EphB2受体的功能及其信号转导途径的初步研究
|
摘要
Eph受体是已知最大的酪氨酸蛋白激酶受体家族。Eph受体及其膜附着配体ephrins在发育过程中呈现不同的表达模式。功能分析表明，该家族在包括神经网络形成，神经管和轴旁中胚层的成型(patterning)，细胞迁移和神经轴突导向、血管发生等方面具有重要功能。Eph受体及其配体也与肿瘤发生有关，因此深入分析这些分子的功能特别是在肿瘤生长中的作用而应用于治疗具有重要的临床意义。EphB2是1994年Kiyokawa等从胃癌组织cDNA文库中筛选出的Eph亚类中又一新基因，在各种肿瘤组织中的表达均高于相应正常组织的几倍。其信号转导途径及作用机理尚需深入研究。
本研究目的是获得与EphB2相互作用并调节其功能的分子。因此，我们首先克隆表达了与EphB2受体结合配体有关的两段cDNA片段(EH and
EL)。从胃癌细胞系SGC7901中提取总RNA，RT-PCR扩增出cDNA，测序证实得到与文献报道一致的目的片段。将cDNA分别克隆入载体pRSETB，pET28a，构建重组表达载体pHRA和pLEA，转化大肠杆菌BL21(DE3)，经IPTG诱导，表达了N端融合His6的融合蛋白。在非变性及变性条件下，利用Ni-NTA金属螯合亲和层析法对表达的蛋白进行纯化，得到纯度94％以上的融合蛋白。ELISA测定表明，His_6融合蛋白在体外可与其天然配体结合。功能分析表明，Ig结构域(EL)可刺激胃癌细胞内蛋白质发生酪氨酸磷酸化状态的变化。通过脂质体介导将缺失激酶区的ΔEphB2转入胃癌SGC7901细胞，流式细胞仪分析表明胃癌细胞发生G_1期阻断，有13％细胞凋亡，提
Eph receptors are the largest known family of receptor tyrosine kinases. The Eph receptors and their membrane-attached ligands,the ephrins,show diverse expression patterns during development.Functional studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes including formation of neuronal network,patterning of neural tube and paraxial mesoderm, guidance of cell migration and axon pathfmding,and vascular formation.Recent studies have also suggested that Eph receptors and ephrins may be involved in carcinogenesis.It is therefore of clinical importance to further. analyze the function of these molecules,particularly their function in tumor cell growth,since manipulation of the activities of these molecules may have therapeutic applications.EphB2 was isolated as a new member of Eph subfamily in 1994 by Kiyokawa from a human gastric cancer cDNA library.Northern blot analysis showed that EphB2 was expressed at higher levels in various tumors of epithelial origin than in corresponding normal tissues. The molecular mechanism by which the EphB2 receptor signaling is propagated, and transmitted remains to be elucidated.
The goal of this study is to identify regulatory proteins of EphB2,which can physically interact and regulate the activities of EphB2.To accomplish this goal, we firstly cloned and expressed two extracelluar domains of EphB2 that are related with EphB2 binding to its ligand. Total RNA was isolated from a human
本研究目的是获得与EphB2相互作用并调节其功能的分子。因此，我们首先克隆表达了与EphB2受体结合配体有关的两段cDNA片段(EH and
EL)。从胃癌细胞系SGC7901中提取总RNA，RT-PCR扩增出cDNA，测序证实得到与文献报道一致的目的片段。将cDNA分别克隆入载体pRSETB，pET28a，构建重组表达载体pHRA和pLEA，转化大肠杆菌BL21(DE3)，经IPTG诱导，表达了N端融合His6的融合蛋白。在非变性及变性条件下，利用Ni-NTA金属螯合亲和层析法对表达的蛋白进行纯化，得到纯度94％以上的融合蛋白。ELISA测定表明，His_6融合蛋白在体外可与其天然配体结合。功能分析表明，Ig结构域(EL)可刺激胃癌细胞内蛋白质发生酪氨酸磷酸化状态的变化。通过脂质体介导将缺失激酶区的ΔEphB2转入胃癌SGC7901细胞，流式细胞仪分析表明胃癌细胞发生G_1期阻断，有13％细胞凋亡，提
Eph receptors are the largest known family of receptor tyrosine kinases. The Eph receptors and their membrane-attached ligands,the ephrins,show diverse expression patterns during development.Functional studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes including formation of neuronal network,patterning of neural tube and paraxial mesoderm, guidance of cell migration and axon pathfmding,and vascular formation.Recent studies have also suggested that Eph receptors and ephrins may be involved in carcinogenesis.It is therefore of clinical importance to further. analyze the function of these molecules,particularly their function in tumor cell growth,since manipulation of the activities of these molecules may have therapeutic applications.EphB2 was isolated as a new member of Eph subfamily in 1994 by Kiyokawa from a human gastric cancer cDNA library.Northern blot analysis showed that EphB2 was expressed at higher levels in various tumors of epithelial origin than in corresponding normal tissues. The molecular mechanism by which the EphB2 receptor signaling is propagated, and transmitted remains to be elucidated.
