Networks of BiologicalSignaling Pathways信号传递网络康海岐 高方远 马欣荣信号传递网络一、生物体内的信号传递l 1. The sense of signal transduction：l intercelluar information exchange,regulation of metobolism, on body level l 2. Type of signals：l neuroregulation: neurotransmitter(乙酰胆碱乙酰胆碱,胺类胺类l 氨基酸氨基酸,调节肽类等调节肽类等),neuroregulatorl chemical signals：cAMP, Ca2+ , hormone，l 3. Mechanisms:l 3.1 pr. pr.,l 3.2 E reaction(p )l 3.3 E activityl 3.4 pr. degradationl 3.5 intracelluar messagerl 3.6 seconder messagerl Ecell信号传递网络一、生物体内的信号传递l 4. Signaling pathways：l 4.1 Ca2+ l 4.2 cAMPl 4.3 tyrosine kinase: EGFR,insulinRl 4.4 other pr. kinase cascade:PKC,PKA,PKGl 4.5 intracelluar protease cascadelSignal transmission occur:l i. Pr.pr. Interactionl ii. Enzymatic reaction: pl iii. Pr. Degradationl iiii. Production of intracellular messager信号传递网络一、生物体内的信号传递l5. cytoplasm membrane receptor：l 5.1 neurotransmitter-dependention channell (依赖神经递质的离子通道)：l nAChR(烟碱型乙酰胆碱受体)l GABA( -氨基丁酸)l GlyR(甘氨酸受体)l 5.2 receptor connecting to signal transduction protein l (G,N protein second messenger activate E.):l mAChR(毒蕈碱型乙酰胆碱受体)l adrenergic -,-receptor (肾上腺素能 -,-受体)l 5.3 growth factor receptor(tyrosine kinase activity)：l PDGFR(血小板衍生的生长因子受体)，l EGFR(表皮生长因子受体)，insulin R(胰岛素受体)信号传递网络PeptideSignalinginPlantsPNAS,Nov.6,2001,vol.98no.23Inplants,onlyafewpeptidehavebeenidentifiedthatactassignalingmolecules. Incontrast,signalingpeptidesaremajorplayersinallaspectsofthelifecycleinanimalsandyeast.suggeststhatsignalingmechanismsacrosstheeukaryotickingdomarefundamentallydifferent.信号传递网络1.1.目前有关植物中信号肽的研究主要基于以下目前有关植物中信号肽的研究主要基于以下5 5种：种：2.2.番茄番茄systemin systemin PSK ENOD40 CLV3 SCR PSK ENOD40 CLV3 SCR 3.3.18 18 aa aa 10-1310-13 aa aa 72-75 72-75 aa aa 53-55 53-55 aaaa 2. 2. 最近分离到另外最近分离到另外3 3种活性信号肽：种活性信号肽：RALFRALF: rapid alkalinization factor, 5 kd; : rapid alkalinization factor, 5 kd; Tobacco systemin: Tobacco systemin: Tob sys I, Tob sys IITob sys I, Tob sys II信号传递网络1 1）tomato systemin:tomato systemin: 由食草动物损伤后引起的系统由食草动物损伤后引起的系统 损伤反应损伤反应( a systemic wounding response)( a systemic wounding response) 在悬浮培养细胞中可以激活促细胞分裂蛋白激酶在悬浮培养细胞中可以激活促细胞分裂蛋白激酶 mitogenmitogen-activated protein(MAP) -activated protein(MAP) kinasekinase 并诱导培养基地碱化并诱导培养基地碱化( (alkalinizationalkalinization) ) 诱导蛋白酶抑制蛋白编码基因的表达诱导蛋白酶抑制蛋白编码基因的表达( (induceinduce expression of expression of proteinaseproteinase-inhibitor -inhibitor protein-encoding genes) protein-encoding genes) 3. 3. 功能：功能：信号传递网络2 2）tobacco systemin tobacco systemin Tob I and Tob II:Tob I and Tob II: 激活激活 MAP kinase MAP kinase，但不诱导蛋白酶抑制蛋白编码，但不诱导蛋白酶抑制蛋白编码 基因的表达基因的表达3 3）RALFRALF (rapid alkalinizaton factor):(rapid alkalinizaton factor): 激活激活 MAPMAP kinase kinase，但不诱导蛋白酶抑制蛋白编码但不诱导蛋白酶抑制蛋白编码 基因的表达基因的表达; ; 快速引起快速引起 medium medium 碱化碱化信号传递网络 From the followings support the idea that peptide and nonpeptide hormone-activated signaling cascades are linked in plants as they are in animals: 植物生长素类似植物生长素类似5羟色胺，乙烯类似一氧化碳，羟色胺，乙烯类似一氧化碳， 油菜素类固醇是类固醇，茉莉酮酸与前列腺素相关；油菜素类固醇是类固醇，茉莉酮酸与前列腺素相关； Systemin-induced wound response is regulated through the octadecanoid pathway, involving jasmonic acid;4. 4. 信号调控网络信号调控网络信号传递网络 PSK-induced cell proliferation requires the hormones auxin or cytokinin; Some of the developmental distortions in roots induced on addition of RALF are reminiscent of impaired nonpeptide hormone-controlled processes. 因此，揭开两种信号因此，揭开两种信号cascades之间关系，将是非常之间关系，将是非常有趣的事。有趣的事。信号传递网络信号传递网络一、生物体内的信号传递l6.2 IP3 system Hermone/neurotransmitterG proteinPLCPIP2IP3+DAGCaMmAChR,mAChR,EGFREGFR, ,insulinRinsulinR,adrenergicR ,adrenergicR ,组胺组胺R,5-R,5-羟色胺羟色胺R,R,多肽激素多肽激素R RCa2+ PKC等蛋白激酶，磷酸酯酶，核苷酸环化酶，离子通道蛋白，肌肉收缩蛋白等依赖Ca2+ /CaM的蛋白。Ca2+ /CaMPKC*使各种受体，膜蛋白，收缩蛋白，细胞骨架蛋白，核蛋白和酶类的丝氨酸或苏氨酸残基磷酸化，从而影响细胞代谢、生长和分化。AAGCcGMP 多种酶及依赖cGMP的蛋白激酶。激活多种酶和依赖cGMP的蛋白激酶而发挥生理作用。