title: Characterization of the recombinant IKK1/IKK2 heterodimer. Mechanisms regulating kinase activity.
abstract: Nuclear factor kappa B (NF-kappaB) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB, initiated by an IkappaB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK1) and IkappaB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. To better understand the role of IKKs in NF-kappaB activation, we have cloned, expressed, purified, and characterized the physiological isoform, the rhIKK1/rhIKK2 heterodimer. We compared its kinetic properties with those of the homodimers rhIKK1 and rhIKK2 and a constitutively active rhIKK2 (S177E, S181E) mutant. We demonstrate activation of these recombinantly expressed IKKs by phosphorylation during expression in a baculoviral system. The K(m) values for ATP and IkappaBalpha peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 micrometer, respectively, which are comparable to those of the IKK2 homodimer. However, the purified rhIKK1/rhIKK2 heterodimer exhibits the highest catalytic efficiency (k(cat)/K(m)) of 47.50 h(-1) micrometer(-1) using an IkappaBalpha peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h(-1) micrometer(-1)), its mutant rhIKK2 (S177E, S181E, 1.18 h(-1) micrometer(-1)), or rhIKK1 (0.02 h(-1) micrometer(-1)). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IkappaBalpha peptide, exhibited competitive inhibitory kinetics, only ADP with the low K(i) of 0.77 micrometer may play a physiological role in regulation of the enzyme activity.
authors.affiliation: Discovery Research, G. D. Searle and Company, the Monsanto Life Science Company, St. Louis, Missouri 63167, USA. quang.k.huynh@monsanto.com
authors.forename: Q K
authors.initials: QK
authors.lastname: Huynh
authors.forename: H
authors.initials: H
authors.lastname: Boddupalli
authors.forename: S A
authors.initials: SA
authors.lastname: Rouw
authors.forename: C M
authors.initials: CM
authors.lastname: Koboldt
authors.forename: T
authors.initials: T
authors.lastname: Hall
authors.forename: C
authors.initials: C
authors.lastname: Sommers
authors.forename: S D
authors.initials: SD
authors.lastname: Hauser
authors.forename: J L
authors.initials: JL
authors.lastname: Pierce
authors.forename: R G
authors.initials: RG
authors.lastname: Combs
authors.forename: B A
authors.initials: BA
authors.lastname: Reitz
authors.forename: J A
authors.initials: JA
authors.lastname: Diaz-Collier
authors.forename: R A
authors.initials: RA
authors.lastname: Weinberg
authors.forename: B L
authors.initials: BL
authors.lastname: Hood
authors.forename: B F
authors.initials: BF
authors.lastname: Kilpatrick
authors.forename: C S
authors.initials: CS
authors.lastname: Tripp
mesh_terms: D002384:Catalysis; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051550:I-kappa B Kinase; D007700:Kinetics; D008970:Molecular Weight; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human