The Multisubunit Ikappa B Kinase Complex Shows Random Sequential Kinetics and Is Activated by the C-terminal Domain of Ikappa Balpha *

James R. BurkeDagger §, Kenneth R. MillerDagger , Marcia K. Wood, and Chester A. Meyers

From The Department of Drug Discovery Research, Bristol-Myers Squibb Pharmaceutical Research Institute, Dagger  Buffalo, New York 14213 and  Princeton, New Jersey 08543

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The multisubunit Ikappa B kinase (IKK) catalyzes the signal-inducible phosphorylation of N-terminal serines of Ikappa B. This phosphorylation is the key step in regulating the subsequent ubiquitination and proteolysis of Ikappa B, which then releases NF-kappa B to promote gene transcription. As measured by 33P incorporation into a GST-Ikappa Balpha fusion protein, varying both the concentration of GST-Ikappa Balpha and [gamma -33P]ATP resulted in a kinetic pattern consistent with a random, sequential binding mechanism. Values of 55 nM and 7 µM were obtained for the dissociation constants of GST-Ikappa Balpha and ATP, respectively. The value of alpha , a factor by which binding of one substrate changes the dissociation constant for the other substrate, was determined to be 0.11. This indicates that the two substrates bind in a cooperative fashion. Peptides corresponding to either amino acids 26-42 (N-terminal peptide) or amino acids 279-303 (C-terminal peptide) of Ikappa Balpha inhibited the IKK-catalyzed phosphorylation of GST-Ikappa Balpha ; the C-terminal peptide, unexpectedly, was more potent. The inhibition by the C-terminal peptide was competitive with respect to GST-Ikappa Balpha and mixed with respect to ATP, which verified the sequential binding mechanism. The C-terminal peptide was also a substrate for the enzyme, and a dissociation constant of 2.9-6.2 µM was obtained. Additionally, the N-terminal peptide was a substrate (Km = 140 µM). Competitive inhibition of the IKK-catalyzed phosphorylation of the C-terminal peptide by the N-terminal peptide indicated that the peptides are phosphorylated by the same active site. Surprisingly, the presence of the C-terminal peptide greatly accelerated the rate of phosphorylation of the N-terminal peptide as represented by a 160-fold increase in the apparent second-order rate constant (kcat/Km). These results are consistent with an allosteric site present within IKK that recognizes the C terminus of Ikappa Balpha and activates the enzyme. This previously unobserved interaction with the C terminus may represent an important mechanism by which the enzyme recognizes and phosphorylates Ikappa B.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The NF-kappa B/Rel family of transcriptional activators regulates the expression of numerous genes involved in inflammatory and immune responses, such as the cytokines tumor necrosis factor-alpha , interleukin-6, interleukin-8, and interleukin-1beta ; the adhesion molecules E-selectin and VCAM-1; and the enzyme nitric oxide synthase (for reviews, see Refs. 1 and 2).

In unstimulated cells, NF-kappa B normally resides in the cytoplasm as an inactive complex with an Ikappa B inhibitory protein. This class of protein includes Ikappa B-alpha , Ikappa B-beta , and Ikappa B-epsilon , which all contain ankyrin repeats necessary for complexation with NF-kappa B (for a review, see Ref. 3). In the case of Ikappa B-alpha , which is the most carefully studied member of this class, stimulation of cells with agents that activate NF-kappa B-dependent gene transcription results in a phosphorylation of Ikappa B-alpha at Ser-32 and Ser-36 (4). This is critical for subsequent ubiquitination and proteolysis of Ikappa B-alpha , which then leaves NF-kappa B free to translocate to the nucleus and promote gene transcription (5-7). Indeed, a mutant in which both Ser-32 and Ser-36 have been changed to alanine prevents signal-induced activation of NF-kappa B and results in an Ikappa B-alpha that is neither phosphorylated, ubiquitinated, nor proteolytically digested (7). Analogous serines have been identified in both Ikappa B-beta and Ikappa B-epsilon , and phosphorylation at these residues appears to regulate the proteolytic degradation of these proteins by a mechanism similar to that of Ikappa B-alpha (8, 9). Because of its important role in the activation of NF-kappa B, the inhibition of this signal-inducible phosphorylation of Ikappa B is an important target for novel anti-inflammatory agents.

