Phosphorylation by protein kinase C decreases catalytic activity of avian phospholipase C-beta

The potential role of protein kinase C (PKC)-promoted phosphorylation has b een examined in the G-protein-regulated inositol lipid signalling pathway. Incubation of [P-32]P-i-labelled turkey erythrocytes with either the P2Y, r eceptor agonist 2-methylthioadenosine triphosphate (2MeSATP) or with PMA re sulted in a marked increase in incorporation of P-32 into the G-protein-act ivated phospholipase C PLC-beta T. Purified PLC-beta T also was phosphoryla ted by PKC in vitro to a stoichiometry (mean +/- S.E.M.) of 1.06 +/- 0.2 mo l of phosphate/mol of PLC-beta T. Phosphorylation by PKC was isoenzyme-spec ific because, under identical conditions, mammalian PLC-beta 2 also was pho sphorylated to a stoichiometry near unity, whereas mammalian PLC-beta 1 was not phosphorylated by PKC, The effects of PKC-promoted phosphorylation on enzyme activity were assessed by reconstituting purified PLC-beta T with tu rkey erythrocyte membranes devoid of endogenous PLC activity. Phosphorylati on resulted in a decrease in basal activity, AlF4-stimulated activity, and activity stimulated by 2MeSATP plus guanosine 5'-[gamma-thio]triphosphate i n the reconstituted membranes. The decreases in enzyme activities were prop ortional to the extent of PKC-promoted phosphorylation. Catalytic activity assessed by using mixed detergent/phospholipid micelles also was decreased by up to 60% by phosphorylation. The effect of phosphorylation on G(q)alpha -stimulated PLC-beta T in reconstitution experiments with purified proteins was not greater than that observed on basal activity alone. Taken together , these results illustrate that PKC phosphorylates PLC-beta T in vivo and t o a physiologically relevant stoichiometry in vitro. Phosphorylation is acc ompanied by a concomitant loss of enzyme activity, reflected as a decrease in overall catalytic activity rather than as a specific modification of G-p rotein-regulated activity.