGF(longbar,triangles).Onlythethirdconditionsucceedsincausingactivationofthefeedbackloop.Why?2、Twoconnectedpathways

(1).ActivationofthefractionalfeedbackloopbyEGFreceptor:(D)ActivationoffeedbackloopbyEGF.B(basal),T(threshold),andA(active).PointArepresentshighactivityforbothPKCandMAPK,whereaspointBrepresentslowactivity.Bothofthesepointsrepresentdistinctsteady-statelevels.Suchasystemwithtwodistinctsteadystatesisabistablesystem.ThebifurcationpointTisimportantbecauseitdefinesthresholdstimulation.2.(1)Activationofthefractionalfeedbackloop

byEGFreceptor:(E)Bistabilityplotforfeedbackloop

Bistabilityispresentoverarangecomparabletotheexperimentaluncertainty,indicatingthatthephenomenonisrobust.(Horizontalstripes:experimentaluncertaintyinconcentration;diagonalstripes,simulatedbistabilityrangeforconcentrations.)MAPKhasaparticularlylargeuncertaintyinconcentrationrangebecauseoflargedifferencesintissuedistributions.2.(1)ActivationofthefractionalfeedbackloopbyEGFreceptor:(F)estimatedexperimetaluncertaintyinEparametersinitiallyactivating:asuprathresholdstimulus,andthenoneofthreeinhibitoryinputswasapplied:10minat8nM(shortbar,circles),20minat4nM(longbar,squares),and20minat8nM

(longbar,triangles.).Onlythethirdconditionisabletoinactivatethefeedbackloop.Thereboundinthefirsttwocasesisduetotwofactors:thepersistenceofAAduetoarelativelyslowtimecourseofremovalandthetimecourseofdephosphorylationofactivatedkinasesintheMAPKcascade.2.(1)ActivationofthefractionalfeedbackloopbyEGFreceptor:(G)InactivationoffeedbackloopbyMKP-1.

2.(1)ActivationofthefractionalfeedbackloopbyEGFreceptor:(H)Thresholdsforinactivationoffeedbackloop.MKPwasappliedforvaryingtimesandamounts.AthighMKPlevels,inactivationoccursmorequickly,butthereisaminimumthresholdofnearly10min.Conversely,whenMKPisappliedforverylongtimes,atleast2nMMKPisrequiredtoinactivatethefeedbackloop.SomeconclusionsforEGFRsignalingpathways(1).100nMEGFcanactivateMAPK.(2).Ca2+activatePLCγ,whichhasmorehighactivityunder0.1uMEGF.(3).100minat5nMEGFactivatedthefeedbackloop.(4).ActivationofMAPKandPKCbyEGFhasathreshold(pointT).(5).ThephenomenonisrobustascomparingwithSimandExptonKmandConc.(6).MPK-1(20min,8nM)caninactivatethefeedbackloop.(7).HighMKPlevel,necessaryfornearly10min.

LongtimeapplicationofMKPrequiresatleast2nMMKP.Aboutbistablesystem(1).Suchabistablesystemhasthepotentialtostoreinformation.Signalingevents

[theinitialstimulation

(amplitudeandduration)]thatpushthelevelsofeitheractivatedPKCoractivatedMAPK

pasttheintersectionpointTwillcausethesystemtoflipfromonestatetoanother.Thisanalysiscanbegeneralizedtoanycombinationofpathwaysinafeedbackloop.(2).Theemergentpropertiesofthisfeedbacksystemdefinenotonlytheamplitudeanddurationoftheextracellularsignalrequiredtoactivatethesystembutalsothemagnitudeanddurationofprocessessuchasphosphataseactionrequiredtodeactivatethesystem.(3).Thesepropertiesmakeafeedbacksystem,onceactivated,capableofdeliveringaconstantoutputinamannerunaffectedbysmallfluctuationscausedbyactivatingordeactivatingevents.Thiscapabilitytodeliverastimulus-triggeredconstantoutputsignalevenafterthestimulusiswithdrawnmayhavenumerousbiologicalconsequences.2.(2)CaMKII

(Ca2+/calmodulin-dependentproteinkinaseII)functionsinLTPofsynapticresponsesinthehippocampus.ThecAMPpathwaygatesCaMKIIsignalingthroughtheregulationofproteinphosphatases.NMDARandCainfluxaremodeledinacompartmentalmodelofaCA1neuronwithaseriesofthreetetanicstimuliat100Hz,lasting1seach,separatedby10min.ThismodelusedmodulesshowninFig.1,C,I,J,M,N,andO(BtoE).Opensquares:fullmodel;Filledtriangles:cAMP(fixedatrestingconcentrations→preventPKAactivity↑).2.(2)(B)ActivationofCaMKII.

