IDENTIFICATION OF AN I-KAPPA-B-ALPHA-ASSOCIATED PROTEIN-KINASE IN A HUMAN MONOCYTIC CELL-LINE AND DETERMINATION OF ITS PHOSPHORYLATION SITES ON I-KAPPA-B-ALPHA

Nuclear factor kappa B (NF-kappa B) is stored in the cytoplasm as an i nactive form through interaction with I kappa B, Stimulation of cells leads to a rapid phosphorylation of I kappa B alpha, which is presumed to be important for the subsequent degradation, We have recently repo rted the establishment of a lipopolysaccharide (LPS)-dependent cell fr ee activation system of NF-kappa B in association with the induction o f I kappa B alpha phosphorylation, In this study, we have identified a kinase in cell extracts from the LPS stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates I kappa B alph a. LPS stimulation transiently enhanced the I kappa B alpha-bound kina se activity in THP-1 cells, Mutational analyses of I kappa B alpha and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in t he C-terminal acidic domain of I kappa B alpha, Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of I kappa B alpha, blocked the LPS induced NF-kappa B activation as well as ind ucible phosphorylation of endogenous I kappa B alpha in a cell-free sy stem using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphoryl ation of the C terminal region of I kappa B alpha and subsequent disso ciation of the NF-kappa B . I kappa B alpha complex.