PDK分子PH结构域与蛋白激酶B在胃癌细胞抗脱落凋亡中的相互作用
摘要
PKB(Protein Kinase B：蛋白酶B)是与PKC结构同源的Ser/Thr蛋白激酶，具有促进细胞增殖，抵抗细胞脱落凋亡的生物学效应。PH结构域(pleckstrin homology domain)是蛋白分子内由约120个氨基酸组成的保守序列，可介导蛋白分子之间的相互识别、结合，进而影响蛋白激酶对底物蛋白分子的活化水平。有研究提示，PKB分子的活化是通过蛋白分子的PH结构域与胞浆膜PDK(phosphoinostide depent protein kinase：磷脂酰肌醇依赖性蛋白激酶)分子的结合、催化而激活的，本实验即研究：①PDK分子对PKB分子的磷酸化调控作用；②PDK影响PKB分子活化后的宿主细胞生物学功能改变的特点。
实验中，构建了PDK分子PH结构域原核表达载体pRSETB-PDK PH，褪皮素(RxR)诱导表达的真核表达载体pIND(SPI)-pVgRxR-PDK PH，腺病毒质粒Ad-PDK PH三个表达载体，分别进行了原核蛋白表达，哺乳动物细胞(人胃癌细胞SGC7901)的稳定转化和裸鼠体内的治疗实验，根据实验所得原核蛋白表达、真核细胞稳定转染后RT-PCR、DNAladder、FCM、Western Blotting和裸鼠体内治疗实验结果，可以明确：
1．PDK PH结构域的原核表达质粒pRSETB-PDK PH可在E-Coli
JM 109(DE3)中稳定表达可溶蛋白。
2.实验结果证实，转染了目的基因PKD PH Domain cDNA序列的
人胃癌细胞株SGC7901在外源刺激IGF(胰岛素类生长因子)存在和目
的基因诱导表达剂RXR诱导下，可在真核表达载体pIND(sPI)中稳定
表达目的蛋白并发挥作用。
3.胰岛素或生长因子诱导的真核细胞生化活动过程中，PDK分子
PH结构域在真核细胞中的过表达可以引起细胞尤其是肿瘤细胞的凋亡
形为，随着PDK分子PH结构域蛋白表达水平的逐步提高，凋亡形为逐
步增强。
4.在胰岛素或生长因子诱导的细胞生化活动过程中，PDK分子PH
结构域在真核细胞中的过表达可以引起PKB分子磷酸化水平的降低，说
明PKB分子的磷酸化活化受PDK分子的调控，两者呈反相相关关系。
5.PDK分子PH结构域目的蛋白的过表达引起的细胞凋亡的机制主
要是下调了起抵抗细胞凋亡作用的PKB分子的磷酸化水平，从而降低其
活性，无法发挥积极的生化作用。
PKB(protein Kinase B) is a kind of Ser/Thr protein Kinase common to PKC structure, functioning to stimulate cellular proliferation and resist apoptosis. Pleckstrin homology domain is a conservative sequence containing about of 120 amino acids. It can mediate the identification and combination between different protein molecules, mediate protein-protein and protein-phospholipid interaction, then affect the activation of protein kinase to substrate protein molecules. Some reports have showed that activation of PKB molecule depends on the excitation induced by the combination and catalysis between Pleckstrin homology domain and plasma membrane's PDK(phosphoinostide depent protein kinase) molecule. This study is aimed to observe l.the phosphorylation regulation of PDK molecule to PKB molecule, 2.the characteristic of PDK affecting the biological function of host cell after PKB molecule's activation.
pRSETB-PDK PH, Prokaryotic express vector of PDK molecule's Pleckstrin homology domain ; pIND(SPI)-pVgRxR-PDK PH, eucaryotic express vector induced by RxR; and adeno virus plasmid Ad-PDK PH were established in the experiment. Prokaryotic protein expression, stable transformation of mammal animal cell(human gastric carcinoma cell SGC7901) and the test in vivo of nude mice were undertaken respectively. According to the results of Prokaryotic protein expression, stable transfection of eucaryotic cell TR-PCR, DNA ladder, FCM, Western Blotting and internal treatment of nude mice, the following conclusion were definite.
