PDF《说明书》

说明书ERK2 结合多肽及其制备 技术领域 本发明涉及一种多肽,尤其是涉及一种具备调节细胞增殖与分化功能的多肽及其制备.

背景技术 接头蛋白是一组分子调节因子,能募集不同的蛋白形成复合物,将上游信号分子的信息 传递下去(Pawson T, et al. Signaling through scaffold, anchoring, and adaptor proteins. Science,19, 2075-80,1997;

Ravichandran KS, et al. Signaling via Shc family adapter proteins. Oncogene, 20(44), 6322-30,2001) .在信号转导复合物形成和完成其生物学功能 的时候,接头蛋白常经历酪氨酸磷酸化、结构改变和亚细胞定位变化(Cheng Y, et al. ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. J. Biol Chem, 277, 19382-19388,2002;

Guri Tzivion,et al. 14-3-3 proteins: Active cofactors in cellular regulation by serine/threonine phosphorylation. J. Biol Chem, 277, 3061-3064,2002;

Mary N. Teruel ,et al. Translocation and reversible localization of signaling proteins: a dynamic future for signal transduction. Cell, 103, 181-184,2000) .成纤维细胞生长 因子受体FGFR是受体酪氨酸激酶家族成员,调节细胞对一系列有关于调节细胞增殖、迁移、 分化和存活的胞内信号的反应(Joseph Schlessinger,et al. Cell signaling by receptor tyrosine kinases. Cell, 103, 211-225,2000;

Kyle A Furge,et al. Met receptor tyrosine kinase: enhanced signaling through adapter proteins. Oncogene,19, 5582-5589,2000;

D. Vaudry, et al. Signaling pathways for PC12 cell differentiation: making the right connections. Science, 296, 1648-1649, 2002).接头蛋白FRS2 (FGFR底物2)被认为是FGFR 信号转导的一个主要的下游介导因子.FRS2通过N端十四烷基化锚定在细胞膜上,通过PTB (Phosphorylated Tyrosine Binding domain)结构域结合于FGFR.FGF(Fibroblast Growth Factor, 成纤维细胞生长因子)刺激后,FRS2迅速地酪氨酸磷酸化,并产生至少6个功能性pY (磷酸化酪氨酸残基)结合位点,从而与蛋白酪氨酸磷酸酶Shp2以及接头蛋白Grb2形成复合 物, Grb2又与Ras (Ras GTPase) 通路激活因子Sos(Son of sevenless)结合. 然后FRS2-Grb2-Sos 复合物激活Ras/MAP kinase(Mitogen Activated Protein Kinase)信号级联通路(H. Kouhara,et al. A lipid-anchored Grb2-binding protein that links FGF-receptor activation to the Ras/MAPK signaling pathway. Cell, 89, 693-702, 1997;

Y. R. Hadari, et al. Binding of Shp2 tyrosine phosphatase to FRS2 is essential for fibroblast growth factor-induced PC12 cell differentiation. Mol. Cell. Biol, 18, 3966-3973, 1998;

S. 说明书H. Ong, et al. FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors. Mol. Cell. Biol, 20, 979-989, 2000).除激活Ras/MAPK信号通路外,FRS2酪氨酸磷酸化这一重 要事件也被报道其通过形成FRS2-Grb2(Growth receptor bound protein 2)-Gab1 (GRB2-associated binding protein 1)复合物而参与PI3(Phosphatidylinositol-3 Kinases)激酶激活,并进一步激活PI3K-Akt(Akt,蛋白激酶B)信号通路.有证据表明FRS2诱导PI3激酶激活,从而导致细胞迁移(Ong SH, et al. Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins. P.N.A.S, 98(11), 6074-6079, 2001).目前,FRS2被报道 在FGFR(Fibroblast Growth Factor, 成纤维细胞生长因子受体), NGFR(Nerve Growth Factor Receptor, 神经生长因子受体), PDGFR(Platelet Derived Growth Factor Receptor,血小 板衍生生长因子受体), EGFR(Epithelial Growth Factor Receptor, 上皮细胞生长因子受 体), BDNFR(Brain Derived Neurotrophic Factor Receptor,脑衍生神经营养因子受体) 和VEGFR(Vascular Endothelial Growth Factor Receptor,血管内皮生长因子受体)信号转 导通路中发挥作用(Y. R. Hadari, et al. Critical role for the docking-protein FRS2a in FGF receptor-mediated signal transduction pathways. P.N.A.S, 98(15), 8578-8583, 2001;

R. M. Melillo, et al. Docking protein FRS2 links the protein tyrosine kinase RET and its oncogenic forms with the Mitogen-Activated Protein Kinase signaling cascade. Mol. Cell. Biol, 21, 4177-4187, 2001;

Yan KS, et al. FRS2 PTB domain conformation regulates interactions with divergent neurotrophic receptors. J Biol Chem, 277(19), 17088-17094, 2002;

Stoletov KV, Fibroblast growth factor receptor substrate

2 participates in vascular endothelial growth factor-induced signaling. FASEB.J ,16(10), 1283-1285, 2002). 早期研究发现FGF刺激不仅能诱导FRS2酪氨酸磷酸化,还会导致其电泳位移改变.其他 刺激因子包括NGF (Nerve Growth Factor) 、 EGF (Epithelial Growth Factor) 、 PDGF (Platelet Derived Growth Factor) 、LPA(Lysophosphatidic acid,溶血磷脂酸)和Thrombin(凝血 酶)也引起FRS2的电泳条带上移,但是这些因子中部分并不能诱导FRS2酪氨酸磷酸化.此现 象提示除酪氨酸磷酸化FRS2可能还有其他修饰改变.然而确切的机制还不清楚. 有丝分裂原激活地蛋白激酶(MAPKs)是许多细胞刺激因子如生长因子、细胞因子和GPCR (G-protein coupled receptor, G蛋白偶联受体)配体,诱导产生的信号转导途径中主要的 下游效应分子.MAPK/ERK(Mitogen Activated Protein Kinase/Extracellular signal-regulated kinase, )激活导致许多不同的胞浆内、胞膜上和核内相应底物磷酸化, 说明书从而调节一组相关基因的转录(Bijan Roshan, et al. Activated ERK2 interacts with and phosphorylates the docking protein GAB1. J Biol Chem, 274, 36362-36368, 1999;

Howe AK, et al. Anchorage-dependent ERK signaling--mechanisms and consequences. Curr Opin Genet Dev, 12(1), 30-35, 2002;

Cans C, et al. Nuclear tyrosine phosphorylation: the beginning of a map. Biochem Pharmacol, 60(8), 1203-1215, 2000;

Treisman. R, et al. Regulation of transcription by MAP kinase cascades. Curr. Opin. Cell. Biol, 8,205-215, 1996).据报道,大约50种蛋白是MAPK/ERK底物,包括下游信号功能蛋白如p90Rsk(90-Kda ribosomal S6 kinase)和上游信号功能蛋白如Sos, Raf 和MEK(MAPK/ERK kinase)(Karin. M, et al. The regulation of AP-1 activity by mitogenactivated protein kinases. J. Biol. Chem, 270, 16483C16486, 1995;

Whitmarsh. A.J, et al. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med, 74, 589C607, 1996;

Madhani. H.D, et al. The riddle of MAP kinase signaling specificity. Trends Genet, 14, 151C155, 1998;

Jacobs D, Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes Dev, 13(2), 163-175, 1998).基于基因组分析和比对的研究发现包含一个保守序列 的广泛存在的ERK2结合位点K/RK/RK/R…I/LXI/L (Tanoue T, et al. A conserved docking motif in MAP kinases common to substrates, activators and regulato........