1、细胞信号转导与肿瘤细胞信号转导与肿瘤中山大学中山大学2005级分子医学博士生课程第四讲级分子医学博士生课程第四讲2005年年9月月27日日黎孟枫 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引言：细胞信号转导与生命过程问题的问题的提出和理论的产生提出和理论的产生 细胞信号转导理论概述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发展中的信号转导的意义 信号转导与肿瘤临床
2、：诊断和治疗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细胞信号转导经典文献举例引言引言信号转导与生命过程信号转导与生命过程问题的提出和理论的产生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信号转导理论建立以前的细胞生物学细胞生物学 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、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)组织生长需要组织生长需要细胞分裂增殖细胞分裂增殖细胞细胞生长因子生长因子细胞周期细胞周期蛋白表达蛋白表达病原体侵入病原体侵入抗感染状态抗感染状态
3、细胞细胞抗原抗原细胞因子细胞因子表达分泌表达分泌细胞过度生长细胞过度生长细胞死亡细胞死亡细胞细胞死亡因子死亡因子胞内致死胞内致死分子表达分子表达细胞骨架蛋细胞骨架蛋白表达、激活白表达、激活牵动细胞移动牵动细胞移动(Cell movement)趋化因子趋化因子细胞粘附细胞粘附细胞存活细胞存活(Survival)抗凋亡因子抗凋亡因子表达、激活表达、激活胞外信号胞外信号信号作用信号作用于细胞于细胞基因表基因表达改变达改变细胞表细胞表型改变型改变细胞信号转导理论概述细胞信号转导理论概述胞外信号分子胞外信号分子(可溶性分子、细胞表面分子、组织基质分子可溶性分子、细胞表面分子、组织基质分子)靶细胞跨膜分子
4、靶细胞跨膜分子(狭义受体如狭义受体如EGFR或广义受体如或广义受体如Integrin)靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成）靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成）靶细胞内信号转导分子化学变化与激活靶细胞内信号转导分子化学变化与激活（如磷酸化、去磷酸化、聚体形成）（如磷酸化、去磷酸化、聚体形成）激活的信号转导分子进入胞核激活的信号转导分子进入胞核进入胞核的转导分子作用于基因转录调控区进入胞核的转导分子作用于基因转录调控区基因表达改变基因表达改变Extracellular Signal Molecules1.Growth Factors2.PDGF(Platelet-D
5、erived Growth Factor),EGF(Epidermal Growth Factor),TGF-(Transforming Growth Factor-),EPO(Erythropoietin),NGF(Nerve Growth Factor),IGF(Insulin-like Growth Factor),TPO(Thrombopoietin)3.2.Cytokines4.IFN-(Interferon-),IFN-(Interferon-),TNF(Tumor Necrosis Factor),Interleukins(1,2,3,4)5.3.Death molecules6
6、.Fas 7.4.Adhesion molecules8.Cadherins,Adhesin9.5.Hormone10.Insulin11.6.StressSignal Transducing Receptors1.Transmembrane receptors that have intrinsic enzymatic activity.2.3.Autophosphorylation4.Phosphorylation of other substratesA)Tyrosine kinases:PDGF-R,insulin-R,EGFR and FGF-RB)Tyrosine phosphat
7、ases:e.g.CD45C)Guanylate cyclases:e.g.natriuretic peptide receptors)D)Serine/Threonine kinases:activin and TGF-receptors2.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.gluca
8、gon,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.,STAT1,3,4,5,6(Signal transducer and activator of transcription)4.Simple receptors:e.g.,ion-c
9、hannels that lead to changes in membrane electric potential信号转导过程中的生物化学信号转导过程中的生物化学 磷酸化反应（酪氨酸激酶、丝磷酸化反应（酪氨酸激酶、丝/苏氨苏氨酸激酶）酸激酶）蛋白质构象改变蛋白质构象改变 去磷酸化反应（磷酸酶）去磷酸化反应（磷酸酶）受体或其他信号转导分子的聚体化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 domain.An intracell
10、ular tyrosine kinase domain.An intracellular regulatory domain.A transmembrane domain.Tyrosine phosphorylationInteract with and phosphorylate Src homology domain 2(SH2)-containing proteins(e.g.,PLC-,Ras,PI-3K,etc)Phosphorylate other kinasesphosphorylate proteins,which upon phosphorylated,can enter t
11、he nuclear and bind DNA regulatory regions.ClassExamplesStructural Features of ClassIEGF receptor,NEU/HER2,HER3cysteine-rich sequencesIIinsulin receptor,IGF-1 receptorcysteine-rich sequences;characterized by disulfide-linked heterotetramersIIIPDGF receptors,c-Kitcontain 5 immunoglobulin-like domains
12、;contain the kinase insertIVFGF receptorscontain 3 immunoglobulin-like domains as well as the kinase insert;acidic domainVvascular endothelial growth factor(VEGF)receptorcontain 7 immunoglobulin-like domains as well as the kinase insert domainVIhepatocyte growth factor(HGF)and scatter factor(SC)rece
13、ptorsheterodimeric 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 oncogeneVIIneurotrophin receptor family(trkA,trkB,trkC)and NGF receptorcontain no or few cysteine-rich
14、 domains;NGFR has leucine rich domainCharacteristics 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)familyMost non-receptor PTKs couple to cellular receptors that lack enzymat
15、ic 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 proteins,e.g.,activins,inhibins and the bone morphogenetic proteins(BMP
