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J Biol Chem, Vol. 274, Issue 46, 32655-32661, November 12, 1999

IB Kinases  and  Show a Random Sequential Kinetic Mechanism and Are Inhibited by Staurosporine and Quercetin^*

Gregory W. Peet and Jun Li

Boehringer Ingelheim Pharmaceuticals, Research and Development Center, Ridgefield, Connecticut 06877-0368

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of transcription factor NF-B is regulated by phosphorylation and subsequent degradation of its inhibitory subunit IB. The signal-induced phosphorylation of IB involves two IB kinases, IKK and IKK. In the present study, we investigated the kinetic mechanisms of IKK and IKK by substrate and product inhibition. For both IKK and IKK, the product ADP was a competitive inhibitor versus ATP and a non-competitive inhibitor versus IB. An alternative peptide substrate, IB-(21-41), was a competitive inhibitor versus IB 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-B 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 IKK and IKK. Daidzein, a quercetin analogue that does not inhibit NF-B activation, showed no significant inhibition of either enzyme. This suggests that the inhibitory properties of quercetin and staurosporine in the NF-B pathway are mediated in part by their inhibition of IKK and IKK. 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 IKK 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 IKK, which is an attractive drug target for inflammatory diseases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The transcription factor NF-B is regulated by the signaling of receptors for inflammatory cytokines such as TNF,¹ interleukin-1, or other external stimuli (1). In resting cells, NF-B is sequestered in the cytoplasm through its association with inhibitory proteins termed IB. When cells are stimulated by TNF or interleukin-1, IB proteins (IB and IB) are rapidly phosphorylated at Ser residues in the N-terminal region (2, 3). Phosphorylated IB and IB are subsequently ubiquitinated and undergo ubiquitin-dependent degradation by the 26 S proteasome (3, 4). Degradation of IB results in the release of NF-B which then translocates to the nucleus where it up-regulates the transcription of target genes (1).

IB and IB are phosphorylated by a 500-900-kDa IB kinase (IKK) (5, 6). Two kinases in the IKK complex, denoted IKK and IKK (or IKK-1 and IKK-2), phosphorylate IB at the specific Ser residues that target the protein for ubiquitination and degradation (5-9). Both IKK and IKK contribute to the activity of the IKK complex and are involved in NF-B activation (5-9). The physiological function of these protein kinases was recently explored by analysis of IKK-deficient or IKK-deficient mice (10-15). Mouse embryonic fibroblast cells that were isolated from IKK(/) embryos showed a marked reduction in TNF- and interleukin-1-induced NF-B activity and enhanced apoptosis in response to TNF (11, 14, 15). In contrast, IKK was not required for activation of IKK and degradation of IB by pro-inflammatory stimuli (10, 12). These results show that IKK, not IKK, is the target for pro-inflammatory stimuli. On the other hand, IKK is essential for development of skin and skeleton during embryogenesis (10, 12, 13). NF-B activation is impaired in the basal layer of epidermal cells in IKK-deficient mice (12). Since IKK and IKK have distinct functions, it is informative to compare the kinetic mechanisms of both kinases. Inhibitors with selectivity between these two kinases would help to elucidate further their different functions in cells and in animal models.

IKK and IKK share ~50% overall homology, and both contain a conserved N-terminal Ser/Thr kinase domain, a leucine-zipper region, and a C-terminal helix-loop-helix (HLH) motif (6-9). Such folding is unique among the known kinases. It has been shown that the HLH domain of IKK is required for its kinase activity and the HLH domain can activate the truncated IKK (HLH deletion) mutant in trans (16). This suggests a functional interaction between the HLH domain and the kinase domain of IKK. IKK and IKK also share a distinguishing feature in that they have a strong preference for Ser versus Thr on the substrates (5, 6). It is important to understand the kinetic mechanisms of these two unique members of the Ser/Thr kinase family.