The goal of this study is to identify regulatory proteins of EphB2,which can physically interact and regulate the activities of EphB2.To accomplish this goal, we firstly cloned and expressed two extracelluar domains of EphB2 that are related with EphB2 binding to its ligand. Total RNA was isolated from a human
引文
1. Unified nomenclature for Eph family receptors and their ligands, the ephrins. Eph Nomenclature Committee [letter] .Cell, 1997; 90(3): 403-404
2. Hirai H,Maru Y.Hagiwara K,et al. A Novel putative tyrosine kinase receptor encoded by the eph gene. Science. 1987;238:1717-1720
3. Flanagan J.G, Vanderhaeghen P,The ephrins and Eph receptors in neural development Annu ,Rev, Neurosci, 1998,21: 309-345
4. Juha-Pekka Himanen, Mark Henkemeyer, Dimitar B NikoIov. Crystal structure of the ligand-binding domain of the receptor tyrosine kinase EphB2. Nature .1998;396:486
5. Smalla M,Schmieder P,Kelly ,M,Ter Laak A,Krause G,Ball L,Wahl M,Bork P, Oschkinat H Solution structure of the receptor tyrosine kinase EphB2 EphB2 SAM domain and identi fication of two Distinct homotypic interaction sites. Protein Sci 1999 ;8( 10): 1954-61
6. Hock B, Bohme B, Karn T et al. PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor.Proc Natl Acad Sci 1998 18;95( 17)9779- 84
7. Stein E, Cerretti DP, Daniel TO.Ligand activation of ELK receptor tyrosine kinase promotes its association with Grb10 and Grb2 in vascular endothelial cells.J Biol Chem. 1996 ;271(38):23588-93.
8. June X. Zou, Bingcheng Wang*, Matthew S ,,An Eph receptor regulates integrin activity through R-Ras PNAsl999, 96( 24) 13813-13818,
9. Ellis C,Kasmi F,Ganju P,et al. A juxtamembrane autophorylation site in the Eph family receptor tyrosine kinases, Sek, mediates high affinity interaction with p59fyn. Oncogene. 1996;12:1727-1736
10. Pandey A, Duan H, Dixit VM. Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase. J Biol Chem 1995 ;270(33): 19201-4
11. Stein E, Huynh-Do U, Lane AA. Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase. J Biol Chem, 1998; 273 (3): 1303-1305.
12. Vincent C. Dodelet, Claudia Pazzagli, Andreas H. Zisch. A Novel Signaling Intermediate, SHEP1, Directly Couples Eph Receptors to R-Ras and RaplA J Biol Chem. 1999;274(45):31941-6.
13. Becker E, Huynh-Do U, Holland S, et al Nck-Interacting Ste20 Kinase Couples Eph Receptors to c- Jun N- Terminal Kinase and Integrin Activation. Mol Cell Biol 2000 Mar l;20(5):1537-1545
14. Choi S,Park S. Phosphorylation at tyr-838 in the kinase domain of EphA8 modululates fyn binding to the tyr-615 Site by enhancing tyrosine linase activity. Oncogene 1999 ;18 (39): 5413-22
15. Kalo MS, Pasquale EB Multiple in Vivo Tyrosine Phosphorylation Sites in EphB Receptors. Biochemistry 1999;38(43): 14396-14408
16.Gale, N.W et al Eph receptors and ligands comprise two major specificity subclasses, and are reciprocally compartmentalised during embryogenesis. Neuron 1996; 17: 9-19
17. Holder N, Klein R. Eph receptors and ephrins :effectors of morphogenesis..Development 1999;126(10);2033-44
18. Bruckner K, Pasquale E B, Klein R . Tyrosine Phosphorylation of Transmembrane Ligands for Eph Receptors . Science 1997; 275: 1640-1643
19. Bruckner K, Pablo Labrador J,Scheiffele P,Herb A,Seeburg PH, Klein R. EphrinB Ligands recruit GRIP family PDZ adaptor proteins into raft membrane mi crodomains Neuron 1999;22 (3):511-24
20. Chong LD, Park EK, Latimer E, Fibroblast Growth Factor Receptor-Mediated Rescue of x-Ephrin Bl-Induced Cell Dissociation in Xenopus Embryos.Mol Cell Biol 2000 ;20 (2):724- 734
21. Birgbauer E, Cowan CA, Sretavan DW, Henkemeyer M. Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina.Development. 2000 ;127 (6): 1231-41
22. Dutting D, Handwerker C, Drescher U Topographic targeting and pathfinding errors of retinal axons following overexpression of ephrinA ligands on retinal ganglion cell axons. Dev Biol 1999;216(1):297-311
23. Helbling PM, Saulnier DM, Brandli AW The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis. Development 2000; 127 (2):269-278
24. Adams RH, Wilkinson GA, Weiss C et al . Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev 1999 ;13 (3):295-306
25. Stein E, Lane AA, Cerretti DP, et al.Eph receptors discriminate specific ligand oligomers o determine alternative signaling complexes, attachment, and assembly responses.Genes Dev. 1998 ;12(5):667-78.