1. Prokaryotic express plasmid pRSETB-PDK PH of PDK's Pleckstrin homology domain could express soluble protein constantly in E coli JM109(DE3);
2. Human gastric carcinoma cell strain SGC7901 transfecting object gene PKD PH Domain cDNA sequence could express object gene protein stably and work well in eucaryotic express vector pIND(SPI) on the condition of the existence of stimulator IGF and express reagent of object gene RxR;
3. In the course of biochemical activity of eucaryotic cell induced by IGF or Growth Factor, overexpression of PDK molecule's Pleckstrin homology domain could cause apoptosis in eucaryotic cell, especially of the tumor cell with good dose-effect relationship;
4. Overexpression of PDK molecule's Pleckstrin homology domain in eucaryotic cell could down-regulete the phosphorylation level of PKB molecule, indicating the opposite regulation of PDK to the phosphorylation of PKB molecule in the course of cellular biochemical activity induced by IGF or Growth Factor;
5. The main mechanism of apoptosis induced by object gene protein overexpression of PDK molecule's Pleckstrin homology domain is to reduce PKB molecule's phosphorylation level of apoptosis-resisting cells, thus decrease their activity and unable to play an active biochemical role.
实验中，构建了PDK分子PH结构域原核表达载体pRSETB-PDK PH，褪皮素(RxR)诱导表达的真核表达载体pIND(SPI)-pVgRxR-PDK PH，腺病毒质粒Ad-PDK PH三个表达载体，分别进行了原核蛋白表达，哺乳动物细胞(人胃癌细胞SGC7901)的稳定转化和裸鼠体内的治疗实验，根据实验所得原核蛋白表达、真核细胞稳定转染后RT-PCR、DNAladder、FCM、Western Blotting和裸鼠体内治疗实验结果，可以明确：
1．PDK PH结构域的原核表达质粒pRSETB-PDK PH可在E-Coli
JM 109(DE3)中稳定表达可溶蛋白。
2.实验结果证实，转染了目的基因PKD PH Domain cDNA序列的
人胃癌细胞株SGC7901在外源刺激IGF(胰岛素类生长因子)存在和目
的基因诱导表达剂RXR诱导下，可在真核表达载体pIND(sPI)中稳定
表达目的蛋白并发挥作用。
3.胰岛素或生长因子诱导的真核细胞生化活动过程中，PDK分子
PH结构域在真核细胞中的过表达可以引起细胞尤其是肿瘤细胞的凋亡
形为，随着PDK分子PH结构域蛋白表达水平的逐步提高，凋亡形为逐
步增强。
4.在胰岛素或生长因子诱导的细胞生化活动过程中，PDK分子PH
结构域在真核细胞中的过表达可以引起PKB分子磷酸化水平的降低，说
明PKB分子的磷酸化活化受PDK分子的调控，两者呈反相相关关系。
5.PDK分子PH结构域目的蛋白的过表达引起的细胞凋亡的机制主
要是下调了起抵抗细胞凋亡作用的PKB分子的磷酸化水平，从而降低其
活性，无法发挥积极的生化作用。
PKB(protein Kinase B) is a kind of Ser/Thr protein Kinase common to PKC structure, functioning to stimulate cellular proliferation and resist apoptosis. Pleckstrin homology domain is a conservative sequence containing about of 120 amino acids. It can mediate the identification and combination between different protein molecules, mediate protein-protein and protein-phospholipid interaction, then affect the activation of protein kinase to substrate protein molecules. Some reports have showed that activation of PKB molecule depends on the excitation induced by the combination and catalysis between Pleckstrin homology domain and plasma membrane's PDK(phosphoinostide depent protein kinase) molecule. This study is aimed to observe l.the phosphorylation regulation of PDK molecule to PKB molecule, 2.the characteristic of PDK affecting the biological function of host cell after PKB molecule's activation.
pRSETB-PDK PH, Prokaryotic express vector of PDK molecule's Pleckstrin homology domain ; pIND(SPI)-pVgRxR-PDK PH, eucaryotic express vector induced by RxR; and adeno virus plasmid Ad-PDK PH were established in the experiment. Prokaryotic protein expression, stable transformation of mammal animal cell(human gastric carcinoma cell SGC7901) and the test in vivo of nude mice were undertaken respectively. According to the results of Prokaryotic protein expression, stable transfection of eucaryotic cell TR-PCR, DNA ladder, FCM, Western Blotting and internal treatment of nude mice, the following conclusion were definite.
