信號通路研究的技術(shù)方法

信號通路研究的技術(shù)方法一、蛋白激酶磷酸化及其活性測定二、蛋白磷酸化位點(diǎn)分析及其功能鑒定三、DNA重組技術(shù)的應(yīng)用四、病毒技術(shù)對于揭示細(xì)胞信號通路的作用五、細(xì)胞信號分子特異性抑制劑的應(yīng)用六、細(xì)胞信號分子的熒光標(biāo)記七、細(xì)胞信號分子的三維結(jié)構(gòu)分析八、蛋白質(zhì)與蛋白質(zhì)相互作用的研究九、報告基因技術(shù)十、蛋白質(zhì)與核酸相互作用技術(shù)十一、對細(xì)胞內(nèi)信號分子的干預(yù)技術(shù)十二、信號分子基因表達(dá)檢測技術(shù)蛋白激酶磷酸化的檢測裂解培養(yǎng)的細(xì)胞獲得裂解上清以免疫共沉淀法獲得蛋白激酶SDS再加入蛋白激酶磷酸化特異性抗體以PhosphoAb-HRPWestern化學(xué)發(fā)光檢測試劑盒測定蛋白激酶的磷酸化程度。一、蛋白激酶磷酸化及其活性測定經(jīng)典蛋白激酶活性分析實(shí)驗流程以免疫共沉淀法獲得蛋白激酶加入該激酶底物和32-ATP,激酶使底物磷酸化放射自顯影,確定磷酸化條帶以免疫共沉淀法獲得蛋白激酶加入該激酶底物,激酶使底物磷酸化再加入底物磷酸化特異性抗體以PhosphoAb-HRPWestern化學(xué)發(fā)光檢測試劑盒測定底物的磷酸化程度,進(jìn)而推出蛋白激酶活性非放射性蛋白激酶活性分析實(shí)驗流程較傳統(tǒng)激酶活性測定方法的優(yōu)點(diǎn)無需接觸放射性物質(zhì)敏感度高:可以檢測到每個磷酸化分子特異性:利用磷酸化特異性抗體可以進(jìn)行位點(diǎn)特異性分析信噪比高低背景系統(tǒng)完整:提供一整套直接從細(xì)胞裂解液中測試酶活的試劑節(jié)約時間:由幾天降到幾分鐘02468101214051020306090120Time(min)RelativekinaseactivityDynamicprocessesofp38activationbyLPSinRAWcellsEffectofindividualMAPKpathwaysonPRAKactivityinintactcellsMKK7(D)GST-ATF2GST-ELK1MKK1(E)MKK6(E)ControlHSP27PRAKControlAniso.ArseniteTNF-80kDa-47kDa-39kDa-80kDa-47kDakDa97.4-68.0-43.9-29.0-18.4-14.3-ControlAniso.ArseniteTNFABThemajorkinasesofp38

andp38P38MAPKinasePathwayPhosphopeptidemapofHSP27hosphorylatedbyPRAKinvitro

ChromatographyElectrophoresispH1.9+MAPKAPK2ChromatographyElectrophoresispH1.9+PRAKChromatographyElectrophoresispH1.9MIX+二、蛋白磷酸化位點(diǎn)分析及其功能鑒定++++----+-HSP27p38

GST-PRAK(93A)GST-PRAK(WT)GST-PRAK(186A)GST-PRAK(212A214A)GST-PRAK(182A)GST-PRAK(WT)GST-PRAK(182D)GST-PRAK(182D212D)+-+-+-FoldofActivation05101520GST-PRAK(182A)GST-PRAK(182D)GST-PRAK(WT)GST-PRAK(93A)GST-PRAK(186A)GST-PRAK(182D212D)GST-PRAK(212A214A)BAElectrophoresispH8.9++p38

-PRAK(182A)+p38

-PRAK(182D)p38

-PRAK(wt)T182istheregulatoryphosphorylationsiteofPRAKPCRprimerPCRprimer三、DNA重組技術(shù)的應(yīng)用活性誘變體無活性誘變體結(jié)構(gòu)與功能的研究JNK2JNK3JNK1p38p38

p38

p38

ERK1ERK2ERK5TherelationshipbetweenthemembersofMAPKfamilyMAPK

DuralPhosphorylationSitesL-12Length

**hp38 DFGLARHTDDEMTGYVATRWYRAPE 25hP38bDFGLARQADEEMTGYVATRWYRAPE 25hp38g DFGLARQADSEMTGYVVTRWYRAPE 25hp38dDFGLARHADAEMTGYVVTRWYRAPE 25hJNK1DFGLARTAGTSFMMTPYVVTRYYRAPE 27hJNK2DFGLARTACTNFMMTPYVVTRYYRAPE 27hJNK3DFGLARTAGTSFMMTPYVVTRYYRAPE 27hERK1DFGLARIADPEHDHTGFLTEYVATRWYRAPE 31hERK2DFGLARVADPDHDHTGFLTEYVATRWYRAPE 31hBMK1DFGMARGLCTSPAEHQYFMTEYVATRWYRAPE 32YHOG1DFGLARIQDPQMTGYVSTRWYRAPE 25YSMK1DFGLARGIHAGFFKCHS--TVQPHITNYVATRWYRAPE 36YMPK1DFGLARGYSENPVENSQFLTEYVATRWYRAPE 32YKSS1DFGLARCLASSSDSRETLVGFMTEYVATRWYRAPE 35YFUS3DFGLARIIDESAADNSEPTGQQSGMTEYVATRWYRAPE 38domainVIIVIIILoop-12(T-Loop)sequenceofMAPkinasesConstructionofp38loop-12toERKlikestructure