26. Kiyokawa E,Takai S,Tanaka M. Overexpression of ERK, an EPH family receptor protein tyrosine kinase, in various human tumors. Cancer Research . 1994;54:3645-3650
27. Maru Y,Hirai H,Takaku F. Overexpression confers an oncogenic potential upon the eph gene. Oncogene. 1990;5:445-447
28. Tang XX,Evans AE,Zhao H,Cnaan A.London W,Cohn SL, Brodeur GM, Ikegaki N. High-level expression of EPHB6, EFNB3, and EFNB3 is associated with low tumor stage and high TrkA expression in human neuroblastomas. Clin Cancer Res 1999 ; 5 (6):1491-6
29. Tang XX.Pleasure DE,Brodeur GM Ikegaki N. A variant transcript encoding and isoform of the human protein tyrosine kinase EPHB2 is generated by alternative splicing and alternative use of polyadenylation signals oncogene 1998; 17(4): 521-6
30. Walker-Daniels J, Coffman K, Azimi M, Rhim JS, Bostwick DG, Snyder P, Kerns BJ, Waters DJ, Kinch MS. Overexpression of the EphA2 tyrosine kinase in prostate cancer. Prostate 1999; 41(4): 275-80
31. Tang XX, Brodeur GM, Campling BG, Ikegaki N. Coexpression of transcripts encoding EPHB receptor protein tyrosine kinases and their ephrin-B ligands in human small cell lung carcinoma. Clin Cancer Res 1999; 5(2): 455-60
32. Mellitzer G, Xu Q, Wilkinson DG et al. Eph receptors and ephrins restrict cell intermingling and communication. Nature 1999; 400(6739): 77-81
33. Oates AC, Lackmann3 M, Power MA, Brennan C, Smith FM, Do C, Evans B, Holder N Boyd AW. An early developmental role for eph-ephrin interaction duing vertebrate gasteulation. Mech Dev 1999; 83(11-2): 77-94
34．J．萨姆布鲁克等著。金冬雁，黎孟枫译。分子克隆实验指南，第二版，科学出版社，1992：880-885
35．严于颖，王海林译，F．奥斯伯，R．E．金斯顿，J．G．塞德曼等著，《精编分子生物学实验指南》，第一版，北京：科学出版社，1998。
36．张晓光 王方 闫小君 胃癌高表达基因ErK的扩增，克隆和鉴定 第四军医大学学报 1997；18(6)：562-565
37．张晓光 董巨莹 茹晓荣 EphB2受体N端Ig类似区的克隆和序列测定 细胞与分子免疫学杂志2000；16(1)：026-028
38．张晓光 刘新平 李福洋 EplaB2配体结合域基因片段的高效表达 中国免疫学杂志2000；16：9-11
39. Smith MHD, Dwin JV. Translational intitation on structured messengers: another role for the Siagle-Dalgarno interaction. J Mol Biol, 1994; 235: 173-184
40. Ralf-Schoefer. The pRSET family of T_7 promoter expression vectors for E. coli. Gene, 1993; 124: 83-85
41. Kroll DJ, Abdel-Hafiz H A-B, Marcell T et al. A multifunction prokarytic protein expression system: overproduction, affinity purifacation,11nd selective detection. DNA and cell biologio. 1993: 12: 441-445
42．俞强 细胞调控的探索 叶金生等主编，军事医学科学出版社，1999，12-15
1.Fields S, Song O: A novel genetic system to detect protein-protein interactions. Nature 1989,340:245-246
2.Brent R,Plashne M; A eukaryolic transcriptional activator bearing the DNA specificity of a prokaryotic repressor Cell 1985,43:729
3. Aronheim A, Zandi E, Hennemann H, Elledge SJ,Karin M:Isolation of an AP-1 repressor by a novel method for detecting protein-orotein interactions.Mol Cell Biol 1997,17:3094-3102.
4.Chien,.CT, Bartel PL, Sternglanz R, Fields S The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc.Natl.Acad. Sci. USA, 1991, 88:9578-9582
5.Durfee T, Becherer K, Chen PL, Yeh SH, Yang Y, Kilburn AE, Lee WH, Elledge SJ. The retinoblastoma protein associates with the protein Phosphatase type 1 catalytic subunit Genes Dev., 1993,7:555-569
6. Le Douarin B, Pierrat B, vom Baur E, Chambon P, Losson R. A new version of the two-hybrid assay for detection of protein-protein interactions. Nucleic Acids Res.,1995,23:876-878
7. James p, Halladay J, Craig EA: Genomic libraries and a host strain designed for highly efficient two-hy-brid selection inteast. Genetics 1996,144:1425-1436.
8. Aronheim A, Zandi E, Hennemann H, Elledge SJ,Karin M:Isolation of an AP-1 repressor by a novel method for detecting protein-orotein interactions.Mol Cell Biol 1997,17:3094-3102.
