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)
2、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),细胞信号转导理论概述,Extracellular Signal Molecules,Growth 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 (Th
3、rombopoietin)2. CytokinesIFN- (Interferon- ), IFN- (Interferon- ), TNF (Tumor Necrosis Factor), Interleukins (1, 2, 3, 4)3. Death moleculesFas 4. Adhesion moleculesCadherins, Adhesin5. HormoneInsulin6. Stress,Signal Transducing Receptors,Transmembrane receptors that have intrinsic enzymatic activity
4、. 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,2. Receptors that are coupled, inside th
5、e 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 li
6、gand-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子的聚体化,Signal Tr
7、ansducers,Receptor Tyrosine Kinases (RTKs) contains: An extracellular ligand binding domain. An intracellular tyrosine kinase domain. An intracellular regulatory domain. A transmembrane domain.,Characteristics of the Common Classes of RTKs,Non-Receptor Protein Tyrosine Kinases (PTKs) Two non-recepto
8、r 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,Receptor Serine/Threonine Kinases (RSTKs),Typica
9、l 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
10、 include c-Myc and Smad,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 orp
11、han 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,Intracellular Hormone Receptors 1. Residing wi
12、thin 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 Trans
13、duction 1. Transmembrane PTPs: e.g., CD45. 2. Intracellular PTPs.,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 expressed and a
14、ctivated 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) 2、免疫沉淀 (p
15、rotein-specific antibody + phospho-AA antibody 3、流式细胞仪分析 4、Luminex分析二、信号转导分子过度表达或过度激活 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,四、信号转导分子的表达或活性抑制 1、Anti-sense 2、RNAi 3、Gene kn
16、ock-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,信号转导在肿瘤发生发展中的意义,Signaling molecules involved in cancer development/prog
17、ression,Receptors Growth factor receptors: EGFR Hormone receptor: ER, AR Angiogenic receptros: VEGF, PDGF, IGF Death receptors The Integrin system,TransducersRasRafRho familyPI-3K/Akt Death transducers STAT-3 Transcription factors c-Myc c-Jun and c-fos STAT-3 ,Biological Effects of Signaling Related
18、 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 transcription factor 1 (FKHR-L1) 14-3-3 binding FK
19、HR-L1 retaining in cytosol abrogation of gene activation by FKHR-L1Akt Bad 14-3-3 binding 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 growth and s
20、urvival,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 diagnosis EGFR: lung, H&N, glioma TGF- receptor (type II): lung, H&N, Ras Androgen receptor and downstream molecules Estrogen receptor and downstream molecules ,Protein phosphorylation and cancer d
21、iagnosis Determination of single phosphorylated signaling molecules: EGFR Phospho-protein profiling: proteomics Phospho-protein based imaging technologySignaling inhibitor and cancer prevention/ therapy EGFR-selective TKi: Iressa EGFR antibodies Farnesylation or Geranylgeranylation inhibitors target
22、ing Ras and Rho VEGFR antibody (and TKi): Avastin,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. Darnell, Jr. , , 1, 1 Laboratory of Molecular Cell Biology, The Rockefeller University, N
23、ew 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 2000,Cell 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,
