Characterization of the recombinant IKK1/IKK2 heterodimer. Mechanisms regulating kinase activity.
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.
Huynh
Q K
QK
Discovery Research, G. D. Searle and Company, the Monsanto Life Science Company, St. Louis, Missouri 63167, USA. quang.k.huynh@monsanto.com
Boddupalli
H
H
Rouw
S A
SA
Koboldt
C M
CM
Hall
T
T
Sommers
C
C
Hauser
S D
SD
Pierce
J L
JL
Combs
R G
RG
Reitz
B A
BA
Diaz-Collier
J A
JA
Weinberg
R A
RA
Hood
B L
BL
Kilpatrick
B F
BF
Tripp
C S
CS
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
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