9. Johnsson N,Varshavsky A :Split ubiquitin as a sen-sor of protein interactions in vivo. Proc Natl Acad Sci UNA 1994,91:10340-10344.
10.MarsoIier M-C,Prioleau M-N,Sentenac A: An RNA polymerase Ill-based two- hybrid system to study RNA polymerase II transcriptional regulators.J Mol Biol 1997,268:243-249.
11. Tsan J,Wang Z,Jin Y, Hwang L,Bash RO,Baer R: Mammalian cells as hosts for two- hybrid studies of protein-protein interaction.In The Yeast Two-Hybrid System,edn 1. Edited by Bartel PL, Fields S.New York: Oxford University Press; 1997:217-232.
12.Rossi F, Charlton CA, Blau HM: Monitoring protein-protein interactions in intact eukaryotic cells by β -galactosidase complementation.proc Natl Acad Sci USA 1997,94:8405-8410.
13.Li JJ, Herskowitz I: Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybid system. Science 1993,262:1870-1874.
14.Vidal M,Brachmann RK,Fattaey A,Harlow E,Boeke JD:Reverse two-hybrid system and one hybrid system to detect dissociation of protein-protein and DNA-protein interaction. Proc Natl Acad Sci USA 1996,93:10315-10320.
15.Brachmann RK, Vidal M,Boeke JD:Dominant-nega-tive p53 mutations selected in yeast hit cancer hot sports. Proc Natl Acad Sci USA 1996,93:4091-4095.
16. Shin HM, Goldman PS, DeMaggio AJ, Holleenberg SM,Goodman RH,Howkstra MF:Apositive genetic selection for disrupting protein-protein interactions;identification of CREB mutations that prevent association with the coactivaor CBP.Proc Natl Acad Sci USA 1996,93:13896-13901.
17. Zhang J,Lautar S: Ayeast three-hybrid method to clone tenary protein compkex componets. Anal Biochem 1996,242:68-72.
18.Licitra EJ,Liu JO:A three-hybrid system for detecting small ligand-protein receptor interactions.proc Natl Acad Sci USA 1996,93:12817-12821
19. Osborne MA, Dalton S, KochanJP: The yeast tribird syetem genetic detection of trans-phosphorylated ITAM-SH2-interaction.Bio-Technology 1995, 13: 1474
20. Putz U, Skehel p,Kuhl D:A tri-hybrid system for the analysis and detection of RNA- protein interac-tions. Nucleic Acids Res 1996,24:4834-4640.
21.Stein E, Huynh-Do U, Lane AA Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase. JBC,1998; 273(3): 1303-1305
22 .Li B.and Fields S: Identification of mutation in p53 that affect its binding to SV40 T antigen by using the yeast two -hybrid system. FASEB J. 1993, 7: 957
23. Staudinger J, Zhou J, Burgess R, Elledge SJ, Olson EN. PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J Cell Biol. 1995 ;128(3):263-71.
24.Yang, M. , Wu Z, Fields S. Protein-peptide interactions analyzed with the yeast two-hybrid system. Nucleic Acids Res. 1995 ;23(7): 1152-6
25 Dixon E.P, Johnstone EM, Liu X, Little SP. An inverse mammalian two-hybrid system for beta secretase and other proteases. Anal Biochem. 1997 ;249(2):239-41.
26 Fromont-Racine M, Rain J-C,Legrain P: Toward a functional analysis of the yeast genome through exhaustive two-hybrid screecs. Nat Genet 1997 16:277-281.
1 .Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985. 228:1315-1317
2 Nambudripad R, Stark W, Makowski L. Neutron diffraction studies of the structure of filamentous bacteriophage Pfl. J. Mol. Biol. 1991. 220: 359-379
3 Kay Bk, Adey NB, He Ys-S et al. An M13 library displaying 38-amino-acid peptides as a source of novel sequences with affinity to selected targets.Gene, 1993; 128: 59-65
4.Maruyama IN, Maruyama HI, Brenner S. λ foo: a λ vector for the expression of foreign proteins. Proc Natl Acad Sci USA, 1994; 91:8273-8277
5.Lindqvist BH, Naderi S. Peptide presentation by bacteriophage T4. Microbiol Rev,1995;17:33-39
6.Lowman HB, Bass SH, Simpson N, et al. Selecting high-affinity binding proteins by monovalent phage display. Biochemistry, 1991; 30: 10832-10838
7.Cwirla S, Peters E, Barrett R et al Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Aad Sci USA, 1990; 87: 6378-6382
8.Clackson T,Well JA.In vitro selection from protein and peptide libraries. TIBS, 1994;12:173-184
9. Lowman HB. Bacteriophage display and discovery of peptide leads for drug development. Annu Rev Biophys Biomol Struct, 1997; 26: 401-424
10. Sparks AB, Adey NB, Quilliam LA. Screening phage-displayed random peptide libraries for SH3 ligands. Methods Enzymol, 1995; 255: 498-509
11. Mcconnell SJ, Kendall ML, Reilly TM et al. Constrained peptide libraries as a tool for finding mimotopes.Gene, 1994; 115-118
