信号传递网络-课件.ppt

1、康海岐 高方远 马欣荣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,调节肽类调节肽类等等),neuroregulatorl chemical signals：cAMP,Ca2+,hormone，l 3.Mechanisms:l 3.1 pr.pr.,l 3.2 E r

2、eaction(p)l 3.3 E activityl 3.4 pr.degradationl 3.5 intracelluar messagerl 3.6 seconder messagerl Ecelll 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.Intera

3、ctionl ii.Enzymatic reaction:pl iii.Pr.Degradationl iiii.Production of intracellular messagerl5.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 s

4、econd 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)Peptide Signaling in Plants PNAS,Nov.6,2001,vol.98 no.23 In plants,only a few peptide have been identified

5、that act as signaling molecules.In contrast,signaling peptides are major players in all aspects of the life cycle in animals and yeast.suggests that signaling mechanisms across the eukaryotic kingdom are fundamentally different.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 system

6、in 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:rapid alkalinization alkalinization factor,5 factor,5 kdkd;Tobacco Tobacco systeminsystemin:Tob Tob sys I,sys I,Tob Tob sys IIsys II1 1）tom

7、ato tomato systeminsystemin: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

8、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：2 2）tobaccotobacco systemin systemin TobTob I and I and Tob Tob II:II: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3 3）RALFRA

9、LF (rapid(rapid alkalinizaton alkalinizaton factor):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 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

10、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 PSK-induced cell proliferation requires the hormones auxin or cytokinin;Some of the developmenta

11、l 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。l6.2 IP3 system Hermone/neurotransmitterG proteinPLCPIP2IP3+DAGCaMmAChRmAChR,EGFREGFR,insulinRinsulinR,adrenergicadrenergicR,R,

12、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。A:EGF,SOSA:EGF,SOSB:GEF,B:GEF,RasRasC:C:cAMPcAMP,AC1,AC2,

13、AC1,AC2D:GD:GE:AA,PLA2 E:AA,PLA2 F:PLCF:PLC,PLC,PLC G:DAG,IP3 G:DAG,IP3 H:MAPK Cascade H:MAPK Cascade I:I:CaMKII CaMKII J:PKA J:PKA K:PKC K:PKC L:Ca,IP3 L:Ca,IP3 M:M:CaM CaM N:N:CaN CaN O:PP1O:PP1 lReaction B:GEF,Reaction B:GEF,RasRaslThe various phosphorylation states of CaMKII have different enzym

14、e 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 m

15、ultiple rate references on the arrows.Reaction I:Reaction I:CaMKII CaMKII 1.Set up model activation of single component.2.generate the model for an individual signaling pathway.3.Obtain a good empirical model which fit the experimental data.4.examine experimentally defined combination of 2 or 3 such

16、 individual signaling pathways.5.test these combined models.(1).Hippocampal CA1 neuron(in GENSIS),(2).NMDARon dendritic spine(树突棘树突棘)on the model (3).Synaptic input(3 tetanic bursts at 100HZ,1s each)LTP Ca2+waveformsGenesis formulation:S+E SE-k3-P+E Vmax=max velocity=k3.Substrate is saturating,so al

17、l of E is in SE form.Substrate is saturating,so all of E is in SE form.So So Vmax Vmax.EtotEtot=SE.k3=SE.k3=EtotEtot.k3.k3 Km=(k3+k2)/k1 k2=k3*4 Kd=Kb/KfIf A*Bhalf*Kf=Chalf=Bhalf*Kb then A=Kb/Kf=Kd Ka=Kf/Kb=1/Kdl (i).Model simple kinetic schemes l that could be calculated analytically,l compare simu

18、lated 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.Reaction K:PKC Reaction K:PKC Refer

19、encesReferencesFigurFigureeReacReac#kfkfkbkbK1150K22E-100.1K31.27053.50 26K40.0000000020.1K510.1K620.2K70.0000010.5K81.3333E-088.63 48K90.0000000010.1K100.000000032ConcsConcs K:PKC K:PKC ReferencesReferencesFigureFigureNameNameConcConcKPKC_inactive11.1.Review:Y.Review:Y.Nishizuka Nishizuka,NatureNat

20、ure 334,661(1988)334,661(1988)2.2.J.D.J.D.Schaechter Schaechter and L.I.and L.I.BenowitzBenowitz,J.J.Neurosci Neurosci.13,4361 13,4361(1993)(1993)3.3.T.T.Shinomura Shinomura,Y.,Y.Asaoka Asaoka,M.,M.OkaOka,K.Yoshida,Y.,K.Yoshida,Y.Nishizuka Nishizuka,Proc.Proc.Natl.Acad.Natl.Acad.Sci Sci.U.S.A.U.S.A.