Several naturally existing kinase inhibitors have been reported to inhibit the NF-B pathway. Quercetin, a flavonoid that occurs in many fruits and vegetables (17), is a nonspecific inhibitor of protein kinases (18) and suppresses TNF-induced NF-B activation (19). The inhibitor blocks the degradation of IB and the consequent translocation of the NF-B p65 subunit (19). Staurosporine, a microbial alkaloid that was isolated from Streptomyces staurosporeus (20), has shown potent inhibition of both tyrosine and Ser/Thr kinases (18, 21). In THP-1 monocytic cells, staurosporine inhibits LPS-dependent NF-B activation, suggesting that staurosporine-sensitive kinase(s) are involved in LPS-mediated NF-B activation (22). The inhibitory effects of quercetin and staurosporine in the NF-B pathway are consistent with their anti-inflammatory responses as observed in various animal models including experimental arthritis and experimental colitis (23-26). However, the molecular target(s) of staurosporine and quercetin in the NF-B signaling cascade have not been identified. Since IKK is essential for activation of NF-B by both TNF and LPS (6-9, 27), it is important to know whether quercetin and staurosporine inhibit IKK and IKK. It was recently shown that high concentrations of the anti-inflammatory agent aspirin inhibits IKK (IC₅₀ = ~50 µM) (28), consistent with its inhibitory effect on the NF-B pathway (29).

Previously, we have demonstrated that purified recombinant IKK and IKK are direct kinases of IB and function independently in vitro (30). We have also shown that both IKK and IKK display a sequential bi bi mechanism (30). However, our previous report did not discriminate between the possibilities of a random sequential or an ordered sequential mechanism. In the current study, we perform product and substrate inhibition experiments that demonstrate that both IKK and IKK proceed by a random sequential mechanism. We also demonstrate that the natural compounds quercetin and staurosporine inhibit both IKK and IKK with compound-specific mechanisms. Thus, the inhibitory effects of quercetin and staurosporine on the NF-B pathway are at least partially through their inhibitions of IKK and IKK.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Expression and Purification-- IKK and IKK were expressed as N-terminal FLAG-tagged fusion proteins in baculovirus. The recombinant FLAG-tagged IKK and IKK were purified to apparent homogeneity by affinity chromatography using M2 anti-FLAG affinity gel (Sigma). The procedures for expression and purification have been described previously (30). IB was expressed as a His₆-tagged thioredoxin fusion protein (TRX-IB-(1-54)) in Escherichia coli and purified by a Ni²⁺-nitrilotriacetic acid affinity column, as described (30).

In Vitro Phosphorylation Assays-- The kinase assays were performed in a plate assay format as described previously (30). Briefly, reactions (55 µl) were performed at 23 °C in 20 mM HEPES, pH 7.5, 10 mM MgCl₂, 2 mM MnCl₂, 100 mM NaCl, 100 µM Na₃VO₄, 20 mM -glycerophosphate, and 1 mM dithiothreitol. The amount of substrates ATP, [-³³P]ATP (2000 Ci/mmol, NEN Life Science Products) and IB are specified for each individual experiment. Samples were analyzed by trichloroacetic acid precipitation on a microtiter plate (Millipore), followed by liquid scintillation counting (30). Assay conditions were controlled so that the degree of phosphorylation of IB was linear with time and concentration of enzyme. The counts represent initial velocity of IKK-catalyzed phosphorylation (<10% of total ATP conversion). All experiments were performed in duplicate.

Kinetic Analysis-- Initial velocity studies were performed with varying concentrations of IB at a constant ATP concentration and several fixed inhibitor concentrations. Conversely, initial velocity studies were performed with varying ATP concentrations at a constant IB concentration and several fixed inhibitor concentrations. All enzyme activity data are reported as the average of duplicate determinations. The initial rate v was recorded as femtomoles of phosphate transferred to IB during the reaction period. Lineweaver-Burk double-reciprocal plots were generated by linear least square fits of the data. Data from inhibition experiments were fitted to either a linear competitive model (Equation 1) or a non-competitive (or mixed inhibition) model (Equation 2) (31-33).

(Eq. 1)

(Eq. 2)

Accordingly, secondary plots were generated by replotting the slopes, the x intercepts, and the y intercepts of the lines as a function of [inhibitor] (32). The values of K_ii and K_is can be determined from the secondary plots. K_is is the apparent K_i value that accounts for the change of the slope. K_ii is the apparent K_i value that accounts for the change of the y intercept.

