【作者】 李婷婷；

【导师】 赵彦艳；

【作者基本信息】 中国医科大学 ， 遗传学， 2009， 博士

【摘要】 前言SH2D4A是本实验室利用外显子捕获技术在人染色体疾病基因富含区域8p22克隆得到的只具有单一SH2结构域的SH2信号蛋白家族的新成员。在前期研究中我们发现SH2D4A亚细胞定位于细胞质,并在许多组织中表达。由100个氨基酸组成的SH2结构域可以介导蛋白质之间相互作用,能特异性识别并结合磷酸化的酪氨酸残基,参与和酪氨酸激酶相关的信号转导过程。具有SH2结构域的蛋白质大部分是含有多种结构域的酶类信号蛋白或接头蛋白,在信号转导过程中除了介导蛋白质之间相互作用,还调节相关的催化亚单位。我们克隆的SH2D4A具有典型的SH2结构域,提示其可能与酪氨酸激酶型受体结合,在酪氨酸激酶的相关信号转导通路中发挥作用,但除SH2结构域外SH2D4A不具有其它结构域,提示它可能具有不同于其他SH2信号蛋白的生物学功能。迄今为止尚无有关SH2D4A生物学功能的详细报道。本研究中我们构建了野生型和缺陷型SH2D4A表达载体,通过MTT和流式细胞术检测SH2D4A对细胞增殖和凋亡的影响。由于胰岛素样生长因子Ⅱ（IGFⅡ）可以通过典型的酪氨酸激酶型受体—胰岛素样生长因子Ⅰ受体（IGFⅠR）促进细胞增殖抑制细胞凋亡,因此我们检测了IGFⅡ刺激下SH2D4A对细胞增殖和凋亡的影响;同时检测了与细胞增殖和凋亡都相关的信号激酶—蛋白激酶C（PKC）以及可以激活PKC的第二信使—Ca²⁺,分析SH2D4A影响细胞增殖和凋亡的可能机制。为发现与SH2D4A相互作用的上游受体蛋白,我们以SH2D4A为诱饵蛋白应用酵母双杂交技术筛选肾脏cDNA文库,筛选发现雌激素受体α亚型（ERα）能够与SH2D4A相互作用,并通过酵母双杂交验证实验、免疫共沉淀、GST pull-down三种不同方法验证了SH2D4A与ERα间的相互作用。由于有报道与SH2D4A结构极为相似的SH2信号家族成员—SH2结构域蛋白1A（SH2D1A）在淋巴细胞信号激活分子（SLAM）介导的信号转导中发挥抑制作用,调节T、B细胞间的协同作用,以及免疫细胞的增殖与凋亡;因此我们推测SH2D4A可能在信号转导通路中发挥相似的抑制作用,参与调节细胞的增殖与凋亡,并检测SH2D4A对ERα介导的信号通路的影响。我们发现磷脂酶Cγ亚型（PLC-γ）与ERα间存在相互作用,并通过对细胞增殖、凋亡和PKC活性的检测,揭示SH2D4A对雌激素（E2）/ERα介导的生物学效应的影响和产生此影响的分子机制。SH2D4A对E2/ERα诱导的细胞增殖的抑制作用提示SH2D4A可能应用于ERs相关肿瘤的治疗。以上研究结果为深入探讨SH2D4A的生物学特性和开发其应用价值奠定了坚实基础。材料与方法一、实验材料1、人胚肾细胞系HEK2932、构建表达载体相关试剂3、MTT检测相关试剂4、流式细胞术检测相关试剂5、PKC活性荧光检测试剂盒6、Fluo-3AM荧光染料7、Western印迹杂交相关试剂8、酵母双杂交实验相关试剂9、免疫共沉淀（IP）相关试剂10、GST pull-down相关试剂11、常用实验设备如荧光分光光度计、紫外分光光度计、酶标仪、流式细胞仪、自动凝胶成像分析仪、全温振荡培养箱等。12、常用数据库及分析软件如基因组数据库、蛋白质分析数据库、凝胶成像及扫描分析软件、引物设计软件（Primer3）、SPSS version 13.0等。二、实验方法1、应用DNA重组技术构建缺失SH2结构域的缺陷型SH2D4A表达载体pSH2D4A-Delet,检测SH2结构域在SH2D4A中的作用。2、通过MTT方法绘制生长曲线,检测SH2D4A对细胞增殖的影响。3、应用流式细胞术通过单染和双染技术检测SH2D4A对细胞周期、细胞增殖和凋亡的影响。4、应用PepTag荧光试剂盒检测SH2D4A对细胞内蛋白激酶C（PKC）活性的影响,及IGFⅡ和E2刺激下SH2D4A对胞内PKC活性的调节。5、应用Fluo-3AM荧光染料检测SH2D4A对细胞内Ca²⁺浓度([Ca²⁺]_i)的影响,及IGFⅡ和E2刺激下SH2D4A对胞内Ca²⁺浓度的调节。6、构建pAD/ERα表达载体;应用酵母双杂交筛选和检测技术,发现和证明酵母细胞中SH2D4A与ERα的相互作用。7、通过酶切、连接和转化等技术构建含有HA标签的野生型SH2D4A表达载体pSH2D4A-HA;应用免疫共沉淀方法,分别利用HA、ERα或磷脂酶C-γ（PLC-γ）抗体进行免疫沉淀,并使用相应抗体为一抗进行Western印迹杂交,分别检测SH2D4A与ERα,PLC-γ与ERα的相互作用,及SH2D4A对PLC-γ与ERα相互作用的影响。8、构建pGST/SH2D4A及pGST/ERα表达载体;应用GST pull-down技术在体外双向鉴定SH2D4A与ERα间的相互作用。结果1、构建缺陷型SH2D4A表达载体、pAD/ERα表达载体、pSH2D4A-HA表达载体、pGST/SH2D4A和pGST/ERα表达载体,为深入研究SH2D4A的生物学特性奠定基础。2、通过MTT和流式细胞术单染和双染技术检测到SH2D4A抑制细胞增殖,促进细胞凋亡,并且SH2D4A对IGFⅡ的促增殖和抗凋亡作用都有抑制作用,但SH2D4A只能抑制E2的促增殖作用,而对E2的抗凋亡作用没有影响。3、应用PepTag试剂盒检测细胞内PKC活性发现SH2D4A能够抑制PKC的激活,并能够阻止IGFⅡ和E2激活胞内PKC。4、通过荧光染料检测细胞内ca²⁺浓度,发现SH2D4A能够抑制IGFⅡ和E2上调Ca²⁺浓度。5、酵母双杂交筛选发现ERα可能与SH2D4A相互作用,酵母双杂交检测实验证实了SH2D4A与ERα间的相互作用。6、转染细胞后提蛋白进行免疫共沉淀实验,实验结果证明SH2D4A与ERα间存在相互作用。7、诱导、纯化融合蛋白GST-SH2D4A和GST-ERα,GST pull-down实验结果证明SH2D4A与ERα的相互作用是直接的,不借助于真核细胞中的其他任何蛋白。8、通过免疫共沉淀实验发现PLC-γ能够与ERα结合,且SH2D4A能够与PLC-γ竞争结合于ERα。结论1、SH2D4A通过影响PKC信号转导通路抑制细胞增殖、促进细胞凋亡。2、SH2D4A与ERα及PLC-γ与ERα之间存在相互作用,而SH2D4A与PLC-γ竞争结合ERα,通过ERα/PLC-γ/PKC信号通路调节细胞增殖。更多还原

