第5章+细胞信号转导与肿瘤

1、1 Rb BRCA1 and BRCA2Summary TSG 2第五章第五章 细胞信号转导与肿瘤细胞信号转导与肿瘤3 General principles of cell communication Growth factor, receptor and cancer Signaling through enzyme-linked cell-surface receptors and cancer Wnt - - catenin signaling pathway and cancer NF-kB- dependent signaling pathway and cancer Signali

2、ng through G-protein- coupled receptors (GPCRs) and cancerOutline 4An overview of the signaling pathwaysEntracellular signaling moleculesReceptorsIntracellular signaling moleculesTarget proteins This overall process in which information carried by extracellular messenger molecules is translated into

3、 changes that occur inside a cell is referred to as signaling transductionn General principles of cell communication5 Our digestive tract begins with the mouth and stomach, continues with the small and large intestines, and ends with the colon. The small intestine: duodenum-十二指肠十二指肠, jejunum-空肠空肠 an

4、d ileum-回肠回肠 The large intestine or colon: ascending colon-升结肠升结肠, transversal colon-横结肠横结肠, descending colon -降结肠降结肠sigmoidal colon -乙状结肠乙状结肠, rectum直肠直肠 The intestinal tractu Normal metazoan cells control each others lives6Maintenance of tissue architectureThe epithelial lining of the small intest

5、ineThe absorptive epithelium of the small intestine is ordered into villi and crypts of Lieberkuhn (李氏肠腺李氏肠腺/隐窝隐窝)Differentiated cells (enterocytes-肠上肠上皮细胞皮细胞, enteroendocrine cells -肠内分泌肠内分泌细胞细胞 and goblet cells-杯状细胞杯状细胞) occupy the villi A fourth differentiated type, the Paneth cell (潘氏细胞潘氏细胞), re

6、sides at the bottom of crypts and secretes antimicrobial agents The remainder of the crypts constitutes the stem (dark blue) / progenitor compartment7 Stem cells in the crypts divided One daughter cell is retained as a stem cell. The other becomes committed to differentiate along one of four pathway

7、s to become an enterocyte (肠肠上皮细胞上皮细胞), enteroendocrine cell, goblet cell or Paneth cell. Cells in the enterocyte lineage divide several more times as they migrate up the crypts, and as they migrate onto the villi, differentiate further into the mature absorptive cells. These cells live only for 3-6

8、 days, die and are shed into the lumen. The life cycle of small intestinal enterocytes 8The wound-healing processThe blood plateletsRelease storage of PDGF by plateletsPDGF attracts fibroblasts into the wound site and then stimulates their proliferation All of these functions depend upon cooperation

9、 among large groups of cells and the cells in a living tissue are constantly talking to one another (growth factors, GFs)Storage of PDGF by plstelets(Large dark spots: a a-granules)9Top: fibroblasts, PDGFLower: mutant fibroblastsEffects of growth factors on cellsTwo monolayers of fibroblasts in Petr

10、i dishes have been wounded by the tip of a pipette (left panels) In vitro model of wound healing10uThe Src protein functions as a tyrosine kinase The first clues to how cell-to-cell signaling via growth factors came from biochemical analysis of the v-src oncogene and the protein that it specifies Cl

11、oning of the src oncogene The nucleotide sequence The amino acid sequence: 533aa, 60KDHow Src operates?n Growth factor, receptor and cancer11Src operates as a protein kinase Phosphorylation of a precipitating antibody molecular by Src Lysates 1a,1b: uninfected chicken embryo fibroblasts (CEFs)2a,2b:

12、 avian leukosis virus (ALV, 禽类白禽类白血病病毒血病病毒)-infected CEFs3a,3b: wild-type RSV-infected CEFs4a,4b: CEFs infected by a transformation-defective mutant of RSV Serum1a,2a,3a,4a: normal rabbit serum1b,2b,3b,4b: serum from a rabbit bearing an RSV-induced tumor Immunoprecipitate cell lysates incubated with

13、 32P-radiolabeled ATP Src was itself a phosphoprotein, carried phosphate groups attached covalently to one or more of its amino acid 12The antibody molecular that was used to immunoprecipitate Src molecules also happened to serve as a substrate for phosphorylation by its kinase13 Src phosphorylated

14、certain tyrosine residues of its protein substrates More than 99% of the phosphoamino acids in normal cells are phosphothreonine or phosphoserine Phosphotyrosine constitutes as little as 0.05 to 0.1% of these cells total phosphoamino acids Substrates specificity of the Src kinaseElectrophoresis Left

15、 panel: chicken embryo fibroblasts (CEFs), almost no phosphotyrosine (arrow) Center panel: src-transformed CEFs, the level of phosphotyrosine is evident 14 Src family: src, yes, fgr, lck, fyn, hlk, bck, yrkHow did the phosphorylation of tyrosine residues result in cell transformation?15 Structure an

