N-tosyl-L-phenylalanine chloromethyl ketone inhibits NF-kappaB activation by blocking specific cysteine residues of IkappaB kinase beta and p65/RelA

Biochemistry. 2009 Aug 4;48(30):7271-8. doi: 10.1021/bi900660f.


N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), a serine/cysteine protease inhibitor, has been reported to inhibit expression of inflammatory mediators by blocking nuclear factor-kappaB (NF-kappaB) activation. We examined the effect of TPCK on the NF-kappaB activation pathway in HeLa cells by measuring the activity of IkappaB kinase (IKK) and p65/RelA-DNA binding. TPCK inhibited tumor necrosis factor-alpha-induced IKK activation and directly blocked IKK activity in vitro. TPCK-induced inhibition of NF-kappaB and IKK activation was abrogated by addition of the thiol-reducing agent dithiothreitol, suggesting that the effect of TPCK occurred through modification of a thiol group in IKK. Consistent with this, an IKKbeta mutant in which Cys-179 was substituted with alanine was not more susceptible to TPCK. Our result also showed that TPCK inhibits the DNA binding of transiently expressed p65/RelA in HeLa cells. Inhibition of p65/RelA-DNA binding was recovered in the presence of dithiothreitol, and substitution of Cys-38 with Ser in p65/RelA rendered the protein resistant to inhibition by TPCK. Mass spectrometry analysis of IKKbeta and p65/RelA isolated from cells treated with TPCK by UPLC-ESI-Q-TOF tandem MS revealed the labeling of Cys-179 of IKKbeta and Cys-38 of p65/RelA with a tosylphenylalanylmethyl group. These results suggest that TPCK inhibits NF-kappaB activation by directly modifying thiol groups on two different targets: Cys-179 of IKKbeta and Cys-38 of p65/RelA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cysteine / metabolism*
  • Enzyme Activation
  • HeLa Cells
  • Humans
  • I-kappa B Kinase* / genetics
  • I-kappa B Kinase* / metabolism
  • Molecular Sequence Data
  • Molecular Structure
  • Mutagenesis, Site-Directed
  • NF-kappa B / genetics
  • NF-kappa B / metabolism*
  • Serine Proteinase Inhibitors / chemistry
  • Serine Proteinase Inhibitors / metabolism*
  • Tosylphenylalanyl Chloromethyl Ketone / chemistry
  • Tosylphenylalanyl Chloromethyl Ketone / metabolism*
  • Transcription Factor RelA* / genetics
  • Transcription Factor RelA* / metabolism
  • Tumor Necrosis Factor-alpha / metabolism


  • NF-kappa B
  • Serine Proteinase Inhibitors
  • Transcription Factor RelA
  • Tumor Necrosis Factor-alpha
  • Tosylphenylalanyl Chloromethyl Ketone
  • I-kappa B Kinase
  • Cysteine