TheinitialincreaseinintracellularCa2+

causedanactivationofCaMKII,AC1,andCaNthroughCaMbindingandofPKAthroughincreaseincAMPproducedthroughactivationofAC1-AC8.

cAMP↑→PKAactivation→PP1↓→CaMKII↑ThepresenceofacAMP-operatedgateleadstoalargeincreaseintheamplitudeoftheCaMKIIresponseandprolongationofitsactivity.Nevertheless,itdoesnotleadtoapersistentactivationofCaMKII.

2.(2)(C)ActivationofPKA.AC1-AC8bindingtoCa/CaM↓producingcAMP.↓PKAactivityrisessharplyOtherwise,itsactivity:don’trise2.(2)(D)ActivityofPP1.[Ca/CaM↑+cAMP(fixed)]→CaNactivation↑→smalltransientscAMPfixed→PKAactivation↓cAMPunfixed→PKAactivation↑→PP1activity↓ActivePP1→dephosphorylateCaMKII(Thr286)→CaMKII↓.2.(2)(E)CaN(PP2B)activationby

Ca/CaMelevation.Thefullmodel–cAMPfixedcurvesoverlapalmosterfectly.

↓CaNuninfluencedbycAMP四、3.Amodelforinteractionbetween4signalingpathways:formanetwork(PKC、MAPKpathways+CaMKII、cAMPpathways)Glu(+postsynapticdepolarization)

→Ca2+influxthroughNMDAR→[Ca2+]↑

→postsynapticPK(CaMKII,PKC,PKA,MAPK)↑四、3.Combinedmodelwithfeedbackloop,synapticinput,andCaMKIIactivityandRegulation.cPLA2(heldactivity)→lessAA→FBOFFMKP(timerofFBinearlyLTPofsynapse)→FBOFFcPLA2(activity↑)→AA↑→FBON四、3.ActivitypromajorenzymesinpathwayFigBtoG

▲：fullmodel(FBON)□：feedbackblocked(FBOFF)

(AAfixedatrestingconcentrations)FBON

:presentfeedbackFBOFF:absencefeedback

四、3.(B)ActivityproPKCFBOFF:→PKC↓FBON→largersuccessivespikes(initialspike+FBON)

→DAG+AA→PKC↑↑四、3.(C)

ActivityproMAPKFBON

→MAPKturnonFBOFF

→MAPKturnoff(initialspike+FBON)

→DAG+AA→PKC↑↑→MAPK↑(steady)

四、3.(D)ActivityproPKA.Ca2+inflow→AC1,8↑→PKA↑Ca2+

→identicalPKA↑FBON:PKC→

AC2↑→cAMP↑→PKA↑↑

sustainedPKC→sustainedPKAactivitySeveralemergentpropertiesofnetwork(1).Extendedsignalduration.(2).Activationoffeedbackloop.(3).Definitionofthresholdstimulationforbiologicaleffects.(4).Multiplesignaloutputs.四、3.(E)ActivityproCaMKII.Ca2+inflow→CaMKII↑Ca2+

→identicalCaMKII↑FBON:PKC→AC2↑→cAMP↑→PKAbaseline↑(twofold)PKA↑→PP1↓→CaMKII↑{

[dephosphorylateCaMKII(Thr286)]→CaMKIIautophosphorylation↓}四、3.(F)ActivityproPP1.Ca2+→PP1↓(overlap:FBON,FBOFF)FBON→PKA↑(sustained)→PP1↓→PP1(sustained)CaMKII↑四、(G)ActivityproCaN(PP2B).FBOFForFBON：

CaN

isnaffected

→