A high molecular mass (500-900 kDa) multisubunit Ikappa B kinase (IKK)1 that phosphorylates at Ser-32 and Ser-36 of Ikappa B-alpha has been isolated from HeLa cells (10-12). The IKK also phosphorylates at Ser-19 and Ser-23 in the N terminus of Ikappa B-beta (13). The kinase activity is greatly enhanced if the cells are first treated with tumor necrosis factor-alpha , which appears to activate a kinase cascade leading to the phosphorylation of the IKK (11, 13).

Recently, two catalytic subunits (termed IKK-alpha and IKK-beta ) of IKK have been identified, cloned, and shown to be widely expressed in human tissues (12-16). IKK-alpha and IKK-beta form homo- and heterodimers with each other, but the active complex appears to be the heterodimer (12, 14). Demonstration that IKK is the kinase involved in the signal-inducible degradation of Ikappa B-alpha was accomplished by both antisense inhibition of IKK-alpha and the use of dominant negative, catalytically inactive mutants of IKK-alpha and IKK-beta (12, 15, 16). Both approaches abrogated cytokine-induced activation of NF-kappa B. The signal-induced activation of IKK appears to proceed through phosphorylation of the IKK-alpha and/or IKK-beta subunits by a mitogen-activated protein kinase kinase (such as mitogen-activated protein kinase/ERK kinase kinase-1 or NF-kappa B-inducing kinase), which greatly enhances the enzymatic activity.

A knowledge of the mechanism is central to the understanding of any enzyme. To this end, we report here a kinetic investigation of the multisubunit IKK that demonstrates, surprisingly, that the enzyme recognizes and is stimulated by elements of the C terminus of Ikappa Balpha .

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- GST-Ikappa Balpha was purchased from Santa Cruz Biotechnology, and the purity was estimated to be 34% by SDS-polyacrylamide gel electrophoresis and Coomassie staining. HeLa S3 cells were obtained from ATCC. Tumor necrosis factor-alpha was purchased from R&D Systems, and [gamma -33P]ATP (1000 Ci/mmol) was purchased from Amersham Pharmacia Biotech.

Peptide Synthesis-- An N-terminal peptide corresponding to amino acids 26-42 of Ikappa B-alpha (LDDRHDSGLDSMKDEEY) was prepared along with a C-terminal peptide corresponding to amino acids 279-303 of Ikappa B-alpha (MLPESEDEESYDTESEFTEFTEDEL). Each peptide was synthesized on Fmoc-Knorr amide resin (Midwest Biotech, Fishers, IN) with an Applied Biosystems (Foster City, CA) model 433A synthesizer and the FastMoc chemistry protocol (0.25 mmol scale) supplied with the instrument. Amino acids were double coupled as their N-alpha -Fmoc- derivatives, and reactive side chains were protected as follows: Asp and Glu, t-butyl ester; Ser and Thr, t-butyl ether; His, triphenylmethyl; Lys, t-butyloxycarbonyl; and Arg, pentamethylchroman-sulfonyl. After the final double coupling cycle, the N-terminal Fmoc group was removed by the two-step treatment with piperidine in N-methylpyrrolidone as described by the manufacturer. The N-terminal free amines were then treated with 10% acetic anhydride, 5% diisopropylamine in N-methylpyrrolidone to yield the N-acetyl derivative. The protected peptidyl resins were simultaneously deprotected and removed from the resin by standard methods, except that extraction of the crude products from the resin was accomplished with successive treatments of 1 N ammonium acetate, water, and acetonitrile. The lyophilized peptides were purified on C18 to apparent homogeneity as judged by reverse phase-HPLC analysis. Predicted peptide molecular weights were verified by electrospray mass spectrometry.

Cell Culture-- HeLa S3 cells were grown as a suspension culture in minimum essential medium (Life Technologies, Inc.). Medium was supplemented with 10% FBS and 50 µg/ml gentamicin.

