DNA-dependent protein kinase phosphorylation of IκBα and IκBβ regulates NF-κB DNA binding properties

Li Liu, Youn Tae Kwak, Françoise Bex, León F. García-Martínez, Xiao Hua Li, Katheryn Meek, William S. Lane, Richard B. Gaynor

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

Regulation of the IκBα and IκBβ proteins is critical for modulating NF-κB-directed gene expression. Both IκBα and IκBβ are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, we utilized a biochemical fractionation scheme to purify a kinase activity which phosphorylates residues in the amino and carboxy termini of both IκBα and IκBβ. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IκBα and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IκBα, murine severe combined immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrated constitutive nuclear NF-κB DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IκBα that was phosphorylated by DNA-PK was a more potent inhibitor of NF-κB binding than nonphosphorylated IκBα. These results suggest that DNA-PK phosphorylation of IκBα increases its interaction with NF-κB to reduce NF-κB DNA binding properties.

Original languageEnglish (US)
Pages (from-to)4221-4234
Number of pages14
JournalMolecular and Cellular Biology
Volume18
Issue number7
StatePublished - Jul 1998

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DNA-Activated Protein Kinase
Phosphorylation
DNA
Severe Combined Immunodeficiency
Phosphotransferases
Serine
Catalytic Domain
Proteins
Threonine
Cell Extracts
Genes
Mass Spectrometry
Gels
High Pressure Liquid Chromatography
Ions
Gene Expression
Cell Line
Peptides

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Liu, L., Kwak, Y. T., Bex, F., García-Martínez, L. F., Li, X. H., Meek, K., ... Gaynor, R. B. (1998). DNA-dependent protein kinase phosphorylation of IκBα and IκBβ regulates NF-κB DNA binding properties. Molecular and Cellular Biology, 18(7), 4221-4234.

DNA-dependent protein kinase phosphorylation of IκBα and IκBβ regulates NF-κB DNA binding properties. / Liu, Li; Kwak, Youn Tae; Bex, Françoise; García-Martínez, León F.; Li, Xiao Hua; Meek, Katheryn; Lane, William S.; Gaynor, Richard B.

In: Molecular and Cellular Biology, Vol. 18, No. 7, 07.1998, p. 4221-4234.

Research output: Contribution to journalArticle

Liu, L, Kwak, YT, Bex, F, García-Martínez, LF, Li, XH, Meek, K, Lane, WS & Gaynor, RB 1998, 'DNA-dependent protein kinase phosphorylation of IκBα and IκBβ regulates NF-κB DNA binding properties', Molecular and Cellular Biology, vol. 18, no. 7, pp. 4221-4234.
Liu, Li ; Kwak, Youn Tae ; Bex, Françoise ; García-Martínez, León F. ; Li, Xiao Hua ; Meek, Katheryn ; Lane, William S. ; Gaynor, Richard B. / DNA-dependent protein kinase phosphorylation of IκBα and IκBβ regulates NF-κB DNA binding properties. In: Molecular and Cellular Biology. 1998 ; Vol. 18, No. 7. pp. 4221-4234.
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