Phosphatases PP1 and PP2A act after the G0/G1 interface in lymphocyte activation.
An understanding of the progressive events required for lymphocyte activation is a prerequisite to understanding the molecular mechanism of immunosuppressive reagents, and is of central relevance to autoimmunity and immune tolerance. Although reversible phosphorylation is a major control mechanism in T cell activation, few facts are known about phosphatases in T cells. It has recently become possible to block selectively the serine/threonine specific protein phosphatases PP1 and PP2A by okadaic acid, a C38 polyether fatty acid produced by dinoflagellates. Here we have used this new and powerful biochemical probe to identify and quantify the activities of PP1 and PP2A at various times during lymphocyte activation. The selected model was mouse T cell activation by concanavalin A because this gave a reproducible experimental system that allowed large scale experiments in defined, serum-free conditions. Our results show the following: (1) levels of PP1 and PP2A in lymphocytes appear to be unusually low, effects being measured at 10 nM okadaic acid compared to 1 microM reported for other cell types; (2) the okadaic acid effect was readily reversible; (3) okadaic acid stimulated mitogenesis when present in early G1 but inhibited mitogenesis in late G1; (4) levels of PP1 and PP2A remained relatively constant over the first 7 h following stimulation; (5) lymphocytes activated in the presence of okadaic acid became resistant to subsequent treatment with FK506; and (6) combined or consecutive treatment with okadaic acid and FK506 resulted in additive drug effects. We conclude that PP1 and PP2A are not involved in the G0-G1 transition as defined by the FK506-sensitive step.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Surgery, University of Cambridge, Addenbrookes Hospital, U.K.
S
S
Metcalfe
J
J
Milner
D000818:Animals; D000900:Anti-Bacterial Agents; D002478:Cells, Cultured; D003208:Concanavalin A; D004247:DNA / Q000096:biosynthesis; D004988:Ethers, Cyclic; D007165:Immunosuppression Therapy; D007399:Interphase; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000166:cytology / Q000201:enzymology*; D016559:Tacrolimus
D000900:Anti-Bacterial Agents; D004988:Ethers, Cyclic; D003208:Concanavalin A; D019319:Okadaic Acid; D004247:DNA; D010749:Phosphoprotein Phosphatases; D016559:Tacrolimus