《信号通路与肿瘤》PPT课件

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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

2、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) 组织生长需要细胞分裂增殖细胞生长因子细胞周期蛋白表达病原体侵入抗感染状态细胞抗原细胞因子表达分泌细胞过度生长细胞死亡细胞死亡因子胞内致死分子表达 细胞骨架蛋白表达、激活牵动细胞移动(Cell movement)趋化因子细胞粘附细胞存活(Survival)抗凋亡因子表达、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 细胞信号转导理论概述 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）化学变化（如磷酸化、二聚体形成）靶细胞

3、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）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基因表达改变 Extracellular Signal Molecules1. Growth FactorsPDGF (Platelet-Derived Growth Factor), EGF (Epidermal Growth Factor), TGF- (Transforming Growth Factor-), EPO (Erythropoietin), NGF (Nerve Growth Factor), IGF (Insulin-like Growth Fac

4、tor), TPO (Thrombopoietin)2. CytokinesIFN- (Interferon- ), IFN- (Interferon- ), TNF (Tumor Necrosis Factor), Interleukins (1, 2, 3, 4)3. Death moleculesFas 4. Adhesion molecules Cadherins, Adhesin5. HormoneInsulin6. Stress Signal Transducing Receptors1. Transmembrane receptors that have intrinsic en

5、zymatic 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- receptors 2. Receptors that are co

6、upled, 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 nucl

7、eus 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 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

8、子的聚体化 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 phosphorylationInteract with and phosphorylate Src homology domain 2 (SH2)-containi

9、ng 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. Class Examples Structural Features of ClassI EGF receptor, NEU/HER2, HER3 cysteine-rich sequencesII insulin receptor, IGF-1 r

10、eceptor cysteine-rich sequences; characterized by disulfide-linked heterotetramersIII PDGF receptors, c-Kit contain 5 immunoglobulin-like domains; contain the kinase insertIV FGF receptors contain 3 immunoglobulin-like domains as well as the kinase insert; acidic domain V vascular endothelial growth

11、 factor (VEGF) receptor contain 7 immunoglobulin-like domains as well as the kinase insert domainVI 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

12、a proto-oncogene that was originally identified as the Met oncogeneVII 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 Non-Receptor Protein Tyrosine Kinases (PTKs) Two

13、 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 enzymatic activity themselves (e.g., CD4, CD8, TCR and all cytokine receptors such as IL-2R Receptor Serine/Threonine Kinases (RST

14、Ks) 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-

15、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 Serine/Threonine Kinases 1) cAMP-dependent protein kinase (PKA) 2) Protein kinase C (PKC) 3) Mitogen activated

16、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) GPCRs that modulate adenylate cyclase activity and produce cAMP 2) GPCRs that activate PLC-g lea

17、ding to hydrolysis of polyphosphoinositides: angiotensin, bradykinin and vasopressin receptors. 3) 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 hormone re

18、ceptors): 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(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）化学变化（如

19、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（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 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 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 exp

20、ressed 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（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)

21、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 kn

22、ock-out4、Dominant negative mutants(1) Ligand-binding site(2) Phosphorylation site(3) Docking site(4) Protein-protein binding site(5) DNA binding site5、Small-molecule inhibitors：e.g., tyrosine kinase 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 deve

23、lopment/progression Receptors1) Growth factor receptors: EGFR2) Hormone receptor: ER, AR3) Angiogenic receptros: VEGF, PDGF, IGF4) Death receptors5) The Integrin system Transducers1) Ras2) Raf3) Rho family4) PI-3K/Akt5) Death transducers6) STAT-37) Transcription factors1) c-Myc2) c-Jun and c-fos3) S

24、TAT-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 resistance Phosphorylation targets of PI-3K Akt Forkhead-related tra

25、nscription 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 cyclin D PDK1 phosphorylation of other kin

26、ases (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 diagnosis1) EGFR: lung, H 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