Isolation of IKK-- A procedure adapted from the one described by Lee et al. (11) was used. HeLa S3 cells that had been collected and resuspended at a concentration of 6 × 108 in 25 ml of medium were treated with 20 ng/ml tumor necrosis factor-alpha for 10 min at 37 °C and then pelleted at 600 × g for 10 min at 4 °C. Cells were washed once with ice-cold PBS and resuspended in ice-cold lysis buffer (50 mM Tris, 1 mM EGTA, pH 7.5). Protease inhibitor mixture (Calbiochem-Novabiochem Corp. La Jolla, CA) was prepared as a 100× stock (50 mM AEBSF, 50 mM EDTA, 100 µM E-64, 100 µM leupeptin, 100 µg/ml aprotinin) and diluted to 1× in ice-cold lysis buffer. Cells were homogenized using a Wheaton Overhead stirrer (Wheaton, Millville, NJ) for 15-20 strokes at setting 3. The cell lysate was clarified at 4600 × g for 10 min. at 4 °C. Supernatant was retained as cell lysate and stored at -80 °C until it was chromatographed.

For anion exchange and size exclusion chromatography, the BioCAD Sprint Perfusion Chromatography System (PerSeptive Biosystems Framingham, MA) was used. Cell lysate was loaded onto a Poros HQ/M column (PerSeptive Biosystems), and the elution buffer (50 mM Tris·Cl, pH 7.5) was increased from 0 to 0.8 M KCl to elute Ikappa B-alpha kinase activity. During elution from the Poros HQ/M column, the flow rate was maintained at a constant 5 ml/min, and fraction size was 3.4 ml, which corresponds to 2 column volumes. Fractions containing significant Ikappa B-alpha kinase activity eluted in the range of 0.15-0.35 M KCl. Three fractions from the Poros HQ/M column were pooled and concentrated using a Amicon YM-3 filter (Amicon, Beverly, MA). Size exclusion chromatography was perfomed by loading the concentrated sample on a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech), and the sample was eluted at a flow rate of 0.75 ml/min with elution buffer (50 mM Tris·Cl, pH 7.5, 150 mM NaCl). Eluted fractions were collected in a fraction size of 0.5 ml. Fractions containing IKK activity eluted in a range of 300-750 kDa. Analysis by SDS-polyacrylamide gel electrophoresis and silver staining showed that the two major bands in this preparation were at 85 and 87 kDa, in agreement with published reports (12, 14).

Assay of IKK-catalyzed Phosphorylation of Ikappa B-alpha -- The Ikappa B-alpha substrate used was a GST-Ikappa Balpha fusion protein. Enzymatic assays were performed by adding IKK at 30 °C to solutions of the GST-Ikappa Balpha and ATP in 50 mM Tris·HCl, 5 mM MgCl2 containing 3.1 µM okadaic acid at pH 8. The specific activity of ATP used in the assay ranged from 215-625 Ci/mmol. After 10 min, the kinase reactions were stopped by the addition of 2× Laemmli sample buffer and heat treated at 90 °C for 3 min. The samples were then loaded on to 10% Tris-glycine gels (Novex, San Diego, CA). After completion of SDS-polyacrylamide gel electrophoresis, gels were dried on a slab gel dryer. The bands were then detected using a 445Si PhosphorImager (Molecular Dynamics), and the radioactivity was quantified using the ImageQuant software while employing a mean background correction factor for each 33P-labeled Ikappa B-alpha band. It should be noted that the amount of radioactivity measured in this way is in arbitrary units and absolute values from one experiment cannot necessarily be compared with those from another experiment. Under these conditions, the degree of phosphorylation of GST-Ikappa Balpha was linear with time and concentration of enzyme.