Materials-- The peptide IB-(21-41) was ordered from Ana Spec Inc. (San Jose, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Various models of kinetic mechanisms have been described for enzymes that catalyze two substrates (34, 35). For IKK and IKK, our previous study had eliminated a ping-pong mechanism and demonstrated that both enzymes followed a sequential bi bi mechanism (30). Scheme I describes the three possible sequential mechanisms: ordered sequential mechanism with ATP binding first (Model 1), ordered sequential mechanism with IB binding first (Model 2), and a random sequential mechanism (Model 3). Validations of these mechanisms are described below.

View larger version (19K): [in this window] [in a new window]   Scheme I.

Inhibition of IKK and IKK by the Product Inhibitor ADP-- First, the kinase activities of IKK and IKK were determined as a function of varying concentrations of ATP at various fixed concentrations of ADP. The Lineweaver-Burk plots of the data for both IKK and IKK followed Michaelis-Menten kinetics (Fig. 1A and 2A). For both IKK and IKK, a series of double-reciprocal straight line plots intersected on the ordinate, indicating a competitive inhibition mechanism (32). Furthermore, the data were plotted as the slope of the reciprocal plot versus the concentration of the inhibitor. The replots for both IKK and IKK are linear (Figs. 1A and 2A, insets), and yielded K_is values of 156 and 147 nM for IKK and IKK, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   The kinetics of inhibition of IKK by ADP. A, double-reciprocal plots of 1/v versus 1/[ATP] were generated at 7 fixed ADP concentrations of 0 nM (open circles), 25 nM (open triangles), 50 nM (open squares), 125 nM (closed circles), 250 nM (closed triangles), 500 nM (closed diamonds), and 1000 nM (closed squares). Reactions were performed at 23 °C for 15 min with 200 ng of IKK, 7 µM IB, 276 nCi of [-33P]ATP, and varying concentrations of ATP as indicated. B, double-reciprocal plots of 1/v versus 1/[IB] were generated at 7 fixed ADP concentrations of 0 nM (open circles), 25 nM (open triangles), 50 nM (open squares), 125 nM (closed circles), 250 nM (closed triangles), 500 nM (closed diamonds), and 1000 nM (closed squares). Reactions were performed at 23 °C for 15 min with 200 ng of IKK, 332 nCi of [-33P]ATP, 200 nM ATP, and varying concentrations of IB as indicated. The initial rate v was recorded as femtomoles of phosphate transferred to IB during the reaction period. Insets, the slopes of the plots in A and B were replotted versus [ADP]. The x intercepts of the plots yielded Kis.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   The kinetics of inhibition of IKK by ADP. A, double-reciprocal plots of 1/v versus 1/[ATP] were generated at 6 fixed ADP concentrations of 0 nM (open circles), 25 nM (open triangles), 50 nM (open squares), 125 nM (closed circles), 250 nM (closed triangles), and 1000 nM (closed diamonds). Reactions were performed at 23 °C for 15 min with 25 ng of IKK, 7 µM IB, 292 nCi of [-33P]ATP, and varying concentrations of ATP as indicated. B, double-reciprocal plots of 1/v versus 1/[IB] were generated at 6 fixed ADP concentrations of 0 nM (open circles), 50 nM (open triangles), 125 nM (open squares), 250 nM (closed circles), 500 nM (closed triangles), and 1000 nM (closed diamonds). Reactions were performed at 23 °C for 15 min with 25 ng of IKK, 342 nCi of [-33P]ATP, 200 nM ATP, and varying concentrations of IB as indicated. Insets, the slopes of the plots in A and B were replotted versus [ADP].

We subsequently investigated the inhibition mechanism of ADP toward the substrate IB. The kinase activities of IKK and IKK were determined as a function of varying concentrations of IB at various fixed concentrations of ADP. The Lineweaver-Burk plots of the data for both IKK and IKK yielded a series of straight lines that crossed on the abscissa, to the left side of the ordinate (Figs. 1B and 2B), indicating a non-competitive inhibition mechanism (32).

As can be seen, the product ADP is a competitive inhibitor of IKK and IKK with respect to ATP and a non-competitive inhibitor with respect to IB. This behavior is incompatible with an ordered sequential mechanism with IB binding first (Scheme I, Model 2), since otherwise ADP would have been an un-competitive inhibitor with respect to IB. However, the results do not exclude a random sequential mechanism or an ordered sequential mechanism with ATP binding first (Scheme I, Model 1 or 3).

