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1、Signal-Transduction Pathways (II) 郑利民郑利民 2005200512 12 珠海珠海Important roles of biosignaling Functional integration of distant organs, tissues and cells requires communication; Signaling is a primal requirement to respond to our environment; The foundation of any complex response pathway lies with cel
2、lular biochemicals.Question:How are they regulated?SopEPKB/AktRac,CDC42PTKPI-3K R调理后吞噬病原侵袭死亡信号死亡信号生存信号生存信号细胞凋亡和粘膜屏障损坏细胞凋亡和粘膜屏障损坏细胞存活细胞存活细胞凋亡是由：细胞凋亡是由： 细胞内细胞内“死亡死亡/ /生存生存”信号之间的精密平衡来决定信号之间的精密平衡来决定 干扰该平衡就可改变病原对细胞凋亡的最终影响干扰该平衡就可改变病原对细胞凋亡的最终影响 Complicated “cross-talk” between bio-signals PART1 Basic chara
3、cteristics of signal transduction2 Four general types of signal transducersPART1 Regulatory mechanisms2 Some diseases caused by defects in the biosignaling pathwaysRegulatory mechanisms of Bio-signals3.1 Phosphorylation as a regulatory mechanism3.2 Regulation of transcription by steroid hormones3.3
4、Regulation of the cell cycle by protein kinasesRegulation of Signaling PathwaysExternalsignalsSecondmessengersModulatorproteinsFunctionTargetproteinsHormones cAMP Odorants cGMPDrugs Ca2+Light DAG IP3Mitogenichormones TyrosineGrowth kinasesfactorsproteinkinaseAnd phosphatasestructuralproteinsAnd enzy
5、mesmetabolicorphysiologicalresponsesPlasma membranePhosphorylation & de-phosphorylation are the most common regulatory mechanisms, mediated by protein kinase and phosphatase, respectively 蛋白激酶蛋白激酶 n n NTP NTP n n NDP NDP 蛋白质蛋白质 蛋白质蛋白质- -n n Pi Pi n n Pi Pi 蛋白磷酸酶蛋白磷酸酶 H H2 2O ONobel Prize in Physiolo
6、gy and Medicine 1992Eldwin G. KrebsEdmond H. Fischer USA “Reversible protein phosphorylation as a biological regulatory mechanismcAMPPKAPhosporylatingcellular proteinsResponseActivation of G ProteinCoupled ReceptorsGEF：Guanine nucleotide Exchange FactorGAP：GTPase Activating Protein Activation of Ras
7、 proteinSHCGrb2GEFRas -GTPFRas -GDPFPiGAPSHCGrb2GEFRas -GTPFRas -GDPFPiGAPRasRas蛋白的上游和下游信号通路蛋白的上游和下游信号通路 PI3K的活化机制及的活化机制及PIP3磷酸酶磷酸酶(PI3K is a downstream target of Ras)PI3KAKT途径对细胞生存的调控机制途径对细胞生存的调控机制The Nobel Prize in Physiology and Medicine 1994for their discovery of G-proteins and the role of these
8、 proteins in signal transduction in cellsAlfred G. Gilman 1941-Martin Rodbell 1925-1998Two-component system or signaling细菌的二元组分细菌的二元组分系统系统NinfaNinfa等等19861986年首年首次报道了大肠杆菌次报道了大肠杆菌营养信号转导的二营养信号转导的二元组分系统元组分系统，大量大量的实验已经证实，的实验已经证实，二元组分系统是细二元组分系统是细菌信号转导的普遍菌信号转导的普遍机制机制。钝化蛋白钝化蛋白活化蛋白活化蛋白 激素激素 受体受体 PIP2 磷脂酶磷脂酶