1. Prokaryotic express plasmid pRSETB-PDK PH of PDK's Pleckstrin homology domain could express soluble protein constantly in E coli JM109(DE3);
2. Human gastric carcinoma cell strain SGC7901 transfecting object gene PKD PH Domain cDNA sequence could express object gene protein stably and work well in eucaryotic express vector pIND(SPI) on the condition of the existence of stimulator IGF and express reagent of object gene RxR;
3. In the course of biochemical activity of eucaryotic cell induced by IGF or Growth Factor, overexpression of PDK molecule's Pleckstrin homology domain could cause apoptosis in eucaryotic cell, especially of the tumor cell with good dose-effect relationship;
4. Overexpression of PDK molecule's Pleckstrin homology domain in eucaryotic cell could down-regulete the phosphorylation level of PKB molecule, indicating the opposite regulation of PDK to the phosphorylation of PKB molecule in the course of cellular biochemical activity induced by IGF or Growth Factor;
5. The main mechanism of apoptosis induced by object gene protein overexpression of PDK molecule's Pleckstrin homology domain is to reduce PKB molecule's phosphorylation level of apoptosis-resisting cells, thus decrease their activity and unable to play an active biochemical role.
引文
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2． Chen JQ ,God win AK, Bellacosa A, et al. Akt2, a putative oncogene encoding a member of a subfa mily of protein-serin/ threonine kinases, is a m plified in hu man ovarian carcinomas. Proc Natl Acad Sci USA, 1992 , 89:9267-9271．
3． Ahmed NN, Franke TF, Bellacosa A, et al . The proteins encoded by c-akt differ in post-translational modification, subcellular localization and oncogenic potential. Oncogene, 1993,8:1957-1963．
4． Haslam RJ, Koide HB , Hemminhd BA . Pleckstrin domain homology. Nature, 1993,363:309-310．
5． Cantley LC , Neel BC . New insight into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/ AKT pathway . Proc Natl Acad Sci USA. 1999, 96: 4240-4245．
6． Burgering BM, Coffer PJ . Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction . Nature, 1995, 376: 599-602．
7． Alessi,D.R., Deak,M., Casamayor,A., Caudwell,F.B., Morrice,N., Norman,D.G., Gaffney,P., MacDougall,C.N., Reese,C.B., Harbison,D., Ashworth,A. and Bownes,M.3-Phosphoinositide-dependent protein kinase-1 (PDK1) : structural and functional homology with the Drosophila DSTPK61 kinase Curr. Biol. 7 (10) , 776-789 (1997)
8． Nunez E,Wei X,Delgado C, Rodriguez-Crespo I, Yelamos B, Gomez-gutierrez J,Peterson D L et al. Cloning,expression, and purification of histidine-tagged preS domain of hepatitis B virus. Protein Expr Purif,2001,21(1) : 183-191
9． August-A, Sadra-A, Dupont-B, et at. re-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase.Proc-Natl-Acad-Sci-U-S-A. 1997 Oct 14; 94(21) : 11227-32
10． Backer-JM, Wjasow-C, Zhang-Y. Vitro binding and phosphorylation of insulin receptor substrate 1 by the insulin receptor. Role of interactions mediated by the
phosphotyrosine-binding domain and the pleckstrin-homology domain. Eur-J-Biochem. 1997 Apr 1; 245(1) : 91-6
11． Bae YS, Cantley LG, Chen CS et al. Activation of phospholipase C-gamma by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998 Feb 20; 273(8) : 4465-9
12． Backer J M, Wjasow C, Zhang Y. In vitro binding and phosphorylation of insulin receptor substrate 1 by the insulin receptor. Role of interactions mediated by the phosphotyrosine-binding domain and the pleckstrin-homology domain. Eur J Biochem,1997,245(1) : 91-96)
13． Blobe-GC; Obeid-LM; Hannun-YA. Regulation of protein kinase C and role in cancer biology. Cancer-Metastasis-Rev. 1994 Dec; 13(3-4) : 411-31
14． Eugene S,Kandel and Nissim Hay.The regulation and Activities of the Multifunctional Serine/Threonine Kinase Akt/PKB.Experimental Cell Research, 1999,253(1) :210-229．
15． Eugene S,Kandel and Nissim Hay.The regulation and Activities of the Multifunctional Serine/Threonine Kinase Akt/PKB.Experimental Cell Research, 1999,253(1) :210-229．
16． Maehama, T. and Dixon, J. E. (1998) . The tumor suppressor. PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3． 4,5-trisphosphate. J. Biol. Chem. 273, 13375-13378 .
