Preliminary Investigation Of The Biological Functions Of Protein Kinase Akt

The protein kinase Akt is a serine/threonine kinase, which is also termed protein kinase B ,based on its homology to protein kinase A and protein kinase C. It is the cellular homolog to the retroviral oncogene viral akt (v-Akt). Akt in mammals comprises three highly homologous members known as Aktl, Akt2, and Akt3. Akt is a muti-domains protein, which includes a N-terminal pleckstrin homology domain ,a central kinase catalytic domain and a regulatory domain at the carboxyl-terminal region. Among them, PH domain can bind PIP3, a lipid second messenger essential for the translocation of Akt/PKB to the plasma membrane where it is phosphorylated and activated. So PH domain play an important role in the activation of Akt. The protein kinase Akt acts as the major downstream target of PI-3K and regulates the biologicalfunction of cells. Akt/PKB can promote protein synthesis and glycogen transportation. Akt/PKB has emerged as a crucial regulator of widely divergent cellular processes including apoptosis, proliferation, differentiation. Akt/PKB appears to play an important role in the progression of human tumors, and its potential machanism is the focus of signal transduction of apoptosis.In order to explore the biological function of protein kinase Akt, the research was composed of three relatively independent parts: the regulation function of Akt in tumorigenesis; the relationship between Akt and NDRG2; analysis of the secondary structure of Akt PH domain.To investigate the regulation function of Akt in tumorigenesis, we tried to intervene in the Akt signal pathway by increasing or reducing endogenous Akt expression, activating Akt or inhibiting Akt activity to investigate the effect on cell growth. Firstly, we successfully reduced endogenous expression of Aktl and Akt2 in breast cancer cell line MCF-7 by RNA interference and detected the result via western blotting and RT-PCR. Then we began to observe MCF-7 cells viability by MTT, cell cycle and apoptosis by flow cytometry, invasion efficiency by soft agar transformation assay. We can not detect significant difference in the biological characters of MCF-7 cells between pSilencer/Akt transfection group and negative control group. Secondly, we inhibited protein kinase Akt and PI-3kinase activity by chemical inhibitors and observe MCF-7 cell viability by MTT. The results showed that PI-3K inhibitors Wortmannin and LY294002, but not Akt inhibitor can weaken MCF-7...