9、C C PKC DAG G蛋白蛋白 IP3 细胞反应细胞反应 CaCa2+2+ IP3敏感通道敏感通道 结合态结合态IP3 内质网或液泡内质网或液泡细胞反应细胞反应 CaCa2+2+细胞内信号转导的双信使系统细胞内信号转导的双信使系统 PI3K的活化机制及的活化机制及PIP3磷酸酶磷酸酶(PI3K is a downstream target of Ras)PI3KAKT途径对细胞生存的调控机制途径对细胞生存的调控机制Most of these complicated signaling pathways are transduced by the phosphorylation and de
10、phosphoralation of signaling protein, regulated by the protein kinases and phosphatses蛋白激酶（蛋白激酶（protein protein kinasekinase，PKPK） 植物和酵母中约有植物和酵母中约有2%-3%2%-3%的基因编码蛋白激酶。的基因编码蛋白激酶。 根据磷酸化靶蛋白的氨基酸残基的种类不同，蛋根据磷酸化靶蛋白的氨基酸残基的种类不同，蛋白激酶有白激酶有丝氨酸丝氨酸/ /苏氨酸激酶苏氨酸激酶、酪氨酸激酶酪氨酸激酶和组氨和组氨酸激酶等三类，它们分别将底物蛋白质的酸激酶等三类，它们分别将底物蛋白质的
11、丝氨酸丝氨酸/ /苏氨酸苏氨酸、酪氨酸酪氨酸和组氨酸残基磷酸化。有的蛋白激和组氨酸残基磷酸化。有的蛋白激酶具有双重底物特异性，既可使丝氨酸或苏氨酸残酶具有双重底物特异性，既可使丝氨酸或苏氨酸残基磷酸化，又可使酪氨酸残基磷酸化基磷酸化，又可使酪氨酸残基磷酸化蛋白磷酸酶蛋白磷酸酶 protein protein phosphatasephosphatase，PPPP 蛋白磷酸酶的分类与蛋白激酶相对应，分蛋白磷酸酶的分类与蛋白激酶相对应，分为丝氨酸为丝氨酸/ /苏氨酸型蛋白磷酸酶和酪氨酸型蛋苏氨酸型蛋白磷酸酶和酪氨酸型蛋白磷酸酶。有些酶具有双重底物特异性白磷酸酶。有些酶具有双重底物特异性 对蛋白磷酸
12、酶的研究还不如蛋白激酶那样对蛋白磷酸酶的研究还不如蛋白激酶那样深入。但两者的协同作用在细胞信号转导中深入。但两者的协同作用在细胞信号转导中的作用是不言而喻的。的作用是不言而喻的。 Phosphorylation as a Regulatory MechanismSignaltransductionAlteredkinaseactivitycGMP cAMP InsulinCa2+ kinase There are 120 kinases in yeast i.e. 2% of their entire genome Some kinases are localized to discrete
13、subcellular regions that are in close proximity to the specific target protein; Many proteins act as targeting or anchoring mediators e.g. AKAP associates tightly with the regulatory subunits of PKA Cells contain many different isoforms of PKC, which are localized by anchoring proteins, e.g. RACKs o
14、r receptors for activated PKC Phosphatases are also often localized closely to ensure that integrated control is possible.Mechanisms by which hormones, retinoids, and Vit. D regulate gene expressionHormone (H), carried to the targettissue on serum binding prot,diffuses across the PM and binds to its
15、 specific receptorprotein (Rec) in the nucleus.H binding changes the conformation of Rec; forms homo or heterodimers and binds to specific R regions called hormone response elements (HREs) in the DNA adjacent to specific genesBinding regulates transcription of theadjacent gene(s), increasing or decr
16、easing the rate of mRNA formation.Altered levels of the hormone regulatedgene product produce the cellular response to the hormone3.3.1 The cell cyclenWhat is cell cycle? nA process of cellular reproduction Four phases: G1, S, G2, MnGap (G1 and G2) phases: Respond to proliferative and anti-prolifera
17、tive signals, synthesize RNA and proteinnSynthetic (S) phase: Replication of the chromosomesnMitotic (M) phase: segregation of chromosomes and other cell constituents into two daughter cellsCell cycle transits orderly and irreversiblyCell cycle transits orderly and irreversiblynThe completion of one
