PHOSPHORYLATION OF THE NC-1.1 RECEPTOR AND REGULATION OF NATURAL CYTOTOXICITY BY PROTEIN-KINASE-C AND CYCLIC GMP-DEPENDENT PROTEIN-KINASE

Natural cytotoxicity (NC) against cancer involves receptor-ligand inte ractions between lymphohemopoietic cells that mediate NC against tumor cells. The only candidate for a receptor on cells mediating NC is NC- 1.1, identified using mAb 1C4. In this study we showed that mAb 1C4 bl ocked NC-1.1(+) cell conjugation to WEHI-164 tumor cells, indicating t hat NC-1.1 is a surface protein required for cell-cell interaction. Af finity-purified NC-1.1 was a 45-kDa monomeric protein. It was a good i n vitro substrate for cyclic GMP (cGMP)-dependent protein kinase (PKG) and protein kinase C (PKC) and a relatively poor substrate for cAMP-d ependent protein kinase (PKA). Phosphopeptide mapping revealed one pho sphopeptide phosphorylated by PKC and PKA, and two additional peptides phosphorylated by PKC, Phosphorylation by PKC or PKA abolished phosph orylation at the PKC sites, while coincubation of NC-1.1 with both PKC and PKC reduced phosphorylation of all sites. NC-1.1 was also a phosp hoprotein after immunoprecipitation from intact spleen cells and its p hosphorylation was increased after cell stimulation with PKC or PKC ac tivators (phorbol esters or 8-bromo-cGMP). The possible consequences o f intracellular signaling were tested in functional assays for NC. Pho rbol ester activation of spleen cells increased NC, while 8-bromo-cGMP and 8-bromo-cAMP had little effect. However, coincubation with both p horbol ester and either 8-bromo-cGMP or 8-bromo-cAMP virtually abolish ed NC without affecting cell conjugation. These results suggest that N C-1.1 is a receptor for a ligand on certain tumor cells and reveal tha t key intracellular signaling pathways involving PKC, PKG, and PKA int eract to effect a coordinated control of NC.