Activation of peripheral large granular lymphocytes with the serine/threonine phosphatase inhibitor, okadaic acid.
The murine polyether fatty acid, okadaic acid, is a potent inhibitor of serine/threonine phosphatases in eukaryotic cells. This compound inhibits both protein phosphatase 1 (PP1) and phosphatase 2A (PP2A). Here we have examined the potential of okadaic acid as an activator of fresh peripheral CD3- large granular lymphocytes (LGL). We demonstrate that overnight exposure of LGL to as little as 1 nM okadaic acid induced an increase in natural killing against the K562 cell line, but does not induce LAK activity. Optimal cytotoxic activation (2-fold) occurred at 0.01-1.0 nM okadaic acid, with a return to baseline levels at 10-20 nM, and inhibition, likely due to toxicity, at 40 nM. In addition, okadaic acid at doses > or = 20 nM induced LGL but not T cells to produce interferon-gamma. Similar to phorbol esters, overnight incubation with okadaic acid causes a dose-dependent reduction in expression of the low-affinity receptor for the Fc portion of IgG (CD16). However, unlike phorbol ester, short-term (5 min) okadaic acid treatment did not block CD16-mediated Ca2+ mobilization in LGL. To address the underlying biochemical mechanisms of okadaic acid activities, the levels of several as-yet-unidentified serine/threonine kinases were assayed after renaturation. Under these conditions, okadaic acid induced similar increases in kinase levels in both T cells and LGL. Taken together, these data suggest an important role for PP1 and PP2A in LGL physiology, and define okadaic acid as a potentially important biological response modifier for the study of LGL and T cell biochemistry, signal transduction, and transcriptional regulation.
McVicar
D W
DW
Laboratory of Experimental Immunology, NCI-FCRDC, Frederick, MD 21702-1201.
Mason
A T
AT
Bere
E W
EW
Ortaldo
J R
JR
D002118:Calcium / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic; D004988:Ethers, Cyclic / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000096:biosynthesis; D007694:Killer Cells, Natural / Q000187:drug effects; D008213:Lymphocyte Activation / Q000187:drug effects*; D016131:Lymphocyte Subsets / Q000187:drug effects*; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017452:Receptors, IgG / Q000096:biosynthesis; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000187:drug effects
D004988:Ethers, Cyclic; D017452:Receptors, IgG; D015375:Receptors, Interleukin-2; D019319:Okadaic Acid; D007371:Interferon-gamma; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D002118:Calcium