12. Wrighton NC, Farrell FX, Chang R et al. Small peptides as potent mimetics of the protein hormone erythropoietin. Science, 1996; 273:458-463
13. Scott JK. Discovering peptide ligands using epitope libraries. Trends Biochem. Sci. 1990. 17:241-245
14. Krook M, Mosbach K, RanstromO. Novel peptides binding to the Fc-portion of immunoglobulins obtained from a combinatorial phage display peptide library. J Immunol Methods, 1998:221(1-2): 151-157
15. Kay B K, Winter Jill, Mccafferty C. Phage display of peptides and proteins:A laboratory Manual. Academic Press. Harcourt Brace & Company Publishers. 1996; p235-237
16. Doorbar J, and Winter G. Isolation of a peptide antagonist to the thrombin receptor using phage display. J. Mol. Biol. 1994. 244: 361-369
17. Hyde-DeRuyscher R, Paige L, Christensen D et al. Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries. Chem Biol 2000; 7(1): 17-25
18. Kimura T, Kaburaki H, Tsujino T, Watanabe Y, Kato H. Signal transduction by the peptide which mimics the activity of thrombopoietin. Biochem Mol Biol Int, 1998, 44(6): 1203-1209
19. Crihalde ND, Chen YC, Golden A, et al. Epitope mapping of anti-HIV and anti-HCV monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library. Gene 1995. 166: 187-195
20. Reece J C, Geysen HM, Rodda SJ. Mapping the major human T helper epitopes of tetanus toxin. The emerging picture. J Immuunology, 1993; 151(11): 6175-6184
21. Sudol M. From SRC homology domains to other signaling modules: proposal of the 'protein recognition code'. Oncogene 1998. 17: 1469-1474
22. Rickles RJ, Botfield MC, Zhou XM, et al. Phage display selection of ligand residues important for Src homology 3 domain binding. Proc. Natl. Acad. Sci. U.S.A. 1995. 92: 10909-10913
23. Wu J, Ma QN, Lam KS et al. Identifying substrate motifs of protein kinases by a random library approach. Biochemistry, 1994; 33(49): 14825-14833
24. Luo K, Zhou P, Lodish HF. The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library Proc Natl Acad Sci, 1995; 92(25): 11761-11765
25. Steward M W, Stanley CM, Obeid OE. A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response. J Virol, 1995; 69(12): 7668-7673
26. Folgori A. A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera. EMBO J, 1994; 13: 2236-2243
27. Estaquier J Gras-Masse H, Boutillon CEur et al. The mixotope: a combinatorial peptide library as a T cell and B cell immunogen., J Immunol, 1994; 24(11): 2789-2795
28 张晓光 药立波 韩炯 噬菌体展示法筛选与EphB2结合的短肽 生物化学与生物物理学报 2000；32(5)：
2. Hirai H,Maru Y.Hagiwara K,et al. A Novel putative tyrosine kinase receptor encoded by the eph gene. Science. 1987;238:1717-1720
3. Flanagan J.G, Vanderhaeghen P,The ephrins and Eph receptors in neural development Annu ,Rev, Neurosci, 1998,21: 309-345
4. Juha-Pekka Himanen, Mark Henkemeyer, Dimitar B NikoIov. Crystal structure of the ligand-binding domain of the receptor tyrosine kinase EphB2. Nature .1998;396:486
5. Smalla M,Schmieder P,Kelly ,M,Ter Laak A,Krause G,Ball L,Wahl M,Bork P, Oschkinat H Solution structure of the receptor tyrosine kinase EphB2 EphB2 SAM domain and identi fication of two Distinct homotypic interaction sites. Protein Sci 1999 ;8( 10): 1954-61
6. Hock B, Bohme B, Karn T et al. PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor.Proc Natl Acad Sci 1998 18;95( 17)9779- 84
7. Stein E, Cerretti DP, Daniel TO.Ligand activation of ELK receptor tyrosine kinase promotes its association with Grb10 and Grb2 in vascular endothelial cells.J Biol Chem. 1996 ;271(38):23588-93.
8. June X. Zou, Bingcheng Wang*, Matthew S ,,An Eph receptor regulates integrin activity through R-Ras PNAsl999, 96( 24) 13813-13818,
9. Ellis C,Kasmi F,Ganju P,et al. A juxtamembrane autophorylation site in the Eph family receptor tyrosine kinases, Sek, mediates high affinity interaction with p59fyn. Oncogene. 1996;12:1727-1736
10. Pandey A, Duan H, Dixit VM. Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase. J Biol Chem 1995 ;270(33): 19201-4
11. Stein E, Huynh-Do U, Lane AA. Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase. J Biol Chem, 1998; 273 (3): 1303-1305.
12. Vincent C. Dodelet, Claudia Pazzagli, Andreas H. Zisch. A Novel Signaling Intermediate, SHEP1, Directly Couples Eph Receptors to R-Ras and RaplA J Biol Chem. 1999;274(45):31941-6.
