Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation

Mol Cell Biol. 1996 Mar;16(3):899-906. doi: 10.1128/MCB.16.3.899.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Casein Kinase II
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Humans
  • I-kappa B Proteins*
  • Molecular Sequence Data
  • NF-kappa B / metabolism
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Tumor Cells, Cultured

Substances

  • DNA-Binding Proteins
  • I kappa B beta protein
  • I-kappa B Proteins
  • NF-kappa B
  • Recombinant Proteins
  • Casein Kinase II
  • Protein-Serine-Threonine Kinases