Phosphorylation/Dephosphorylation Possibly Regulate Cellular PLA₂ and Pl- PLC-_γ1 Activity: Implication for the Mechanism of H₂O₂ and Glucocorticoid Action

Summary

The possible involvement of phosphorylation/dephosphorylation processes in cellular regulation of PLA₂ and PI-PLC-γl activity was examined in mast cells. For this purpose, we chose two agents that have opposite effects on protein phosphatase activity, a) H₂O₂, a well known activator of cellular PLA₂ inhibits protein phosphatase; and b) dexamethasone, which suppresses enhanced PLA₂ activity, induces protein phosphatase biosynthesis. H₂O₂ rapidly (observed at 5 min) and significantly reduced acid phosphatase (APase) activity (50% using p-nitrophenyl phosphate as a substrate) in several cell types, in doses comparable to those which activate PLA₂ (3–4 fold). The inhibitiion of APase activity preceded the stimulation of PLA ₂ activity which is observed only after 30 min incubation. H₂O₂ -induced inhibition of APase activity was independent of external Ca²⁺, whereas its activation of PLA₂ was absolutely dependent on external Ca²⁺ and high [Ca²⁺]_i. This stimulatory action of H₂O₂ is thus compatible with its known effect on elevation of [Ca²⁺]_i in various cell types. Down regulation of protein kinase C (PKC) or the action of protein kinase inhibitors (staurosporine and K252a in nanomolar concentration), largely prevented the PLA₂ activation, but had no effect on the APase inhibition. Dexamethasone had a delayed effect in the opposite direction; 6 hours after it induced APase activity (240%), it prevented (50%) PLA₂ activation by Ca²⁺ ionophore and other agonists. Hence we suggest that inhibition of APase activity leads to gradual accumulation of phosphorylated, active PLA₂, while induction of APase leads to dephosphorylation of PLA₂ and its inactivation. Since PLA₂ is a key enzyme for inflammation, allergy or radiation damage, symptoms associated with these states could result, at least partly, from prevention of protein phosphatase activity on dephosphorylation of PLA₂. In contrast, the antiinflammatory and antiallergic action of glucocorticoids could be partly related to the induction of protein phosphatase, resulting in dephosphorylation and desensitization of such enzymes such as PLA₂and PI-PLC-γ1.