信号通路和肿瘤

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209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第1页引言：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2页引言信号转导与生命过程问题提出和理论产生第3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

2、结构和亚细胞结构（脂质双层膜结构、细胞器)第4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5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6页第7页第8页细胞信号转导理论概述第9页第10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

3、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11页Extracellular Signal MoleculesGrowth FactorsPDGF (Platelet-Derived Growth Factor), EGF (Epidermal Growth Factor), TGF- (Transforming Growth Factor-), EPO (Erythropoietin), NGF (Nerve Growth Factor), IGF (Insulin-like Growth Factor

4、), TPO (Thrombopoietin)2. CytokinesIFN- (Interferon- ), IFN- (Interferon- ), TNF (Tumor Necrosis Factor), Interleukins (1, 2, 3, 4)3. Death moleculesFas 4. Adhesion moleculesCadherins, Adhesin5. HormoneInsulin6. Stress第12页Signal Transducing ReceptorsTransmembrane receptors that have intrinsic enzyma

5、tic activity. AutophosphorylationPhosphorylation of other 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第13页2. Receptors that are coup

6、led, 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 nucleu

7、s 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-channels that lead to changes in membrane electric potential第14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

8、子聚体化第15页Signal TransducersReceptor Tyrosine Kinases (RTKs) contains:An extracellular ligand binding domain. An intracellular tyrosine kinase domain. An intracellular regulatory domain. A transmembrane domain.Tyrosine phosphorylationInteract with and phosphorylate Src homology domain 2 (SH2)-containi

9、ng proteins (e.g., PLC-, Ras, PI-3K, etc)Phosphorylate other kinasesphosphorylate proteins, which upon phosphorylated, can enter the nuclear and bind DNA regulatory regions.第16页ClassExamplesStructural Features of ClassIEGF receptor, NEU/HER2, HER3cysteine-rich sequencesIIinsulin receptor, IGF-1 rece

10、ptorcysteine-rich sequences; characterized by disulfide-linked heterotetramersIIIPDGF receptors, c-Kitcontain 5 immunoglobulin-like domains; contain the kinase insertIVFGF receptorscontain 3 immunoglobulin-like domains as well as the kinase insert; acidic domainVvascular endothelial growth factor (V

11、EGF) receptorcontain 7 immunoglobulin-like domains as well as the kinase insert domainVIhepatocyte growth factor (HGF) and scatter factor (SC) receptorsheterodimeric like the class II receptors except that one of the two protein subunits is completely extracellular. The HGF receptor is a proto-oncog

12、ene that was originally identified as the Met oncogeneVIIneurotrophin receptor family (trkA, trkB, trkC) and NGF receptorcontain no or few cysteine-rich domains; NGFR has leucine rich domainCharacteristics of the Common Classes of RTKs 第17页Non-Receptor Protein Tyrosine Kinases (PTKs) Two non-recepto

13、r PTK families: 1) The archetypapl PTK familty: Src-related proteins2) Janus kinase (Jak) familyMost 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18页第19页第20页第21页Receptor Serine/Threonine Kinases

14、(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 T

15、GF- 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 第22页第23页Non-Receptor Serine/Threonine Kinases 1) cAMP-dependent protein kinase (PKA) 2) Protein kinase C (PKC) 3) Mitoge

16、n 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 activate PLC-

17、g leading to hydrolysis of polyphosphoinositides: angiotensin, bradykinin and vasopressin receptors. Photoreceptor第24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 hormo

18、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. 第25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

19、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26页信号转导研究中重大理论问题及热点领域第27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28页Cross-Talk第29页信号转导效应特异性When and Where?Cooperation with other signaling pathways?Pre-existing transcription co-factors differe

20、ntially 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30页替换通路（Alternative Pathways)第31页信号转导研究方法与工具第32页一、蛋白质磷酸化状态检测1、免疫印迹 (phospho-protein speci

21、fic antibodies)2、免疫沉淀 (protein-specific antibody + phospho-AA antibody3、流式细胞仪分析4、Luminex分析二、信号转导分子过分表示或过分激活1、Overexpression by gene transduction2、Constitutively activated mutants三、基因转录活性测定1、Electrophoretic mobility shift analysis (EMSA)、Reporter gene expression detection第33页四、信号转导分子表示或活性抑制1、Anti-sen

22、se2、RNAi3、Gene knock-out4、Dominant negative mutantsLigand-binding sitePhosphorylation siteDocking siteProtein-protein binding siteDNA binding site5、Small-molecule inhibitors：e.g., tyrosine kinase inhibitor (TKi)6、Inhibitory oligopeptides第34页信号转导在肿瘤发生发展中意义第35页Signaling molecules involved in cancer de

23、velopment/progressionReceptorsGrowth factor receptors: EGFRHormone receptor: ER, ARAngiogenic receptros: VEGF, PDGF, IGFDeath receptorsThe Integrin system第36页Transducers Ras Raf Rho family PI-3K/AktDeath transducersSTAT-3Transcription factorsc-Mycc-Jun and c-fosSTAT-3第37页Biological Effects of Signal

24、ing Related to Cancer Development/ProgressionCell immobilizationAbrogation of apoptosisActivation of cell cycle and removal of cell cycle checkpointsAngiogenesisCell invasionMetastasisDrug resistance第38页第39页Phosphorylation targets of PI-3KAkt Forkhead-related transcription factor 1(FKHR-L1) 14-3-3 b

25、inding 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 and cyclin DPDK1 phosphorylation of other kinases (p70 S6-kinasse, CISK, PKC) Cell grow

26、th and survival第40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 diagnosisEGFR: lung, H&N, gliomaTGF- receptor (type II): lung, H&N, RasAndrogen receptor and downstream moleculesEstrogen receptor and downstream molecules第41页Protein phosphorylation and cancer diagnosisDetermination of single phosphorylated signaling molecules: EGFRPhospho-protein profiling: proteomicsPhospho-protein based imaging technologySignaling inhibitor and cancer prevention/ therapyEGF