18、 phase is required for the beginning of the next.nPeriodic activation of specific cyclin-Cdk complexes nA cycle of protein destruction eliminates proteins used in the preceding phase as well as proteins that would inhibit progression into the next phase.Cyclin-dependent kinases (Cdks)nCdks drive cel
19、l cycle progression by phosphorylating a group of substrates- Cdks present a unique pattern of activity in each phase of the cell cycleCdk1 (Cdc2)Cdk2Cdk3Cdk4 Cdk5Cdk6Cdk7G1G2, MLate G1, SG1, S, G2, MRegulation of Cdks activity Cyclin: Activating subunit. nCyclins were named because of their cyclic
20、expression during the cell cycleMitogen StimulationFour mechanisms regulate CDK activity: 1 phosphorylation or dephosphorylation, 2 controlled degradation of cyclin subunit, 3 periodic synthesis of CDKs and cyclins, 4 action of specific CDK inhibiting proteins.Regulation of Cdks activity Phosphoryla
21、tion and dephospho. in Cdk1 Activating phosphorylation site: Thr 161Inhibitory phosphorylation site: Thr 14, Thr 15PPGrowth controlling signals are conveyed by stimulatory or inhibitory pathwayssome target proteins of CDKs:Laminin - Breakdown of nuclear envelope before segregation of sister chromati
22、ds in mitosis is partly due to Pi-laminin by CDKActin myosin contractile machinery-Involved in pinching a dividing cell into two equal parts during cytokinesisRetinoblastoma protein, pRb-participates in a mechanism that arrests cell division in G1 CDKs Regulate Cell Division by Phosphorylating Criti
23、cal Proteins3.3.3 Oncogenes, Tumor Suppressor Genes (TSGs), and Programmed Cell DeathOncogenes: encode defective signaling proteins ( e.g., growth factors, receptors, G proteins, protein kinases, or transcription regulators), continually giving the signal for cell division; TSG: encode regulatory pr
24、oteins; normally inhibit cell division; mutations in these genes are genetically recessiveCancer: the result of an accumulation of mutations in oncogenes and TSGs.Oncogene-encoded defective EGF receptorFrom normal epithelial cell to cancerSummary Four basic characters of biosignalsFour schemes of in
25、tercellular signaling Four general types of signal transducers gated ion channels; receptor enzymes; G-protein coupled receptorsSteroid ReceptorsG proteins: Heterotrimeric & monomeric; Activation and deactivation mechanismsMajor second messengers: cAMP, IP3, DAG, Ca2+, cGMP.Using an example to expla
