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 further phosphorylated and subsequently degraded, allowing NF-kappa B to translocate to the nucleus and transactivate target genes. To identify the kinase(s) involved in I kappa B alpha phosphorylation, we first performed an I kappa B alpha in-gel kinase assay. Two kinase activities of 35 and 42 kDa were identified in cellular extracts from Jurkat T and U937 promonocytic cell lines. Specific inhibitors and immunodepletion studies identified the I kappa B alpha kinase activities as those of the alpha and alpha' subunits of casein kinase II (CKII). Immunoprecipitation studies demonstrated that CKII and I kappa B alpha physically associate in vivo. Moreover, phosphopeptide maps of I kappa B alpha phosphorylated in vitro by cellular extracts and in vivo in resting Jurkat T cells contained the same pattern of phosphopeptides as observed in maps of I kappa B alpha phosphorylated in vitro by purified CKII. Sequence analysis revealed that purified CKII and the kinase activity within 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 uninfected and human immunodeficiency virus (HIV)-infected U937 cells. Immunodepletion of CKII from these extracts abrogated both the basal and enhanced HIV-induced degradation of I kappa B alpha. These studies provide new evidence that the protein kinase CKII physically associates with I kappa B alpha in vivo, induces multisite (serine/threonine) phosphorylation, and is required for the basal and HIV-induced degradation of I kappa B alpha in vitro.
McElhinny
J A
JA
Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Trushin
S A
SA
Bren
G D
GD
Chester
N
N
Paya
C V
CV
D001483:Base Sequence; D047390:Casein Kinase II; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; D011994:Recombinant Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases