title: Phosphorylation of an Mr = 29,000 protein by IL-1 is susceptible to partial down-regulation after endothelial cell activation.
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.
authors.affiliation: Department of Molecular and Experimental Medicine, Scripps Clinic and Research Foundation, La Jolla, CA 92037.
authors.forename: E G
authors.initials: EG
authors.lastname: Levin
authors.forename: L
authors.initials: L
authors.lastname: Santell
mesh_terms: D002478:Cells, Cultured; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000378:metabolism*; D004988:Ethers, Cyclic / Q000494:pharmacology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D008970:Molecular Weight; D019319:Okadaic Acid; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D004988:Ethers, Cyclic; D007375:Interleukin-1; D010750:Phosphoproteins; D019319:Okadaic Acid