Functional Prediction And Analysis Of P55PIK Protein

Phosphatidylinositol 3 kinase(PI3K)is an intracellular phosphatidylinositol kinase.Class IA PI3 K is a heterodimer consisting of a regulatory subunit and a catalytic subunit.The catalytic subunits include p110α、p110β and p110δ,while the regulatory subunits consist of p85α,p85β,p55α,p55γ(p55PIK),and p50α.It has been reported that the regulatory subunits have no enzymatic activity,but are needed to form a stable complex binding to the N-terminus of the p110 catalytic subunit by i SH2 domain,thus active the PI3 K.As an important regulatory subunit of PI3 K,p55PIK involves in progression of cell proliferation,differentiation and DNA synthesis.This regulatory subunit is closely associated with the formation of tumor,and its unique N-terminal structure was thought to play an important role in carcinogenesis.The shorter isoform of p55 PIK,p55PIK was revealed to be generated by alternative initiation of translation while the function of p50 PIK was not yet fully understood.Objective: In our currernt study,we sought to investigate the evolutionary conservation of p55 PIK protein,especially of the N terminal of p55 PIK in differernt phylum.Moreover,differernt isoforms of p55 PIK were identified and functional significance of each isoform was examined by using cell biology analysis and structrure biologiy based predication.In our work,the mechanisms of p55 PIK and its isoforms invloed in cell cycle regulation and DNA damage response were especially concerned.In sum,our study will suggest the distinct significance of p55 PIK isoforms and provide a potential target for further cancer therapy.Methods: Bioinformation analysis was conducted to examine the evolutionary conservation of p55 PIK in different phylum.Vectors for overexpression or RNA interfere were constructed and were introduced into cells by transfection or infection based on lentivirus.Protein levels of p55 PIK and isoforms were detected using Western blotting and effects of overexpression or knock down of p55 PIK or isoforms were evaluated with flow cytometry and Ed U staining.The subcellular localization was observed using immunofluorescence analysis.Results: Phylogenetic results showed that p55 PIK protein is highly conserved in amphibians,reptiles,fish,birds,mammals and humans,but not in lower invertebrates sucha as Drosophila and Caenorhabditis elegans.The plasmids of plenti-p55,plenti-p50,plenti-p48 and 3flag-p55,3flag-p50,3flag-p48 were successfully constructed by molecular cloning;The results of cell cycle assay showed that p55 PIK could promote cell cycle,p50 PIK had no obvious effect on cell cycle,and p55 PIK,p50PIK and p48 PIK were both localized in the nucleus.Conclusion: The results of the phylogenetic tree revealed that the p55 PIK protein,particularly the N-terminal peptide might have a specific function in higher vertebrates.All isoform of p55 PIK,including p55 PIK,p50PIK and p48 PIK were revealed to be localized in nucleus,suggesting their special function beyond PI3 K.p55PIK and p50 PIK were explored to have different effects on cell cycle,indicating that N24 has specific functions in cells.