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Phosphorylation of an Mr = 29,000 protein by IL-1 is susceptible to partial down-regulation after endothelial cell
1Department of Molecular and Experimental Medicine, Scripps Clinic and Research Foundation, La Jolla, CA 92037.
Abstract:
IL-1 treatment of human endothelial cells leads to the rapid phosphorylation of a Mr = 29,000 (P29) set of proteins to 18 times that of control cultures. Approximately 80% of the phosphorylated P29 (pP29) disappeared within 60 min although the remaining component was stable and remained for at least another 2 h. IL-1R antagonist protein blocked phosphorylation completely. Secondary treatment of IL-1 failed to increase the level of pP29 above that remaining after 1 h although other unrelated agonists that stimulated pP29 generation could. Removal of the cytokine and incubation of the cells in agonist-free medium for 2 h resulted in the total loss of the remaining pP29. Readdition of IL-1 2 h after washout restimulated P29 phosphorylation but only back to the lower level. Maximum rephosphorylation could not be attained until 16 h after IL-1 removal. Protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and staurosporine, the calcium chelators bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester and EGTA, and the calmodulin inhibitor N-(6-aminohexyl)-1-naphthalene-sulfonamide had no effect on IL-I-induced phosphorylation. However, when cultures were treated with the protein phosphatase inhibitor okadaic acid alone, the level of pP29 increased after 1 h and the presence of okadaic acid during prolonged IL-1 treatment blocked the decline in pP29. The protein synthesis inhibitors puromycin, emetine, and cycloheximide also blocked the decline in pP29 during IL-1 treatment. These data suggest that IL-1-stimulated P29 phosphorylation is made up of two components, one susceptible to prolonged down-regulation even in the absence of the cytokine and one refractory to desensitization but that remains active only in the presence of IL-1. IL-1-induced changes in pP29 levels may be dependent on the relative activities of protein kinase and protein phosphatase activities.
Insights
Interleukin-1 (IL-1) rapidly increases protein P29 phosphorylation in endothelial cells, with two distinct components: one sensitive to IL-1 withdrawal and another requiring IL-1 for sustained activity. This suggests a balance between protein kinase and phosphatase activities regulates IL-1 signaling.
Area of Science:
- Cellular signaling
- Molecular biology
- Inflammation research
Background:
- Interleukin-1 (IL-1) is a key inflammatory cytokine.
- IL-1 signaling involves post-translational modifications like protein phosphorylation.
- Understanding IL-1-induced protein phosphorylation is crucial for inflammatory pathway elucidation.
Purpose of the Study:
- To investigate the dynamics of IL-1-induced phosphorylation of a Mr = 29,000 protein (P29) in human endothelial cells.
- To identify the regulatory mechanisms controlling P29 phosphorylation and dephosphorylation.
- To explore the role of protein kinases, phosphatases, and protein synthesis in IL-1 signaling.
Main Methods:
- Treatment of human endothelial cells with IL-1 and IL-1 receptor antagonist.
- Analysis of protein phosphorylation using Western blotting or similar techniques.
- Assessment of the effects of protein kinase inhibitors, calcium chelators, calmodulin inhibitors, protein phosphatase inhibitors (okadaic acid), and protein synthesis inhibitors (puromycin, emetine, cycloheximide).
Main Results:
- IL-1 treatment caused a rapid 18-fold increase in P29 phosphorylation (pP29), with 80% disappearing within 60 minutes.
- A stable pP29 component remained for hours, but was lost upon IL-1 removal and required 16 hours for full restimulation.
- Protein kinase inhibitors had no effect, but okadaic acid and protein synthesis inhibitors blocked the decline of pP29, suggesting phosphatase and protein synthesis involvement.
Conclusions:
- IL-1-stimulated P29 phosphorylation comprises two components: one desensitizing and one IL-1-dependent.
- The regulation of pP29 levels appears to depend on the interplay between protein kinase and protein phosphatase activities.
- Protein synthesis is necessary for the sustained downregulation of IL-1-induced P29 phosphorylation.