激活蛋白激酶活性，自身与tyrosine残基磷酸化,促进cell生长和分化。信号传递网络二、海马趾CA1神经元区室化模型中的15个信号途径A:EGF,SOSB:GEF,RasC:cAMP,AC1,AC2D:GE:AA,PLA2F:PLC,PLCG:DAG,IP3H:MAPKCascadeI:CaMKIIJ:PKAK:PKCL:Ca,IP3M:CaMN:CaNO:PP1信号传递网络ReactionA:EGF,SOS信号传递网络 lReactionB:GEF,Ras信号传递网络ReactionC:cAMP,AC1,AC2信号传递网络ReactionD:G 信号传递网络ReactionE:AA,PLA2信号传递网络ReactionsF,G:ReactionsF,G:PLCPLC,PLC,PLC ,DAG,IP3,DAG,IP3信号传递网络ReactionH:MAPKCascade信号传递网络lThe various phosphorylation states of CaMKII have different enzyme kinetics, and each of these were explicitly modeled. For simplicity the autophosphorylation steps are represented by a single enzyme arrow in this figure, with CaMKII_a as the combined activity of the various phosphorylation states. The individual kinetic terms used in the model are indicated by the multiple rate references on the arrows. ReactionI:CaMKII信号传递网络ReactionJ:PKA信号传递网络ReactionK:PKC信号传递网络ReactionL:Ca,IP3信号传递网络ReactionM:CaM信号传递网络ReactionN:CaN信号传递网络ReactionO:PP1信号传递网络三三、establishingtheindividualpathways1.steps1.Setupmodelactivationofsinglecomponent.2.generatethemodelforanindividualsignalingpathway.3.Obtainagoodempiricalmodelwhichfittheexperimentaldata.4.examineexperimentallydefinedcombinationof2or3suchindividualsignalingpathways.5.testthesecombinedmodels.信号传递网络2.Materialsandmethord(1).HippocampalCA1neuron(inGENSIS),(2).NMDARondendriticspine(树突棘树突棘)onthemodel(3).Synapticinput(3tetanicburstsat100HZ,1seach)LTPCa2+waveforms信号传递网络3.ComputationformulationGenesisformulation:S+ESE-k3-P+EVmax=maxvelocity=k3.Substrateissaturating,soallofEisinSEform.SoVmax.Etot=SE.k3=Etot.k3Km=(k3+k2)/k1k2=k3*4Kd=Kb/KfIfA*Bhalf*Kf=Chalf=Bhalf*KbthenA=Kb/Kf=KdKa=Kf/Kb=1/Kd信号传递网络4.verificationl (i). Model simple kinetic schemes l that could be calculated analytically, l compare simulated results with analytical results. l (ii). Use the law of mass conservation and l microscopic reversibility principles(微观可逆性原理) l test accuracy in complex reaction schemes. l (iii). Run the same model at different time steps, l compare the resulting simulated values.信号传递网络5.ProteinKinaseCmodelingexample信号传递网络Simulationparameters:PKCReactionK:PKCReferencesFigureReac#kfkbK1150K22E-100.1K31.27053.5026K40.0000000020.1K510.1K620.2K70.0000010.5K81.3333E-088.6348K90.0000000010.1K100.000000032信号传递网络ReferencesConcsK:PKCReferencesFigureNameConcKPKC_inactive11.Review:Y. Nishizuka,Nature334,661(1988)2. J. D. Schaechter and L. 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).信号传递网络A.BlockdiagramofactivationforPKCA.BlockdiagramofactivationforPKCpathwaybyCa2+,AAandDAG.pathwaybyCa2+,AAandDAG.lbuiltupsimulationsiteratively:lFirst:matchedAAactivationofPKCatzeroCa.lThen: matched activation ofPKCwithCaatzeroAA,lThird:matchedthecurvesinBwith1uMCaandvaryingAA.lFour:testthematchforC,withvaryingCaand50uMAA.lLast:incorporatedDAGinteractionsintothemodel.信号传递网络B:ActivationofPKCbyAA,withB:ActivationofPKCbyAA,with(triangles)orwithout(squares)1mM(triangles)orwithout(squares)1mMCa2+.Ca2+.lOpen symbols and dashed lines represent simulations, solid symbols and solid lines are experimental data. Shows:Ca2+ is necessary for the activation of PKC.experimentalconcentration-effectcurvesfromtwomainsources:J. D. Schaechter and L. 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) 信号传递网络C:ActivationofPKCbyCa2+,withC:ActivationofPKCbyCa2+,with(triangles)orwithout(squares)50mM(triangles)orwithout(squares)50mMAA.AA.lThecurveinthepresenceof 50 mM AA (triangles)was predicted from theparameters obtained bymatching the curves in BandthecurvewithoutAA(squares) in C, withoutfurtheradjustment.信号传递网络D:ActivationofPKCbyDAG,withD:ActivationofPKCbyDAG,with(triangles)orwithout(squares)50mM(triangles)orwithout(squares)50mMAA.AA.lBoth curves in D were obtained in the presence of 1 mM Ca2+. Due to different methods for estimating DAG concentrations the levels of DAG used in the model are scaled 15-fold up with respect to the experimental conditions from Shinomura et al. 