The Study Of Regulatory Function Of PDK PH Domain And PKB Protein Kinase On Anti-apoptosis In Gastric Cancer Cell SGC7901

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.