信号通路异常与疾病及技术方法课件.ppt

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 Phosphopeptide map of HSP27 hosphorylated by PRAK in vitro Chromatography Electrophoresis pH1.9 + MAPKAPK2 Chromatography Electrophoresis pH1.9 + PRAK Chromatography Electrophoresis pH1.9 MIX + 二、蛋白磷酸化位点分析及其功能鉴定 + + + + - - - - + - 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) T182 is the regulatory phosphorylation site of PRAK PCR primer PCR primer 三、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 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 DFGLARHTDDEMTGYVATRWYRAPE 25 hP38b DFGLARQADEEMTGYVATRWYRAPE 25 hp38g DFGLARQADSEMTGYVVTRWYRAPE 25 hp38d DFGLARHADAEMTGYVVTRWYRAPE 25 hJNK1 DFGLARTAGTSFMMTPYVVTRYYRAPE 27 hJNK2 DFGLARTACTNFMMTPYVVTRYYRAPE 27 hJNK3 DFGLARTAGTSFMMTPYVVTRYYRAPE 27 hERK1 DFGLARIADPEHDHTGFLTEYVATRWYRAPE 31 hERK2 DFGLARVADPDHDHTGFLTEYVATRWYRAPE 31 hBMK1 DFGMARGLCTSPAEHQYFMTEYVATRWYRAPE 32 YHOG1 DFGLARIQDPQMTGYVSTRWYRAPE 25 YSMK1 DFGLARGIHAGFFKCHS--TVQPHITNYVATRWYRAPE 36 YMPK1 DFGLARGYSENPVENSQFLTEYVATRWYRAPE 32 YKSS1 DFGLARCLASSSDSRETLVGFMTEYVATRWYRAPE 35 YFUS3 DFGLARIIDESAADNSEPTGQQSGMTEYVATRWYRAPE 38 domain VII