16、d function of SrcInactive state (C) Active state16 Src and tumor V-Src: the C-terminal oligopeptide domain containing the tyrosine 527 residue has been replaced by another, unrelated amino acid sequenceHow dose Rous sarcoma virus induce the cell transformation?17uThe EGF receptor functions as a tyro

17、sine kinase Epidermal growth factor (EGF), the first of the growth factors to be discovered Ability to provoke premature eye opening in newborn mice Have mitogenic effects when applied to a variety of epithelial cell(上皮细胞上皮细胞) types EGF served as the ligand for its cognate receptor- the still-hypoth

18、etical EGF receptor (EGF-R)18Structure of EGF-R protein(1186aa, 65KDa)Ectodomain: 621aaTransmembrane domain: 23aaC-terminus: 542aa (包括包括4个磷个磷酸化位点酸化位点: 一个紧靠膜内侧的一个紧靠膜内侧的654位位Thr，其余，其余3个位于个位于1068，1148和和1173Tyr自身磷酸化位点和一个自身磷酸化位点和一个TPK功能区功能区) Isolation of the EGF-R protein from an epidermoid(上皮上皮) carcino

19、ma of the uterus, expressing EGF-R levels as much as 100-higher than normal The sequence of cytoplasic domain is similarity with the already-known sequence of the Src protein19 Subsequent sequencing efforts revealed overall sequence similarities among a variety of tyrosine kinases, many of which can

20、 function as oncoproteinsComparison of the amino acid sequences of the cytoplasmic domain20l Formation of phosphotyrosine on the EGF-R following ligand additionThe use of a fluorescent reagent that binds specifically to a phosphotyrosine residue on the EGF-R enables the visualization of receptor act

21、ivation following ligand bindingA monkey kidney cellAG1478: a chemical inhibitor of the EGF-RTransphosphorylation underlies the operations of receptor tyrosine kinases Blue: receptor activity above the basal level Red: receptor activity below the basal level21l Apparent autophosphorylation of the EG

22、F receptorlane 1: In the absence of EGF lane 2: exposed to EGF, radiolabeled protein can be immunoprecipitated with an anti-phosphotyrosine antiserum (red)(B) Electrophoresis Human A431 epidermoid carcinoma cells Overexpression the EGF-R Incubated in 32PO4-containing medium Immunoprecipitated with a

23、n anti-phosphotyrosine antiserum 22l Receptor dimerization following ligand binding From the structure of many growth factor ligands, dimeric: homodimers, heterodimers Many transmembrane proteins constructed like the EGF and PDGF receptors have lateral mobility in the plane of the plasma membrane23

24、Alternative mechanisms of growth factor-induced receptor dimerizationVEGF dimer(red, blue)Two FGF-2 are bound to a common heparin molecule(stick figure) Receptor monomers respond to ligand binding by exposing domains that mediate their “back-to-back dimerization”X-ray crystallography24A altered grow

25、th factor receptor can function as an oncoproteinAvian erythroblastosis virus (AEV, 鸟类成红细胞增多症病毒鸟类成红细胞增多症病毒 ), a transforming retrovirus that rapidly induces a leukemia of the red blood cell precursors (an erythroleukemia)The erbB oncogene of AEV was found to lack sequences present in the N-terminal

26、ectodomain of the EGF receptorThe EGF receptor and v-ErbB25EGFR and v-erbB26 Deregulation of receptor firing Truncation of the ectodomainAmino acid substitutionsExposure of pregnant rats to the mutagen ethylnitrosourea (乙乙基亚硝基脲基亚硝基脲, ENU) results in the birth of pups that often succumb to neuroectod

27、ermal (神经外胚层神经外胚层) tumors 3 to 6 months A point mutation in the transmembrane domain of this receptor (ValGlu)The constitutive dimerization of the two receptors, even in the absence of ligand binding HER2/ErbB2/Neu27 Deregulation of receptor firingAuto-stimulatory or autocrine signaling loop Cancer

28、cells have a greatly reduced dependence on growth factors for their growth and survival28Red: immunostained for expression of the EGF receptorGreen: stained for the expression of TGF-a a (a ligand of the EGF-R)Yellow: expressing both receptor and ligand Blue: nucleiAn invasive human breast cancer An

29、 example of auto-stimulatory or autocrine signaling loop2930uA growth factor gene can become an oncogene: the case of sis PDGF protein was isolated and its amino acid sequence determined. PDGF stimulates largely mesenchymal cells because the PDGF-R was found to be expressed on the surfaces of mesenc

30、hymal cells. In 1983, the B chain of PDGF was found to be closely related in sequence to the oncoprotein (p28) encoded by the v-sis oncogene of simian scarcoma virus( (猿猴猿猴肉瘤病毒肉瘤病毒).). Growth factor: protein that is able to stimulate the growth and / or proliferation of a cell by binding to a specif

31、ic cell surface receptor displayed by that cell31The sis-encoded oncoproteins transform cells The product of sis is a secretory protein The product of c-sis and the chain of PDGF- 38 99 v-sis (220aa) 258 NH2 COOH 99 207 258 PDGF-B 图 19- sis 基因产物的结构和 PDGF 比较32A variety of human tumor cells are known

