信号通路和肿瘤课件

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细胞信号转导与肿瘤中山大学2005级分子医学博士生引言：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：细胞信号转导与生命过程问题的提出和理论的产生引言信号转导与生命过程问题的提出和理论的产生引言信号转导与生命过程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

2、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)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组织生长需要细胞分裂增殖细胞生长因子细胞周期病原体侵入抗感染细胞骨架蛋白表达、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

3、改变细胞表型改变细胞骨架蛋牵动细胞移动趋化因子细胞粘附细胞存活抗凋亡因子胞外信号通路和肿瘤课件信号通路和肿瘤课件细胞信号转导理论概述细胞信号转导理论概述信号通路和肿瘤课件胞外信号分子(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Extracellular Signal Molecule

4、sGrowth 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. CytokinesIFN- (Interferon- ), IFN- (Interferon- ), TNF (Tumor Necrosis Fa

5、ctor), Interleukins (1, 2, 3, 4)3. Death moleculesFas 4. Adhesion moleculesCadherins, Adhesin5. HormoneInsulin6. StressExtracellular Signal MoleculesSignal Transducing ReceptorsTransmembrane receptors that have intrinsic enzymatic activity. AutophosphorylationPhosphorylation of other substratesA) Ty

6、rosine 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 receptorsSignal Transducing ReceptorsTr2. Receptors that are coupled, inside the cell, to GTP-binding and hydrol

7、yzing 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 af

8、fects 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 potential2. Receptors that are coupled,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信号转导过程中的生物化学磷酸化

9、反应（酪氨酸激酶、丝/苏氨酸激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)-c

10、ontaining proteins (e.g., PLC-, Ras, PI-3K, etc)Phosphorylate other kinasesphosphorylate proteins, which upon phosphorylated, can enter the nuclear and bind DNA regulatory regions.Signal TransducersReceptor TyrClassExamplesStructural Features of ClassIEGF receptor, NEU/HER2, HER3cysteine-rich sequen

11、cesIIinsulin receptor, IGF-1 receptorcysteine-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 domainVvasc

12、ular endothelial growth factor (VEGF) 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.

13、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 domains; NGFR has leucine rich domainCharacteristics of the Common Classes of RTKs ClassExamplesStructural FeaturN

14、on-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 enzymatic activity themselves (e.g., CD4, CD8, TCR and all cytokine receptors such

15、as IL-2R Non-Receptor Protein Tyrosine 信号通路和肿瘤课件信号通路和肿瘤课件信号通路和肿瘤课件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

16、 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 Receptor Serine/Thre

17、onine Kina信号通路和肿瘤课件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 Receptors1. 1000 GPCRs, most of which are orphan re

18、ceptors)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. PhotoreceptorNon-Receptor Serine/Threonine Intracellular Hormone Rec

19、eptors 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). * Phospha

20、tases in Signal Transduction 1. Transmembrane PTPs: e.g., CD45. 2. Intracellular PTPs. Intracellular Hormone Receptor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胞外信号分子(可溶性分子、细胞表面分子、组织基质分子)

21、靶细胞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信号转导通路的调控磷酸化去磷酸化调控Cross-TalkCross-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

22、 types?Pre-existing co-activators of target proteins?Subcellular localization of transducers?Optimal level (or a threshold) of phosphorylation/dephosphorylation?信号转导效应的特异性When and Where?替代通路（Alternative Pathways)替代通路（Alternative Pathways)信号转导的研究方法与工具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 s

23、pecific 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、基因转录活性测定1、Electrophoretic mobility shift analysis (EMSA)、Reporter gene expression detection一、蛋白质磷酸化状态的检测四、信号转导分子的表达或

24、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 kinase inhibitor (TKi)6、Inhibitory oligopeptides四、信号转导分子的表达或活性抑制信号转导在肿瘤发生发展中的意义信号转导在肿瘤发生发展中的意义Sig

25、naling molecules involved in cancer development/progressionReceptorsGrowth factor receptors: EGFRHormone receptor: ER, ARAngiogenic receptros: VEGF, PDGF, IGFDeath receptorsThe Integrin systemSignaling molecules involved iTransducers Ras Raf Rho family PI-3K/AktDeath transducersSTAT-3Transcription f

26、actorsc-Mycc-Jun and c-fosSTAT-3TransducersBiological Effects of Signaling Related to Cancer Development/ProgressionCell immobilizationAbrogation of apoptosisActivation of cell cycle and removal of cell cycle checkpointsAngiogenesisCell invasionMetastasisDrug resistanceBiological Effects of Signalin

27、信号通路和肿瘤课件Phosphorylation targets of PI-3KAkt 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 activ

28、ation of c-Myc and cyclin DPDK1 phosphorylation of other kinases (p70 S6-kinasse, CISK, PKC) Cell growth and survivalPhosphorylation targets of PI-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): lun

29、g, H&N, RasAndrogen receptor and downstream moleculesEstrogen receptor and downstream molecules信号转导与肿瘤临床诊断、预防与治疗ExpressionProtein phosphorylation and cancer diagnosisDetermination of single phosphorylated signaling molecules: EGFRPhospho-protein profiling: proteomicsPhospho-protein based imaging technologySignaling inhibitor and cancer prevention/ therapyEGFR-selective TK