Characterization of the recombinant IKK1/IKK2 heterodimer - Mechanisms regulating kinase activity

被引：56

作者：

Huynh, QK ^{[1
]}

Boddupalli, H ^{[1
]}

Rouw, SA ^{[1
]}

Koboldt, CM ^{[1
]}

Hall, T ^{[1
]}

Sommers, C ^{[1
]}

Hauser, SD ^{[1
]}

Pierce, JL ^{[1
]}

Combs, RG ^{[1
]}

Reitz, BA ^{[1
]}

Diaz-Collier, JA ^{[1
]}

Weinberg, RA ^{[1
]}

Hood, BL ^{[1
]}

Kilpatrick, BF ^{[1
]}

Tripp, CS ^{[1
]}

机构：

[1] Monsanto Life Sci Co, Dept Discovery Pharmacol, Searle Discovery Res, GD Searle & Co, St Louis, MO 63167 USA

来源：

JOURNAL OF BIOLOGICAL CHEMISTRY | 2000年 / 275卷 / 34期

关键词：

D O I：

10.1074/jbc.M000296200

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Nuclear factor kappa B (NP-kappa B) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-kappa B activation depends on phosphorylation and degradation of its inhibitor protein, I kappa B, initiated by an I kappa B kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of I kappa B kinase 1 (IKK1) and I kappa B kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-kappa B essential modulator. To better understand the role of IKKs in NF-kappa B 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 I kappa Ba peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 mu M, 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) mu M-1 using an I kappa B alpha peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h(-1) mu M-1), its mutant rhIKK2 (S177E, S181E, 1.18 h(-1) mu M-1), or rhIKK1 (0.02 h(-1) mu M-1). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated I kappa B alpha peptide, exhibited competitive inhibitory kinetics, only ADP with the low K-i of 0.77 mu M may play a physiological role in regulation of the enzyme activity.

引用

页码：25883 / 25891

页数：9

共 35 条

[1] ADENOSINE CYCLIC 3',5'-MONOPHOSPHATE DEPENDENT PROTEIN-KINASE - A NEW FLUORESCENCE DISPLACEMENT TITRATION TECHNIQUE FOR CHARACTERIZING THE NUCLEOTIDE BINDING-SITE ON THE CATALYTIC SUBUNIT [J].