N-tosyl-L-phenylalanine chloromethyl ketone inhibits NF-kappaB activation by blocking specific cysteine residues of IkappaB kinase beta and p65/RelA.
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.
Ha
Kyung-Ho
KH
Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea.
Byun
Mi-Sun
MS
Choi
Jin
J
Jeong
Jaeho
J
Lee
Kong-Joo
KJ
Jue
Dae-Myung
DM
D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000737:chemistry / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016328:NF-kappa B; D015842:Serine Proteinase Inhibitors; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D014108:Tosylphenylalanyl Chloromethyl Ketone; D051550:I-kappa B Kinase; D003545:Cysteine