26、in the cascade of events: a single molecule of hormone activates catalyst that in turn activates another catalyst, and so on, results in signal amplification and cell responseThe cell cycle and its regulationOncogenes, TSGs, and CancerPART1 Basic characteristics of signal transduction2 Four general
27、types of signal transducersPART1 Regulatory mechanisms2 Some diseases caused by defects in the biosignaling pathways信号转导异常的原因和机制信号转导异常的原因和机制一、信号转导异常一、信号转导异常1.1.生物学因素：生物学因素： 干扰细胞内信号转导通路干扰细胞内信号转导通路: :如霍乱毒素如霍乱毒素2. 2. 理化因素：理化因素：电离辐射或机械刺影响信号转导成分而致癌电离辐射或机械刺影响信号转导成分而致癌3.3.遗传因素：信号蛋白数量遗传因素：信号蛋白数量/ /功能改变（突变）功
28、能改变（突变）4.4.免疫学因素：刺激型和阻断型抗受体抗体免疫学因素：刺激型和阻断型抗受体抗体5.5.内环境因素内环境因素二、信号转导异常的发生环节二、信号转导异常的发生环节V2RGsACcAMPATPPKAADHH2OH2O配体、受体或受体后信号通路的任何一个环节配体、受体或受体后信号通路的任何一个环节出现障碍都会影响到最终效应，使细胞增殖、出现障碍都会影响到最终效应，使细胞增殖、分化、凋亡、代谢或功能失常，并导致疾病。分化、凋亡、代谢或功能失常，并导致疾病。以尿崩症为例以尿崩症为例ADHADH作用的三个环节异常均可导致尿崩症：作用的三个环节异常均可导致尿崩症： ADHADH分泌减少分泌减少
29、 中枢性尿崩症中枢性尿崩症 ADH-VADH-V2 2受体变异受体变异 肾小管上皮细胞水通道蛋白（肾小管上皮细胞水通道蛋白（AQPAQP2 2）异常）异常集合管上皮细胞对集合管上皮细胞对ADHADH的反应性降低的反应性降低家族性尿崩症家族性尿崩症不同受体介导的信号转导通路存在不同受体介导的信号转导通路存在cross-talk 并非所有的信号转导蛋白异常都能导致疾病并非所有的信号转导蛋白异常都能导致疾病细胞信号转导异常与疾病细胞信号转导异常与疾病v受体、信号转导障碍与疾病受体、信号转导障碍与疾病v受体数量减少受体数量减少v受体亲和力降低受体亲和力降低v受体阻断型抗体的作用受体阻断型抗体的作用v协
30、同因子或辅助因子缺陷协同因子或辅助因子缺陷v受体功能缺陷受体功能缺陷v受体后信号转导蛋白缺陷受体后信号转导蛋白缺陷特特定定信信号号转转导导过过程程减减弱弱或或中中断断激激素素抵抵抗抗征征雄激素受体缺陷与雄激素抵抗征雄激素受体缺陷与雄激素抵抗征原因和机制：原因和机制：AR减少或失活突变减少或失活突变男性假两性畸形男性假两性畸形特发性无精症症特发性无精症症延髓脊髓性肌萎缩延髓脊髓性肌萎缩胰岛素受体与胰岛素抵抗性糖尿病胰岛素受体与胰岛素抵抗性糖尿病1.1.遗传性胰岛素受体异常，包括受体的遗传性胰岛素受体异常，包括受体的合成减少合成减少与配体的亲和力降低，如与配体的亲和力降低，如ArgArg73573
31、5突变为突变为SerSerTPKTPK活性降低，如活性降低，如GlyGly10081008 突变等突变等2.2.自身免疫性胰岛素受体异常自身免疫性胰岛素受体异常血液中存在抗胰岛素受体的抗体血液中存在抗胰岛素受体的抗体二、受体、信号转导过度激活与疾病二、受体、信号转导过度激活与疾病v某些信号转导蛋白过度表达某些信号转导蛋白过度表达v某些信号转导蛋白组成型激活突变某些信号转导蛋白组成型激活突变v刺激型抗受体抗体刺激型抗受体抗体生长激素（生长激素（GHGH）分泌过多的垂体腺瘤中，）分泌过多的垂体腺瘤中，30%30%是由于编码是由于编码GsGs的基因突变所致，从而抑制了的基因突变所致，从而抑制了GTP
32、GTP酶活性，使酶活性，使GsGs处于持续激活状态，处于持续激活状态，cAMPcAMP含含量增多，垂体细胞生长和分泌功能活跃。量增多，垂体细胞生长和分泌功能活跃。通通路路过过度度激激活活如肢端肥大症和巨人症如肢端肥大症和巨人症霍乱毒素选择性的催化霍乱毒素选择性的催化GsGs亚基上的亚基上的ArgArg201201核糖核糖化，使化，使GTPGTP酶活性丧失，不能将酶活性丧失，不能将GTPGTP水解成水解成GDPGDP，从，从而使而使GsGs处于不可逆激活状态处于不可逆激活状态, ,不断刺激不断刺激ACAC生成生成cAMPcAMP, ,胞浆中的胞浆中的cAMPcAMP含量可增加含量可增加至正常的至
33、正常的100100倍以上，倍以上，导致小肠上皮细胞膜蛋白构型改变，大量导致小肠上皮细胞膜蛋白构型改变，大量ClCl- -和和水分子持续转运入肠腔，引起严重腹泻和脱水。水分子持续转运入肠腔，引起严重腹泻和脱水。肠肠 腔腔GsCTCTcAMP Cl-H2ONa+CT：Cholera Toxin三、多环节信号转导异常与疾病三、多环节信号转导异常与疾病v 肿瘤肿瘤v1. 1. 促细胞增殖的信号转导过强促细胞增殖的信号转导过强v 生长因子产生增多生长因子产生增多v受体的改变受体的改变v生长因子受体表达异常增多生长因子受体表达异常增多v突变使受体组成型激活突变使受体组成型激活v细胞内信号转导蛋白的改变细胞
34、内信号转导蛋白的改变v 如如RasRas突变突变2. 2. 抑制细胞增殖的信号转导过弱抑制细胞增殖的信号转导过弱生长抑制因子受体减少、丧失生长抑制因子受体减少、丧失受体后信号转导通路异常受体后信号转导通路异常细胞的生长负调控机制减弱或丧失细胞的生长负调控机制减弱或丧失GEF：Guanine nucleotide Exchange FactorGAP：GTPase Activating Protein Activation of Ras proteinGEF：Guanine nucleotide Exchange FactorGAP：GTPase Activating Protein Mutat
