N-Tosyl-L-phenylalanine Chloromethyl Ketone Inhibits NF-κB Activation by Blocking Specific Cysteine Residues of IκB Kinase β 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-kappa B (NF-kappa B) activation. We examined the effect of TPCK on the NF-kappa B activation pathway in HeLa cells by measuring the activity of I kappa B kinase (IKK) and p65/Re1A-DNA binding. TPCK inhibited tumor necrosis factor-alpha-induced IKK activation and directly blocked IKK activity in vitro. TPCK-induced inhibition of NF-kappa B 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 IKK beta 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/Re1A in HeLa cells. Inhibition of p65/Re1A-DNA binding was recovered in the presence of dithiothreitol, and substitution of Cys-38 with Ser in p65/Re1A rendered the protein resistant to inhibition by TPCK. Mass spectrometry analysis of IKK beta and p65/Re1A isolated from cells treated with TPCK by UPLC-ESI-Q-TOF tandem MS revealed the labeling of Cys-179 of IKK beta and Cys-38 of p65/Re1A with a tosylphenylalanylmethyl group. These results suggest that TPCK inhibits NF-kappa B activation by directly modifying thiol groups on two different targets: Cys-179 of IKK beta and Cys-38 of p65/Re1A.