【Abstract】 IntroductionSH2 domain containing 4A（SH2D4A） is a novel protein of the SH2 signaling protein family that only contains a single SH2 domain.We cloned the SH2D4A gene from the human chromosome 8p22 region,and indicated SH2D4A protein is located in the cytoplasm and is expressed ubiquitously.The SH2 domain is the prototype for protein-protein interaction modules,binds to a phosphotyrosine,and mediates the formation of multiprotein complexes in protein tyrosine kinase pathways.SH2 domains are typically found in multidomain signaling enzymes or in adaptor proteins,where they mediate protein-protein interactions and regulate associated catalytic subunits.The lack of additional domains in SH2D4A suggests that it is unlikely to function in either of these roles.However,only a few reports of SH2D4A have been published so far,and its detailed biological function is still poorly understood.To investigate the biological function of SH2D4A,we constructed SH2D4A expression vector and its SH2 domain-deleted expression vector,and detected the effect of SH2D4A on cell proliferation and apoptosis using MTT assay and Flow-cytometric analyses and protein kinase C（PKC） activity assay.The data showed that SH2 domain is the functional domain of SH2D4A,and SH2D4A inhibited cell proliferation and promoted cell apoptosis.Furthermore,SH2D4A suppressed the insulin-like growth factorⅡ（IGFⅡ）-induced Ca²⁺release and PKC activation.Therefore,these findings suggested that SH2D4A inhibited cell proliferation and promoted cell apoptosis by suppression of PKC signaling pathway.We further performed a yeast two-hybrid screen using SH2D4A as the bait and found SH2D4A directly bind to estrogen receptorα（ERα）.Co-immunoprecipitation and GST pull-down assays further confirmed their interaction.We also demonstrated an inhibitory effect of SH2D4A on estrogen-induced cell proliferation.We showed that this effect was due to suppression of a non-genomic action of ERαvia an ERα/phospholipase C-γ（PLC-γ）/PKC signaling pathway.In addition,we confirmed a new relationship among SH2D4A,ERα,and PLC-γ.SH2D4A may be useful for the development of a new anti-cancer drug acting as an ER signaling modulator.Our findings provide insight into the biological function of SH2D4A.Materials and MethodsMaterials1、Human embryonic kidney cell line（HEK293）2、Reagents for construction expression vector3、Reagents for Microculture tetrazolium（MTT） assay.4、Reagents for Flow-cytometric analyses.5、Reagents for PKC activity assay.6、Fluorescent probe Fluo-3/AM7、Reagents for Western blot8、Reagents for Yeast two-hybrid screen and assay9、Reagents for Co-immunoprecipitation10、Reagents for GST pull-down assay11、Equipments for experiments:fluorospectrophotometer,UV spectrophotometer, microplate spectrophotometer,flowcytometry,gel-formatter,oscillatory incubator et al.12、Database and software:genome data base,protein analytic data base,analytic