The catalytic subunits of IkappaB kinase, IKK-1 and IKK-2, contain non-equivalent active sites when expressed as homodimers.


Journal Article

The signal-inducible phosphorylation of serines 32 and 36 of IkappaBalpha is the key step in regulating the subsequent ubiquitination and proteolysis of IkappaBalpha which then releases NF-kappaB to promote gene transcription. The multisubunit IkappaB kinase responsible for this phosphorylation contains two catalytic subunits, termed IKK-1 and IKK-2. It has been shown that both subunits catalyze the phosphorylation of IkappaBalpha as well as an autophosphorylation at a C-terminal cluster of serines. With baculovirus/insect cell-expressed homodimeric IKK-1 or IKK-2, inhibitors such as ADP or a peptide inhibitor (corresponding to amino acid residues 26-42 of IkappaBalpha with Ser-32 and Ser-36 changed to aspartates) inhibited autophosphorylation and IkappaBalpha phosphorylation reactions with different potencies. ADP was more potent against IkappaBalpha phosphorylation as compared to autophosphorylation, while the peptide inhibitor showed the opposite effect. Pseudo-Dixon plots of the inhibition with ADP were linear while non-linear plots were obtained with the peptide inhibitor, suggesting a cooperative effect in the case of the latter. Using different concentrations of IKK-1, autophosphorylation was shown to be intramolecular. These results indicated that there were non-equivalent active sites present within the preparations of recombinant homodimers of IKK-1 and IKK-2. The peptide inhibitor showed equivalent inhibitory effects with wild-type IKK-1 and the S176E/S180E mutant. In contrast, ADP showed equipotent inhibition against the S176E/S180E mutant-catalyzed autophosphorylation and IkappaBalpha phosphorylation reactions. A model is proposed in which the phosphorylation state of the activation loop of IKK-1 or IKK-2 affects the active site conformation of the enzyme such that the two forms catalyze the autophosphorylation and IkappaBalpha phosphorylation reactions with different affinities. In addition, the two active sites within the dimer appear to act in a cooperative fashion so that binding of peptide inhibitor at one active site affects the conformation of the other active site.

Full Text

Duke Authors

Cited Authors

  • Burke, JR; Strnad, J

Published Date

  • May 24, 2002

Published In

Volume / Issue

  • 293 / 5

Start / End Page

  • 1508 - 1513

PubMed ID

  • 12054687

Pubmed Central ID

  • 12054687

International Standard Serial Number (ISSN)

  • 0006-291X

Digital Object Identifier (DOI)

  • 10.1016/S0006-291X(02)00417-5


  • eng

Conference Location

  • United States