Gold compound auranofin inhibits IκB kinase (IKK) by modifying Cys-179 of IKKβ subunit

Antirheumatic gold compounds have been shown to inhibit NF-κB activation by blocking IκB kinase (IKK) activity. To examine the possible inhibitory mechanism of gold compounds, we expressed wild type and mutant forms of IKKα and β subunits in COS-7 cells and determined the effect of gold on the activity of these enzymes both in vivo and in vitro. Substitution of Cys-179 of IKKβ with alanine (C179A) rendered the enzyme to become resistant to inhibition by a gold compound auranofin, however, similar protective effect was not observed with an equivalent level of IKKα (C178A) mutant expressed in the cells. Auranofin inhibited constitutively active IKKα and β and variants; IKKα (S176E, S180E) or IKKβ (S177E, S181E), suggesting that gold directly cause inhibition of activated enzyme. The different inhibitory effect of auranofin on IKKα (C178A) and IKKβ (C179A) mutants indicates that gold could inhibit the two subunits of IKK in a different mode, and the inhibition of NF-κB and IKK activation induced by inflammatory signals in gold-treated cells appears through its interaction with Cys-179 of IKKβ.

[1]  Ebrahim Zandi,et al.  Direct Phosphorylation of IκB by IKKα and IKKβ: Discrimination Between Free and NF-κB-Bound Substrate , 1998 .

[2]  E. Zandi,et al.  Bridging the Gap: Composition, Regulation, and Physiological Function of the IκB Kinase Complex , 1999, Molecular and Cellular Biology.

[3]  Michael Karin,et al.  Positive and Negative Regulation of IκB Kinase Activity Through IKKβ Subunit Phosphorylation , 1999 .

[4]  J. Bondeson,et al.  Auranofin inhibits the induction of interleukin 1β and tumor necrosis factor α mRNA in macrophages , 1995 .

[5]  Zhaodan Cao,et al.  NF-κB-inducing kinase activates IKK-α by phosphorylation of Ser-176 , 1998 .

[6]  J. Ziegler,et al.  The gold complexes. , 1990, Bailliere's clinical rheumatology.

[7]  P. Baeuerle,et al.  Function and activation of NF-kappa B in the immune system. , 1994, Annual review of immunology.

[8]  G. Natoli,et al.  Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IκB kinase , 2000, Nature.

[9]  K. Jeon,et al.  Thiol-Reactive Metal Compounds Inhibit NF-κB Activation by Blocking IκB Kinase1 , 2000, Journal of Immunology.

[10]  R. Tsien,et al.  Inhibition of NF-κB Activation by Arsenite through Reaction with a Critical Cysteine in the Activation Loop of IκB Kinase* , 2000, The Journal of Biological Chemistry.

[11]  P. B. Chock,et al.  Roles of Superoxide Radical Anion in Signal Transduction Mediated by Reversible Regulation of Protein-tyrosine Phosphatase 1B* , 1999, The Journal of Biological Chemistry.

[12]  G. W. Peet,et al.  Recombinant IκB Kinases α and β Are Direct Kinases of IκBα* , 1998, The Journal of Biological Chemistry.

[13]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[14]  S. Ghosh,et al.  Signal transduction through NF-κB , 1998 .

[15]  Yukio Nakamura,et al.  Genetic approaches in mice to understand Rel/NF-κB and IκB function: transgenics and knockouts , 1999, Oncogene.

[16]  David M. Rothwarf,et al.  A cytokine-responsive IκB kinase that activates the transcription factor NF-κB , 1997, Nature.

[17]  K. Philipson,et al.  Partial rescue of the Na+-Ca2+ exchanger (NCX1) knock-out mouse by transgenic expression of NCX1 , 2003, Experimental & Molecular Medicine.

[18]  G. Panayi,et al.  Intramuscular gold decreases cytokine expression and macrophage numbers in the rheumatoid synovial membrane. , 1994, Annals of the rheumatic diseases.

[19]  M. Karin,et al.  Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. , 1997, The New England journal of medicine.

[20]  T. Maniatis,et al.  MEKK1 activates both IκB kinase α and IκB kinase β , 1998 .

[21]  K. Hoe,et al.  Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation , 2003, Experimental and Molecular Medicine.