16、s).17 RSTKs isolated are in 2 subfamilies:type I and type II receptors.Nuclear proteins responding to TGF-activation include c-Myc and SmadLigands bind to the type II receptors Complexed with type I receptors Type II R phosphorylates type I receptor Initiation of signaling cascade Non-Receptor Serin
17、e/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 Receptors1.1000 GPCRs,most of which are orphan receptors)2.Three different classes of GPCR:1)GPCR
18、s that modulate adenylate cyclase activity and produce cAMP 2)GPCRs that activate PLC-g leading to hydrolysis of polyphosphoinositides:angiotensin,bradykinin and vasopressin receptors.3)Photoreceptor Intracellular Hormone Receptors 1.Residing within the cytoplasm.2.The steroid/thyroid hormone recept
19、or 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.胞外信号分子
20、胞外信号分子(可溶性分子、细胞表面分子、组织基质分子可溶性分子、细胞表面分子、组织基质分子)靶细胞跨膜分子靶细胞跨膜分子(狭义受体如狭义受体如EGFR或广义受体如或广义受体如Integrin)靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成）靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成）靶细胞内信号转导分子化学变化与激活靶细胞内信号转导分子化学变化与激活（如磷酸化、去磷酸化、聚体形成）（如磷酸化、去磷酸化、聚体形成）激活的信号转导分子进入胞核激活的信号转导分子进入胞核进入胞核的转导分子作用于基因转录调控区进入胞核的转导分子作用于基因转录调控区基因表达改变基因表达改变信号转导研究中的重大
21、信号转导研究中的重大理论问题及热点领域理论问题及热点领域信号转导通路的调控信号转导通路的调控 磷酸化磷酸化去磷酸化调控去磷酸化调控 信号转导分子消长的调控（分子半衰期）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不同通路之间的效应调控 胞内内源性抑制物的调控功能胞内内源性抑制物的调控功能Cross-Talk信号转导效应的特异性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
22、 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?替代通路替代通路（Alternative Pathways)信号转导的研究方法信号转导的研究方法与工具与工具一、蛋白质磷酸化状态的检测一、蛋白质磷酸化状态的检测1、免疫印迹(phospho-protein specif
23、ic 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、基因转录活性测定三、基因转录活性测定1、Electrophoretic mobility shift analysis(EMSA)、Reporter gene expression detection四、信号转导分
24、子的表达或活性抑制四、信号转导分子的表达或活性抑制1、Anti-sense2、RNAi3、Gene knock-out4、Dominant negative mutants(1)Ligand-binding site(2)Phosphorylation site(3)Docking site(4)Protein-protein binding site(5)DNA binding site(6)5、Small-molecule inhibitors：e.g.,tyrosine kinase inhibitor(TKi)(7)6、Inhibitory oligopeptides信号转导在肿瘤发生
25、信号转导在肿瘤发生发展中的意义发展中的意义Signaling molecules involved in cancer development/progressionReceptors1)Growth factor receptors:EGFR2)Hormone receptor:ER,AR3)Angiogenic receptros:VEGF,PDGF,IGF4)Death receptors5)The Integrin systemTransducers1)Ras2)Raf3)Rho family4)PI-3K/Akt5)Death transducers6)STAT-37)Transcr
26、iption factors1)c-Myc2)c-Jun and c-fos3)STAT-34)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 resistancePhosphorylation ta
27、rgets 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 14-3-3 binding Release of Bcl-2 and Bcl-X Cell survival Akt GSK3 GSK3 catalytic activity turned off Permitting activation of c-Myc and cycli
28、n D PDK1 phosphorylation of other kinases(p70 S6-kinasse,CISK,PKC)Cell growth and survival信号转导与肿瘤临床信号转导与肿瘤临床诊断、预防与治疗诊断、预防与治疗Expression level,mutations and antibodies of signaling molecules in cancer diagnosis1)EGFR:lung,H&N,glioma2)TGF-receptor(type II):lung,H&N,3)Ras4)Androgen receptor and downstre
29、am molecules5)Estrogen receptor and downstream molecules6)Protein phosphorylation and cancer diagnosis1)Determination of single phosphorylated signaling molecules:EGFR2)Phospho-protein profiling:proteomics3)Phospho-protein based imaging technologySignaling inhibitor and cancer prevention/therapy1)EG
30、FR-selective TKi:Iressa2)EGFR antibodies3)Farnesylation or Geranylgeranylation inhibitors targeting Ras and Rho4)VEGFR antibody(and TKi):Avastin经典文献举例经典文献举例Stat3 as an Oncogene Jacqueline F.Bromberg1,Melissa H.Wrzeszczynska1,Geeta Devgan1,Yanxiang Zhao2,Richard G.Pestell3,Chris Albanese3 and James E
31、.Darnell,Jr.,1,1 Laboratory of Molecular 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 2000Cell Volume 98,Issue 3,295-303 信号转导理论在各生命科学领信号转导理论在各生命科学领域中的普遍意义域中的普遍意义以本课程中各讲为例：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
