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Phosphorylation by protein kinase C decreases catalytic activity of avian phospholipase C-beta
T M Filtz1, M L Cunningham, K J Stanig
1Mary Ellen Jones Building, University of North Carolina School of Medicine, Department of Pharmacology, Chapel Hill, NC 27599, USA.
Abstract:
The potential role of protein kinase C (PKC)-promoted phosphorylation has been examined in the G-protein-regulated inositol lipid signalling pathway. Incubation of [32P]Pi-labelled turkey erythrocytes with either the P2Y1 receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) or with PMA resulted in a marked increase in incorporation of 32P into the G-protein-activated phospholipase C PLC-betaT. Purified PLC-betaT also was phosphorylated by PKC in vitro to a stoichiometry (mean+/-S. E.M.) of 1.06+/-0.2 mol of phosphate/mol of PLC-betaT. Phosphorylation by PKC was isoenzyme-specific because, under identical conditions, mammalian PLC-beta2 also was phosphorylated to a stoichiometry near unity, whereas mammalian PLC-beta1 was not phosphorylated by PKC. The effects of PKC-promoted phosphorylation on enzyme activity were assessed by reconstituting purified PLC-betaT with turkey erythrocyte membranes devoid of endogenous PLC activity. Phosphorylation resulted in a decrease in basal activity, AlF4(-)-stimulated activity, and activity stimulated by 2MeSATP plus guanosine 5'-[gamma-thio]triphosphate in the reconstituted membranes. The decreases in enzyme activities were proportional 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 Gqalpha-stimulated PLC-betaT 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-betaT in vivo and to a physiologically relevant stoichiometry in vitro. Phosphorylation is accompanied by a concomitant loss of enzyme activity, reflected as a decrease in overall catalytic activity rather than as a specific modification of G-protein-regulated activity.
Insights
Protein kinase C (PKC) phosphorylates phospholipase C-betaT (PLC-betaT), reducing its enzyme activity. This phosphorylation decreases overall catalytic function rather than specifically altering G-protein regulation in the inositol lipid signaling pathway.
Area of Science:
- Biochemistry
- Cell Signaling
- Enzymology
Background:
- G-protein-coupled receptors initiate intracellular signaling cascades.
- Inositol lipid signaling pathways are crucial for cellular communication.
- Protein kinase C (PKC) plays a regulatory role in various cellular processes through phosphorylation.
Purpose of the Study:
- To investigate the role of PKC-promoted phosphorylation in the G-protein-regulated inositol lipid signaling pathway.
- To determine the effect of PKC phosphorylation on phospholipase C-betaT (PLC-betaT) activity and regulation.
Main Methods:
- Incubation of [32P]Pi-labeled turkey erythrocytes with agonists or PMA to assess in vivo phosphorylation.
- In vitro phosphorylation of purified PLC-betaT by PKC.
- Reconstitution experiments with purified PLC-betaT and erythrocyte membranes to assess enzyme activity.
- Assay of catalytic activity using detergent/phospholipid micelles.
Main Results:
- PKC-promoted phosphorylation of PLC-betaT was observed both in vivo and in vitro.
- Phosphorylation by PKC was isoenzyme-specific, affecting PLC-betaT and PLC-beta2 but not PLC-beta1.
- Phosphorylation led to a significant decrease in basal, stimulated, and catalytic activities of PLC-betaT.
- The reduction in enzyme activity was proportional to the extent of phosphorylation.
Conclusions:
- PKC phosphorylates PLC-betaT in vivo and in vitro to a physiologically relevant stoichiometry.
- PKC-mediated phosphorylation reduces the overall catalytic activity of PLC-betaT.
- This phosphorylation event impacts enzyme function by decreasing general catalytic efficiency, not by specifically altering G-protein-mediated regulation.