信号通路与肿瘤市公开课一等奖省优质课赛课一等奖课件.pptx

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,引言：细胞信号转导与生命过程问题提出和理论产生,细胞信号转导理论概述,信号转导研究中重大理论问题及热点领域,信号转导研究方法与工具,信号转导理论研究及应用举例：在肿瘤发生发展中信号转导意义,信号转导与肿瘤临床：诊疗和治疗,细胞信号转导经典文件举例,第1页,引言,信号转导与生命过程,问题提出和理论产生,第2页,细胞信号转导理论建立以前细胞生物学,细胞显微结构（胞膜、胞浆、胞核）,细胞生理功效（生存、“活性”、分裂增殖、胞间连接、吞饮、分泌、迁移、死亡),细胞组分生物化学（脂、糖、核酸、蛋白）,细胞超微结构和亚细胞结构（脂质双层膜结构、细胞器),第3页,组织生长需要,细胞分裂增殖,细胞,生长因子,细胞周期,蛋白表示,病原体侵入,抗感染状态,细胞,抗原,细胞因子,表示分泌,细胞过分生长,细胞死亡,细胞,死亡因子,胞内致死,分子表示,第4页,细胞骨架蛋,白表示、激活,牵动细胞移动,(Cell movement),趋化因子,细胞粘附,细胞存活,(Survival),抗凋亡因子,表示、激活,胞外信号,信号作用,于细胞,基因表,达改变,细胞表,型改变,第5页,第6页,第7页,细胞信号转导理论概述,第8页,第9页,胞外信号分子(可溶性分子、细胞表面分子、组织基质分子),靶细胞跨膜分子(狭义受体如EGFR或广义受体如Integrin),靶细胞受体（胞内段）化学改变（如磷酸化、二聚体形成）,靶细胞内信号转导分子化学改变与激活,（如磷酸化、去磷酸化、聚体形成）,激活信号转导分子进入胞核,进入胞核转导分子作用于基因转录调控区,基因表示改变,第10页,Extracellular Signal Molecules,Growth Factors,PDGF(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.Cytokines,IFN-,(Interferon-,),IFN-,(Interferon-,),TNF(Tumor Necrosis Factor),Interleukins(1,2,3,4),3.Death molecules,Fas,4.,Adhesion molecules,Cadherins,Adhesin,5.Hormone,Insulin,6.Stress,第11页,Signal Transducing Receptors,Transmembrane,receptors that have intrinsic enzymatic activity.,A,utophosphorylation,P,hosphorylation of other substrates,A)Tyrosine kinases:PDGF-R,insulin-R,EGFR and FGF-R,B)Tyrosine phosphatases:e.g.CD45,C)Guanylate cyclases:e.g.natriuretic peptide receptors),D)Serine/Threonine kinases:activin and TGF-,b,receptors,第12页,2.Receptors that are coupled,inside the cell,to GTP-binding and hydrolyzing proteins(G-proteins).,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 transcription,e.g.,STAT1,3,4,5,6(Signal transducer and activator of transcription),4.Simple receptors:,e.g.,ion-channels that lead to changes in membrane electric potential,第13页,信号转导过程中生物化学,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）,受体或其它信号转导分子聚体化,第14页,Signal Transducers,Receptor Tyrosine Kinases(RTKs),contains:,An extracellular ligand binding domain.,An intracellular tyrosine kinase domain.,An intracellular regulatory domain.,A transmembrane domain.,Tyrosine phosphorylation,Interact with and phosphorylate,Src homology domain 2,(SH2)-containing proteins,(e.g.,PLC-,Ras,PI-3K,etc),Phosphorylate other kinases,phosphorylate proteins,which,upon phosphorylated,can enter,the nuclear and bind,DNA regulatory,regions.,第15页,Class,Examples,Structural Features of Class,I,EGF receptor,NEU/HER2,HER3,cysteine-rich sequences,II,insulin receptor,IGF-1 receptor,cysteine-rich sequences;characterized by disulfide-linked heterotetramers,III,PDGF receptors,c-Kit,contain 5 immunoglobulin-like domains;contain the kinase insert,IV,FGF receptors,contain 3 immunoglobulin-like domains as well as the kinase insert;acidic domain,V,vascular endothelial growth factor(VEGF)receptor,contain 7 immunoglobulin-like domains as well as the kinase insert domain,VI,hepatocyte growth factor(HGF)and scatter factor(SC)receptors,heterodimeric like the class II receptors except that one of the two protein subunits is completely extracellular.The HGF receptor is a proto-oncogene that was originally identified as the Met oncogene,VII,neurotrophin receptor family(trkA,trkB,trkC)and NGF receptor,contain no or few cysteine-rich domains;NGFR has leucine rich domain,Characteristics of the Common Classes of RTKs,第16页,Non-Receptor Protein Tyrosine Kinases(PTKs),Two non-receptor PTK families:,1)The archetypapl PTK familty:Src-related proteins,2)Janus kinase(Jak)family,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,第17页,第18页,第19页,第20页,Receptor Serine/Threonine Kinases(RSTKs),Typical example:Receptors for the TGF-,superfamily of ligands,The TGF-,superfamily include 30 multifunctional 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,Ligands bind,to the type,II receptors,Complexed,with type I,receptors,Type II R,phosphorylates,type I receptor,Initiation of,signaling,cascade,第21页,第22页,Non-Receptor