21、88,5149.88,5149(1991)(1991)U.Kikkawa,Y.Takai,R.Minakuchi,S.Inohara,Y.Nishizuka,J.Biol.Chem.257,13341(1982).lbuilt up simulations iteratively:lFirst:matched AA activation of PKC at zero Ca.lThen:matched activation of PKC with Ca at zero AA,lThird:matched the curves in B with 1 uM Ca and varying AA.lF

22、our:test the match for C,with varying Ca and 50 uM AA.lL a s t:i n c o r p o r a t e d D A G interactions into the model.lOpen symbols and dashed lines represent simulations,solid symbols and solid lines are experimental data.Shows:Ca2+is necessary for the activation of PKC.experimental concentratio

23、n-effect curves from two main sources: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)lThe curve in the presence of 50 mM AA(triangles)was p r e d i c t e d f r o m t h e parameters obtained by matching t

24、he curves in B and the curve without AA(squares)in C,without further adjustment.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 condition

25、s from Shinomura et al.lFirst: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 inte

26、racting 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.lFig

27、.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).l(B)Predict

28、ed(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 phosphoryl

29、ationland inactivation of SoS.l(C)Concentration-effect curves.lDashed lines are model data,and solid lines are experimental data.The y axis represents activation.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,tri

30、angles).lOnly the third condition succeeds in causing activation of the feedback loop.Why?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 wit

31、h two distinct steady states is a bistable system.The bifurcation point T is important because it defines threshold stimulation.l Bistability is present overla range comparable to the experimental uncertainty,indicating that thelphenomenon is robust.l(Horizontal stripes:experimental uncertainty in c

32、oncentration;diagonallstripes,simulated bistability range for concentrations.)lMAPK has a particularly large uncertainty in concentration range because of large differences in tissue distributions.linitially activating:a suprathreshold stimulus,and then one of three inhibitory inputs was applied:10

33、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 an

34、d the time course ofldephosphorylation of activated kinases in the MAPK cascade.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

35、 required to inactivate the feedback loop.l(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 compari

36、ng 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.l(1).Such a bistable system has the potential to store information.Signaling events the initial stimulat

37、ion(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 feed

38、back 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 deli

39、vering 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.lThe cAMP pathway gates CaMKII sign

40、aling 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 mode

41、l;l Filled triangles:cAMP(fixed at resting concentrations prevent PKA activity).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 p

42、resence 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.AC1-AC8 binding to Ca/CaM producing cAMP.PKA activity rises sharply Otherwise,its activity:dont risel

43、Ca/CaM +cAMP(fixed)CaN activation smalltransientslcAMP fixed PKA activationlcAMP unfixed PKAactivation PP1 activitylActive PP1 dephosphorylate CaMKII(Thr286)CaMKII.l The full modelcAMP fixed curves overlap almost erfectly.l l CaN uninfluenced by cAMP(PKC、MAPK pathways+CaMKII、cAMP pathways)Glu(+posts

44、ynaptic depolarization)Ca2+influx through NMDAR Ca2+postsynaptic PK(CaMKII,PKC,PKA,MAPK)cPLA2(held activity)less AA FBOFFMKP(timer of FB in early LTP of synapse)FBOFFcPLA2(activity)AA FBONlFig B to G：full model(FBON)：feedback blocked(FBOFF)(AA fixed at resting concentrations)lFBON :present feedbackl

45、FBOFF:absence feedback lFBOFF:PKClFBON larger successive spikes l(initial spike+FBON)DAG+AAPKClFBON MAPK turn onlFBOFF MAPK turn offl(initial spike+FBON)DAG+AAl PKCMAPK(steady)l lCa2+inflowAC1,8PKA Ca2+identical PKA lFBON:PKC AC2 cAMPPKA l sustained PKCsustained l PKA activityl(1).Extended signal du

46、ration.l(2).Activation of feedback loop.l(3).Definition of threshold stimulation forl biological effects.l(4).Multiple signal outputs.lCa2+inflowCaMKIIlCa2+identical CaMKII lFBON:PKC AC2 cAMPPKA baseline(twofold)lPKAPP1CaMKIIl dephosphorylate CaMKII(Thr286)CaMKII autophosphorylationlCa2+PP1l (overla

47、p:FBON,FBOFF)lFBONPKA(sustained)PP1 PP1(sustained)lCaMKIIlFBOFF or FBON：l CaN is naffectedl its effect on PP1 limited to the duration of the initial signals.l(1).Networksustained PK activity(after initial stimulus)l correspond to early LTPl(2).MKP induction Other transcriptional events be initiated

48、l gene 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.l(1).Such a m

49、odel 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

50、 diversity.l CaMAC1,PKCAC2(connection,sustain CaMKII activation)l(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