Activation of Go T cells with ionomycin and PMA rapidly increases the rate of protein synthesis. We are able to study newly synthesized proteins by metabolically labeling cells with 35S-methionine and analyzing cell extracts by SDS PAGE. Treatment with ionomycin did not induce protein synthesis, but treatment with PMA by itself did. In the presence of immunosuppressants FK506 or rapamycin, protein synthesis induced by ionomycin+PMA was reduced by 40-50%. Rapamycin treatment affected overall synthesis proteins, but one protein in particular appeared to be strongly affected. Inhibition of synthesis of this protein was dependent upon activation with both ionomycin and PMA. This protein was found to be cytoplasmic actin. Northern Blot analysis two hours after activation in the presence of FK506 or rapamycin revealed that rapamycin inhibited induction of actin mRNA whereas treatment with FK506 increased actin mRNA levels. Treatment with PMA by itself induced high levels of actin mRNA that was not affected by FK506 or rapamycin treatment. Actinomycin D inhibited these effects demonstrating that regulation occurs at the transcriptional level. This demonstrates that separate downstream pathways are affected by either FK506 or rapamycin after activation with ionomycin + PMA. These effects were not observed with PMA treatment showing the importance of Ca+2 dependent pathways on this effect. Translating the monosome/polysomal fraction from activated T cells with rabbit reticulocyte lysate allows us to study the synthesis of proteins from mRNA associated with this fraction. Synthesis of some of these proteins occurred rapidly after activation for certain proteins (within 15 min), peaked at 30 min and decreased thereafter. Induction of other proteins occurred with different kinetics. Treatment of T cells with cycloheximide enabled us to examine the effect that inhibition of protein synthesis has on mRNAs associated with the ribosomal fraction. Little effect was observed by treating the cells only for the time of activation. If cells, however, were pretreated with cycloheximide for 2 hr prior to activation, in vitro synthesis of certain proteins increased. This probably indicates stabilization of mRNAs for these proteins. Synthesis of other proteins, however, decreased with cycloheximide treatment, potentially indicating the importance of protein synthesis for stabilization of other mRNAs. We are in the process of identifying and cloning the cDNAs from these mRNAs, so that we can study how their expression is regulated during T cell activation.
