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 catfish nonspecific cytotoxic cells (NCC) with anti-receptor monoclonal antibody or with a synthetic peptide activates cytotoxicity and initiates signalling responses. Receptor linked signalling was associated with the production of increased levels of expression of 50-60 and 20-30 kDa phosphoproteins determined by immunoprecipitation with anti-phosphoserine 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 produced the same pattern of phosphoprotein expression as peptide or mab. Maximum 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 inhibitor MDL 29,152 produced 50% inhibition of NCC lysis of IM-9 target cells, and completely inhibited serine phosphorylation of peptide activated cells but had no effect on tyrosine phosphorylation of the phosphointermediates. Genistein pretreatment of NCC inhibited cytotoxicity and tyrosine phosphorylation. Sequential immunprecipitation of the phosphointermediate demonstrated that the phosphorylated serine and tyrosine residues were on the same 50-60 kDa protein. These data indicate that both proximal and distal signalling events required for NCC activation may be associated with ATPase phosphorylation.
Jaso-Friedmann
L
L
Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens 30602, USA.
Leary
J H
JH
Evans
D L
DL
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D019833:Genistein / Q000494:pharmacology; D007059:Ictaluridae; D007546:Isoquinolines / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology; D008969:Molecular Sequence Data; D010081:Oxazolone / Q000031:analogs & derivatives / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011804:Quinolines / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007546:Isoquinolines; D010455:Peptides; D011804:Quinolines; D013449:Sulfonamides; C073398:MDL 29152; D010081:Oxazolone; D010768:Phosphoserine; D012694:Serine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C043204:N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; D019833:Genistein; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate