Cysteine-179 of I kappa B kinase beta plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines

I kappa B kinase beta (IKK beta) subunit of IKK complex is essential for the activation of NF-kappa B in response to various proinflammatory signals. Cys-179 in the activation loop of IKK beta is known to be the target site for IKK inhibitors such as cyclopentenone prostaglandins, arsenite, and antirheumatic gold compounds. Here we show that a mutant IKK beta in which Cys-179 is substituted with alanine had decreased activity when it was expressed in HEK-293 cells, and TNF stimulation did not restore the activity. Phosphorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKK beta activation was reduced in the IKK beta (C179A) mutant. The activity of IKK beta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappa B inducing kinase (NIK) and MAPK/extracellular signal-regulated kinase kinase kinase 1(MEKK1) or when the serine residues were replaced with phospho-mimetic glutamate. The IKK beta (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate ATP in reaction mixture. Our results suggest that Cys-179 of IKK beta plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.

Gold compound auranofin inhibits I kappaB kinase (IKK) by modifying Cys-179 of IKK beta subunit

Antirheumatic gold compounds have been shown to inhibit NF-kB activation by blocking IkB kinase (IKK) activity. To examine the possible inhibitory mechanism of gold compounds, we expressed wild type and mutant forms of IKk alpha and beta 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 IKK beta 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 IKK alpha (C178A) mutant expressed in the cells. Auranofin inhibited constitutively active IKK alpha and beta and variants; IKK alpha (S176E, S180E) or IKK beta (S177E, S181E), suggesting that gold directly cause inhibition of activated enzyme. The different inhibitory effect of auranofin on IKK alpha (C178A) and IKK beta (C179A) mutants indicates that gold could inhibit the two subunits of IKK in a different mode, and the inhibition of NF- kB and IKK activation induced by inflammatory signals in gold-treated cells appears through its interaction with Cys-179 of IKK beta.

Dual effect of oxidative stress on NF-kappaB activation in HeLa cells

Reactive oxygen species (ROS) has been implicated as an inducer of NF-kappaB activity in numbers of cell types where exposure of cells to ROS such as H2O2 leads to NF-kappaB activation. In contrast, exposure to oxidative stress in certain cell types induced reduction of tumor necrosis factor (TNF)-induced NF-kappaB activation. And various thiol-modifying agents including gold compounds and cyclopentenone prostaglandins inhibit NF-kappaB activation by blocking IkappaB kinase (IKK). To understand such conflicting effect of oxidative stress on NF-kappaB activation, HeLa cells were incubated with H2O2 or diamide and TNF-induced expression of NF-kappaB reporter gene was measured. NF-kappaB activation was significantly blocked by these oxidizing agents, and the inhibition was accompanied with reduced nuclear NF-kappaB and inappropriate cytosolic IkappaB degradation. H2O2 and diamide also inhibited IKK activation in HeLa and RAW 264.7 cells stimulated with TNF and lipopolysaccharide, respectively, and directly blocked IKK activity in vitro. In cells treated with H2O2 alone, nuclear NF-kappaB was induced after 2 h without detectible degradation of cytosolic IkBa or activation of IKK. Our results suggest that ROS has a dual effect on NF-kappaB activation in the same HeLa cells: it inhibits acute IKK-mediated NF-kappaB activation induced by inflammatory signals, while longer-term exposure to ROS induces NF-kappaB activity through an IKK-independent pathway.