Peptides as Substrates-- When using peptides as substrates for the IKK, enzymatic assays were performed by adding the enzyme at 30 °C to solutions containing peptide and [gamma -33P]ATP (1000 Ci/mmol) in 50 mM Tris·HCl, 5 mM MgCl2 containing 3.1 µM okadaic acid at pH 8. After 60-90 min, the kinase reactions were frozen until analyzed by HPLC. HPLC analysis employed a Vydac C18 reverse phase 4.6 × 250-mm column (218TP52) with a 36-min gradient from 100% H2O (with 0.1% trifluoroacetic acid) to 100% CH3CN (with 0.1% trifluoroacetic acid). Under these conditions, the N-terminal and C-terminal peptides had retention times of 28 and 31 min, respectively. The amount of IKK-catalyzed incorporation of 33P into each peptide was quantitated by liquid scintillation counting.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Kinetic Mechanism of IKK-- There are several kinetic mechanisms that have been described for two substrate enzyme systems. Fig. 1 shows a Hanes plot of IKK velocity as a function of the concentration of Ikappa B-alpha at different ATP concentrations. This analysis is best fit to a random, sequential binding mechanism in which the enzyme binds both substrates prior to product release. This is in contrast to a ping-pong mechanism, in which one product is released before the second substrate binds, which would give a Hanes plot with lines intersecting at the y axis (17). Verification of a random versus ordered binding mechanism comes from the use of inhibitors and is presented below. This sequential mechanism is defined in Scheme 1, where KIkappa Balpha and alpha KIkappa Balpha are the dissociation constants for Ikappa B-alpha in the absence and presence, respectively, of ATP in the active site; KATP and alpha KATP are the dissociation constants for ATP in the absence and presence, respectively, of Ikappa B-alpha in the active site. Using a nonlinear regression analysis of the data (18), values of 55 ± 25 nM, 7.3 ± 3.4 µM, and 0.11 were obtained for KIkappa Balpha , KATP, and alpha , respectively. A value of alpha <1 demonstrates that the binding of one substrate increases the affinity for the second substrate (17).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Initial velocity patterns for IKK with varying levels of ATP and Ikappa B-alpha . Hanes plot of the initial rate of IKK-catalyzed phosphorylation of Ikappa B-alpha at ATP concentrations of 200 (closed triangles), 400 (open triangles), 800 (closed circles), and 1600 (open circles) nM ATP. The solid lines represent nonlinear regression fits to the sequential kinetics (18). [IKK] = 39 µg/ml. See "Experimental Procedures" for details.


View larger version (8K):
[in this window]
[in a new window]
 
Scheme 1.   Random sequential binding mechanism of IKK.

Peptide Analogs of Ikappa B-alpha as Inhibitors and Substrates of IKK-- In an effort to verify the sequential mechanism represented in Scheme 1, peptides corresponding to either amino acids 26-42 of Ikappa B-alpha (termed the N-terminal peptide) or amino acids 279-303 of Ikappa B-alpha (a C-terminal peptide) were tested as inhibitors of the the IKK-catalyzed phosphorylation of Ikappa B-alpha . With the N-terminal peptide, a dose-dependent inhibition was obtained that yielded a linear Dixon plot, as shown in Fig. 2. This is not surprising because this N-terminal peptide corresponds to the amino acid sequence around the phosphorylation sites (Ser-32/Ser-36) of Ikappa B-alpha . Indeed, this peptide is also phosphorylated by the enzyme (see below). Unexpectedly, a C-terminal peptide also inhibited the enzyme, and Fig. 2 demonstrates that it was even more potent than the N-terminal peptide.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibition by peptides of the IKK-catalyzed phosphorylation of Ikappa B-alpha . Dixon plot of the initial rate of IKK-catalyzed phosphorylation of Ikappa B-alpha using the following peptides as inhibitors: closed circles, N-terminal peptide; open circles, C-terminal peptide. [Ikappa B-alpha ] = 51 nM; [ATP] = 2.3 µM; [IKK] = 39 µg/ml. See "Experimental Procedures" for details.

As represented in Fig. 3, the inhibition of the IKK-catalyzed phosphorylation of Ikappa B-alpha by the C-terminal peptide was shown to be competitive with respect to Ikappa B-alpha . This would be expected from a mechanism detailed in Scheme 2, in which the binding of the inhibitor (i.e. peptide) competes with the binding of Ikappa B-alpha but not with the binding of ATP. Verification of this mechanism comes from the inhibition observed with the C-terminal peptide while keeping the concentration of Ikappa B-alpha fixed and varying the concentration of ATP. As shown in Fig. 4, the inhibition pattern observed in this case is mixed type. As expected from Scheme 2, infinitely large concentrations of ATP are unable to completely overcome the inhibition produced by the C-terminal peptide, an observation that unequivocally rules out an ordered binding mechanism that would have shown competitive inhibition (17, 19).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Competitive inhibition by the C-terminal peptide with respect to Ikappa B-alpha . Varying concentrations of Ikappa B-alpha were used in the assay at a fixed ATP concentration of 1100 nM with C-terminal peptide concentrations of 0 (open circles), 2 (closed circles), 10 (open triangles), and 20 (closed triangles) µM. The solid lines represent a nonlinear regression fit to competitive inhibition (18). [IKK] = 39 µg/ml. See "Experimental Procedures" for details.


