Inhibition of Calcineurin Phosphatase Activity

Introduction

Protein phosphatases dephosphorylate a number of critical cellular regulators to promote or inhibit their activity. Calcineurin (protein phosphatase 2B) is a protein phosphatase involved in T cell activation and is specifically inhibited by the immunosuppressive drugs, cyclosporin A and FK506 (tacrolimus). The immunosuppressive activity of cyclosporins has been correlated with their ability to form complexes with cyclophilins that inhibit calcineurin phosphatase activity and prevent incorporation of cyclophilin into viral particles. Similarly, FK506 binds to FKBP12 causing it to become a potent inhibitor of calcineurin phosphatase activity. R&D Systems currently offers recombinant human Cyclophilin A (Catalog # 3589-CA), recombinant human Calcineurin (Catalog # 3160-CA), and recombinant human FKBP12 (Catalog # 3777-FK) for the study of calcineurin phosphatase activity. The activities of Cyclophilin A and FKBP12 were measured by their ability to inhibit calcineurin phosphatase activity in the presence of either Cyclosporin A or FK506, respectively.

Materials

Note: The enzymes, immunosuppressive agents, and dilutions of each vary depending on the enzyme that is being assayed. Consult the specific assay protocol for the dilutions that should be used in each assay.

• Assay Buffer: 20 mM Tris, 10 mM MgCl₂, 0.1 mM CaCl₂, 1 mg/mL BSA, pH 7.5
• Inhibitors of Calcineurin phosphatase activity: Recombinant human Cyclophilin A (MW: 18 kDa; R&D Systems, Catalog # 3589-CA), Recombinat human FKBP12 (MW; 13 kDa; R&D Systems, Catalog # 3777-FK)
• Recombinant human Calcineurin (R&D Systems, Catalog # 3160-CA)
• Substrate: Serine/Threonine Phosphatase substrate I (DLDVPIPGRFDRRVS(PO3)VAAE) (R&D Systems, Catalog # ES012), MW: 2192 Da. Prepare a 1 mM stock in diH₂O
• Calmodulin (MW: 16.8 kDa) (Sigma, Catalog # P2277). Prepare a 10 mM stock in assay buffer.
• EDTA (Sigma, Catalog # E6758), prepare a 0.1 M stock in diH₂O (pH 8.0)
• Immunosuppressive drugs: Cyclosporin A (Sigma, Catalog # C1832) or FK506 (Sigma, Catalog # F4679). Prepare a 1 mM stock in DMSO.
• Incubator set at 37° C able to shake samples
• Malachite Green Phosphate Detection Kit (R&D Systems, Catalog # DYC996)
  • Malachite Green Reagent A: 1.75% Ammonium molybdate, 3 M H₂SO₄
  • Malachite Green Reagent B: 0.07% Malachite Green oxalate, 0.35% Polyvinyl alcohol
  • Phosphate Standard: KH₂PO₄ MW 136, 1M = 6.8 g/50 mL
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Sealers (Corning, Catalog # 3095)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Assay Protocols