Using immunosuppressants, FK506 and rapamycin, to block critical pathways in the activation of T cells, as well as other known inhibitors of signaling pathways, the pathways linking upstream signals to downstream events at the level of translation have been determined. Signal transduction and the control of translation in T cells. Identification of signaling pathways important for the global regulation of the overall increase in translation during T cell is in progress. We are in the process of identifying which translation initiation factors are regulated by each signaling pathway, so that a total picture of the signaling pathways and events controlling the increase in translation during T cell activation can be constructed. Analysis of protein changes resulting from specific signal transduction pathways during the activation of primary human peripheral blood lymphocytes. In vivo changes in the synthesis of proteins, which occurred during the early period of T cell activation, were analyzed by 1D and 2D PAGE. A sensitive method based upon the in vitro translation of polysome-associated mRNAs (PAmRNAs) was developed that increased our ability to analyze these protein changes. Using these methods, the effect of immunosuppressants FK506 or rapamycin, or specific protein kinase inhibitors on the synthesis of proteins was examined. Synthesis of a number of proteins was found to be selectively affected by these conditions. Isolation and identification have been accomplished for several of these proteins. One protein, p33 was identified as BAP37 a protein possibly important for senescence. Another protein, p72 was not found in the existing data bases or EST sequence data bases and thus far judged to be a novel protein. These protein changes were correlated with changes in mRNA expression. Biological relevance based upon identification of these proteins is also being pursued by in situ immunofluorescence. Development of protein isolation methods for peptide sequencing and protein identification. These procedures enabled the isolation of the gene products from in vitro synthesis of PAmRNAs. Identification of these proteins was then possible by mass spectrometry. Mass spectrometry was performed using MALDI-TOF and electrospray LCQ.
