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The specificity of 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an ATP/GTP competitive inhibitor of protein kinase casein kinase-2 (CK2), has been examined against a panel of 33 protein kinases, either Ser/Thr-or Tyr-specific. In the presence of 10 W WM TBB (and 100 W WM ATP) only CK2 was drastically inhibited ( s 85%) whereas three kinases (phosphorylase kinase, glycogen synthase kinase 3L L and cyclin-dependent kinase 2/cyclin A) underwent moderate inhibition, with IC 50 values one^two orders of magnitude higher than CK2 (IC 50 = 0.9 W WM). TBB also inhibits endogenous CK2 in cultured Jurkat cells. A CK2 mutant in which Val66 has been replaced by alanine is much less susceptible to inhibition by TBB as well as by another ATP competitive inhibitor, emodin. These data show that TBB is a quite selective inhibitor of CK2, that can be used in cell-based assays. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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Nrf2 is a key transcription factor in the cellular response to oxidative stress. In this study we first identify two phosphorylated forms of endogenous human Nrf2 after chemically-induced oxidative stress and provide evidence that protein kinase CK2-mediated sequential phosphorylation plays potential role in Nrf2 activation and degradation. Human Nrf2 has a predicted molecular mass of 66 kDa. However, immunoblots showed that two bands at 98 and 118 kDa, which are identified as phosphorylated forms, are increased in response to Nrf2 inducers. In addition, human Nrf2 was found to be a substrate for CK2 which mediated two steps of phosphorylation, resulting in two forms of Nrf2 migrating with differing Mr at 98 kDa (Nrf2-98) and 118 kDa (Nrf2-118). Our results support a role in which calmodulin binding regulates CK2 activity, in that cold (25 °C) in Ca 2+ -free media (cold/Ca 2+ -free) decreased both cellular calcium levels and CK2-calmodulin binding and induced Nrf2-118 formation, the latter of which was prevented by CK2 specific inhibitors. Gel-shift assays showed that the Nrf2-118 generated under cold/Ca 2+ -free conditions does not bind to the antioxidant response element, indicating that Nrf2-98 has transcriptional activity. In contrast, Nrf2-118 is more susceptible to degradation. These results provide evidence for phosphorylation by CK2 as a critical controlling factor in Nrf2-mediated cellular antioxidant response.

A search has been initiated for lead inhibitors of the nonstructural protein 3 (NS3)-associated NTPase/helicase activities of hepatitis C virus, the related West Nile virus, Japanese encephalitis virus and the human mitochondrial Suv3 enzyme. Random screening of a broad range of unrelated low-molecular mass compounds, employing both RNA and DNA substrates, revealed that 4,5,6,7-tetrabromobenzotriazole (TBBT) hitherto known as a potent highly selective inhibitor of protein kinase 2, is a good inhibitor of the helicase, but not NTPase, activity of hepatitis C virus NTPase/helicase. The IC 50 is approximately 20 lM with a DNA substrate, but only 60 lM with an RNA substrate. Several related analogues of TBBT were enzymeand/or substrate-specific inhibitors. For example, 5,6-dichloro-1-(b-D-ribofuranosyl)benzotriazole (DRBT) was a good, and selective, inhibitor of the West Nile virus enzyme with an RNA substrate (IC 50 0.3 lM), but much weaker with a DNA substrate (IC 50 3 lM). Preincubation of the enzymes, but not substrates, with DRBT enhanced inhibitory potency, e.g. the IC 50 vs the hepatitis C virus helicase activity was reduced from 1.5 to 0.1 lM. No effect of preincubation was noted with TBBT, suggesting a different mode of interaction with the enzyme. The tetrachloro congener of TBBT, 4,5,6,7,-tetrachlorobenzotriazole (TCBT; a much weaker inhibitor of casein kinase 2) is also a much weaker inhibitor than TBBT of all four helicases. Kinetic studies, supplemented by comparison of ATP-binding sites, indicated that, unlike the case with casein kinase 2, the mode of action of the inhibitors vs the helicases is not by interaction with the catalytic ATP-binding site, but rather by occupation of an allosteric nucleoside/nucleotide binding site. The halogeno benzimidazoles and benzotriazoles included in this study are excellent lead compounds for the development of more potent inhibitors of hepatitis C virus and other viral NTPase/helicases.

Carbon monoxide (CO) is a putative gaseous neurotransmitter that lacks vesicular storage and must be synthesized rapidly following neuronal depolarization. We show that the biosynthetic enzyme for CO, heme oxygenase-2 (HO2), is activated during neuronal stimulation by phosphorylation by CK2 (formerly casein kinase 2). Phorbol ester treatment of hippocampal cultures results in the phosphorylation and activation of HO2 by CK2, implicating protein kinase C (PKC) in CK2 stimulation. Odorant treatment of olfactory receptor neurons augments HO2 phosphorylation and activity as well as cyclic guanosine monophosphate (cGMP) levels, with all of these effects selectively blocked by CK2 inhibitors. Likewise, CO-mediated nonadrenergic, noncholinergic (NANC) relaxation of the internal anal sphincter requires CK2 activity. Our findings provide a molecular mechanism for the rapid neuronal activation of CO biosynthesis, as required for a gaseous neurotransmitter.