Protein synthesis is essential for immune function and proliferation of the activated T cell. Activation of quiescent Go T cells with ionomycin and PMA rapidly increases the rate of protein synthesis. We are able to study these induced proteins by metabolically labeling cells with 35S-methionine and analyzing them by SDS gel electrophoresis. Treatment with ionomycin alone did not induce protein synthesis, but treatment with PMA alone did. In the presence of immunosuppressants FK506 or rapamycin, protein synthesis induced by ionomycin+PMA was reduced by 40- 50%. Rapamycin treatment affected synthesis of some specific proteins and this inhibition appeared to be dependent upon activation in the presence of both ionomycin and PMA. Isolating the monosome/polysomal fraction from activated T cells and translating resuspended ribosomal fraction with rabbit reticulocyte lysates, demonstrated the rapid increase in synthesis of proteins from mRNAs associated with this fraction. Synthesis of some of these proteins occurred rapidly (within 15 min) for certain proteins, peaked at 30 min and decreased thereafter. Induction of other proteins occurred with different kinetics. With this system, we are also able to study the effect of inhibiting protein synthesis during T cell activation by treating T cells with cycloheximide during activation. Little effect was observed by treating the cells only for the time of activation. If cells, however, were pretreated with CHX for 2 hr prior to activation, in vitro synthesis of certain proteins increased. This probably indicates a stabilization of mRNAs for these proteins. Synthesis of other proteins, however, decreased with CHX treatment. It is important to identify and clone these proteins of interest and to study how the synthesis of these proteins is regulated during T cell activation.
