RECEPTOR-ASSOCIATED PHOSPHORYLATION FOLLOWING MONOCLONAL-ANTIBODY OR SYNTHETIC PEPTIDE BINDING TO NONSPECIFIC CYTOTOXIC-CELLS

We have previously shown that crosslinkage of a receptor protein on ca tfish nonspecific cytotoxic cells (NCC) with anti-receptor monoclonal antibody or with a synthetic peptide activates cytotoxicity and initia tes signalling responses. Receptor linked signalling was associated wi th the production of increased levels of expression of 50-60 and 20-30 kDa phosphoproteins determined by immunoprecipitation with anti-phosp hoserine and anti-phosphotyrosine mabs. These proteins are components of a macromolecular protein complex (>200 kDa) determined by reducing and nonreducing SDS-PAGE. The calcium ionophore A23187 treatment produ ced the same pattern of phosphoprotein expression as peptide or mab. M aximum phosphoserine expression occurred at 15'-30' post-mab binding. We now show that synthetic peptide or mab treatment initiated the same serine and tyrosine phosphorylation profiles. The PKC specific inhibi tor MDL 29, 152 produced 50% inhibition of NCC lysis of IM-9 target ce lls, and completely inhibited serine phosphorylation of peptide activa ted cells but had no effect on tyrosine phosphorylation of the phospho intermediates. Genistein pretreatment of NCC inhibited cytotoxicity an d tyrosine phosphorylation. Sequential immunoprecipitation of the phos phointermediate demonstrated that the phosphorylated serine and tyrosi ne residues were on the same 50-60 kDa protein. These data indicate th at both proximal and distal signalling events required for NCC activat ion may be associated with ATPase phosphorylation.