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 interactions 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 blocked NC-1.1+ cell conjugation to WEHI-164 tumor cells, indicating that NC-1.1 is a surface protein required for cell-cell interaction. Affinity-purified NC-1.1 was a 45-kDa monomeric protein. It was a good in vitro substrate for cyclic GMP (cGMP)-dependent protein kinase (PKG) and protein kinase C (PKC) and a relatively poor substrate for cAMP-dependent protein kinase (PKA). Phosphopeptide mapping revealed one phosphopeptide phosphorylated by PKG and PKA, and two additional peptides phosphorylated by PKC. Phosphorylation by PKG or PKA abolished phosphorylation at the PKC sites, while coincubation of NC-1.1 with both PKG and PKC reduced phosphorylation of all sites. NC-1.1 was also a phosphoprotein after immunoprecipitation from intact spleen cells and its phosphorylation was increased after cell stimulation with PKC or PKG activators (phorbol esters or 8-bromo-cGMP). The possible consequences of intracellular signaling were tested in functional assays for NC. Phorbol ester activation of spleen cells increased NC, while 8-bromo-cGMP and 8-bromo-cAMP had little effect. However, coincubation with both phorbol ester and either 8-bromo-cGMP or 8-bromo-cAMP virtually abolished NC without affecting cell conjugation. These results suggest that NC-1.1 is a receptor for a ligand on certain tumor cells and reveal that key intracellular signaling pathways involving PKC, PKG, and PKA interact to effect a coordinated control of NC.
Holmgreen
S P
SP
Faculty of Medicine, University of Newcastle, New South Wales, Australia.
Wang
X
X
Clarke
G R
GR
Noltorp
R S
RS
Roberts
T K
TK
Burton
R C
RC
Robinson
P J
PJ
Smart
Y C
YC
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000276:immunology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000494:pharmacology*; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D005260:Female; D007694:Killer Cells, Natural / Q000201:enzymology / Q000276:immunology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000494:pharmacology*; D011971:Receptors, Immunologic / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D011971:Receptors, Immunologic; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C