p38...DFGLARHTDDEMTGYVATRWYRAPE...p38(E)...DFGLARHTDDEMTEYVATRWYRAPE...p38(6+)...DFGLARHTDDEHDHTGFMTGYVATRWYRAPE...p38(6+E)...DFGLARHTDDEHDHTGFMTEYVATRWYRAPE...p38(VAP)...DFGLARVADPEMTGYVATRWYRAPE...p38(DL)...DFGLARHTDDDLTGYVATRWYRAPE...p38(VAPD6+LE)...DFGLARVADPDHDHTGFLTEYVATRWYRAPE...ERK2...DFGLARVADPDHDHTGFLTEYVATRWYRAPE...MAPKMKKSubstratesLoop-12isakeystructuretodeterminetheselectionofsubstrate

(...TXY...)四、病毒技術(shù)對于揭示細(xì)胞信號通路的作用腺病毒逆轉(zhuǎn)錄病毒幾種MAPK通路抑制劑的化學(xué)結(jié)構(gòu)示意圖:ONH2OCH3OPD98059HNNSOFCH3NOHNFNHNSB202190SB203580CH=CH

CH=CH

OHOCH3OHOCH3OOCurcumin五、細(xì)胞信號分子特異性抑制劑的應(yīng)用EffectsofSB203580andPD98059onendogenousPRAKactivityHSP27TNF

+++---------Arsenite+++---------PMA+++---------SB203580_---+--+---+PD98059----+--+--+-LPSEGFControl六、細(xì)胞信號分子的熒光標(biāo)記p38p38

p38

p38

p38p38

p38

p38

LPS、UV刺激心肌細(xì)胞共聚焦顯微鏡大體掃描LPSControlUV蛋白質(zhì)三維結(jié)構(gòu)分析對于揭示信號分子的功能的作用七、細(xì)胞信號分子的三維結(jié)構(gòu)分析PHTHSH3SH2KinaseBtkSH3SH2KinaseSrc蛋白激酶結(jié)構(gòu)域八、蛋白質(zhì)與蛋白質(zhì)相互作用的研究1.蛋白質(zhì)結(jié)合實(shí)驗2.免疫共沉淀實(shí)驗3.FRET和BRET熒光能量共振轉(zhuǎn)移(Fluorescenceresonanceenergytransfer)生物發(fā)光共振能量轉(zhuǎn)移(bioluminescenceresonanceenergytransfer)4.酵母雙雜合系統(tǒng)IdentificationofMEF2Casasubstrateforp38第一步附著第二步激活第三步緊密粘附第四步滲出ECEC：趨化因子受體：PECAM：白細(xì)胞整合素：選擇素：選擇素配體：白細(xì)胞激活物：Ig家族成員白細(xì)胞的滲出過程

5.噬菌體展示技術(shù)九、報告基因技術(shù)研究細(xì)胞信號轉(zhuǎn)導(dǎo)通路通過轉(zhuǎn)錄因子對基因啟動子轉(zhuǎn)錄活性的影響。051015RelativeluciferaseactivityControlLPSEGFLPS+FHPIp38isinvolvedintheenhancementofTNF-

promotertransactivityInductionofc-JunthroughMEF2Cphosphorylationbyp38EMSA十、蛋白質(zhì)與核酸相互作用技術(shù)研究細(xì)胞內(nèi)蛋白質(zhì)尤其是轉(zhuǎn)錄因子與特定核酸序列的相互作用。反義核酸技術(shù)的應(yīng)用十一、對細(xì)胞內(nèi)信號分子的干預(yù)技術(shù)研究細(xì)胞內(nèi)蛋白質(zhì)尤其是轉(zhuǎn)錄因子與特定核酸序列的相互作用。RNAiRNAiFigure1.Effectsofmex-3RNAinterferenceonlevelsoftheendogenousmRNA.NomarskiDICmicrographsshowinsituhybridizationof4-cellstageembryos.(A)Negativecontrolshowinglackofstainingintheabsenceofthehybridizationprobe.(B)Embryofromuninjectedparentshowingnormalpatternofendogenousmex-3RNA(purplestaining).(C)Embryofromparentinjectedwithpurifiedmex-3antisenseRNA.Theseembryos(andtheparentanimals)retainmex-3mRNA,althoughlevelsmaybesomewhatlessthanwildtype.(D)Late4-cellstageembryofromaparentinjectedwithdsRNAcorrespondingtomex-3;nomex-3RNAisdetected.(TemplatesusedforinterferingRNAandinsituprobeswerelargelynon-overlapping.)

十二、信號分子基因表達(dá)檢測技術(shù)研究細(xì)胞內(nèi)mRNA表達(dá)水平。HeartBrainPlacentaLungLiverSkeletalMuscleKidne