13. Becker E, Huynh-Do U, Holland S, et al Nck-Interacting Ste20 Kinase Couples Eph Receptors to c- Jun N- Terminal Kinase and Integrin Activation. Mol Cell Biol 2000 Mar l;20(5):1537-1545
14. Choi S,Park S. Phosphorylation at tyr-838 in the kinase domain of EphA8 modululates fyn binding to the tyr-615 Site by enhancing tyrosine linase activity. Oncogene 1999 ;18 (39): 5413-22
15. Kalo MS, Pasquale EB Multiple in Vivo Tyrosine Phosphorylation Sites in EphB Receptors. Biochemistry 1999;38(43): 14396-14408
16.Gale, N.W et al Eph receptors and ligands comprise two major specificity subclasses, and are reciprocally compartmentalised during embryogenesis. Neuron 1996; 17: 9-19
17. Holder N, Klein R. Eph receptors and ephrins :effectors of morphogenesis..Development 1999;126(10);2033-44
18. Bruckner K, Pasquale E B, Klein R . Tyrosine Phosphorylation of Transmembrane Ligands for Eph Receptors . Science 1997; 275: 1640-1643
19. Bruckner K, Pablo Labrador J,Scheiffele P,Herb A,Seeburg PH, Klein R. EphrinB Ligands recruit GRIP family PDZ adaptor proteins into raft membrane mi crodomains Neuron 1999;22 (3):511-24
20. Chong LD, Park EK, Latimer E, Fibroblast Growth Factor Receptor-Mediated Rescue of x-Ephrin Bl-Induced Cell Dissociation in Xenopus Embryos.Mol Cell Biol 2000 ;20 (2):724- 734
21. Birgbauer E, Cowan CA, Sretavan DW, Henkemeyer M. Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina.Development. 2000 ;127 (6): 1231-41
22. Dutting D, Handwerker C, Drescher U Topographic targeting and pathfinding errors of retinal axons following overexpression of ephrinA ligands on retinal ganglion cell axons. Dev Biol 1999;216(1):297-311
23. Helbling PM, Saulnier DM, Brandli AW The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis. Development 2000; 127 (2):269-278
24. Adams RH, Wilkinson GA, Weiss C et al . Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev 1999 ;13 (3):295-306
25. Stein E, Lane AA, Cerretti DP, et al.Eph receptors discriminate specific ligand oligomers o determine alternative signaling complexes, attachment, and assembly responses.Genes Dev. 1998 ;12(5):667-78.
26. Kiyokawa E,Takai S,Tanaka M. Overexpression of ERK, an EPH family receptor protein tyrosine kinase, in various human tumors. Cancer Research . 1994;54:3645-3650
27. Maru Y,Hirai H,Takaku F. Overexpression confers an oncogenic potential upon the eph gene. Oncogene. 1990;5:445-447
28. Tang XX,Evans AE,Zhao H,Cnaan A.London W,Cohn SL, Brodeur GM, Ikegaki N. High-level expression of EPHB6, EFNB3, and EFNB3 is associated with low tumor stage and high TrkA expression in human neuroblastomas. Clin Cancer Res 1999 ; 5 (6):1491-6
29. Tang XX.Pleasure DE,Brodeur GM Ikegaki N. A variant transcript encoding and isoform of the human protein tyrosine kinase EPHB2 is generated by alternative splicing and alternative use of polyadenylation signals oncogene 1998; 17(4): 521-6
30. Walker-Daniels J, Coffman K, Azimi M, Rhim JS, Bostwick DG, Snyder P, Kerns BJ, Waters DJ, Kinch MS. Overexpression of the EphA2 tyrosine kinase in prostate cancer. Prostate 1999; 41(4): 275-80
31. Tang XX, Brodeur GM, Campling BG, Ikegaki N. Coexpression of transcripts encoding EPHB receptor protein tyrosine kinases and their ephrin-B ligands in human small cell lung carcinoma. Clin Cancer Res 1999; 5(2): 455-60
32. Mellitzer G, Xu Q, Wilkinson DG et al. Eph receptors and ephrins restrict cell intermingling and communication. Nature 1999; 400(6739): 77-81
33. Oates AC, Lackmann3 M, Power MA, Brennan C, Smith FM, Do C, Evans B, Holder N Boyd AW. An early developmental role for eph-ephrin interaction duing vertebrate gasteulation. Mech Dev 1999; 83(11-2): 77-94
34．J．萨姆布鲁克等著。金冬雁，黎孟枫译。分子克隆实验指南，第二版，科学出版社，1992：880-885
35．严于颖，王海林译，F．奥斯伯，R．E．金斯顿，J．G．塞德曼等著，《精编分子生物学实验指南》，第一版，北京：科学出版社，1998。
36．张晓光 王方 闫小君 胃癌高表达基因ErK的扩增，克隆和鉴定 第四军医大学学报 1997；18(6)：562-565
37．张晓光 董巨莹 茹晓荣 EphB2受体N端Ig类似区的克隆和序列测定 细胞与分子免疫学杂志2000；16(1)：026-028
38．张晓光 刘新平 李福洋 EplaB2配体结合域基因片段的高效表达 中国免疫学杂志2000；16：9-11
39. Smith MHD, Dwin JV. Translational intitation on structured messengers: another role for the Siagle-Dalgarno interaction. J Mol Biol, 1994; 235: 173-184
40. Ralf-Schoefer. The pRSET family of T_7 promoter expression vectors for E. coli. Gene, 1993; 124: 83-85
41. Kroll DJ, Abdel-Hafiz H A-B, Marcell T et al. A multifunction prokarytic protein expression system: overproduction, affinity purifacation,11nd selective detection. DNA and cell biologio. 1993: 12: 441-445
42．俞强 细胞调控的探索 叶金生等主编，军事医学科学出版社，1999，12-15
1.Fields S, Song O: A novel genetic system to detect protein-protein interactions. Nature 1989,340:245-246
2.Brent R,Plashne M; A eukaryolic transcriptional activator bearing the DNA specificity of a prokaryotic repressor Cell 1985,43:729
3. Aronheim A, Zandi E, Hennemann H, Elledge SJ,Karin M:Isolation of an AP-1 repressor by a novel method for detecting protein-orotein interactions.Mol Cell Biol 1997,17:3094-3102.
