细胞信号转导的技术方法.ppt

,First Military Medical University,细胞信号转导的技术方法,蛋白激酶磷酸化及其活性测定,蛋白激酶磷酸化的检测,裂解培养的细胞获得裂解上清,以免疫共沉淀法获得蛋白激酶,SDS-PAGE,再加入蛋白激酶磷酸化特异性抗体,以,Phospho,Ab,-HRP Western,化学发光检测试剂盒测定蛋白激酶的磷酸化程度。,经典蛋白激酶活性分析实验流程,以免疫共沉淀法获得蛋白激酶,加入该激酶底物和,32,-ATP,，,激酶使底物磷酸化,放射自显影，确定磷酸化条带,以免疫共沉淀法获得蛋白激酶,加入该激酶底物，激酶使底物磷酸化,再加入底物磷酸化特异性抗体,以,Phospho,Ab,-HRP Western,化学发光检测试剂盒测定底物的磷酸化程度，进而推出蛋白激酶活性,非放射性蛋白激酶活性分析实验流程,较传统激酶活性测定方法的优点,无需接触放射性物质,敏感度高：可以检测到每个磷酸化分子,特异性：利用磷酸化特异性抗体可以进行位点特异性分析,信噪比高,低背景,系统完整：提供一整套直接从细胞裂解液中测试酶活的试剂,节约时间：由几天降到几分钟,0,2,4,6,8,1,0,1,2,1,4,0 5 10 20 30 60 90 120,Time(min),Relative,kinase,activity,Dynamic processes of p38 activation by LPS in RAW cells,Effect of individual MAPK pathways on PRAK activity in intact cells,MKK7(D),GST-ATF2,GST-ELK1,MKK1(E),MKK6(E),Control,HSP27,PRAK,Control,Aniso,.,Arsenite,TNF,-80kDa,-47kDa,-39kDa,-80kDa,-47kDa,kDa,97.4-,68.0-,43.9-,29.0-,18.4-,14.3-,Control,Aniso,.,Arsenite,TNF,A,B,The major,kinases,of p38,and p38,P38 MAP,Kinase,Pathway,蛋白磷酸化位点分析及其功能鉴定,Phosphopeptide,map of HSP27,phosphorylated,by PRAK,in vitro,Chromatography,Electrophoresis pH1.9,+,MAPKAPK2,Chromatography,Electrophoresis pH1.9,+,PRAK,Chromatography,Electrophoresis pH1.9,MIX,+,+,+,+,+,-,-,-,-,+,-,HSP27,p38,GST-PRAK(93A),GST-PRAK(WT),GST-PRAK(186A),GST-PRAK(212A 214A),GST-PRAK(182A),GST-PRAK(WT),GST-PRAK(182D),GST-PRAK(182D 212D),+,-,+,-,+,-,Fold of Activation,0,5,10,15,20,GST-PRAK(182A),GST-PRAK(182D),GST-PRAK(WT),GST-PRAK(93A),GST-PRAK(186A),GST-PRAK(182D 212D),GST-PRAK(212A 214A),B,A,Electrophoresis pH 8.9,+,+,p38,-PRAK(182A),+,p38,-PRAK(182D),p38,-PRAK(wt),T,182,is the regulatory,phosphorylation,site of PRAK,DNA,重组技术的应用,活性诱变体,无活性诱变体,结构与功能的研究,PCR primer,PCR primer,JNK2,JNK3,JNK1,p38,p38,p38,p38,ERK1,ERK2,ERK5,The relationship between the members of MAPK family,MAPK,Dural,Phosphorylation,Sites L-12 Length,*,hp38,DFGLARHTDD-EM,T,G,Y,VATRWYRAPE,25,hP38,b,DFGLARQADE-EM,T,G,Y,VATRWYRAPE,25,hp38,g,DFGLARQADS-EM,T,G,Y,VVTRWYRAPE,25,hp38,d,DFGLARHADA-EM,T,G,Y,VVTRWYRAPE,25,hJNK1,DFGLARTAGTS-FMM,T,P,Y,VVTRYYRAPE,27,hJNK2,DFGLARTACTN-FMM,T,P,Y,VVTRYYRAPE,27,hJNK3,DFGLARTAGTS-FMM,T,P,Y,VVTRYYRAPE,27,hERK1,DFGLARIADPEHDH-TGFL,T,E,Y,VATRWYRAPE,31,hERK2,DFGLARVADPDHDH-TGFL,T,E,Y,VATRWYRAPE,31,hBMK1,DFGMARGLCTSPAEH-QYFM,T,E,Y,VATRWYRAPE,32,YHOG1,DFGLARIQDP-QM,T,G,Y,VSTRWYRAPE,25,YSMK1,DFGLARGIHAGFFKCHS-TVQPHI,T,N,Y,VATRWYRAPE,36,YMPK1,DFGLARGYSENPVEN-SQFL,T,E,Y,VATRWYRAPE,32,YKSS1,DFGLARCLASSSDSRET-LVGFM,T,E,Y,VATRWYRAPE,35,YFUS3,DFGLARIIDESAADNSEPTGQQSGM,T,E,Y,VATRWYRAPE,38,domain VII VIII,Loop-12(T-Loop)sequence of MAP,kinases,Construction of p38 loop-12 to ERK like structure,p38 .DFGLARHTDDE-M,T,G,Y,VATRWYRAPE.,p38(E).DFGLARHTDDE-M,T,E,YVATRWYRAPE.,p38(6+).DFGLARHTDDE,HDHTGF,M,T,G,Y,VATRWYRAPE.,p38(6+E).DFGLARHTDDE,HDHTGF,M,T,E,Y,VATRWYRAPE.,p38(VAP).DFGLAR,VA,D,P,E-M,T,G,Y,VATRWYRAPE.,p38(DL).DFGLARHTDD,D,-,L,T,G,Y,VATRWYRAPE.,p38(VAPD6+LE).DFGLAR,VA,D,PDHDHTGFL,T,E,Y,VATRWYRAPE.,ERK2 .DFGLARVADPDHDHTGFL,T,E,Y,VATRWYRAPE.,MAPK,MKK,Substrates,Loop-12 is a key structure to determine the selection of substrate,(.,T,X,Y,.)-,病毒技术对于揭示细胞信号通路的作用,腺病毒,逆转录病毒,细胞信号分子特异性抑制剂的应用,几种,MAPK,通路抑制剂的化学结构示意图,:,O,NH,2,OCH,3,O,PD98059,HN,N,S,O,F,CH,3,N,OH,N,F,N,H,N,SB,202190,SB,203580,CH=CH,CH=CH,OH,OCH,3,OH,OCH,3,O,O,Curcumin,Effects of SB203580 and PD98059 on endogenous PRAK activity,HSP27,TNF,+,+,+,-,-,-,-,-,-,-,-,-,Arsenite,+,+,+,-,-,-,-,-,-,-,-,-,PMA,+,+,+,-,-,-,-,-,-,-,-,-,SB203580,_,-,-,-,+,-,-,+,-,-,-,+,PD98059,-,-,-,-,+,-,-,+,-,-,+,-,细胞信号分子的荧光标记,LPS,EGF,Control,p38,p38,p38,p38,p38,p38,p38,p38,LPS,、,UV,刺激心肌细胞共聚焦显微镜大体扫描,LPS,Control,UV,细胞信号分子的三维结构分析,蛋白质三维结构分析对于揭示信号分子的功能的作用,蛋白质与蛋白质相互作用的研究,PH,TH,SH3,SH2,Kinase,Btk,SH3,SH2,Kinase,Src,蛋白激酶结构域,1,、蛋白质结合实验,2,、免疫共沉淀实验,2,、,FRET,和,BRET,与信号分子相互作用蛋白质分子的相互作用,酵母双杂合系统,Identification of MEF2C as a substrate for p38,第一步 附着,第二步激活,第三步,紧密粘附,第四步渗出,EC,EC,：,趋化因子受体,：PECAM,：白细胞整合素,：选择素,：选择素配体,：白细胞激活物,：,Ig,家族成员,图,12.1,白细胞的渗出过程,噬菌体展示技术,报告基因技术,研究细胞信号转导通路通过转录因子对基因启动子转录活性的影响。,0,5,1,0,1,5,Relative,luciferase,activity,Control,LPS,EGF,LPS+FHPI,p38 is involved in the enhancement of TNF-,promoter,transactivity,Induction of c-Jun through MEF2C,phosphorylation,by p38,蛋白质与核酸相互作用技术,研究细胞内蛋白质尤其是转录因子与特定核酸序列的相互作用。,EMSA,对细胞内信号分子的干预技术,研究细胞内蛋白质尤其是转录因子与特定核酸序列的相互作用。,反义核酸技术的应用,RNAi,RNAi,Figure 1.,Effects of,mex,-3,RNA interference on levels of the endogenous,mRNA,.,Nomarski,DIC micrographs show,in,situ,hybridization of 4-cell stage embryos.(,A,)Negative control showing lack of staining in the absence of the hybridization probe.(,B,)Embryo from,uninjected,parent showing normal pattern of endogenous,mex,-3,RNA(purple staining).(,C,)Embryo from parent injected with purified,mex,-3,antisense,RNA.These embryos(and the parent animals)retain,mex,-3,mRNA,although levels may be somewhat less than wild type.(,D,)Late 4-cell stage embryo from a parent injected with,dsRNA,corresponding to,mex,-3,;no,mex,-3,RNA is detected.(Templates used for interfering RNA and,in,situ,probes were largely non-overlapping.),信号分子基因表达检测技术,研究细胞内,mRNA,表达水平。,Heart,Brain,Placenta,Lung,Liver,Skeletal Muscle,Kidney,Pancreas,p38,p38,p38,p38,kb,3.5-,2.5-,1.8-,2.0-,Tissue distribution of,mRNA,of p38,isoforms,p38,a、,b、g、d,ATF2,MEF2C,CHOP10,SAP1,TNF-,a,mRNA,etc.,monocyte,macrophage,Rt,LPS,MKK3/,6,MKKKs,nucleus,TK,CD14,PRAK,sHSP,Cytoskeleton,?,LBP,？,THANKS,FOR YOUR ATTENTION,