32、to produce and release substantial amounts of growth factors to which these cells can also respond autocrine3334The key to the puzzle of receptor signalingDomain structure of the Src proteinYellow, orange: the catalytic SH1 domainRed stick: the ATP-binding siteBlue, light green : SH2 and SH3 domain,

33、 involved in substrate recognition and regulation of catalytic activityDark green: connecting sequenceX-ray crystallographic analysesuTyrosine phosphorylation controls the location and thereby the actions of many cytoplasmic signaling proteins 35Structure and function of SH2 groups 100 animo acid re

34、sidues, acts as an intracellular “receptor” The ligand for this SH2 receptor: a short oligopeptide sequence that contains both a phosphorylated tyrosine and a specific oligopeptide sequence 3 to 6 residues long that flanks the phosphotyrosine on its C-terminal site The human genome is estimated to e

35、ncode at least 117 distinct SH2 groups (different affinity for binding a particular phosphotyrosine together with a flank oligopeptide sequence )X-ray crystallography36Attraction of signal-transducing proteins by phosphorylated receptorsThe discoveries about SH2 groups finally solved the puzzle of h

36、ow tyrosine kinase receptors are able to emit signaling37 SH3 domain bind specifically to certain proline-rich sequence domains in partner proteinsThrough analyses of the human genome sequence, 253 distinct SH3 domains, each part of a larger protein, have been uncoveredProtein interaction domains as

37、 modular units of protein structure38PTB: phosphotyrosine binding39SH2 groups explain how growth factor receptors active Ras and acquire signaling specificity To explain how the receptorSos Ras cascade40n Signaling through enzyme-linked cell-surface receptors and cancerSix classes of enzyme linked r

38、eceptorsReceptor tyrosine kinaseTyrosine-kinase-associated receptorsRecptorlike tyrosine phosphatasesReceptor Ser/Thr kinasesReceptor guanylyl cyclasesHistidine-kinase-associated receptors411、Receptor tyrosine kinasel Extracellular signal proteins42 EGFR(表皮生长因子受体家族表皮生长因子受体家族); Insulin-R(胰岛素受体家族胰岛素受体

39、家族); PDGFR(血血小板衍生的生长因子受体家族小板衍生的生长因子受体家族); FGFR(纤维细胞生长因子受体家族纤维细胞生长因子受体家族); NGFR(神经细胞生长因子受体家族神经细胞生长因子受体家族); HGFR(肝细胞生长因子受体家族肝细胞生长因子受体家族); VEGFR(血管内皮细胞生长因子受体家族血管内皮细胞生长因子受体家族)l Receptor tyrosine kinase43lLigand binding leads to autophosphorylation of RTK44 Three major downstream signaling cascade radiat

40、e from the RaslThe Ras protein stands in the middle of a complex signaling cascade 45RTK-ras pathway Grb2 ( growth-factor-receptor-bound protein 2) Sos: son of sevenless GAP: GTP酶活化蛋白酶活化蛋白46MNK1/2: Mitogen activated protein kinase (MAPK)-interacting kinase MSK1/2: Mitogen-and stress-activated kinase

41、 RSK: 90-KDa ribosomal S6 protein kinase PPARr: peroxisome-proliferator activated receptor-rELK-1: Ets转录因子家族成转录因子家族成员员4748 H-ras ( Harvey ras or Ha-ras) K-ras ( Kiersten ras or Ki-ras) N-rasUnderstanding the biochemistry of the Ras C- termini: all carry covalently attached lipid tails, composed of f

42、arnesyl(法尼烯法尼烯), palmitoyl(软软脂酰脂酰), or geranyl(香叶醇香叶醇) groups Enable the Ras proteins to become anchored to the cytoplasmic face of the plasma membrane49 Ras was found to bind and hydrolyze guanosine nucleotides(鸟核苷酸鸟核苷酸)The Ras signaling cycleLike a light switch The Ras protein, an apparent compone

43、nt of the downstream signaling cascade, functions as a G protein50 Ras: point and tumor51X-ray crystallography Point mutation or deletion?To explain why substitutions of these resides affect the GTPase activity of RasThe structure of the Ras protein52GAP (GTP酶激活蛋白酶激活蛋白)SOSOncogenic Ras: activated Ra

44、s in GTP- bound form53 Ras regulationGuanine nucleotide-exchange factors (GEFs; green ovals); GTPase activating proteins (GAPs; orange ovals)54( (FT: 法尼基转移酶；法尼基转移酶；FTI: 法尼基转移酶抑制剂法尼基转移酶抑制剂) )55The second pathway downstream of Ras controls inositol lipids and the Akt/PKB pathwayThe PI3 kinase pathway56 PI(3)KCatalytic subunit p110Adaptor/regulatory subunit p85(heterodimers)Biochemistry of lipid bilayers(PI3K:磷酸肌醇磷酸肌醇3激酶激酶)57 PathwayPDK1: phosphatatidylinositol-dependent protein