Cysteine-179 of IkappaB kinase beta plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines.
IkappaB kinase beta (IKKbeta) subunit of IKK complex is essential for the activation of NF-kappaB in response to various proinflammatory signals. Cys-179 in the activation loop of IKKbeta 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 IKKbeta 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 IKKbeta activation was reduced in the IKKbeta (C179A) mutant. The activity of IKKbeta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappaB 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 IKKbeta (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 IKKbeta plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.
Byun
Mi-Sun
MS
Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea.
Choi
Jin
J
Jue
Dae-Myung
DM
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution / Q000502:physiology; D020134:Catalytic Domain; D002478:Cells, Cultured; D003545:Cysteine / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000737:chemistry*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D012694:Serine / Q000378:metabolism*; D014162:Transfection
D050505:Mutant Proteins; D012694:Serine; D000255:Adenosine Triphosphate; D051550:I-kappa B Kinase; D020930:MAP Kinase Kinase Kinases; D003545:Cysteine