Cysteine-179 of IκB kinase β plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines

Abstract

IκB kinase β (IKKβ) subunit of IKK complex is essential for the activation of NF-κB in response to various proinflammatory signals. Cys-179 in the activation loop of IKKβ 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 IKKβ 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 IKKβ activation was reduced in the IKKβ (C179A) mutant. The activity of IKKβ (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-κB 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 IKKβ (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 IKKβ plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.

Anti-angiogenic effects of dietary isothiocyanates: Mechanisms of action and implications for human health

“…For example, the DNA binding domains of the p50 and p65 NF-B subunits contain redox-regulated cysteine residues (Cys 62 and Cys 38 , respectively) that have been shown to be covalently modified by andrographolide and picroliv, respectively, thereby preventing DNA binding [50,51]. Also, IKK contains a redox regulated cysteine residue (Cys 179 ) that is essential for activity [52], and is modulated by other agents such as curcumin and butein (3,4,2,4-tetrahydroxychalcone) [53,54]. Thus, these cysteine residues are candidate sites for thiocarbamoylation by ITCs, although direct evidence for covalent binding to NF-B subunits by this class of molecule is lacking.…”

Piperlongumine Chemosensitizes Tumor Cells through Interaction with Cysteine 179 of IκBα Kinase, Leading to Suppression of NF-κB–Regulated Gene Products

“…IKKb, a subunit of the IKK complex, is essential for the activation of NF-kB in response to various proinflammatory stimuli. Cys-179 in the activation loop of this IKKb plays a major role in IKK activation as mutation of this residue to alanine decreased its activity (24). Mutation of Cys179 to alanine in IKKb also caused reduced phosphorylation of serine residues at positions 177 and 181, required for IKKb activation.…”

Chemical biology of inflammatory cytokine signaling

“…53,54 Indeed, it has been shown that Cys-179 of IKKb is critical for enzyme activation by promoting the phosphorylation of serines in the activation loop. 55 The IKKb mutant in which Cys-179 is replaced with alanine exhibited reduced kinase activity in response to physiological stimuli, whereas it exerted enzymatic activity at levels similar to wild-type IKKb when co-expressed with mitogen-activated protein (MAP) kinase kinase kinase, such as NF-kB-inducing kinase. [53][54][55] The difference in sensitivity between wild-type IKKb and its mutant IKKb (C179A) in cell culture or in their purified forms clearly revealed that a large number of small molecules directly inhibit IKKb activity by the modification of Cys-179.…”