NF-B is sequestered in the cytoplasm by the inhibitory IB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IBs leading to their degradation that results in NF-B activation. IKK-1 and IKK-2 are two direct IB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and compared their enzymatic properties with those of rhIKK-2. We show that rhIKK-i and rhTBK-1 are enzymatically similar to each other. We demonstrate by phosphopeptide mapping and site-specific mutagenesis that rhIKK-i and rhTBK-1 are phosphorylated on serine 172 in the mitogen-activated protein kinase kinase activation loop and that this phosphorylation is necessary for kinase activity. Also, rhIKK-i and rhTBK-1 have differential peptide substrate specificities compared with rhIKK-2, the mitogen-activated protein kinase kinase activation loop of IKK-2 being a more favorable substrate than the IB␣ peptide. Finally, using analogs of ATP, we demonstrate unique differences in the ATP-binding sites of rhIKK-i, rhTBK-1, and rhIKK-2. Thus, although these IKKs are structurally similar, their enzymatic properties may provide insights into their unique functions.

Nuclear factor kappa B (NF-B) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-B activation depends on phosphorylation and degradation of its inhibitor protein, IB, initiated by an IB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IB kinase 1 (IKK1) and IB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-B essential modulator. To better understand the role of IKKs in NF-B activation, we have cloned, expressed, purified, and characterized the physiological isoform, the rhIKK1/rhIKK2 heterodimer. We compared its kinetic properties with those of the homodimers rhIKK1 and rhIKK2 and a constitutively active rhIKK2 (S177E, S181E) mutant. We demonstrate activation of these recombinantly expressed IKKs by phosphorylation during expression in a baculoviral system. The K m values for ATP and IB␣ peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 M, respectively, which are comparable to those of the IKK2 homodimer. However, the purified rhIKK1/rhIKK2 heterodimer exhibits the highest catalytic efficiency (k cat / K m ) of 47.50 h ؊1 M ؊1 using an IB␣ peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h ؊1 M ؊1 ), its mutant rhIKK2 (S177E, S181E, 1.18 h ؊1 M ؊1 ), or rhIKK1 (0.02 h ؊1 M ؊1 ). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IB␣ peptide, exhibited competitive inhibitory kinetics, only ADP with the low K i of 0.77 M may play a physiological role in regulation of the enzyme activity.

Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme in hexosamine biosynthesis, an important pathway for cellular glucose sensing. Human GFA has two potential sites for phosphorylation by cAMP-dependent protein kinase A (PKA). To test whether GFA activity is regulated by cAMP-dependent phosphorylation, rat aortic smooth muscle cells were treated in vivo with cAMP-elevating agents, 10 micromol/l forskolin, 1 mmol/l 8-Br-cAMP, or 3-isobutyl-1-methylxanthine. All treatments resulted in rapid and significant increases (2- to 2.4-fold) in GFA activity assayed in cytosolic extracts. Maximal effects of forskolin were observed at 10 micromol/l and 60 min. Preincubation of cells with cycloheximide did not abolish the effect of forskolin. Incubation of cytosolic extracts at 37 degrees C for 10 min in a buffer without phosphatase inhibitors led to a 79% decrease of GFA activity. This loss of activity was inhibited by the addition of phosphatase inhibitors (5 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, or 5 mmol/l EDTA, but not 100 nmol/l okadaic acid), suggesting that GFA undergoes rapid dephosphorylation by endogenous phosphatases. Purified GFA is phosphorylated in vitro by purified PKA, resulting in a 1.7-fold increase in GFA activity. Treatment of GFA with purified protein kinase C had no effect. We conclude that GFA activity may be modulated by cAMP-dependent phosphorylation.

Plants exhibit an altered pattern of protein synthesis in response to pathogen invasion and abiotic stress. One of these ;pathogenesis-related' proteins has been identified as chitinase, which is capable of inhibiting fungal growth in vitro. This observation has led to the suggestion that the in vivo role of chitinases is to protect plants against fungal invasion. Here, we report the purification and characterization of a basic chitinase from Arabidopsis thaliana (L.) Heynh. Columbia wild type. The purified enzyme has a molecular mass of approximately 32 kilodaltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and an apparent pl of approximately 8.7 as determined by isoelectric focusing. The purified protein is an effective inhibitor of the growth of Trichoderma reesei in vitro but does not affect the growth of several other fungi. Amino acid composition analysis of the intact protein as well as amino acid composition analysis and automatic Edman degradation of isolated tryptic fragments of the enzyme indicate that it may be identical to the product of a chitinase gene isolated from an Arabidopsis genomic library (Samac DA, Hironaka CM, Yallaly PE, Shah DM [1990] Plant Physiol 93: 907-914).

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