Inhibition of IKK and IKK by a Peptide Analogue of IB-- The peptide corresponding to amino acids 21-41 of IB would compete with IB for binding to the enzymes, since the peptide can be phosphorylated by both IKK and IKK (6, 30). Thus, this peptide is an alternative substrate for IKK and IKK with respect to IB. Since the 21-amino acid peptide is not retained during trichloroacetic acid precipitation and membrane filtration in the phosphorylation assay (data not shown), the assay only monitors the appearance of the radioactive ³³P on recombinant protein Trx-IB. Therefore, we are able to use this peptide as an alternative substrate inhibitor to study the kinetic mechanisms of IKK and IKK. In an effort to further elucidate the sequential mechanism (Scheme I, Model 1 or Model 3), we inhibited the phosphorylation of IB with this peptide using approaches similar to that employed for the ADP inhibition studies as described above. As shown in Figs. 3A and 4A, double-reciprocal plots of 1/v versus 1/[IB] at various fixed peptide concentrations yielded straight lines that crossed on the ordinate, confirming its being a competitive inhibitor toward the substrate IB for both IKK and IKK. The apparent K_is values of 139 and 90 µM for IKK and IKK, respectively, were obtained from linear secondary plots (Figs. 3A and 4A, insets).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Inhibition of IKK by peptide IB-(21-41). A, double-reciprocal plots of 1/v versus 1/[IB] were generated at 5 fixed peptide concentrations of 0 µM (open triangles), 38 µM (closed circles), 100 µM (closed diamonds), 200 µM (closed triangles), and 400 µM (closed squares). Reactions were performed at 23 °C for 15 min with 200 ng of IKK, 533 nCi of [-33P]ATP, 200 nM ATP, and varying concentrations of IB as indicated. B, double-reciprocal plots of 1/v versus 1/[ATP] were generated at 5 fixed peptide concentrations of 0 µM (open triangles), 50 µM (closed circles), 100 µM (closed diamonds), 200 µM (closed triangles), and 500 µM (closed squares). Reactions were performed at 23 °C for 15 min with 200 ng of IKK, 7 µM IB, 563 nCi of [-33P]ATP, and varying concentrations of ATP as indicated. Insets, the slopes of the plots in A and B were replotted versus [peptide].

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Inhibition of IKK by peptide IB-(21-41). A, double-reciprocal plots of 1/v versus 1/[IB] were generated at 5 fixed peptide concentrations of 0 µM (closed circles), 62.5 µM (open circles), 125 µM (closed triangles), 250 µM (open triangles), and 500 µM (closed squares). Reactions were performed at 23 °C for 15 min with 25 ng of IKK, 546 nCi of [-33P]ATP, 200 nM ATP, and varying concentrations of IB as indicated. B, double-reciprocal plots of 1/v versus 1/[ATP] were generated at 5 fixed peptide concentrations of 0 µM (closed circles), 50 µM (open circles), 100 µM (closed triangles), 200 µM (open triangles), and 500 µM (closed squares). Reactions were performed at 23 °C for 15 min with 25 ng of IKK, 2 µM IB, 566 nCi of [-33P]ATP, and varying concentrations of ATP as indicated. Insets, the slopes of the plots in A and B were replotted versus [peptide].

The kinase activities of IKK and IKK were also measured as a function of varying concentrations of ATP at several different fixed concentrations of peptide IB-(21-41). The Lineweaver-Burk plots of the data for both IKK and IKK yielded a series of straight lines that intersected on the abscissa, to the left side of the ordinate, indicating a non-competitive inhibition mechanism (Figs. 3B and 4B).

The different patterns of product inhibition and substrate inhibition for bi bi sequential reactions have been derived (34, 35). The inhibition patterns obtained for IKK and IKK in this study are summarized in Table I. The fact that the product ADP was a competitive inhibitor versus ATP but a non-competitive inhibitor versus IB indicates either a random sequential mechanism (Scheme I, Model 3) or an ordered sequential mechanism with ATP binding first (Scheme I, Model 1). The peptide IB-(21-41) behaves as a competitive inhibitor versus IB but as a non-competitive inhibitor versus ATP. This eliminates the possibility of an ordered sequential mechanism with ATP binding first (Scheme I, Model 1), which would give an un-competitive inhibition pattern with respect to ATP. In conclusion, the kinetics of IKK and IKK follow a random-ordered sequential bi bi mechanism (Scheme I, Model 3).

