Phosphorylation of a cytosolic 65-kDa protein induced by interleukin 1 in glucocorticoid pretreated normal human peripheral blood mononuclear leukocytes.

The authors have previously observed that glucocorticoids dramatically increase the number of interleukin 1 (IL-1) receptors on normal human peripheral blood mononuclear cells (PBMC) (from approximately 100 to 2000 receptors/cell) without significant change in the binding affinity (Kd = approximately 2.6 x 10(-10) M). We, therefore, used such a receptor-enriched glucocorticoid-pretreated PBMC to investigate whether IL-1 induces/increases the phosphorylation of any cell-associated proteins, including possible autophosphorylation of IL-1 receptors. Extraction of 125I-labeled IL-1 alpha cross-linked to IL-1 receptor on steroid-treated PBMC yielded two bands estimated to be 60 and 70 kDa in molecular mass. No molecules were significantly cross-linked with 125I-labeled IL-1 alpha on untreated PBMC. Carrier-free recombinant human IL-1 alpha induced phosphorylation of an acidic 65-kDa protein (pp65) at serine residues within 5 min more effectively in glucocorticoid-treated PBMC than in untreated PBMC. Fractionation of extracts of IL-1-stimulated prednisolone-pretreated PBMC by ultracentrifugation showed that pp65 is located in the cytosol, suggesting that pp65 is not the IL-1 receptor itself. Protein kinase inhibitors, HA1004 and W-7, but not H-7, significantly inhibited the induction of the phosphorylation of 65-kDa protein by IL-1. These data indicate that the glucocorticoid-induced IL-1 receptor is functional and either contains or is closely associated with a serine kinase that is distinct from protein kinase C.