View larger version (12K):
[in this window]
[in a new window]
 
Scheme 2.   Equilibria in a random sequential mechanism showing an inhibitor (I) that competes with Ikappa B-alpha but allows ATP to bind (17). Here, KI and beta KI represent the dissociation constants of the inhibitor in the absence and presence, respectively, of ATP; and KATP and beta KATP represent the dissociation constants of ATP in the absence and presence, respectively, of inhibitor.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Mixed inhibition by the C-terminal peptide with respect to ATP. Varying concentrations of ATP were used in the assay at a fixed Ikappa B-alpha concentration of 127.5 nM with C-terminal peptide concentrations of 0 (open circles), 25 (closed circles), and 50 (open triangles) µM. The solid lines represent nonlinear regression fits to mixed type inhibition (18). [IKK] = 39 µg/ml. See "Experimental Procedures" for details.

Values for KI and beta  were estimated from the data represented in Figs. 3 and 4 and from the values of KATP, KIkappa Balpha , and alpha  determined above. First, by obtaining from Fig. 4 a value of 20.5 ± 8.9 µM for the apparent Ki (Kiapp'), the value of the intrinsic KI was calculated to be 6.2 ± 3.4 µM from Equation 1 (17). Then, the apparent Ki (Kiapp") of 4.4 ± 0.9 µM from Fig. 3 was used in Equation 2 (taken from Ref. 17) to obtain a value for beta  of 0.3 ± 0.5. This value, within experimental error, was the same as that obtained for alpha . Because the value for beta  is less than 1, it demonstrates that the binding of the peptide also increases the affinity for ATP.
K<SUB>i</SUB><UP>app</UP>′=K<SUB>I</SUB><FENCE>1+<FR><NU>[<UP>I&kgr;B</UP>]</NU><DE>K<SUB><UP>I&kgr;B</UP></SUB></DE></FR></FENCE> (Eq. 1)
K<SUB>i</SUB><UP>app</UP> (Eq. 2)
Even more surprisingly, the C-terminal peptide was phosphorylated by IKK. A hyperbolic plot (not shown) of rate versus peptide concentration was obtained that yielded an apparent Km of 2.9 ± 1.0 µM for the peptide under these conditions. Because the concentration of ATP used was only 8 nM, this apparent Km is probably a close approximation of the true Km. Indeed, this value agrees well with the KI value of 6.2 ± 3.4 µM calculated for the C-terminal peptide as an inhibitor.

Although the N-terminal peptide was also a substrate for the IKK, it bound with considerably less affinity to the enzyme under these conditions, giving a Km of 140 ± 28 µM. The Vmax for phosphorylation of this N-terminal peptide is approximately <FR><NU>1</NU><DE>10</DE></FR> the Vmax for phosphorylation of Ikappa B-alpha under identical conditions (results not shown). An analog of the N-terminal peptide, which had residues corresponding to Ser-32 and Ser-36 of Ikappa B-alpha changed to alanines, was not phosphorylated under these conditions. However, this mutant peptide inhibited the IKK-catalyzed phosphorylation of Ikappa B-alpha with a potency roughly equal to that shown by the N-terminal peptide (results not shown).

In an attempt to verify that the C-terminal and N-terminal peptides are binding to (and being phosphorylated by) the same active site, the inhibition by the N-terminal peptide of C-terminal peptide phosphorylation was measured. As shown in Fig. 5, the data fit to competitive inhibition, giving an apparent KI value (Kiapp") of 12 ± 2 µM for the N-terminal peptide.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Competitive inhibition by the N-terminal peptide of IKK-catalyzed phosphorylation of the C-terminal peptide. The indicated concentrations of C-terminal peptide were used in the assay at a fixed ATP concentration of 11 nM (1000 Ci/mmol) with N-terminal peptide concentrations of 0 (open circles), 84 (closed circles), and 169 (open triangles) µM. The solid lines represent a nonlinear regression fit to competitive inhibition (18). [IKK] = 22 µg/ml. See "Experimental Procedures" for details.

