Preliminary characterization of the role of protein serine/ threonine phosphatases in the regulation of human lung mast cell function

Abstract

1 Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-dependent release of mediators from human lung mast cells (HLMC). The concentration of okadaic acid required to inhibit by 50% (IC 50 ) the IgE-dependent release of histamine was 0.2 mM. Okadaic acid also inhibited the IgE-mediated generation of prostaglandin D 2 (PGD 2 ) and sulphopeptidoleukotrienes (sLT) with IC 50 values of 0.2 mM and 0.6 mM respectively. 2 The IgE-mediated generation of histamine, PGD 2 and sLT was inhibited by okadaic acid and two analogues of okadaic acid, okadaol and okadaone, with the following rank order of activity; okadaic acid4okadaol4okadaone. This order of activity for the inhibition of mediator release parallels the activity of these compounds as inhibitors of isolated PPs. 3 Extracts of HLMC liberated 32 P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by the PP inhibitors (all at 3 mM), okadaic acid (73+4% inhibition, P50.0005), okadaol (26+7% inhibition, P50.05) and okadaone (8+7% inhibition, P=0.52). The rank order of activity of okadaic acid4okadaol4okadaone parallels the activity of these compounds as inhibitors of isolated PPs. 4 Dephosphorylation of radiolabelled glycogen phosphorylase by extracts of HLMC was inhibited by 15+3% (P50.001) by a low (2 nM) concentration of okadaic acid and by 88+4% (P50.0005) by a higher (5 mM) concentration of okadaic acid. Because 2 nM okadaic acid may act selectively to inhibit PP2A whereas 5 mM okadaic acid inhibits both PP1 and PP2A, these data suggest that both PP1 and PP2A are present in HLMC. 5 Inhibitor 2, a PP1-selective inhibitor, attenuated (71+3% inhibition, P50.05) PP activity in extracts of HLMC suggesting that HLMC contain PP1 and that it may constitute 71% of the phosphorylase PP activity in extracts of HLMC. 6 Radiolabelled casein, a PP2A-restricted substrate, was dephosphorylated by extracts of puri®ed HLMC and this activity was inhibited (81+8% inhibition, P50.005) by 2 nM okadaic acid suggesting that PP2A is resident in HLMC. 7 Collectively, these data suggest that both PP1 and PP2A are resident in HLMC. However, although the data suggest that okadaic acid regulates responses in HLMC by interacting with PPs, it has not been possible to determine whether either PP1 or PP2A or both PPs are involved in the okadaic acid-induced inhibition of mediator release from HLMC.

Positive and negative regulatory mechanisms in high-affinity IgE receptor-mediated mast cell activation

“…This increased cytokine production was mediated by an increase in the phosphorylation of p38 MAPK following PP2A inhibition by okadaic acid [8]. In addition, the administration of okadaic acid to human lung mast cells resulted in the inhibition of IgE-dependent histamine release and prostaglandin D2 and sulphopeptidoleukotriene generation [85]. Thus, protein serine/threonine phosphatases, in particular PP2A, can both positively and negatively regulate mast cell functions.…”

ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation

“…5C). 29 Moreover, the catalytically inactive PPP1 mutant (H248K), 30 could not decrease the phosphorylation of ATG16L1. These data suggest that PPP1, but not PPP2CA, dephosphorylates CSNK2-phosphorylated ATG16L1 in vitro, which indicates that PPP1 may be the major phosphatase responsible for the dephosphorylation of ATG16L1.…”

“…For inhibitor-2 (New England Biolabs, P0755) treatment, 2.5 units of inhibitor-2 were added. The reaction was incubated at 30 C for 30 min. The beads and supernatant fraction were carefully separated by centrifugation.…”

“…(C) In vitro CSNK2 kinase assay. Immunoprecipitated CSNK2 subunits were incubated with GST-ATG16L1 protein at 30 C for 30 min in kinase buffer. The reaction products were then subjected to western blotting using an anti-phosphoserine/ threonine antibody to analyze the ability of CSNK2 to phosphorylate ATG16L1.…”

Effects of fostriecin on β2-adrenoceptor-driven responses in human mast cells

“…With respect to recycling, it appears that dephosphorylation of the receptor, by a PP2A-like phosphatase, is involved in ensuring receptor re-expression. Our previous studies (Peirce et al 1997) provided a framework for the study here to determine the role, if any, of PP2A in desensitization/re-sensitization of mast cells. Those studies (and later ones) had shown that mast cells expressed PP2A but that inhibition of PP2A with the highly selective inhibitor fostriecin had no effect on IgE-mediated histamine release (Bastan et al 2001).…”