Tissue-specific Expression Of 14-3-3ε And αN-catenin And Their Interaction

Objective: 14-3-3 is a highly conserved protein family, which is ubiquitously expressed in all eukaryotic species. Recent years, it was found that 14-3-3 proteins can interact with various proto-oncogene proteins, cytoskeletal proteins and signaling proteins, and play an important role in cell signaling transduction, cell cycle regulation, cell apoptosis and gene expression regulation.Catenin is a newly found protein family in recent years. As cellular adhesion molecule and cell signaling protein, it extensively exists in many kinds of tissues. Our previous studies found that 14-3-3εmay interact withαN-catenin by yeast two-hybrid. In this study, we identified the tissue distribution of these two proteins, constructed prokaryotic expression plasmid containing full coding sequence of 14-3-3ε, expressed 14-3-3εprotein, and further confirmed their interaction in vitro.Method:1 Tissue-specific expression of 14-3-3εandαN-catenin gene1.1 The distribution of 14-3-3εandαN-catenin mRNA at rat different tissuesTotal RNA of rat different tissues (artery, brain, lung, heart, liver, kidney, testis, intestine, spleen) was isolated using Trizol, identified by agarose gel electrophoresis, and assayed by OD260 and OD260/280. The level of 14-3-3εandαN-catenin mRNA was examined by RT-PCR.1.2 The distribution of 14-3-3εandαN-catenin protein at rat different tissuesTotal protein of rat different tissues (artery, brain, lung, heart, liver, kidney, testis, intestine, spleen) was extracted, separated by SDS-PAGE, and analyzed by Western blot with anti-14-3-3εand anti-αN-catenin antibody.2 Construction and identification of prokaryotic expression plasmid pGEX-4T-1-14-3-3εThe cDNA fragment coding 14-3-3εprotein was cloned into the pGEX-4T-1 vector. Recombinant plasmid was confirmed by analysis of restriction enzymes and DNA sequencing. After the inserted fragments proved correct, we named the plasmid as pGEX-4T-1-14-3-3ε.3 Expression and purification of GST-14-3-3εfusion protein The recombinant plasmid was transformed into BL21 E.coli strain. Transformed E.coli was induced by different IPTG concentrations and incubation times to identify the optimal induction condition. After induction, the cells were collected, and sonicated. The supernatant was purified via GST-Sepharose 4B affinity resin.4 GST pull-down analysis determined the interaction between 14-3-3εandαN-catenin in vitroThe protein extracted from rat artery was incubated with GST-Sepharose 4B containing GST-14-3-3εfusion protein. The pellets were collected and analyzed by Western blot with anti-αN-catenin antibody.Results:1 The distribution of 14-3-3εandαN-catenin mRNA at rat different tissuesThe results of RT-PCR showed, at transcription level, 14-3-3εwere expressed in all of nine tissues, and it was more abundant in artery, lung, kidney, testis and intestine.αN-catenin mRNA in various tissues was obviously less than 14-3-3ε, and it was not detected in kidney. Both mRNAs was plenty in artery.2 The distribution of 14-3-3εandαN-catenin protein at rat different tissuesThe results of Western blot showed, at translation level, 14-3-3εwas expressed abundantly in all of nine tissues, butαN-catenin protein was much less than 14-3-3ε. It existed in artery, brain, testis, intestine and spleen.3 Construction of prokaryotic expression plasmid pGEX- 4T-1-14-3-3εThe results of restriction enzyme analysis and DNA sequencing showed that the inserted fragment was correct in pGEX-4T-1-14-3-3ε.4 Expression of GST-14-3-3εfusion protein GST-14-3-3εfusion protein (MW 54 kD) could be expressed in E.coli transformed by pGEX-4T-1-14-3-3εafter IPTG induction. 5 Purification of GST-14-3-3εfusion proteinThe supernatant of bacteria lysates was purified via GST- Sepharose 4B affinity resin. SDS-PAGE analysis showed a 54 kD protein band.6 GST pull-down analysis confirmed the interaction between 14-3-3εandαN-catenin in vitroThe result of GST pull-down analysis showed thatαN-catenin was associated with GST-14-3-3εfusion protein, but not GST protein. It indicated the specificity of the interaction betweenαN-catenin and 14-3-3εin vitro.Conclusions:1 At transcription and translation levels, the distribution spectra of 14-3-3εandαN-catenin are established.2 Prokaryotic expression plasmid pGEX-4T-1-14-3-3εis successfully constructed.3 GST-14-3-3εfusion protein is effectively expressed in E.coli.4 GST-14-3-3εfusion protein is purified via GST-Sepharose 4B affinity resin, and the purity is electrophoretically pure.5 GST pull-down analysis confirmed the physical interaction between 14-3-3εandαN-catenin in vitro.