17． Myers, M. P.. Pass , I., Batty , I.H., Van der Kaay, J., Stolarov. J. P., Hemmings, B. A., Wigler, M. H., Downes, C. P., and Tonks, N. K.(1998) . The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc. Natl. Acad. Sci. USA 95, 13513-13518．
18． Alessi, D.R., Caudwell, F.B., Andjelkovic, M., Hemmings. B.A.,and Cohen, P.( 1996) . Molecular basis for the substrate specificity of protein kinase B: Comparison with MAPKAP kinase-1 and p70 S6 kinase . Fed . Eur. Biochem. Soc. 399,333-338．
19． Alessi, D., James, S., Downes, C.P., Holmes , A. ,Gaffney , P., Reese, C., and Cohen. P.(1997) . Characterizaation of a 3-phos-phoinositide-dependent protein kinase which phosphorylates and activates protein kinase Ba.Curr .Biol.7． 261-269 .
20． Alessi, D.R., Deak ,M., Casamayor, A.,Caudwell, F.B., Morrice , N., Norman, D.G., Gaffney, P., Reese, C.B., MacDougall, C.N., Harbison, D., Ashworth , A., and Bownes,
M.(1997) .3-Phosphoinositide-dependent protein kinase-1 (PDK1) : Structural and functional homology with the Drosophila DSTPK61 kinase . Curr. Biol. 7, 776-789 .
21． J.Sambrook E.F. Fritsch T. Maniatis.et al. Molecular Cloning, Cold Spring Harbor Laboratary Press,2000．
22． Myers, M. P., Pass , I., Batty , I.H., Van der Kaay, J., Stolarov, J. P., Hemmings, B. A., Wigler, M. H., Downes, C. P., and Tonks, N. K.(1998) . The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc. Natl. Acad. Sci. USA 95, 13513-13518
23． Tanaka,K.,Adachi,H.,Konishi,H.,Iwamstsu,A.,Ohdawa,K.,Shirae,T.,Nagata,S.,Kikka wa,U.,and Fudui,Y.(1999) .identification of protein dinase B(PKB)as a phosphatidylinositol3,4,5-trisphosphate binding protein in Dictyostelium discoidium.Biosci.Biotecgnol.Biochem.63,368-372．
24． Frandi,T.F.,Yang,s.,Chan,T.O.,Datta,K,Kazlauskas,A.,Morrison,D.K.,Kaplan,K.R.,an d Tsichlis,P.N.( 1995) .The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase.Cell 81,727-736．
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2． Chen JQ ,God win AK, Bellacosa A, et al. Akt2, a putative oncogene encoding a member of a subfa mily of protein-serin/ threonine kinases, is a m plified in hu man ovarian carcinomas. Proc Natl Acad Sci USA, 1992 , 89:9267-9271．
3． Ahmed NN, Franke TF, Bellacosa A, et al . The proteins encoded by c-akt differ in post-translational modification, subcellular localization and oncogenic potential. Oncogene, 1993,8:1957-1963．
4． Haslam RJ, Koide HB , Hemminhd BA . Pleckstrin domain homology. Nature, 1993,363:309-310．
5． Cantley LC , Neel BC . New insight into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/ AKT pathway . Proc Natl Acad Sci USA. 1999, 96: 4240-4245．
6． Burgering BM, Coffer PJ . Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction . Nature, 1995, 376: 599-602．
7． Alessi,D.R., Deak,M., Casamayor,A., Caudwell,F.B., Morrice,N., Norman,D.G., Gaffney,P., MacDougall,C.N., Reese,C.B., Harbison,D., Ashworth,A. and Bownes,M.3-Phosphoinositide-dependent protein kinase-1 (PDK1) : structural and functional homology with the Drosophila DSTPK61 kinase Curr. Biol. 7 (10) , 776-789 (1997)
8． Nunez E,Wei X,Delgado C, Rodriguez-Crespo I, Yelamos B, Gomez-gutierrez J,Peterson D L et al. Cloning,expression, and purification of histidine-tagged preS domain of hepatitis B virus. Protein Expr Purif,2001,21(1) : 183-191
9． August-A, Sadra-A, Dupont-B, et at. re-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase.Proc-Natl-Acad-Sci-U-S-A. 1997 Oct 14; 94(21) : 11227-32