View this table: [in this window] [in a new window]   Table I Kinetic parameters of IKK and IKK from data presented in Figs. 1-4

Staurosporine Is an ATP-competitive Inhibitor of IKK and IKK-- The natural kinase inhibitor staurosporine has been implicated to inhibit the NF-B pathway since it blocks LPS-stimulated NF-B activation in THP-1 monocytic cells (22). Since LPS activates NF-B through IKK in THP-1 cells (27), we decided to test whether staurosporine inhibits IKK or IKK. Staurosporine inhibited both IKK and IKK in a dose-dependent manner, with an apparent IC₅₀ of 0.85 and 1.6 µM for IKK and IKK, respectively (Fig. 5A). The effect of staurosporine on the initial velocity patterns for IKK and IKK are shown in Fig. 5, B and C. Double-reciprocal plots of 1/v versus 1/[ATP] at different fixed concentrations of staurosporine intersect on the ordinate, indicating that the inhibitor is competitive with ATP for both IKK and IKK (Fig. 5, B and C). As represented in Fig. 5D, increased concentrations of IB did not reduce the inhibition of IKK and IKK by staurosporine, indicating that staurosporine is non-competitive with IB. This is consistent with staurosporine being a competitive inhibitor with ATP (Fig. 5, B and C). Global fitting of the data in Fig. 5, B and C, to a competitive inhibition model (EnzFitter program, Biosoft) yielded K_i values of 86 ± 17 and 172 ± 39 nM for IKK and IKK, respectively. The potent inhibition of IKK and IKK by staurosporine is consistent with its potent inhibition of NF-B activation (22).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Inhibition of IKK and IKK by staurosporine. A, IC50 plots of IKK (200 ng, closed circles) and IKK (100 ng, closed triangles). IC50 assays employed a 5-min preincubation of enzyme plus inhibitor at 23 °C prior to initiation of reaction with substrates. Data are presented as a percentage of control activity (no inhibitor). For IKK, reactions were performed at 23 °C for 60 min with 550 nCi of [-33P]ATP, 250 nM ATP, and 7 µM IB. For IKK, reactions were performed at 23 °C for 30 min with 550 nCi of [-33P]ATP, 250 nM ATP, and 2 µM IB. The IC50 curves were generated by SigmaPlot regression fitting using the equation: y = 100  (Imaxxn/(IC50n + xn)) (x = [compound], y = % activity, and Imax is the maximum percentage of inhibition). B and C, inhibition pattern of staurosporine with respect to ATP. Double-reciprocal plots of 1/v versus 1/[ATP] were generated at 4 fixed staurosporine concentrations of 0 nM (closed triangles), 125 nM (open circles), 250 nM (closed circles), and 500 nM (closed squares). Reactions were performed at 23 °C for 15 min with 7 µM IB, 554 nCi of [-33P]ATP, varying concentrations of ATP as indicated, and either 200 ng of IKK (B) or 25 ng of IKK (C). D, effect of various concentrations of IB on staurosporine-mediated inhibition of IKK and IKK. Both IKK (200 ng, closed circles) and IKK (25 ng, closed triangles) were assayed in the presence or absence of 500 nM staurosporine. Reactions were performed at 23 °C for 15 min with 200 nM ATP, 550 nCi of [-33P]ATP, and varying concentrations of IB as indicated. Data are presented as percentage of inhibition by staurosporine.

IKK and IKK Are Inhibited by Quercetin-- Quercetin has been reported as an inhibitor of both tyrosine kinases and Ser/Thr kinases (18, 36). Since quercetin inhibits TNF-induced nuclear translocation of NF-B (19), we investigated whether it acts upon IKK and IKK. Quercetin inhibited both IKK and IKK (Fig. 6, A and B), with an apparent IC₅₀ value of 11 and 4 µM, respectively. Daidzein, a structural analogue of quercetin (Scheme II), showed no significant inhibitory effects on the activities of IKK and IKK (Fig. 6, A and B). Since daidzein failed to block TNF-mediated NF-B activation at 80 µg/ml (19), this result is consistent with IKK and IKK being involved as molecular targets of quercetin in the TNF pathway.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Inhibition of IKK and IKK by quercetin and daidzein. Kinase assays were performed with a 5-min preincubation of enzyme plus inhibitor at 23 °C. Data are presented as a percentage of control activity (no inhibitor). A, IC50 plots of IKK with inhibitor quercetin (open circles) or daidzein (closed circles). Reactions were performed at 23 °C for 60 min with 212 ng of IKK, 7 µM IB, 24 nCi of [-33P]ATP, and 250 nM ATP. B, IC50 plots of IKK with inhibitor quercetin (open circles) or daidzein (closed circles). Reactions were performed at 23 °C for 30 min with 100 ng of IKK, 2 µM IB, 30 nCi of [-33P]ATP, and 250 nM ATP. The IC50 curves were generated by SigmaPlot as described in Fig. 5A.