Surprisingly, the C-terminal peptide did not inhibit the IKK-catalyzed phosphorylation of the N-terminal peptide. Instead, the presence of the C-terminal peptide greatly increased the rate of phosphorylation of the N-terminal peptide as shown in Fig. 6. As shown in Fig. 6 and Table I, this effect resulted from both a decrease in the apparent Km and an increase in the kcat (Vmax).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Activation of the IKK-catalyzed phosphorylation of the N-terminal peptide by the presence of the C-terminal peptide. The indicated concentrations of N-terminal peptide were used in the assay at a fixed ATP concentration of 11 nM (1000 Ci/mmol) with C-terminal peptide concentrations of 0 (open circles), 1.2 (closed circles), 3.7 (open triangles), and 12.4 (closed triangles) µM. The solid lines represent nonlinear regression fits to hyperbolic kinetics at each concentration of C-terminal peptide (18). [IKK] = 22 µg/ml. See "Experimental Procedures" for details.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Effect of the presence of the C-terminal peptide on the IKK-catalyzed phosphorylation of the N-terminal peptide
The data from Fig. 8 were used to calculate Vmax and apparent N-terminal peptide Km values for the phosphorylation of the N-terminal peptide at each C-terminal peptide concentration using nonlinear regression fits to hyperbolic kinetics (18).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The results presented here clearly demonstrate that the IKK proceeds through a random sequential binding mechanism in which both substrates bind before the first product is released (Scheme 1). During the course of this work, however, a number of surprising observations were made, including the finding that the enzyme recognizes a peptide corresponding to amino acids 279-303 of the C terminus of Ikappa B-alpha . Indeed, this C-terminal peptide inhibited the IKK-catalyzed phosphorylation of Ikappa B-alpha . Recognition by the enzyme of the C terminus of Ikappa B-alpha is surprising because IKK has been shown to phosphorylate at Ser-32 and Ser-36 within the N terminus of Ikappa B-alpha (13). The inhibition shown by this peptide, however, does explain the findings of Kuno et al. (20), in which this same C-terminal peptide was shown to block both LPS-induced NF-kappa B activation and phosphorylation of Ikappa B-alpha in a cell-free system.

Our additional finding that the C-terminal peptide is phosphorylated by IKK corroborates a recent report that showed that a small amount of Ikappa B-alpha phosphorylation catalyzed by IKK occurs within residues 264-314 of the C terminus (12). The present results also show that the enzyme phosphorylated an N-terminal peptide corresponding to amino acids 26-42 of Ikappa B-alpha . Competitive inhibition of C-terminal peptide phosphorylation by the N-terminal peptide (Fig. 5) indicated that these peptides are not phosphorylated by two different enzymes within the IKK preparation but, instead, are binding to the same active site.

The most surprising observation was that the rate of IKK-catalyzed phosphorylation of the N-terminal peptide was greatly increased in the presence of the C-terminal peptide. This effect resulted from both an increase in the kcat (Vmax) and a decrease in the apparent Km (Fig. 6 and Table I). A small but discernible increase in the rate of Ikappa B-alpha phosphorylation was also observed at low concentrations of the C-terminal peptide (results not shown). The activation by the C-terminal peptide presumably occurs through its binding to a regulatory site other than the active site. The activation of the active site may result from a conformational change in the enzyme induced by C-terminal peptide binding to this putative regulatory site.

This observation that the C terminus of Ikappa B-alpha activates the IKK-catalyzed phosphorylation of the N terminus can be used to reconcile the variable dissociation constants measured for the N-terminal peptide. For instance, when the N-terminal peptide was used as a substrate in the absence of the C-terminal peptide, a Km of 140 ± 28 µM was determined. When the N-terminal peptide was used as an inhibitor of C-terminal peptide phosphorylation, a considerably smaller dissociation constant (apparent KI) of 12 ± 2 µM was obtained. This is consistent with the C-terminal peptide increasing the affinity of the enzyme for the N-terminal peptide. In addition, the data represented in Fig. 2 can be used with the KI determined for the C-terminal peptide to estimate a dissociation constant (KI) of 43 ± 27 µM for the N-terminal peptide when used as an inhibitor of Ikappa B-alpha phosphorylation. This dissociation constant for the N-terminal peptide is also smaller than the Km value of 140 µM because the C terminus of Ikappa B-alpha is able to activate the IKK to bind the N-terminal peptide.

