Phosphorylation of serine 276 is essential for p65 NF-kappaB subunit-dependent cellular responses

Biochem Biophys Res Commun. 2003 Jan 24;300(4):807-12. doi: 10.1016/s0006-291x(02)02932-7.

Abstract

Phosphorylation of several serine residues especially in the transactivation (TA) domain of p65 NF-kappaB subunit has been suggested to be important for its transcriptional activity. However, the responsible phosphorylation site of p65 remains controversial. To investigate the biological significance of phosphorylation and to determine the critical phosphorylation sites of p65, we reconstituted murine embryonic fibroblasts (MEFs) from p65(-/-) mice with various serine to alanine (SA)-substituted mutants of p65. Unexpectedly, mutants in the TA domain, including S529A, S536A, and S529A/S536A, completely rescued the defect of p65(-/-) MEFs as assessed by tumor necrosis factor (TNF)- or interleukin-1 (IL-1)-induced IL-6 production and protection from TNF-induced cell death. On the other hand, S276A mutant had an impaired ability to rescue these responses. Moreover, TNF-induced phosphorylation of p65 was severely impaired in S276A mutant, indicating that S276 is the major phosphorylation site of p65 and its phosphorylation is essential for p65-dependent cellular responses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / physiology
  • Alanine / metabolism
  • Animals
  • Cell Death / drug effects
  • Cell Death / physiology
  • Cells, Cultured
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / physiology
  • Glycogen Synthase Kinase 3 / metabolism
  • Glycogen Synthase Kinase 3 beta
  • Humans
  • Interleukin-1 / pharmacology
  • Interleukin-6 / metabolism
  • Mice
  • Mice, Knockout
  • NF-kappa B / genetics*
  • NF-kappa B / metabolism*
  • Phosphorylation
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Serine / metabolism*
  • Transcription Factor RelA
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Interleukin-1
  • Interleukin-6
  • NF-kappa B
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Transcription Factor RelA
  • Tumor Necrosis Factor-alpha
  • Serine
  • Tbk1 protein, mouse
  • Glycogen Synthase Kinase 3 beta
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • TBK1 protein, human
  • Cyclic AMP-Dependent Protein Kinases
  • Glycogen Synthase Kinase 3
  • Alanine