View larger version (16K): [in this window] [in a new window]   Scheme II.

We further investigated the inhibition mechanism of quercetin on IKK and IKK. We first examined kinase inhibition by quercetin in the presence of various amounts of ATP. Fig. 7, A and B, shows double-reciprocal plots of 1/v versus 1/[ATP] at several fixed concentrations of quercetin. The Lineweaver-Burk plots of the data for both IKK and IKK are linear, indicating Michaelis-Menten kinetics at each individual concentration of quercetin (Fig. 7, A and B). For both IKK and IKK, quercetin significantly reduced the apparent V_max (1/y intercept) and increased the apparent K_m (1/x intercept), indicating a mixed type inhibition mechanism. However, both series of double-reciprocal plots did not intersect at a single point to the left of the ordinates (Fig. 7, A and B), suggesting a more complicated mechanism than the standard linear mixed type inhibition mechanism (33). In contrast to that observed for staurosporine (Fig. 5D), the inhibition of IKK and IKK by quercetin was protected by increased amounts of substrate IB (Fig. 7C). This result is consistent with quercetin being a non-exclusive inhibitor with respect to ATP and IB as indicated by Fig. 7, A and B. These observations suggest that the binding site of quercetin may overlap with both the ATP- and IB-binding sites.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Substrate protection of quercetin-mediated inhibition of IKK and IKK. A and B, inhibition pattern of quercetin with respect to ATP. Double-reciprocal plots of 1/v versus 1/[ATP] were generated at various fixed concentrations of quercetin. Kinase assays were conducted with a 5-min preincubation of enzyme plus quercetin and ATP prior to initiation of the reaction with IB. A, IKK was assayed with 4 fixed concentrations of quercetin at 0 µM (open triangles), 12.5 µM (closed circles), 25 µM (open circles), and 50 µM (closed squares). Reactions were performed at 23 °C for 15 min with 200 ng of IKK, 7 µM IB, 550 nCi of [-33P]ATP, and varying concentrations of ATP as indicated. B, IKK was assayed with 5 fixed concentrations of quercetin at 0 µM (open triangles), 1 µM (closed circles), 2 µM (open circles), 4 µM (closed squares), and 8 µM (closed diamonds). Reactions were performed at 23 °C for 15 min with 25 ng of IKK, 2 µM IB, 700 nCi of [-33P]ATP, and varying concentrations of ATP as indicated. C, effect of various concentrations of IB on quercetin-mediated inhibition of IKK and IKK. For IKK (closed circles), kinase assays were performed at 23 °C for 10 min in the presence of 200 µM quercetin, 200 nM ATP, 550 nCi of [-33P]ATP, and variable concentrations of IB as indicated. For IKK (closed triangles), kinase assays were performed at 23 °C for 15 min in the presence of 50 µM quercetin, 200 nM ATP, 553 nCi of [-33P]ATP, and variable concentrations of IB as indicated. Data are presented as percentage of inhibition by quercetin.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous kinetic studies of IKK and IKK did not discriminate between a random sequential or an ordered sequential mechanism (30). The results of the present inhibition studies clearly demonstrate that both IKK and IKK proceed through a random sequential mechanism. The equilibria shown in Scheme III describe the kinetic parameters in a random sequential bi bi system. In our previous report (30), we had fitted the two-substrate profiling data of IKK and IKK to a random sequential model as described in Scheme III. As a result, for IKK, values of 85 nM, 25 µM, 0.09/min, and 1.0 were obtained for K_ATP, K_IB, k_cat, and , respectively. For IKK, values of 130 nM, 1.4 µM, 0.30/min, and 1.0 were obtained for K_ATP, K_IB, k_cat, and , respectively (30). Thus, as we have proven the random sequential model in this study, the kinetic mechanisms and parameters of IKK and IKK are now complete. Since the native 500-900-kDa IKK complex is composed of both IKK and IKK (6, 7), the kinetics of the IKK complex is likely to proceed through a random sequential mechanism. Consistent with this assumption, it has been shown that a multisubunit IB kinase complex isolated from HeLa cells displays a random sequential mechanism (38), although it has not been demonstrated whether it is the same IKK complex that contains IKK and IKK.