Serine/Threonine Kinases,1)cAMP-dependent protein kinase(PKA),2)Protein kinase C(PKC),3)Mitogen activated protein kinases(MAPK or ERK)(requiring phosphorylation of both tyrosine and threonine),G-Protein Coupled Receptors,1.1000 GPCRs,most of which are orphan receptors),2.Three different classes of GPCR:,GPCRs that modulate adenylate cyclase activity and produce cAMP,GPCRs that activate PLC-g leading to hydrolysis of polyphosphoinositides:angiotensin,bradykinin and vasopressin receptors.,Photoreceptor,第23页,Intracellular Hormone Receptors,1.Residing within the cytoplasm.,2.The,steroid/thyroid hormone receptor,superfamily(e.g.glucocorticoid,vitamin D,retinoic acid and thyroid hormone 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.,第24页,胞外信号分子(可溶性分子、细胞表面分子、组织基质分子),靶细胞跨膜分子(狭义受体如EGFR或广义受体如Integrin),靶细胞受体（胞内段）化学改变（如磷酸化、二聚体形成）,靶细胞内信号转导分子化学改变与激活,（如磷酸化、去磷酸化、聚体形成）,激活信号转导分子进入胞核,进入胞核转导分子作用于基因转录调控区,基因表示改变,第25页,信号转导研究中重大理论问题及热点领域,第26页,信号转导通路调控,磷酸化去磷酸化调控,信号转导分子消长调控（分子半衰期）,不一样通路之间效应调控,胞内内源性抑制物调控功效,第27页,Cross-Talk,第28页,信号转导效应特异性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 phosphorylation/dephosphorylation?,第29页,替换通路（Alternative Pathways),第30页,信号转导研究方法与工具,第31页,一、蛋白质磷酸化状态检测,1、免疫印迹(phospho-protein specific antibodies),2、免疫沉淀(protein-specific antibody+phospho-AA antibody,3、流式细胞仪分析,4、Luminex分析,二、信号转导分子过分表示或过分激活,1、Overexpression by gene transduction,2、Constitutively activated mutants,三、基因转录活性测定,1、Electrophoretic mobility shift analysis(EMSA),、Reporter gene expression detection,第32页,四、信号转导分子表示或活性抑制,1、Anti-sense,2、RNAi,3、Gene knock-out,4、Dominant negative mutants,Ligand-binding site,Phosphorylation site,Docking site,Protein-protein binding site,DNA binding site,5、Small-molecule inhibitors：e.g.,tyrosine kinase inhibitor(TKi),6、Inhibitory oligopeptides,第33页,信号转导在肿瘤发生发展中意义,第34页,Signaling molecules involved in cancer development/progression,Receptors,Growth factor receptors:EGFR,Hormone receptor:ER,AR,Angiogenic receptros:VEGF,PDGF,IGF,Death receptors,The Integrin system,第35页,Transducers,Ras,Raf,Rho family,PI-3K/Akt,Death transducers,STAT-3,Transcription factors,c-Myc,c-Jun and c-fos,STAT-3,第36页,Biological Effects of Signaling Related to Cancer Development/Progression,Cell immobilization,Abrogation of apoptosis,Activation of cell cycle and removal of cell cycle checkpoints,Angiogenesis,Cell invasion,Metastasis,Drug resistance,第37页,第38页,Phosphorylation 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-L1,Akt,Bad,14-3-3binding,Release of Bcl-2 and Bcl-X Cell survival,Akt GSK3 GSK3 catalytic activity turned off Permitting activation of c-Myc and cyclin D,PDK1 phosphorylation of other kinases(p70 S6-kinasse,CISK,PKC)Cell growth and survival,第39页,信号转导与肿瘤临床,诊疗、预防与治疗,Expression level,mutations and antibodies of signaling molecules in cancer diagnosis,EGFR:lung,H&N,glioma,TGF-,receptor(type II):lung,H&N,Ras,Androgen receptor and downstream molecules,Estrogen receptor and downstream molecules,第40页,Protein phosphorylation and cancer diagnosis,Determination of single phosphorylated signaling molecules:EGFR,Phospho-protein profiling:proteomics,Phospho-protein based imaging technology,Signaling inhibitor and cancer prevention/therapy,EGFR-selective TKi:Iressa,EGFR antibodies,Farnesylation or Geranylgeranylation inhibitors targeting Ras and Rho,VEGFR antibody(and TKi):Avastin,第41页,经典文件举例,第42页,Stat3,as an Oncogene,Jacqueline F.Bromberg,1,Melissa H.Wrzeszczynska,1,Geeta Devgan,1,Yanxiang Zhao,2,Richard G.Pestell,3,Chris Albanese,3,and James E.Darnell,Jr.,1,1,Laboratory of Molecular Cell Biology,The Rockefeller University,New York,New York 10021-6399,USA,2,Laboratory of Molecular Biophysics,The Rockefeller University,New York,New York 10021-6399,USA,3,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,Cell,Volume 98,Issue 3,295-303,第43页,第44页,第45页,第46页,信号转导理论在各生命科学领域中普遍意义,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,第47页,