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1、细胞信号转导与肿瘤中山大学2005级分子医学博士生课程第四讲2005年9月27日,黎孟枫 医学博士中山大学基础医学院微生物学教研室,引言：细胞信号转导与生命过程问题的提出和理论的产生细胞信号转导理论概述信号转导研究中的重大理论问题及热点领域信号转导的研究方法与工具信号转导理论研究及应用举例：在肿瘤发生发展中的信号转导的意义信号转导与肿瘤临床：诊断和治疗细胞信号转导经典文献举例,引言,信号转导与生命过程问题的提出和理论的产生,细胞信号转导理论建立以前的细胞生物学,细胞的显微结构（胞膜、胞浆、胞核）细胞的生理功能（生存、“活性”、分裂增殖、胞间连接、吞饮、分泌、迁移、死亡)细胞组分的生物化学（脂、
2、糖、核酸、蛋白）细胞的超微结构和亚细胞结构（脂质双层膜结构、细胞器),细胞信号转导理论概述,Extracellular Signal Molecules,Growth FactorsPDGF(Platelet-Derived Growth Factor),EGF(Epidermal Growth Factor),TGF-(Transforming Growth Factor-),EPO(Erythropoietin),NGF(Nerve Growth Factor),IGF(Insulin-like Growth Factor),TPO(Thrombopoietin)2.CytokinesIF
3、N-(Interferon-),IFN-(Interferon-),TNF(Tumor Necrosis Factor),Interleukins(1,2,3,4)3.Death moleculesFas 4.Adhesion moleculesCadherins,Adhesin5.HormoneInsulin6.Stress,Signal Transducing Receptors,Transmembrane receptors that have intrinsic enzymatic activity.AutophosphorylationPhosphorylation of other
4、 substratesA)Tyrosine kinases:PDGF-R,insulin-R,EGFR and FGF-RB)Tyrosine phosphatases:e.g.CD45C)Guanylate cyclases:e.g.natriuretic peptide receptors)D)Serine/Threonine kinases:activin and TGF-b receptors,2.Receptors that are coupled,inside the cell,to GTP-binding and hydrolyzing proteins(G-proteins).
5、e.g.,adrenergic receptors,odorant receptors,and certain hormone receptors(e.g.glucagon,angiotensin,vasopressin and bradykinin).3.Receptors that are found intracellularly and upon ligand binding migrate to the nucleus where the ligand-receptor complex directly affects gene transcriptione.g.,STAT1,3,4
6、,5,6(Signal transducer and activator of transcription)4.Simple receptors:e.g.,ion-channels that lead to changes in membrane electric potential,信号转导过程中的生物化学,磷酸化反应（酪氨酸激酶、丝/苏氨酸激酶）蛋白质构象改变去磷酸化反应（磷酸酶）受体或其他信号转导分子的聚体化,Signal Transducers,Receptor Tyrosine Kinases(RTKs)contains:An extracellular ligand binding
7、 domain.An intracellular tyrosine kinase domain.An intracellular regulatory domain.A transmembrane domain.,Characteristics of the Common Classes of RTKs,Non-Receptor Protein Tyrosine Kinases(PTKs)Two non-receptor PTK families:1)The archetypapl PTK familty:Src-related proteins2)Janus kinase(Jak)famil
8、y,Most non-receptor PTKs couple to cellular receptors that lack enzymatic activity themselves(e.g.,CD4,CD8,TCR and all cytokine receptors such as IL-2R,Receptor Serine/Threonine Kinases(RSTKs),Typical example:Receptors for the TGF-superfamily of ligands The TGF-superfamily include 30 multifunctional
9、 proteins,e.g.,activins,inhibins and the bone morphogenetic proteins(BMPs).17 RSTKs isolated are in 2 subfamilies:type I and type II receptors.Nuclear proteins responding to TGF-activation include c-Myc and Smad,Non-Receptor Serine/Threonine Kinases 1)cAMP-dependent protein kinase(PKA)2)Protein kina
10、se C(PKC)3)Mitogen activated protein kinases(MAPK or ERK)(requiring phosphorylation of both tyrosine and threonine)G-Protein Coupled Receptors1.1000 GPCRs,most of which are orphan receptors)2.Three different classes of GPCR:GPCRs that modulate adenylate cyclase activity and produce cAMP GPCRs that a
11、ctivate PLC-g leading to hydrolysis of polyphosphoinositides:angiotensin,bradykinin and vasopressin receptors.Photoreceptor,Intracellular Hormone Receptors 1.Residing within the cytoplasm.,2.The steroid/thyroid hormone receptor superfamily(e.g.glucocorticoid,vitamin D,retinoic acid and thyroid hormo
