Akt1 Protein Complex Purification And Identification Of Interacting Proteins Function

Systematic study of components and dynamic process of signaling transduction pathway net, which also has been the focus of study of "mics" presently, is the key to understanding the regulation and function mechanism of its own. Insulin signaling pathway net is in charge of physical metabolism and cellular growth, study of the dynamic change of protein complexes composed of important molecules of which will help us to understand the molecular mechanism of its regulation in all-round way.Tandem Affinity Purification (TAP) is a new technology for study of protein complexes, whose biggest advantage compared with other technics of purification is it can enrich protein complexes close to the physiological level efficiently. Taking advantage of advanced experiences of study of protein complexes in signaling transduction pathway from abroad and using TAP-MS, we have undertaken the research on insulin signaling pathway net.First, we successfully introduced the Ecdysone-lnducible Mammalian Expression System into the Tandem Affinity Purification system by means of reconstruction of expression plasmid and construction of stable human hepatoma G2(HepG2) cell line capable of expressing ecdysone receptor. As a result, we can control the expression level of the bait close to the native physiological level through controlling the dose of inducing agent. This technique may be applied widely to the study of high eukaryotic cell protein complexes.Meanwhile, we selected eleven molecules in insulin signaling pathway as the target genes and constructed their expression vectors of plND-HisA/5, which is the foundation of subsequent study.In order to examine the feasibility of TAP-MS in our experiment, we first selected Akt1 as the target of our study. Akt1, also known as PKB, is one of the key molecules in PI3K/Akt signaling pathway. It regulates a diverse array of cellular functions, including intermediary metabolism, cell migration, gene expression, cell growth and survival. Purification and identification of Akt1 complex will shed light on the function and regulation mechanisms of it. Based on the HepG2 cell line which expressed ecdysone receptor, we constructed stable cell line expressing Akt1-TAP. Then we separated and purified Akt1 protein complex from HepG2. After mass spectrometry(MS) analysis of the complex, 87 proteins including the bait protein were found. To estimate our data, we have used several independent bioinformatics analysis to evaluate the potential biological relevance of the identified interactions. By systematically searching the PubMed databases for biological functions of the molecules, we found 4 interactions have been reported and 16 interactions whose partners show linkage of the corresponding gene symbols. By analysis GO annotation, we found 14 interactions whose partners appear on the same GO hierarchy level which is from a depth of four onward in the GO hierarchy. By integrating with phenotypic profiling(phenome) data, we found 20 interactions whose partners show similar gene knockout phenotype. By searching the protein sequences, we found 30 interactions which have the Akt1 potential phosphorylation sites. These results indicate that our interactions are of high condifidence to same extent.Using bioinformatics analysis information, we established a scoring system for confidence evaluation. Each interaction was scored with this system, resulting in 36 interactions with medium and high confidence. Then we confirmed the interactions of six proteins of them with Akt1 using co-immunoprecipitation assay, the results of which indicated that five proteins (ANAPC2,MYLK2,PEX19,AIF and HEB) could interact with Akt1. Further study of Akt1/AIF and Akt1/HEB suggested that the cellular biological functions of HEB and AIF have relationship to Akt1. The results indicated that activated Akt1 could inhibits the nuclear translocation of AIF under apoptosis stimulation. It is probably a new mechanism of Akt1 regulation cellular survival. HEB may be one of the substrates of Akt1 phosphorylation in addition.In conclusion, we purified Akt1 complex with TAP-MS and obtained some interactions of Akt1 with high confidence. We also found some potential substrates of Akt1 and potential kinases which can activate Akt1. This will help us to understand the complicated regulations for Akt1 functions. At the same time, TAP-MS strategy will help us to carry out the study of insulin signaling pathway.