4.Chien,.CT, Bartel PL, Sternglanz R, Fields S The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc.Natl.Acad. Sci. USA, 1991, 88:9578-9582
5.Durfee T, Becherer K, Chen PL, Yeh SH, Yang Y, Kilburn AE, Lee WH, Elledge SJ. The retinoblastoma protein associates with the protein Phosphatase type 1 catalytic subunit Genes Dev., 1993,7:555-569
6. Le Douarin B, Pierrat B, vom Baur E, Chambon P, Losson R. A new version of the two-hybrid assay for detection of protein-protein interactions. Nucleic Acids Res.,1995,23:876-878
7. James p, Halladay J, Craig EA: Genomic libraries and a host strain designed for highly efficient two-hy-brid selection inteast. Genetics 1996,144:1425-1436.
8. Aronheim A, Zandi E, Hennemann H, Elledge SJ,Karin M:Isolation of an AP-1 repressor by a novel method for detecting protein-orotein interactions.Mol Cell Biol 1997,17:3094-3102.
9. Johnsson N,Varshavsky A :Split ubiquitin as a sen-sor of protein interactions in vivo. Proc Natl Acad Sci UNA 1994,91:10340-10344.
10.MarsoIier M-C,Prioleau M-N,Sentenac A: An RNA polymerase Ill-based two- hybrid system to study RNA polymerase II transcriptional regulators.J Mol Biol 1997,268:243-249.
11. Tsan J,Wang Z,Jin Y, Hwang L,Bash RO,Baer R: Mammalian cells as hosts for two- hybrid studies of protein-protein interaction.In The Yeast Two-Hybrid System,edn 1. Edited by Bartel PL, Fields S.New York: Oxford University Press; 1997:217-232.
12.Rossi F, Charlton CA, Blau HM: Monitoring protein-protein interactions in intact eukaryotic cells by β -galactosidase complementation.proc Natl Acad Sci USA 1997,94:8405-8410.
13.Li JJ, Herskowitz I: Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybid system. Science 1993,262:1870-1874.
14.Vidal M,Brachmann RK,Fattaey A,Harlow E,Boeke JD:Reverse two-hybrid system and one hybrid system to detect dissociation of protein-protein and DNA-protein interaction. Proc Natl Acad Sci USA 1996,93:10315-10320.
15.Brachmann RK, Vidal M,Boeke JD:Dominant-nega-tive p53 mutations selected in yeast hit cancer hot sports. Proc Natl Acad Sci USA 1996,93:4091-4095.
16. Shin HM, Goldman PS, DeMaggio AJ, Holleenberg SM,Goodman RH,Howkstra MF:Apositive genetic selection for disrupting protein-protein interactions;identification of CREB mutations that prevent association with the coactivaor CBP.Proc Natl Acad Sci USA 1996,93:13896-13901.
17. Zhang J,Lautar S: Ayeast three-hybrid method to clone tenary protein compkex componets. Anal Biochem 1996,242:68-72.
18.Licitra EJ,Liu JO:A three-hybrid system for detecting small ligand-protein receptor interactions.proc Natl Acad Sci USA 1996,93:12817-12821
19. Osborne MA, Dalton S, KochanJP: The yeast tribird syetem genetic detection of trans-phosphorylated ITAM-SH2-interaction.Bio-Technology 1995, 13: 1474
20. Putz U, Skehel p,Kuhl D:A tri-hybrid system for the analysis and detection of RNA- protein interac-tions. Nucleic Acids Res 1996,24:4834-4640.
21.Stein E, Huynh-Do U, Lane AA Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase. JBC,1998; 273(3): 1303-1305
22 .Li B.and Fields S: Identification of mutation in p53 that affect its binding to SV40 T antigen by using the yeast two -hybrid system. FASEB J. 1993, 7: 957
23. Staudinger J, Zhou J, Burgess R, Elledge SJ, Olson EN. PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J Cell Biol. 1995 ;128(3):263-71.