software of imaging and scanning,Primer3,SPSS version 13.0 et al.Methods1、To investigate the biological function of SH2 domain in SH2D4A,we constructed SH2 domain-deleted expression vector pSH2D4A-Delet by enzymatic digestion,ligation and transform technique.2、The MTT assay was performed to test the effect of SH2D4A in cell rpoliferation.3、Flow-cytometric analyses was used to observe the influence of in cell cycle,cell proliferation and cell apoptosis.4、The IGFⅡor estrogen-induced activation of PKC was measured by the PepTag Protein Kinase Assay.5、Changes in Ca²⁺ concentration were measured by the Ca²⁺-sensitive fluorescent probe Fluo-3/AM.6、Through constructed pAD/ERα,yeast two-hybrid screen and assay were performed to investigate the interaction between SH2D4A and ERa.7、After constructed pSH2D4A-HA,co-immunoprecipitation was performed to investigate the relationship among SH2D4A,ERαand PLC-γ.8、After constructed pGST/SH2D4A and pGST/ERα,GST pull-down was performed to confirmed the directly interaction between SH2D4A and ERα.Results1、Constructions of expression vectors,such as pAD/ERα,pSH2D4A-HA, pGST/SH2D4A and pGST/ERα,provided the bases for investigating the biological function of SH2D4A.2、SH2D4A was found to inhibite cell proliferation and promote apoptosis. SH2D4A also suppressed IGFⅡ-induced cell proliferation and its anti-apoptosis effect. But SH2D4A only arrested estrogen-induced cell proliferation and didn’t influence its anti-apoptosis effect.3、The activity of PKC was measured by PepTag Protein Kinase Assay.SH2D4A was observed to inhibit the activation of PKC,and suppressed the IGFⅡor estrogen-induced activation of PKC4、The concentration of intracellular Ca²⁺ was tested using a fluorescent probe. SH2D4A arrested the IGFⅡor estrogen-induced release of Ca²⁺. 5、We found the interaction between ERαand SH2D4A in yeast two-hybrid screen, and verified this interaction via the yeast two-hybrid assay.SH2D4A interacted with ERαin yeast cell.6、We further investigated the interaction between SH2D4A and ERαin vivo using co-immunoprecipitation assay.The data confirmed the interaction of SH2D4A with ERαin eukaryotic cells.7、After expression-inducion and purification of fusion protein,GST pull-down assays were used to determine the interaction between SH2D4A and ERαin vitro.The interaction between SH2D4A and ERαwas direct and independent on additional proteins in the cells.8、In the co-immunoprecipitation assay,PLC-γinteracted with ERαin bidirectional reactions,and this interaction was suppressed by SH2D4A.Conclusion1、SH2D4A regulated cell proliferation and apoptosis by suppression of PKC signaling pathway.2、SH2D4A directly interacted with ERα;PLC-γ,interacted with ERα;SH2D4A competed with PLC-γfor binding to ERαand regulated cell proliferation via an ERα/PLC-γ/PKC signal pathway.更多还原