Screening Of Key Domain Of Affecting Cell Growth For HAph2 Gene

Human Aph2 gene(hAph2) was firstly identified and cloned from a human placenta cDNA library in our laboratory(GenBank Access No.AF258563),and bioinformatics analysis revealed that hAPH2 protein shares 96%identity with mouse APH2.hAph2,locating at the chromosome 10q24.1,has five isoforms including hAph2α,hAph2β,hAph2γ,hAph2δand hAph2ε,and encodes ZDHHC16(zinc finger, DHHC-type containing 16) proteins which contains DHHC domain and DPG domain, and the former is also known as NEW1.In this study,We focus on the expression of hAph2 in COS-7 cells and its influence on the growth of cells.After forty-eight hours of transfection,We utilized Flow Cytometry(FCM),western blotting and fluorescent immunocytochemistry to analyse the expression of wild hAph2α-GFP,hAph2β-GFP, hAph2γ-GFP,and hAph2δ-GFP in COS-7 cells.The results showed that hAPH2 proteins,which could induce apoptosis in COS-7 cells.Ectopic expressed hAph2 has polymer and monomer forms and mainly located at perinuclear cytoplasm.To further investigate the mechanism of hAph2-induced apoptosis in COS-7 cells,we also constructed the mutants of hAph2,that is,hAph2β⊿ADHHC-GFP and hAph2β⊿DPG-GFP.Then we utilized westem blotting to detect Caspases,MAPK superfamily members and other proteins related to apoptosis.The results showed that the expression of hAph2 increased the active caspases,and slightly activated JNK and Fas-FasL signal pathway.Our data showed that the expression forms of hAph2 in COS-7 cells,possible mechanism of hAph2-indueed apoptosis in COS-7 cells,and suggested both DHHC domain and DPG domain could protect COS-7 cells from apoptosis.We also utilized Co-Immuno-precipitation(Co-IP) and irnmunofluorescent subcellular co-localization to select proteins interacted with hAPH2βprotein,and indicated that hAPH2βcan interact with c-Abl in SMMC-7721 HCC cells.