信号传递网络四、develope the network model in stageslFirst : model individual pathwayslThen: examin experimentally defined combinations of two or three such individual pathways and test these combined models against published data. lThird: repeat this process using larger assemblies of pathways until the entire network model of interacting pathways waslformed. lPathways were linked by two kinds of interactions: l (i) Second messengers such as AA and DAG, produced by one pathway were used as inputs to other pathways. l(ii) Enzymes whose activation was regulated by one pathway were coupled to substrates belonging to other pathways.信号传递网络1、one Signaling pathways exampleS(1).(1).EGFs stimulation of MAPK1,2EGFs stimulation of MAPK1,2lFig. 2. EGF receptor signaling pathways.l(A). Block diagram of signalinglpathways. Rectangles represent enzymes, and circles represent messengerlmolecules. This model used modules shown in Fig. 1, reaction A(EGF), B(Ca2+/CaM), E(AA,PLA2), H(PKC),F(PLC,DAG,IP3), H(MAPK ascade), K(PKC), I(CaMKII), L(Ca,IP3). 信号传递网络Fig.2B the time course of activation of MAPK by EGFl (B) Predicted (open triangle) and experimental (filled triangles) time course of response of MAPK to a steady EGF stimulus of 100 nM.lthe y axis represents fractional activation.l The fall in the MAPK activity after the initial stimulation is due to a combination of EGF receptor internalization and MAPK phosphorylationland inactivation of SoS.信号传递网络1、one Signaling pathways exampleS (2).(2). Activation of Activation of PLC by Ca2+by Ca2+ in the presence in the presence (triangles) or absence (squares) of EGF.(triangles) or absence (squares) of EGF.l (C) Concentration-effect curves.lDashed lines are model data, and solid lines are experimental data. The y axis represents activation. 信号传递网络lThree stimulus conditions: l10 min at 5 nM EGF (short bar, circles), l100 min at 2 nM EGF (long bar,squares), l100 min at 5 nM EGF (long bar, triangles). lOnly the third condition succeeds in causing activation of the feedback loop. Why?2 2、Two connected pathwaysTwo connected pathways(1). Activation of the fractional feedback loop by EGF (1). Activation of the fractional feedback loop by EGF receptor : receptor : (D)(D) Activation of feedback loop by EGF. Activation of feedback loop by EGF.信号传递网络lB (basal), T (threshold), and A(active).lPoint A represents high activity forlboth PKC and MAPK, whereas point B represents low activity. Both of these points represent distinct steady-state levels. Such a system with two distinct steady states is a bistable system. The bifurcation point T is important because it defines threshold stimulation.2.(1) Activation of the fractional feedback loop2.(1) Activation of the fractional feedback loop by EGF receptor : by EGF receptor : (E) Bistability plot for feedback loop (E) Bistability plot for feedback loop 信号传递网络l Bistability is present overla range comparable to the experimental uncertainty, indicating that thelphenomenon is robust. l(Horizontal stripes: experimental uncertainty in concentration; diagonallstripes, simulated bistability range for concentrations.) lMAPK has a particularly large uncertainty in concentration range because of large differences in tissue distributions.2.(1) Activation of the fractional feedback loop by 2.