Although the apparent Km for the N-terminal peptide in the absence of the C-terminal peptide is 140 µM, the apparent Km in the presence of the C-terminal peptide is reduced to a value of 21 µM (Table I). In fact, this apparent Km in the presence of the C-terminal peptide is an overestimate of the "true" dissociation constant under these conditions. This is because the C-terminal peptide is competitively inhibiting the binding of the N-terminal peptide at the active site even though C-terminal binding to the putative regulatory site is enhancing N-terminal peptide binding. Thus, the value of the true dissociation constant (Km) for the N-terminal peptide is approximately 4 µM when activated by C-terminal peptide binding to a regulatory site (i.e. Kmapp = Km(1 + [I]/KI), where [I] and KI equal the concentration and active-site dissociation constant, respectively, of the C-terminal peptide (21)). Therefore, the presence of the C-terminal peptide increases the apparent second-order rate constant (kcat/Km) for the phosphorylation of the N-terminal peptide by a factor of at least 160 as compared with the value in the absence of the C-terminal peptide (i.e., 291/4 versus 64/140).

The Vmax values for N-terminal peptide phosphorylation were plotted at various C-terminal peptide concentrations to give a hyperbolic dependence (Fig. 7). This implies that the dissociation constant for the binding of the C-terminal peptide to the regulatory site is approximately 1 µM. Thus, binding of the C terminus to this putative regulatory site may provide most of the overall free energy of binding of Ikappa B-alpha to the enzyme. Moreover, this model may account for the specificity of phosphorylation of Ikappa B-alpha at Ser-32/Ser-36 because activation of the active site would only occur when the C terminus of Ikappa B-alpha is bound to the enzyme. In fact, when the C terminus of Ikappa B-alpha is bound to the regulatory site, the N terminus may then be optimally positioned to interact with the now-activated active site.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Plot of Vmax for IKK-catalyzed N-terminal peptide phosphorylation versus concentration of C-terminal peptide. The values from Table I were used. The solid line represents a nonlinear regression fit to the following equation: Vmax = Vmax° + ([C-terminal peptide]·Vmaxsat/(KD + [C-terminal peptide])), where KD is the dissociation constant for the C-terminal peptide binding to the regulatory site; Vmax is the value at various C-terminal peptide concentrations; Vmax° is the Vmax value in the absence of C-terminal peptide; and Vmaxsat is the maximum increase in the Vmax value at a saturating concentration of the C-terminal peptide.

This finding that the IKK recognizes and is activated by the C terminus of Ikappa B-alpha also has important ramifications for our understanding of both the regulation of the degradation of Ikappa B-alpha and the activation of NF-kappa B. Indeed, the presence and phosphorylation state of the C terminus of Ikappa B-alpha has been shown to play a role in the signal-induced degradation of Ikappa B-alpha (22-24). An analogous C-terminal region rich in proline, glutamate, aspartate, serine, and threonine residues (termed the PEST domain) has also been identified in Ikappa B-beta (8, 25). Interestingly, a recent report has indicated that recombinantly transferring the C terminus (along with the N terminus) of Ikappa B-alpha to an unrelated protein, such as GST, enables GST to be phosphorylated and degraded in a signal-responsive way (27), although this may result from a recognition of the C terminus by the proteasome (26). Transferring only the N terminus of Ikappa B-alpha to GST did not result in a recombinant protein that underwent signal-induced proteolysis.

Because the IKK is composed of multiple subunits, it is not clear whether activation by the C terminus of Ikappa B-alpha results from binding to an allosteric site on the IKK-alpha and/or IKK-beta catalytic subunits or through binding to another subunit within the complex, which then allosterically affects these catalytic kinase subunit(s). Because it has been reported that IKK-alpha and IKK-beta need to form a heterodimer for maximal enzyme activity (15), an intriguing possibility is that the binding of the C terminus of Ikappa B-alpha to one of the catalytic subunits (IKK-alpha , for instance) allosterically affects the other subunit (IKK-beta in this example) to accelerate the N-terminal phosphorylation of a second Ikappa B-alpha molecule. We are currently addressing these questions through the use of purified IKK-alpha and IKK-beta catalytic subunits.