View larger version (9K): [in this window] [in a new window]   Scheme III.   Equilibria in a random sequential mechanism. Left, without inhibitor; right, with an inhibitor that competes with substrate A.

The product ADP is a potent inhibitor for both IKK and IKK, with a K_i value of 125 and 136 nM, respectively (Table I). These values are slightly higher than the corresponding K_ATP values (85 nM for IKK and 130 nM for IKK) (30). This suggests that the binding of ATP to IKK and IKK is predominantly mediated by the ADP portion of the molecule. It should be noted that, within the kinase family, a distinguishing feature for IKK and IKK is their low K_m (ATP) value for ATP (~100 nM) (30). In comparison, much higher K_m (ATP) values have been reported for other Ser/Thr protein kinases, such as cAMP-dependent protein kinase (K_m = 10 µM) (39) and p38 mitogen-activated protein kinase (K_m = 23 µM) (40). Similarly, IKK and IKK have unprecedented low K_i (ADP) values within the kinase family. The high affinity of IKK and IKK to substrate ATP would allow for the design of substrate-based inhibitors. The low K_i values of ADP are in support of such a feasibility. Adenosyl-based compounds such as sulfonylbenzoyl adenosine have been previously designed and found to inhibit tyrosine kinases (41, 42). In addition, based on structure homology modeling, 2'-thioadenosine has been successfully designed to inhibit selectively the ErbB tyrosine kinase subfamily (43). Similar rational approaches are applicable to design selective inhibitors of IKK and IKK.

As expected, the peptide IB-(21-41) is a competitive inhibitor with respect to IB. The K_i value of this peptide for IKK is lower than the K_i value for IKK, consistent with the observation that IKK has a higher affinity to substrate IB than IKK (30). However, the K_i values (29 µM for IKK and 136 µM for IKK, Table I) are significantly higher as compared with the K_IB values (1.4 µM for IKK and 25 µM for IKK) (30), suggesting that the IB-binding site of both kinases includes residues outside the 21-41 peptide motif of IB.

Based on the model shown in Scheme III,  represents the factor by which the K_i is changed by the binding of the second substrate. IKK has a  value of 1.0 for the inhibitor IB-(21-41) (Table I), indicating that the binding of the peptide inhibitor to IKK has no effect on the affinity for ATP. This can be visualized in Fig. 4B as the double-reciprocal plots intersected on the abscissa, indicating that the concentration of the peptide inhibitor has no effect on the apparent K_m for ATP. Similarly, a  value of 1.0 was obtained for the inhibitor ADP (Table I). These results are consistent with the  value of 1.0 for IKK (30). For IKK,  values of 0.7 and 1.0 were obtained for inhibitors ADP and IB-(21-41), respectively (Table I). These values, with allowance for experimental error, are comparable to the 1.0  value for IKK (30). Taken together, for both IKK and IKK, the binding of one substrate has no effect on the affinity for the other substrate.

The native cytokine-inducible IKK complex contains both IKK and IKK (5, 6). By using purified recombinant IKK or IKK, we have previously demonstrated that IKK and IKK are direct kinases of IB but that they have no synergistic kinase activity (30). Since these two kinases share ~50% homology, it is possible to inhibit both kinases with a small molecule compound. This possibility is supported by our observation that staurosporine and quercetin are potent inhibitors of both kinases. On the other hand, IKK and IKK have distinct physiological functions (10-15). Specific inhibition of each individual kinase may be preferred. Inhibitors that show selectivity between these kinases would allow characterization of their physiological functions in vivo.