(1) Activation of the fractional feedback loop by EGF receptor :EGF receptor : (F (F) estimated experimetal uncertainty in E ) estimated experimetal uncertainty in E parameters parameters 信号传递网络linitially activating: a suprathreshold stimulus, and then one of three inhibitory inputs was applied: 10 min at 8 nM (short bar, circles), 20 min at 4 nM (long bar, squares), and 20 min at 8 nM (long bar, triangles.).lOnly the third condition is able to inactivate the feedback loop.lThe rebound in the first two cases is due to two factors: the persistence of AA due to a relatively slow time course of removal and the time course ofldephosphorylation of activated kinases in the MAPK cascade.2.(1)Activation of the fractional feedback loop by EGF 2.(1)Activation of the fractional feedback loop by EGF receptor: receptor: (G) Inactivation of feedback loop by MKP-1. (G) Inactivation of feedback loop by MKP-1. 信号传递网络2.(1) Activation of the fractional feedback loop by EGF 2.(1) Activation of the fractional feedback loop by EGF receptor: receptor: (H) Thresholds for inactivation of feedback loop. (H) Thresholds for inactivation of feedback loop. lMKP was applied for varying timesland amounts. At high MKP levels, inactivation occurs more quickly, but there is a minimum threshold of nearly 10 min. Conversely, when MKP is applied for very long times, at least 2 nM MKP is required to inactivate the feedback loop.信号传递网络Some conclusions for EGFR signaling pathwaysl(1).100 nM EGF can activate MAPK.l(2).Ca2+ activate PLC,which has more high activity under 0.1uM EGF.l(3).100 min at 5 nM EGF activated the feedback loop.l(4).Activation of MAPK and PKC by EGF has a threshold(point T).l(5).The phenomenon is robust as comparing with Sim and Expt on Km and Conc.l(6).MPK-1(20 min,8nM) can inactivate the feedback loop.l(7).High MKP level ,necessary for nearly 10 min.l Long time application of MKP requires at least 2nM MKP.信号传递网络About bistable systeml(1). Such a bistable system has the potential to store information. Signaling events the initial stimulation (amplitude and duration) that push the levels of either activated PKC or activated MAPK past the intersection point T will cause the system to flip from one state to another. This analysis can be generalized to any combination of pathways in a feedback loop.l(2). The emergent properties of this feedback system define not only the amplitude and duration of the extracellular signal required to activate the system but also the magnitude and duration of processes such as phosphatase action required to deactivate the system. l(3). These properties make a feedback system, once activated, capable of delivering a constant output in a manner unaffected by small fluctuations caused by activating or deactivating events. lThis capability to deliver a stimulus-triggered constant output signal even after the stimulus is withdrawn may have numerous biological consequences.信号传递网络2.(2) 2.(2) CaMKIICaMKII ( (Ca2+/calmodulin-dependent protein kinase II )Ca2+/calmodulin-dependent protein kinase II ) functions in LTP of synaptic responses in the functions in LTP of synaptic responses in the hippocampus.hippocampus.lThe cAMP pathway gates CaMKII signaling through the regulationlof protein phosphatases.lNMDAR and Ca influx are modeled in a compartmental model of a CA1 neuron with a series of three tetaniclstimuli at 100 Hz, lasting 1 s each, separated by 10 min. This model used modules shown in Fig. 1, C,lI, J, M, N, and O(B to E).lOpen squares: full model;l Filled triangles:cAMP(fixed at resting concentrations prevent PKA activity ). 信号传递网络2.(2) (B) Activation of CaMKII. 2.(2) (B) Activation of CaMKII. l The initial increase in intracellular Ca2+ caused an activation of CaMKII, AC1,and CaN through CaM binding and of PKA through increase in cAMP produced through activation of AC1-AC8. lcAMP PKA activation PP1 CaMKII lThe presence of a cAMP-operated gate leads to a large increase in the amplitude of the CaMKII response and prolongation of its activity. Nevertheless, it does not lead to a persistent activation of CaMKII. 