CASEIN KINASE-II PHOSPHORYLATES I-KAPPA-B-ALPHA AT S-283, S-289, S-293, AND T-291 AND IS REQUIRED FOR ITS DEGRADATION

The phosphoprotein I kappa B alpha exists in the cytoplasm of resting cells bound to the ubiquitous transcription factor NF-kappa B (p50-p65 ). In response to specific cellular stimulation, I kappa B alpha is fu rther phosphorylated and subsequently degraded, allowing NF-KB to tran slocate to the nucleus and transactivate target genes. To identify the kinase(s) involved in I kappa B alpha phosphorylation, we first perfo rmed an I kappa B alpha in-gel kinase assay. Two kinase activities of 35 and 32 kDa were identified in cellular extracts from Jurkat T and U 937 promonocytic cell lines. Specific inhibitors and immunodepletion s tudies identified the I kappa B alpha kinase activities as those of th e alpha and alpha ' subunits of casein kinase II (CKII). Immunoprecipi tation studies demonstrated that CKII and I kappa B alpha physically a ssociate in vivo. Moreover, phosphopeptide maps of I kappa B alpha pho sphorylated in vitro by cellular extracts and in vivo in resting Jurka t T cells contained the same pattern of phosphopeptides as observed in maps of I kappa B alpha phosphorylated in vitro by purified CKII. Seq uence analysis revealed that purified CKII and the kinase activity wit hin cell extracts phosphorylated I kappa B alpha at its C terminus at S-283, S-288, S-293, and T-291. The functional role of CKII was tested in an in vitro I kappa B alpha degradation assay with extracts from u ninfected and human immunodeficiency virus (HIV)-infected U937 cells. Immunodepletion of CKII from these extracts abrogated both the basal a nd enhanced HIV-induced degradation of I kappa B alpha. These studies provide new evidence that the protein kinase CKII physically associate s with I kappa B alpha in vivo, induces multisite (serine/threonine) p hosphorylation, and is required for the basal and HIV-induced degradat ion of I kappa B alpha in vitro.