CHARACTERIZATION OF PROTEIN-SERINE THREONINE PHOSPHATASE-ACTIVITIES IN HUMAN LUNG MAST-CELLS AND BASOPHILS

1 The serine/threonine protein phosphatase (PP) inhibitors, okadaic ac id and calyculin, attenuated the IgE-mediated release of histamine fro m human lung mast cells (HLMC) and basophils in a dose-dependent manne r whereas an alternative PP inhibitor, microcystin, was ineffective. C alyculin was more potent than okadaic acid in both cell types. The con centration required to inhibit by 50% (IC50) the release of histamine was 15 (HLMC) and 50 nM (basophils) for calyculin and 200 (HLMC) and 3 00 nM (basophils) for okadaic acid. 2 Lysates of purified HLMC and bas ophils dephosphorylated radiolabelled glycogen phosphorylase, a substr ate for both PP1 and PP2A. The PP activity in lysates of both cell typ es was inhibited in a dose-dependent fashion by the PP inhibitors with the following rank order of activity, calyculin (approximate IC50; 0. 02-0.1 nM)greater than or equal to microcystin (0.1 nM)>okadaic acid ( 70 nM). 3 The PP1-selective inhibitor, inhibitor-2 (I-2), attenuated t he dephosphorylation of glycogen phosphorylase in lysates of both HLMC and basophils. I-2 (20 nM) inhibited the glycogen phosphorylase PP ac tivity by 71+/-3% and 49+/-13% in HLMC and basophil extracts, respecti vely. There were, approximately, 6 fold greater levels of I-2-sensitiv e activity in HLMC than in basophils. Qualitatively similar results we re obtained with an alternative PP1-selective inhibitor, inhibitor-1 ( I-1). 4 Lysates derived from HLMC and basophils dephosphorylated radio labelled casein which is a PP2A-restricted substrate. HLMC lysates con tained, approximately, 2.5 fold higher levels of casein PP activity th an basophil lysates. 5 These data indicate that HLMC and basophils bot h contain PPI and PP2A. The data suggest that, on a per cell basis, HL MC have higher levels of both PPI and PP2A. Moreover, the ratio of PP1 to PP2A is higher in HLMC than in basophils.