Pathways of signaling in nonspecific cytotoxic cells: effects of protein kinase and phosphatase inhibitors and evidence for membrane tyrosine phosphorylation.

The present study was done to examine the role of protein kinases (PK) and phosphatases in nonspecific cytotoxic cell (NCC) activation processes. Treatment of NCC with 30 or 60 microM H-7 prior to adding IM-9 target cells significantly inhibited cytotoxicity. A similar concentration and time-dependent inhibition response was observed following treatment with H-8. The calcium and cyclic nucleotide antagonist HA1004, however, produced a significant increase in cytotoxicity at 30 and 60 microM concentrations. Treatment with genistein produced almost 100% inhibition of NCC lysis of IM-9 targets at a 142 micrograms/ml concentration. Studies were also carried out to determine the effects of these cytotoxicity modulators on stimulus-induced responses. In all cases where the costimulus consisted of the calcium ionophore A23187, cytotoxicity was markedly increased. In experiments using subsaturating concentrations of mab 5C6 as the costimulus, genistein completely reversed (inhibited) the modulating effects of this mab. Additional evidence for protein phosphorylation was obtained by immunoprecipitation and Western blot analysis (using antiphosphotyrosine mab 4G10) of membranes from mab 5C6-stimulated NCC. The role of protein phosphorylation on cytotoxic activity was further analyzed by the use of phosphatase inhibitors. Preincubation of NCC with either sodium orthovanadate or sodium fluoride significantly increased cytotoxicity. Lithium chloride had little or no effect on the killing of IM-9 target cells. These data indicate that multiple second messenger pathways participate in NCC lytic responses, the most crucial of which appears to facilitate phosphorylation of tyrosine residues on membrane proteins.