10． Backer-JM, Wjasow-C, Zhang-Y. Vitro binding and phosphorylation of insulin receptor substrate 1 by the insulin receptor. Role of interactions mediated by the
phosphotyrosine-binding domain and the pleckstrin-homology domain. Eur-J-Biochem. 1997 Apr 1; 245(1) : 91-6
11． Bae YS, Cantley LG, Chen CS et al. Activation of phospholipase C-gamma by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998 Feb 20; 273(8) : 4465-9
12． Backer J M, Wjasow C, Zhang Y. In vitro binding and phosphorylation of insulin receptor substrate 1 by the insulin receptor. Role of interactions mediated by the phosphotyrosine-binding domain and the pleckstrin-homology domain. Eur J Biochem,1997,245(1) : 91-96)
13． Blobe-GC; Obeid-LM; Hannun-YA. Regulation of protein kinase C and role in cancer biology. Cancer-Metastasis-Rev. 1994 Dec; 13(3-4) : 411-31
14． Eugene S,Kandel and Nissim Hay.The regulation and Activities of the Multifunctional Serine/Threonine Kinase Akt/PKB.Experimental Cell Research, 1999,253(1) :210-229．
15． Eugene S,Kandel and Nissim Hay.The regulation and Activities of the Multifunctional Serine/Threonine Kinase Akt/PKB.Experimental Cell Research, 1999,253(1) :210-229．
16． Maehama, T. and Dixon, J. E. (1998) . The tumor suppressor. PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3． 4,5-trisphosphate. J. Biol. Chem. 273, 13375-13378 .
17． Myers, M. P.. Pass , I., Batty , I.H., Van der Kaay, J., Stolarov. J. P., Hemmings, B. A., Wigler, M. H., Downes, C. P., and Tonks, N. K.(1998) . The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc. Natl. Acad. Sci. USA 95, 13513-13518．
18． Alessi, D.R., Caudwell, F.B., Andjelkovic, M., Hemmings. B.A.,and Cohen, P.( 1996) . Molecular basis for the substrate specificity of protein kinase B: Comparison with MAPKAP kinase-1 and p70 S6 kinase . Fed . Eur. Biochem. Soc. 399,333-338．
19． Alessi, D., James, S., Downes, C.P., Holmes , A. ,Gaffney , P., Reese, C., and Cohen. P.(1997) . Characterizaation of a 3-phos-phoinositide-dependent protein kinase which phosphorylates and activates protein kinase Ba.Curr .Biol.7． 261-269 .
20． Alessi, D.R., Deak ,M., Casamayor, A.,Caudwell, F.B., Morrice , N., Norman, D.G., Gaffney, P., Reese, C.B., MacDougall, C.N., Harbison, D., Ashworth , A., and Bownes,
M.(1997) .3-Phosphoinositide-dependent protein kinase-1 (PDK1) : Structural and functional homology with the Drosophila DSTPK61 kinase . Curr. Biol. 7, 776-789 .
21． J.Sambrook E.F. Fritsch T. Maniatis.et al. Molecular Cloning, Cold Spring Harbor Laboratary Press,2000．
22． Myers, M. P., Pass , I., Batty , I.H., Van der Kaay, J., Stolarov, J. P., Hemmings, B. A., Wigler, M. H., Downes, C. P., and Tonks, N. K.(1998) . The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc. Natl. Acad. Sci. USA 95, 13513-13518
23． Tanaka,K.,Adachi,H.,Konishi,H.,Iwamstsu,A.,Ohdawa,K.,Shirae,T.,Nagata,S.,Kikka wa,U.,and Fudui,Y.(1999) .identification of protein dinase B(PKB)as a phosphatidylinositol3,4,5-trisphosphate binding protein in Dictyostelium discoidium.Biosci.Biotecgnol.Biochem.63,368-372．
24． Frandi,T.F.,Yang,s.,Chan,T.O.,Datta,K,Kazlauskas,A.,Morrison,D.K.,Kaplan,K.R.,an d Tsichlis,P.N.( 1995) .The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase.Cell 81,727-736．
25． Cross,D.A.,Aless,D.R.,Dohen,P., Andjelkovich,M.,and Hemmings,B.A.(1995) . Inhibition of glycogen synthase kinase-3 by insulin mediated by pritein kinase B.Naturi 378,785-789．
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