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Assaying Protein Kinase Activity with Radiolabeled ATP
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A serine/threonine kinase activity is closely associated with a 65-kDa phosphoprotein specifically recognized by the

J Ostrowski1, J E Sims, C H Sibley

  • 1Department of Medicine, University of Washington, Seattle 98195.

The Journal of Biological Chemistry
|July 5, 1991
PubMed

Insights

Researchers identified a 65-kDa nuclear phosphoprotein that binds the kappa B enhancer, a key element in gene transcription. This protein, phosphorylated by a nuclear kinase, may play a role in how growth factors regulate gene expression.

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Protein Biochemistry

Background:

  • The immunoglobulin kappa light chain enhancer (kappa B) is a critical cis-acting transcriptional element.
  • kappa B binds transcription factors, including the NF-kappa B family.

Purpose of the Study:

  • To isolate and characterize proteins that bind the kappa B enhancer.
  • To investigate the phosphorylation and kinase activity associated with kappa B-binding proteins.

Main Methods:

  • Affinity purification using kappa B motif-coupled agarose beads.
  • Southwestern blot analysis to confirm direct DNA binding.
  • In vivo and in vitro phosphorylation assays.
  • Anion-exchange and hydrophobic interaction chromatography.

Main Results:

  • A 65-kDa nuclear phosphoprotein specifically binding the kappa B element was isolated.
  • This protein is phosphorylated by a nuclear serine/threonine kinase, activity stimulated by IL-1 alpha and LPS.
  • Kinase activity correlated with NF-kappa B binding in B and T cell lines.
  • The 65-kDa protein and kinase activity remained associated after chromatography, suggesting autophosphorylation or close association.

Conclusions:

  • A novel 65-kDa kappa B-associated phosphoprotein and its associated kinase activity were identified.
  • This system may represent a new mechanism for growth factor-mediated gene expression regulation.
  • The protein is likely autophosphorylated or phosphorylated by a tightly associated kinase.

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