Calcineurin protein phosphatase activity in peripheral blood lymphocytes

F. Rusnak, A. H. Beressi, A. Haddy, A. Tefferi

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

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, R(II) 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 Ca 2+ 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.

Original languageEnglish (US)
Pages (from-to)309-313
Number of pages5
JournalBone Marrow Transplantation
Volume17
Issue number3
StatePublished - Mar 1 1996

Keywords

  • Cyclophilin
  • Cyclosporin A
  • FK506
  • FK506-binding protein, immunosuppression

ASJC Scopus subject areas

  • Hematology
  • Transplantation

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