Regulation of natural killer cell-mediated cytotoxicity by serine/threonine phosphatases: identification of a calyculin A-sensitive serine/threonine kinase.
We have recently reported that Ser/Thr phosphatases play a key role in regulating natural killer (NK) cell lytic activity and that calyculin A and okadaic acid affect this activity differently [Bajpai and Brahmi (1994) J. Biol. Chem. 269, 18864-18869]. Here, we investigate a mechanism that might account for this differential action of calyculin A and okadaic acid on NK cells. Calyculin A specifically inhibited the lytic activity of YT-INDY, an NK-like cell line, and hyperphosphorylated 60 and 78 kDa proteins. The kinetics of appearance of these two proteins was correlated with the loss of lytic activity. In contrast, okadaic acid did not significantly affect either of these activities. The 78 kDa protein is localized in the cytosolic compartment whereas the 60 kDa protein is distributed equally between the membrane and the cytosolic fractions. Both proteins display a kinase activity and are phosphorylated mainly at serine and threonine residues but not at tyrosine residues. The activation of these kinases is specific to calyculin A treatment; it is independent of protein kinase C, protein kinase A, Ca2+, phosphotyrosine phosphatase and protein synthesis de novo. In conclusion, we have demonstrated that calyculin A, but not okadaic acid, hyper-phosphorylates two proteins with Ser/Thr kinase activity, thus explaining the differential regulation of NK cells by these two Ser/Thr phosphatase inhibitors.
Bajpai
A
A
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, USA.
Brahmi
Z
Z
D018106:CD28 Antigens / Q000378:metabolism; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007476:Ionophores / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000201:enzymology / Q000276:immunology*; D007700:Kinetics; D008387:Marine Toxins; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D018106:CD28 Antigens; D004791:Enzyme Inhibitors; D007476:Ionophores; D008387:Marine Toxins; D010080:Oxazoles; D019319:Okadaic Acid; D014443:Tyrosine; C059041:calyculin A; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate