Probing kinase activation and substrate specificity with an engineered monomeric IKK2.

Catalytic subunits of the IκB kinase (IKK), IKK1/IKKα, and IKK2/IKKß function in vivo as dimers in association with the necessary scaffolding subunit NEMO/IKKγ. Recent X-ray crystal structures of IKK2 suggested that dimerization might be mediated by a smaller protein-protein interaction than previously thought. Here, we report that removal of a portion of the scaffold dimerization domain (SDD) of human IKK2 yields a kinase subunit that remains monomeric in solution. Expression in baculovirus-infected Sf9 insect cells and purification of this engineered monomeric human IKK2 enzyme allows for in vitro analysis of its substrate specificity and mechanism of activation. We find that the monomeric enzyme, which contains all of the amino-terminal kinase and ubiquitin-like domains as well as the more proximal portions of the SDD, functions in vitro to direct phosphorylation exclusively to residues S32 and S36 of its IκBα substrate. Thus, the NF-κB-inducing potential of IKK2 is preserved in the engineered monomer. Furthermore, we observe that our engineered IKK2 monomer readily autophosphorylates activation loop serines 177 and 181 in trans. However, when residues that were previously observed to interfere with IKK2 trans autophosphorylation in transfected cells are mutated within the context of the monomer, the resulting Sf9 cell expressed and purified proteins were significantly impaired in their trans autophosphorylation activity in vitro. This study further defines the determinants of substrate specificity and provides additional evidence in support of a model in which activation via trans autophosphorylation of activation loop serines in IKK2 requires transient assembly of higher-order oligomers.

The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity.

NF-kappaB activation in response to pro-inflammatory stimuli relies upon phosphorylation of IkappaB alpha at serines 32 and 36 by the beta subunit of the IkappaB kinase complex (IKK). In this study, we build upon the observation that highly purified human IKKbeta subunit preparations retain this specificity in vitro. We show that IKKbeta constructs that lack their carboxy-terminus beginning at the leucine zipper motif fail to phosphorylate IkappaB alpha at Ser-32 and Ser-36. Rather, these constructs, which contain the entire IKKbeta subunit kinase domain, phosphorylate serine and threonine residues contained within the IkappaB alpha carboxy-terminal PEST region. Furthermore, removal of the leucine zipper and helix-loop-helix regions converts IKKbeta to monomer. We propose that the helix-loop-helix of the human IKKbeta subunit is necessary for restricting substrate specificity toward Ser-32 and Ser-36 in IkappaB alpha and that in the absence of its carboxy-terminal protein structural motifs the human IKKbeta subunit kinase domain exhibits a CK2-like phosphorylation specificity.