12、ne receptors):bind steroid/thyroid hormone,translocate to nuclear and bind specific DNA sequences hormone response elements(HREs).,*Phosphatases in Signal Transduction 1.Transmembrane PTPs:e.g.,CD45.2.Intracellular PTPs.,信号转导研究中的重大理论问题及热点领域,信号转导通路的调控,磷酸化去磷酸化调控信号转导分子消长的调控（分子半衰期）不同通路之间的效应调控胞内内源性抑制物的调控
13、功能,Cross-Talk,信号转导效应的特异性When and Where?,Cooperation with other signaling pathways?Pre-existing transcription co-factors differentially expressed and activated in different cell types?Pre-existing co-activators of target proteins?Subcellular localization of transducers?Optimal level(or a threshold)of
14、 phosphorylation/dephosphorylation?,替代通路（Alternative Pathways),信号转导的研究方法与工具,一、蛋白质磷酸化状态的检测1、免疫印迹(phospho-protein specific antibodies)2、免疫沉淀(protein-specific antibody+phospho-AA antibody3、流式细胞仪分析4、Luminex分析二、信号转导分子过度表达或过度激活1、Overexpression by gene transduction2、Constitutively activated mutants三、基因转录活性
15、测定1、Electrophoretic mobility shift analysis(EMSA)、Reporter gene expression detection,四、信号转导分子的表达或活性抑制1、Anti-sense2、RNAi3、Gene knock-out4、Dominant negative mutantsLigand-binding sitePhosphorylation siteDocking siteProtein-protein binding siteDNA binding site5、Small-molecule inhibitors：e.g.,tyrosine k
16、inase inhibitor(TKi)6、Inhibitory oligopeptides,信号转导在肿瘤发生发展中的意义,Signaling molecules involved in cancer development/progression,ReceptorsGrowth factor receptors:EGFRHormone receptor:ER,ARAngiogenic receptros:VEGF,PDGF,IGFDeath receptorsThe Integrin system,Transducers Ras Raf Rho family PI-3K/AktDeath
17、transducersSTAT-3Transcription factorsc-Mycc-Jun and c-fosSTAT-3,Biological Effects of Signaling Related to Cancer Development/Progression,Cell immobilizationAbrogation of apoptosisActivation of cell cycle and removal of cell cycle checkpointsAngiogenesisCell invasionMetastasisDrug resistance,Phosph
18、orylation targets of PI-3K,Akt Forkhead-related transcription factor 1(FKHR-L1)14-3-3 binding FKHR-L1 retaining in cytosol abrogation of gene activation by FKHR-L1Akt Bad 14-3-3binding Release of Bcl-2 and Bcl-X Cell survivalAkt GSK3 GSK3 catalytic activity turned off Permitting activation of c-Myc
19、and cyclin DPDK1 phosphorylation of other kinases(p70 S6-kinasse,CISK,PKC)Cell growth and survival,信号转导与肿瘤临床诊断、预防与治疗,Expression level,mutations and antibodies of signaling molecules in cancer diagnosisEGFR:lung,H&N,gliomaTGF-receptor(type II):lung,H&N,RasAndrogen receptor and downstream moleculesEst
20、rogen receptor and downstream molecules,Protein phosphorylation and cancer diagnosisDetermination of single phosphorylated signaling molecules:EGFRPhospho-protein profiling:proteomicsPhospho-protein based imaging technologySignaling inhibitor and cancer prevention/therapyEGFR-selective TKi:IressaEGF
21、R antibodiesFarnesylation or Geranylgeranylation inhibitors targeting Ras and RhoVEGFR antibody(and TKi):Avastin,经典文献举例,Stat3 as an Oncogene Jacqueline F.Bromberg1,Melissa H.Wrzeszczynska1,Geeta Devgan1,Yanxiang Zhao2,Richard G.Pestell3,Chris Albanese3 and James E.Darnell,Jr.,1,1 Laboratory of Molec
22、ular Cell Biology,The Rockefeller University,New York,New York 10021-6399,USA2 Laboratory of Molecular Biophysics,The Rockefeller University,New York,New York 10021-6399,USA3 The Albert Einstein Cancer Center,Department of Developmental and Molecular Biology,and Department of Medicine,Albert Einstein College of Medicine,Bronx,New York 10461,USA Received 30 March 1999;Revised 23 June 1999.Available online 27 September 2000,Cell Volume 98,Issue 3,295-303,信号转导理论在各生命科学领域中的普遍意义,以本课程中各讲为例：干细胞蛋白质组学细胞凋亡肿瘤转移血管增生,