信号传递网络2.(2) 2.(2) (C) Activation of PKA.(C) Activation of PKA.AC1-AC8 binding to Ca/CaM producing cAMP. PKA activity rises sharply Otherwise,its activity: dont rise信号传递网络2.(2) (D) Activity of PP1.2.(2) (D) Activity of PP1.l Ca/CaM + cAMP(fixed) CaN activation smalltransientslcAMP fixed PKA activationlcAMP unfixed PKAactivation PP1 activitylActive PP1 dephosphorylate CaMKII(Thr286) CaMKII .信号传递网络 2.(2) (E) CaN (PP2B) activation by 2.(2) (E) CaN (PP2B) activation byCa/CaM elevation.Ca/CaM elevation.l The full modelcAMP fixed curves overlap almost erfectly.l l CaN uninfluenced by cAMP信号传递网络四、3. A model for interaction between 4 signaling pathways: form a network(PKC、MAPKpathways+CaMKII、cAMPpathways)Glu(+postsynapticdepolarization)Ca2+influxthroughNMDARCa2+postsynapticPK(CaMKII,PKC,PKA,MAPK)信号传递网络四、3. Combined model with feedback loop, synaptic input, and CaMKII activity and Regulation.cPLA2(held activity) lessAAFBOFFMKP(timer of FB in early LTP of synapse)FBOFFcPLA2(activity) AAFBON信号传递网络四、3. Activity pro major enzymes in pathway lFig B to G ：full model(FBON) ：feedback blocked(FBOFF) (AA fixed at resting concentrations)lFBON : present feedbacklFBOFF : absence feedback 信号传递网络四、3. (B) Activity pro PKClFBOFF :PKClFBON larger successive spikes l(initialspike+FBON ) DAG+AAPKC信号传递网络四、3. (C)Activity pro MAPKlFBON MAPKturnonlFBOFF MAPKturnoffl(initialspike+FBON ) DAG+AAlPKCMAPK(steady)l 信号传递网络四、3. (D) Activity pro PKA. lCa2+ inflowAC1,8PKA Ca2+ identical PKA lFBON : PKC AC2 cAMPPKA lsustainedPKCsustainedlPKAactivity信号传递网络Several emergent properties of networkl(1).Extended signal duration.l(2).Activation of feedback loop.l(3).Definition of threshold stimulation forl biological effects.l(4).Multiple signal outputs.信号传递网络四、3. (E) Activity pro CaMKII.lCa2+ inflowCaMKIIlCa2+ identical CaMKII lFBON : PKC AC2 cAMPPKA baseline (twofold) lPKAPP1CaMKIIl dephosphorylate CaMKII(Thr286) CaMKIIautophosphorylation信号传递网络四、3. (F) Activity pro PP1. lCa2+PP1l(overlap:FBON,FBOFF)lFBONPKA(sustained)PP1PP1(sustained)lCaMKII信号传递网络四、(G) Activity pro CaN (PP2B).lFBOFF or FBON：l CaN is naffectedlitseffect on PP1 limited to the duration of the initial signals.信号传递网络On Networkl(1).Networksustained PK activity(afterinitialstimulus)l correspond to early LTPl(2). MKP inductionOther transcriptional events be initiated lgene productsreach the active synapse with MKPl(3).FBloop may gate incorporation of these products into the cytoskeleton. l act as bridge between extremly short stimuli and longer term synaptic change and also between local synaptic events and cell wide production of synaptic proteins. 信号传递网络On the modell(1).Such a model facilitates “thought experiments” on l involved signaling pathways to predict hierarchies.l(2). The model also provides a framework for understandingl biological consequences of multiple modes ofl stimulating a single component.l(3).Such models provide insights into the possible roles of l isoform diversity. l CaMAC1,PKCAC2(connection,sustainCaMKIIactivation) 信号传递网络On the modell (4).Limitations: l The biochemical parameters are not unaltered with the cell. Given these uncertainties, models such as these should not be considered as definitive descriptions of networks within the cell, but rather as one approach that allows us to understand the capabilities of complex systems and devise experiments to test these capabilities.l(5). Conclusion: l simple biochemical reactions can, with appropriate coupling, be used to store information. Thus, reactions within signaling pathways may constitute one locus for the biochemical basis for learning and memory.信号传递网络信号传递网络