Modulation of natural killer cell activity by surface phosphorylation reactions

Abstract

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 μ M [γ- 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 μ M 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 0X-34, an antibody which binds and inhibits killing, as well as upon exposure to 0X-18, which also binds but does not inhibit NK activity. These findings are indicative of the activation of a kinase with high affinity for [γ- 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 [γ- 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.