24.Yang, M. , Wu Z, Fields S. Protein-peptide interactions analyzed with the yeast two-hybrid system. Nucleic Acids Res. 1995 ;23(7): 1152-6
25 Dixon E.P, Johnstone EM, Liu X, Little SP. An inverse mammalian two-hybrid system for beta secretase and other proteases. Anal Biochem. 1997 ;249(2):239-41.
26 Fromont-Racine M, Rain J-C,Legrain P: Toward a functional analysis of the yeast genome through exhaustive two-hybrid screecs. Nat Genet 1997 16:277-281.
1 .Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985. 228:1315-1317
2 Nambudripad R, Stark W, Makowski L. Neutron diffraction studies of the structure of filamentous bacteriophage Pfl. J. Mol. Biol. 1991. 220: 359-379
3 Kay Bk, Adey NB, He Ys-S et al. An M13 library displaying 38-amino-acid peptides as a source of novel sequences with affinity to selected targets.Gene, 1993; 128: 59-65
4.Maruyama IN, Maruyama HI, Brenner S. λ foo: a λ vector for the expression of foreign proteins. Proc Natl Acad Sci USA, 1994; 91:8273-8277
5.Lindqvist BH, Naderi S. Peptide presentation by bacteriophage T4. Microbiol Rev,1995;17:33-39
6.Lowman HB, Bass SH, Simpson N, et al. Selecting high-affinity binding proteins by monovalent phage display. Biochemistry, 1991; 30: 10832-10838
7.Cwirla S, Peters E, Barrett R et al Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Aad Sci USA, 1990; 87: 6378-6382
8.Clackson T,Well JA.In vitro selection from protein and peptide libraries. TIBS, 1994;12:173-184
9. Lowman HB. Bacteriophage display and discovery of peptide leads for drug development. Annu Rev Biophys Biomol Struct, 1997; 26: 401-424
10. Sparks AB, Adey NB, Quilliam LA. Screening phage-displayed random peptide libraries for SH3 ligands. Methods Enzymol, 1995; 255: 498-509
11. Mcconnell SJ, Kendall ML, Reilly TM et al. Constrained peptide libraries as a tool for finding mimotopes.Gene, 1994; 115-118
12. Wrighton NC, Farrell FX, Chang R et al. Small peptides as potent mimetics of the protein hormone erythropoietin. Science, 1996; 273:458-463
13. Scott JK. Discovering peptide ligands using epitope libraries. Trends Biochem. Sci. 1990. 17:241-245
14. Krook M, Mosbach K, RanstromO. Novel peptides binding to the Fc-portion of immunoglobulins obtained from a combinatorial phage display peptide library. J Immunol Methods, 1998:221(1-2): 151-157
15. Kay B K, Winter Jill, Mccafferty C. Phage display of peptides and proteins:A laboratory Manual. Academic Press. Harcourt Brace & Company Publishers. 1996; p235-237
16. Doorbar J, and Winter G. Isolation of a peptide antagonist to the thrombin receptor using phage display. J. Mol. Biol. 1994. 244: 361-369
17. Hyde-DeRuyscher R, Paige L, Christensen D et al. Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries. Chem Biol 2000; 7(1): 17-25
18. Kimura T, Kaburaki H, Tsujino T, Watanabe Y, Kato H. Signal transduction by the peptide which mimics the activity of thrombopoietin. Biochem Mol Biol Int, 1998, 44(6): 1203-1209
19. Crihalde ND, Chen YC, Golden A, et al. Epitope mapping of anti-HIV and anti-HCV monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library. Gene 1995. 166: 187-195
20. Reece J C, Geysen HM, Rodda SJ. Mapping the major human T helper epitopes of tetanus toxin. The emerging picture. J Immuunology, 1993; 151(11): 6175-6184
21. Sudol M. From SRC homology domains to other signaling modules: proposal of the 'protein recognition code'. Oncogene 1998. 17: 1469-1474
22. Rickles RJ, Botfield MC, Zhou XM, et al. Phage display selection of ligand residues important for Src homology 3 domain binding. Proc. Natl. Acad. Sci. U.S.A. 1995. 92: 10909-10913
23. Wu J, Ma QN, Lam KS et al. Identifying substrate motifs of protein kinases by a random library approach. Biochemistry, 1994; 33(49): 14825-14833
24. Luo K, Zhou P, Lodish HF. The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library Proc Natl Acad Sci, 1995; 92(25): 11761-11765
25. Steward M W, Stanley CM, Obeid OE. A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response. J Virol, 1995; 69(12): 7668-7673
26. Folgori A. A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera. EMBO J, 1994; 13: 2236-2243
27. Estaquier J Gras-Masse H, Boutillon CEur et al. The mixotope: a combinatorial peptide library as a T cell and B cell immunogen., J Immunol, 1994; 24(11): 2789-2795
28 张晓光 药立波 韩炯 噬菌体展示法筛选与EphB2结合的短肽 生物化学与生物物理学报 2000；32(5)：