Calcineurin protein phosphatase activity in peripheral blood lymphocytes.
The protein phosphatase activity of peripheral blood T lymphocytes (PBLs) was examined to quantify the contribution of calcineurin and other members of the family of serine/threonine protein phosphatases. Using selective phosphatase inhibitors, the fractional phosphatase activities of calcineurin, protein phosphatases 1 (PP1), 2A (PP2A), and 2C (PP2C) were determined. Okadaic acid was used to inhibit the activity of both PP1 and PP2A while cyclosporin A/cyclophilin or trifluoperazine were used as a specific inhibitors of the calmodulin-dependent phosphatase calcineurin. Using a [32P]labeled 19-residue phosphopeptide substrate, RII peptide, it was found that PP1 and PP2A comprise the majority of the total phosphatase activity in PBLs with okadaic acid inhibiting 80% of the phosphatase activity. The remaining 20% of the phosphatase activity can be attributed primarily to calcineurin since it was Ca2+ dependent, sensitive to inhibition by the calmodulin antagonist trifluoperazine, and inhibited by the complex of cyclosporin A (CsA) and cyclophilin. These results indicate that PBL extracts contain little PP2C activity. In addition, PBLs treated with CsA had measurably lower calcineurin activity in cell lysates. The measurement of calcineurin activity may provide a useful means of assessing the extent of immunosuppression during drug therapy.
Rusnak
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Division of Hematology and Internal Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Beressi
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AH
Haddy
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Tefferi
A
A
D000595:Amino Acid Sequence; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000097:blood*; D016572:Cyclosporine / Q000494:pharmacology; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000097:blood*; D013601:T-Lymphocytes / Q000201:enzymology*; D014268:Trifluoperazine / Q000494:pharmacology
D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D007166:Immunosuppressive Agents; D019319:Okadaic Acid; D014268:Trifluoperazine; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases