The kinetics of association and phosphorylation of I kappa B isoforms by Ikappa B kinase 2 correlate with their cellular regulation in human endothelial cells

Activation of the transcription factor NF-kappa B depends on the specific d ual phosphorylation of its inhibitor protein I kappa B by the homologous cy tokine-inducible I kappa B kinases 1 and 2 (IKK1/2). Various I kappa B isof orms exist: I kappa B alpha, I kappa B beta 1/2 (two alternative splice var iants), and I kappa B epsilon. However, the individual relevance and the sp ecific regulation of these isoforms is not well-understood. We have studied the direct interaction of recombinant I kappa B alpha, I kappa B beta 1, I kappa B beta 2, and I kappa B epsilon with the recombinant homodimeric IKK 2. Fluorescence-based active site titration revealed that each IKK2 dimer c ontains two binding sites for I kappa B. By using surface plasmon resonance analysis, we found that all I kappa B proteins interact with the IKK2 dime r following a noncooperative binding mechanism. Further, the four I kappa B proteins bind to the kinase with equilibrium dissociation constants (K-D) in the range of 50-300 nM; the association rate constants for all I kappa B isoforms with IKK2 were between 6.0 x 10(3) and 22.5 x 10(3) M-1 s(-1), an d the dissociation rate constants were between 1.25 x 10(-3) and 1.75 x 10( -3) s(-1). This high-affinity binding suggests that the previously observed preassociation of all analyzed I kappa B proteins with the biochemically p urified 700 kDa I kappa B kinase (IKK) complex is based on a direct enzyme- substrate association between the various I kappa B isoforms and the IKK pr oteins. The apparent catalytic efficiencies (k(cat)/K-M) of IKK2 for I kapp a B alpha, I kappa B beta 1, I kappa B beta 2, and I kappa B epsilon were 2 2 x 10(3), 10 x 10(3), 5.4 x 10(3), and 8.5 x 10(3) s(-1) M-1, respectively , with K-M values ranging between 1.7 x 10(-6) and 3.2 x 10(-6) M and k(cat ) values ranging between 1.5 x 10(-2) and 3.7 x 10(-2) s(-1). The relative affinities and catalytic efficiencies of IKK2 for the I kappa B isoforms we re also reflected by the kinetics observed for the TNF-induced, phosphoryla tion-dependent degradation of the or, beta 1, beta 2, and epsilon isoforms of I kappa B in human umbilical vein endothelial cells. Therefore, differen tial regulation of the I kappa B isoforms in some cell types is not a direc t result of the IKK activity, but appears to be due to parallel events.