Modulation of natural killer cell activity by surface phosphorylation reactions.
To determine whether phosphorylation of cell surface proteins is involved in NK cell activity, the phosphorylation patterns of a rat NK cell line (RNK-16) incubated with 12.5 microM [gamma-32P]ATP were characterized before and after exposure to YAC-1 cells, which serve as targets for killing, and K562 cells, which are not killed by RNK-16 cells. By 51Cr release assays, the inhibitory effect of ATP on RNK-16 killing activity previously reported was corroborated. RNK-16 cells prelabeled with 12.5 microM ATP show enhanced labeling of a 70- to 72,000-Da protein after exposure to unlabeled target YAC-1 cells but not after exposure to K562 cells. A protein of similar apparent molecular size is also labeled upon exposure of RNK-16 cells to OX-34, an antibody which binds and inhibits killing, as well as upon exposure to OX-18, which also binds but does not inhibit NK activity. These findings are indicative of the activation of a kinase with high affinity for [gamma-32P]ATP, which phosphorylates an endogenous surface substrate of 70-72,000 Da upon binding of macromolecules to the RNK-16 cells. RNK-16 cells, previously labeled with micromolars [gamma-32P]ATP and subsequently treated with millimolars unlabeled ATP, showed loss of label from a 110,000-Da protein component, indicative of the rapid turnover of a phosphate group on a surface protein. Thus, extracellular ATP enhances the phosphorylation of a 70- to 72,000-Da component upon binding of RNK-16 cells to target cells or upon binding of antibodies at micromolar concentrations of ATP and catalyzes the loss of phosphate from a 110,000-Da component at millimolar concentrations of ATP. These findings reflect a complex repertoire of surface phosphorylation changes which occur in RNK-16 cells.
Myers
L K
LK
Department of Pediatrics, College of Medicine, University of Tennessee, Memphis 38163.
Kang
E S
ES
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D009000:Monocytes / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured / Q000276:immunology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D008565:Membrane Proteins; D010750:Phosphoproteins; D011494:Protein Kinases; D000469:Alkaline Phosphatase