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Characterization of protein kinase C activity in interferon gamma treated murine peritoneal macrophages
Abstract:
Macrophage activation for tumoricidal and microbicidal functions can be achieved in part by treatment with recombinant interferon gamma (IFN gamma) in vitro. We have previously demonstrated that IFN gamma treatment of murine peritoneal macrophages results in a two- to five-fold increase in the activity of Ca++, phospholipid dependent protein kinase C (Hamilton et al., J. Biol. Chem., 260:1378, 1985). We now report that this effect was not dependent upon continuing protein synthesis since treatment with cycloheximide under conditions where normal protein synthesis was inhibited by greater than 95% had no effect upon the development of increased enzyme activity. Examination of Ca++ and phospholipid requirements revealed no differences between enzyme isolated from control or IFN gamma treated cells. Similarly, protein kinase C from control and IFN gamma-treated cells could not be distinguished in terms of the diacylglycerol (DG) or phorbol diester (PMA) concentration required for stimulation of activity. Kinetic analysis of the ATP (as substrate) concentration dependence revealed that both control and treated enzyme preparations (either basal or stimulated) had comparable Km values. Maximum velocity (Vmax) was increased both by IFN gamma treatment and also by stimulation with DG or PMA. The major difference which could be discerned between protein kinase C derived from control versus IFN gamma-treated macrophages was the magnitude of the response to DG or PMA; IFN gamma treatment increased the stimulation index (i.e., ratio of basal to stimulated activity) by a factor of two to four fold. These results suggest that IFN gamma treatment leads to reversible modulation of existing protein kinase C resulting in increased catalytic efficiency when exposed to an appropriate stimulant.
Insights
Interferon gamma (IFN gamma) enhances macrophage tumoricidal functions by modulating protein kinase C activity. This modulation increases enzyme responsiveness to stimulants without affecting protein synthesis or enzyme requirements.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Macrophage activation is crucial for anti-tumor and anti-microbial immunity.
- Recombinant interferon gamma (IFN gamma) is known to activate macrophages.
- Previous studies showed IFN gamma increases Ca++, phospholipid-dependent protein kinase C (PKC) activity in murine macrophages.
Purpose of the Study:
- To investigate the mechanism by which IFN gamma treatment affects PKC activity in macrophages.
- To determine if continuing protein synthesis is required for IFN gamma-induced PKC modulation.
- To characterize the kinetic and regulatory differences in PKC from IFN gamma-treated versus control macrophages.
Main Methods:
- Murine peritoneal macrophages were treated with IFN gamma.
- Protein synthesis was inhibited using cycloheximide.
- Protein kinase C activity was measured under varying conditions (Ca++, phospholipid, diacylglycerol, phorbol diester, ATP concentrations).
- Kinetic parameters (Km, Vmax) and stimulation indices were analyzed.
Main Results:
- IFN gamma-induced increase in PKC activity was independent of ongoing protein synthesis.
- No differences were observed in Ca++, phospholipid, or diacylglycerol/phorbol diester concentration requirements between control and treated cells.
- Kinetic analysis showed comparable Km values for ATP but increased Vmax in IFN gamma-treated cells.
- IFN gamma treatment resulted in a 2-4 fold increase in the stimulation index (ratio of basal to stimulated activity) for PKC.
Conclusions:
- IFN gamma treatment reversibly modulates existing protein kinase C in macrophages.
- This modulation enhances the enzyme's catalytic efficiency upon stimulation with diacylglycerol or phorbol diester.
- The findings suggest a mechanism for enhanced macrophage effector functions mediated by PKC regulation.