MODULATION OF THE EFFECTOR FUNCTIONS OF CYTOLYTIC LYMPHOCYTES-T WITH SYNTHETIC PEPTIDE INHIBITORS OF PROTEIN-KINASES

The hypothesis was tested that it is possible to influence cellular responses of intact cells using synthetic peptide substrates, pseudosubstrates, and inhibitors of protein kinases. Using cytotoxic T-cells (CTL), we demonstrate here that some basic amino acid-containing synthetic peptide substrates of protein kinases [e.g., of cGMP-dependent protein kinase (peptide PKG-S), synthetic peptide inhibitor of cGMP-dependent protein kinase (peptide PKG-I), and peptide corresponding to the tyrosine phosphorylation site in pp60src (peptide RR-src)] were strongly inhibitory in T-cell receptor (TCR) and T-cell growth factor, interleukin 2 (IL-2)-triggered proliferation of CTL. These peptides also inhibited other cellular responses of CTL. Peptides which contain basic amino acids, but do not have substrate specificity determinants for protein kinase, were not inhibitory. The inhibition with peptides is not due to their toxicity, since no cell death was observed by the trypan blue exclusion test and by lactate dehydrogenase release. Use of the granule exocytosis assay provided opportunities to clarify the mechanism of the peptide action. Tested peptides inhibited not only cell-surface ligand-induced CTL activation, but also affected cell-surface receptor-independent CTL activation (granule exocytosis and gamma-interferon secretion) induced by the synergistic action of the protein kinase C activator (PMA) and ionophore A23187. It was found that minor changes in amino acid composition or amino acid position in the synthetic peptides dramatically change their ability to affect lymphocytes. Pretreatment with peptide PKG-S at 37-degrees-C, but not at 22 or 4-degrees-C, for at least 90 min, followed by washing, was sufficient to observe inhibition of CTL function (a small effect was observed even after 60 min of pretreatment). Results of the peptide binding and of intracellular localization studies support the view that studied peptides exert their effect after gaining access to the cell interior. Percoll gradient fractionation of CTL disrupted by nitrogen cavitation revealed that the largest proportion of radiolabel was in cytosol. The requirement for the relatively high concentration of peptides needed to demonstrate their effects may reflect their partial degradation by peptidases. The presented data support the model in which specific features of amino acid composition allow for the peptide internalization and interference with cellular functions.