    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: Bristol-Myers Squibb, 100 Forest Ave., Buffalo, NY 14213. Tel.: 716-887-7615; Fax: 716-887-3203.

1 The abbreviations used are: IKK, Ikappa B kinase; GST, glutathione S-transferase; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Siebenlist, U., Franzoso, G., and Brown, K. (1994) Annu. Rev. Cell Biol. 10, 405-455[CrossRef]
  2. Bauerle, P. A., and Baltimore, D. (1997) Cell 87, 13-20[CrossRef]
  3. Whiteside, S. T., and Israel, A. (1997) Semin. Cancer Biol. 8, 75-82[CrossRef][Medline] [Order article via Infotrieve]
  4. Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., and Siebenlist, U. (1995) Science 267, 1485-1488[Medline] [Order article via Infotrieve]
  5. Finco, T. S., Beg, A. A., and Baldwin, A. S., Jr. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11884-11888[Abstract/Free Full Text]
  6. Baldi, L., Brown, K., Franzoso, G., and Siebenlist, U. (1996) J. Biol. Chem. 271, 376-379[Abstract/Free Full Text]
  7. Roff, M., Thompson, J., Rodriguez, M. S., Jacque, J.-M., Baleux, F., Arenzana-Seisdedos, F., and Hay, R. T. (1996) J. Biol. Chem. 271, 7844-7850[Abstract/Free Full Text]
  8. Weil, R., Laurent-Winter, C., and Israel, A. (1997) J. Biol. Chem. 272, 9942-9949[Abstract/Free Full Text]
  9. Whiteside, S. T., Epinat, J.-C., Rice, N. R., and Israel, A. (1997) EMBO J. 16, 1413-1426[Abstract/Free Full Text]
  10. Chen, Z. J., Parent, L., and Maniatis, T. (1996) Cell 84, 853-862[Medline] [Order article via Infotrieve]
  11. Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88, 213-222[Medline] [Order article via Infotrieve]
  12. DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548-554[CrossRef][Medline] [Order article via Infotrieve]
  13. Régnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. (1997) Cell 90, 373-383[Medline] [Order article via Infotrieve]
  14. Zandi, E., Rothwarf, D., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve]
  15. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J. W., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997) Science 278, 860-866[Abstract/Free Full Text]
  16. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science 278, 866-869[Abstract/Free Full Text]
  17. Segel, I. H. (1975) Enzyme Kinetics, pp. 274-291, John Wiley & Sons, New York
  18. Cleland, W. W. (1979) Methods Enzymol. 63, 103-138[Medline] [Order article via Infotrieve]
  19. Segel, I. H. (1975) Enzyme Kinetics, pp. 328-329, John Wiley & Sons, New York
  20. Kuno, K., Ishikawa, Y., Ernst, M. K., Ogata, M., Rice, N. R., Mukaida, N., and Matsushima, K. (1995) J. Biol. Chem. 270, 27914-27919[Abstract/Free Full Text]
  21. Fersht, A. (1985) Enzyme Structure and Mechanism, pp. 107-109, W. H. Freeman & Sons, New York
  22. Beauparlant, P., Lin, R., and Hiscott, J. (1996) J. Biol. Chem. 271, 10690-10696[Abstract/Free Full Text]
  23. Aoki, R., Sano, Y., Yamamoto, T., and Inoue, J. (1996) Oncogene 12, 1159-1164[Medline] [Order article via Infotrieve]
  24. Schwarz, E. M., Van Antwerp, D., and Verma, I. M. (1996) Mol. Cell. Biol. 16, 3554-3559[Abstract]
  25. Thompson, J. E., Phillips, R. J., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1995) Cell 80, 573-582[Medline] [Order article via Infotrieve]
  26. Kroll, M., Conconi, M., Desterro, M. J. P., Marin, A., Thomas, D., Friguet, B., Hay, R. T., Virelizier, J.-L., Arenzana-Seisdedos, F., and Rodriguez, M. S. (1997) Oncogene 15, 1841-1850[CrossRef][Medline] [Order article via Infotrieve]
  27. Brown, K., Franzoso, G., Baldi, L., Carlson, L., Mills, L., Lin, Y. C., Gerstberger, S., and Siebenlist, U. (1997) Mol. Cell. Biol. 17, 3021-3027[Abstract]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.