Staurosporine inhibits widely divergent members of the protein kinase family (21). This suggests that staurosporine functions by binding to a region that is conserved throughout the protein kinase family. The inhibition of the mammalian small heat-shock protein (HSP25) kinase by staurosporine and its analogue K252a is competitive with respect to ATP (44). In addition, an ATP-competitive mechanism has been observed in the inhibition of protein kinase C and cAMP-dependent protein kinase by the staurosporine analogue K252a (45). The same mechanism is now shown in the inhibition of IKK and IKK by staurosporine. This is not surprising since both IKK and IKK contain a conserved catalytic kinase domain at the N-terminal region which includes the conserved ATP-binding site (5-9). At this time, staurosporine is the most potent compound inhibitor of IKK (K_i = 86 nM) and IKK (K_i = 172 nM) ever reported. Such potent inhibitions by staurosporine provide a starting point for building more selective inhibitors of IKK and IKK. In fact, several staurosporine derivatives such as CGP 41251 (4'-N-benzoyl staurosporine) and Ro 318425 show significant selectivity for protein kinase C over cAMP-dependent protein kinase and epidermal growth factor receptor tyrosine kinase (26, 46). The inhibition mechanism of quercetin on various kinases appears to be diverse. Quercetin inhibits pp60^Src tyrosine kinase as an ATP-competitive inhibitor (47). In contrast, the inhibition of phosphatidylinositol 3-kinase I and phosphatidylinositol 3-kinase II by quercetin is non-competitive versus ATP (48). In our studies of IKK and IKK, quercetin showed a mixed inhibition mechanism toward ATP (Fig. 7). The binding site of quercetin is likely to overlap with both the ATP and IB binding pockets.

Several tyrosine kinase inhibitors, such as quercetin, genistein, staurosporine, and herbimycin, are able to inhibit NF-B activation (19, 22). Thus, it has been implicated that tyrosine kinase(s) are involved in NF-B regulation. However, there is a lack of direct evidence that tyrosine kinases participate in the NF-B pathway. We have now shown that quercetin and staurosporine inhibit IKK and IKK, the two key regulated serine kinases in the NF-B pathway, consistent with their inhibitory effects on NF-B activation. In addition, IKK and IKK were not inhibited by daidzein (Fig. 6), a quercetin analogue without inhibitory effects on TNF-induced NF-B activation (19). The tyrosine kinase inhibitor genistein also inhibits IKK.² Since kinase inhibitors usually have poor selectivity, their inhibitory effects on certain signaling pathways are likely to be a combination of inhibitions of several kinase targets within multiple signaling cascades. This study suggests that the inhibitory effects of staurosporine and quercetin on NF-B activation are at least partially due to the inhibition of IKK and IKK. As NF-B is a key cellular regulator of the inflammatory response, the anti-inflammatory properties of quercetin and staurosporine (23-26) may be partially due to their inhibition of IKK and IKK. A correlation between the anti-inflammatory effects and the inhibition of IKK has been observed for aspirin and salicylate (28).

The recent in vivo knock-out studies of IKK imply that IKK is a valid target for inflammatory diseases (11, 14, 15). Thus high throughput screening for inhibitors of IKK could yield small molecules of therapeutic value. Here we have demonstrated the kinetic mechanism of both IKK and IKK to be random sequential, with each substrate binding independently of the other. This characterized kinetic mechanism will help in the evaluation of potential drug leads. Based on the potent inhibition of IKK by ADP, staurosporine, and quercetin, these compounds may be considered starting points for designing specific inhibitors. The different inhibition mechanisms of staurosporine and quercetin also indicate that potent inhibition of the enzyme can be achieved by targeting different parts of the ATP-binding site. However, it is challenging to create tight-binding inhibitors that are selective between IKK and IKK, the two homologous kinases that have similar kinetic mechanisms. Comparison of x-ray crystal structures of both kinases will help us to accomplish this goal.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Boehringer Ingelheim Pharmaceuticals, Research and Development Center, 900 Ridgebury Rd., Ridgefield, CT 06877-0368. Tel.: 203-798-5714; Fax: 203-791-6906; E-mail: jli@rdg.boehringer-ingelheim.com.

² G. Peet and J. Li, unpublished data.

	ABBREVIATIONS

The abbreviations used are: HLH, helix-loop-helix; IKK, IB kinase; TNF, tumor necrosis factor; TRX, thioredoxin; LPS, lipopolysaccharide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES