Novel NEMO/IkappaB kinase and NF-kappa B target genes at the pre-B to immature B cell transition.

The IkappaB kinase (IKK) signaling complex is responsible for activating NF-kappaB-dependent gene expression programs. Even though NF-kappaB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-kappaB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cells versus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKgamma/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-kappaB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118, Pea-15/MAT1, CD82, CD40L, Wnt10a, Notch 1, R-ras, Rgs-16, PAC-1, ISG15, and CD36) and five repressed (CCR2, VpreB, lambda5, SLPI, and CMAP/Cystatin7) genes, respectively, were bona fide NF-kappaB targets by virtue of their response to a transdominant IkappaBalphaSR (super repressor). MyD118 and ISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-kappaB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

IkappaB kinases alpha and beta show a random sequential kinetic mechanism and are inhibited by staurosporine and quercetin.

Activation of transcription factor NF-kappaB is regulated by phosphorylation and subsequent degradation of its inhibitory subunit IkappaB. The signal-induced phosphorylation of IkappaB involves two IkappaB kinases, IKKalpha and IKKbeta. In the present study, we investigated the kinetic mechanisms of IKKalpha and IKKbeta by substrate and product inhibition. For both IKKalpha and IKKbeta, the product ADP was a competitive inhibitor versus ATP and a non-competitive inhibitor versus IkappaBalpha. An alternative peptide substrate, IkappaBalpha-(21-41), was a competitive inhibitor versus IkappaBalpha and a non-competitive inhibitor versus ATP for both kinases. These results rigorously eliminate the possibility of an ordered sequential mechanism and demonstrate that both kinases have a random sequential bi bi mechanism. Two natural compounds, quercetin and staurosporine, had previously been shown to inhibit the NF-kappaB pathway, but the molecular target(s) of these compounds in the event had not been established. Here we demonstrate that quercetin and staurosporine potently inhibit both IKKalpha and IKKbeta. Daidzein, a quercetin analogue that does not inhibit NF-kappaB activation, showed no significant inhibition of either enzyme. This suggests that the inhibitory properties of quercetin and staurosporine in the NF-kappaB pathway are mediated in part by their inhibition of IKKalpha and IKKbeta. Mechanism studies reveal that staurosporine is a competitive inhibitor versus ATP, whereas quercetin serves as a mixed type inhibitor versus ATP. The strong inhibition of IKKbeta by staurosporine (K(i) = 172 nM) and ADP (K(i) = 136 nM) provides a rationale and structural framework for designing potent ATP-site inhibitors of IKKbeta, which is an attractive drug target for inflammatory diseases.

Recombinant IkappaB kinases alpha and beta are direct kinases of Ikappa Balpha.

Activation of the transcription factor NF-kappaB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK), catalyzes the phosphorylation of IkappaB. The two kinase components of the IKK complex, IKKalpha and IKKbeta, were overexpressed in insect cells and purified to homogeneity. Both purified IKKalpha and IKKbeta specifically catalyzed the phosphorylation of the regulatory serine residues of Ikappa Balpha. Hence, IKKalpha and IKKbeta were functional catalytic subunits of the IKK complex. Purified IKKalpha and IKKbeta also preferentially phosphorylated serine as opposed to threonine residues of Ikappa Balpha, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKalpha and IKKbeta revealed that the kinase activity of IKKbeta on Ikappa Balpha is 50-60-fold higher than that of IKKalpha. The primary difference between the two activities is the Km for Ikappa Balpha. The kinetics of both IKKalpha and IKKbeta followed a sequential Bi Bi mechanism. No synergistic effects on Ikappa Balpha phosphorylation were detected between IKKalpha and IKKbeta. Thus, in vitro, IKKalpha and IKKbeta are two independent kinases of Ikappa Balpha.

Non-sialate inhibitor of influenza A/WSN/33 neuraminidase.

An N1 strain of influenza A virus neuraminidase (A/WSN/33 NA) was purified and used to screen for inhibitors. As a result, a well-known tuberculostatic, 4'-formylacetanilide thiosemicarbazone (or thiacetazone), was identified. Thiacetazone is a non-sialate compound and inhibits the enzyme in a noncompetitive manner with respect to the substrate sialic acid. Mechanistic studies indicate that the inhibition was due to the competition of thiacetazone with Ca2+, which maintains N1 neuraminidase in an active conformation. The Ki for the inhibition was estimated to be about 4 microM. Equilibrium exchange experiments revealed that when purified A/WSN/33 NA was incubated with 5 microM 45CaCl2, 2 mol of 45Ca2+ ion was exchanged into each mole of NA tetramer and subsequently displaced from the enzyme upon the introduction of the inhibitor. Inhibition of plaque formation by thiacetazone in an MDCK cell culture that had been infected with the influenza A/WSN/33 virus was demonstrated. Thiacetazone was highly specific for A/WSN/33 neuraminidase, since little effect was noted when it was tested against NAs from the other strains of influenza virus or from bacteria. This compound might represent a group of non-sialate inhibitors of influenza NA that bind to a noncatalytic or an allosteric site on the enzyme.