Detection of early gene expression changes during activation of human primary lymphocytes by in vitro synthesis of proteins from polysome-associated mRNAs

The rapid increase in protein synthesis during the mitogenic stimulation of human peripheral blood lymphocyte is the result of global and specific tra nslational control mechanisms. To study some of these mechanisms, we examin ed the in vitro translatability of mRNAs associated with the polyribosome f raction. Polyribosome fractions were isolated from lymphocytes after activa tion with ionomycin and the phorbol ester PMA. The associated PAmRNAs were translated in the presence of mRNA-depleted rabbit reticulocyte lysate and [S-35]Met, and the protein products were analyzed by SDS-PAGE and autoradio graphy. There was little synthesis of protein from the PAmRNAs isolated fro m unactivated T cells, but the PAmRNAs isolated from activated T cells show ed a rapid increase in translatability. Translation of the PAmRNAs was sens itive to edeine and m7GTP, suggesting their cap-dependent translation. With activation, the majority of proteins showed increasing in vitro translatio n, but two proteins, p72 and p33, were found to have increased synthesis wi thin 30 min, which decreased in 1 h. Transcription inhibitors were used to ascertain if regulation of their expression was transcriptional or translat ional. To identify these proteins, we used biotinylated lysine during the i n vitro translation reaction, and we extracted the biotinylated protein by using streptavidin magnetic beads. The protein product was analyzed by mass spectrometry. p33 was identified as a prohibitin-like protein (BAP37), but the identification of p72 was not found in the databases. The distinct up- regulation acid down-regulation of their protein expression suggest their t ightly controlled regulation during early T cell activation.