35、ion of Ras led to its constitutive activationRhoRho蛋白在肿瘤进展过程中的作用蛋白在肿瘤进展过程中的作用 To be, or not to be, that is a question!Apoptosis is a tightly regulated process.Apoptosis is a tightly regulated process.1. The shift of balance between death & survival1. The shift of balance between death & survival sig
36、nals determined the cell fate ( signals determined the cell fate (命运）命运）2. Usually required a number of events (or genes)2. Usually required a number of events (or genes) to control apoptosis to control apoptosisE.g., activation of one E.g., activation of one oncogenesoncogenes induce apoptosis indu
37、ce apoptosisin most cases, inhibit apoptosis only with the in most cases, inhibit apoptosis only with the support from other signals.support from other signals. ( (Together we stand, separate we diedTogether we stand, separate we died) ) 双向调控基因双向调控基因 c-Mycc-Myc的作用模型的作用模型的作用模型的作用模型肿瘤形成肿瘤形成必须是在单个细胞中发生
38、必须是在单个细胞中发生多重遗传突变多重遗传突变单个癌基因激活和抑癌基因失活不足以引起肿瘤单个癌基因激活和抑癌基因失活不足以引起肿瘤细胞的显著扩增，细胞的显著扩增，因为细胞对凋亡的易感性也同时增加；凋亡抑制因为细胞对凋亡的易感性也同时增加；凋亡抑制是肿瘤细胞能够生长到足以威胁宿主所必须的；是肿瘤细胞能够生长到足以威胁宿主所必须的；例如：例如：Rb的失活仅会促进的失活仅会促进p53依赖的凋亡，但不会引起肿依赖的凋亡，但不会引起肿瘤，除非细胞的凋亡过程被抑制；瘤，除非细胞的凋亡过程被抑制；Apoptosis and oncogenesisRegulation of cellular signal t
39、ransduction in prevention and treatment of diseases以信号转导蛋白为靶分子对疾病以信号转导蛋白为靶分子对疾病进行防治进行防治STI571 as a paradigm (范例）范例）for cancer therapyIn 1960, described the presence of a consistent In 1960, described the presence of a consistent chromosomal abnormality in CMLchromosomal abnormality in CML（一种白血病（一种白血
40、病) )，so-called Philadelphia chromosome (Ph)so-called Philadelphia chromosome (Ph)A reciprocal translocation between the long arms A reciprocal translocation between the long arms of chromosomes 9 and 22, t(9:22)(q34;q11),of chromosomes 9 and 22, t(9:22)(q34;q11),The molecular consequence of this eve
41、nt is the The molecular consequence of this event is the generation of a generation of a chimericchimeric bcr-ablbcr-abl gene (22:9)gene (22:9)The tyrosine The tyrosine kinasekinase activity of activity of Bcr-AblBcr-Abl stimulates stimulates a variety of signaling pathways, leading to a variety of signaling pathways, leading to alterations in survival properties of cellsalterations in survival properties of cellsSTI571STI571an an AblAbl-specific tyrosine -specific tyrosine kinasekinase inhibitor inhibitorBefore the discovery of this drug, most CML patients died within 6 month具备勇气和智慧具备勇气和智慧
