From The Department of Drug Discovery Research, Bristol-Myers
Squibb Pharmaceutical Research Institute,
Buffalo,
New York 14213 and ¶ Princeton, New Jersey 08543
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INTRODUCTION |
The NF-
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-
, interleukin-6, interleukin-8, and interleukin-1
; 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-
B normally resides in the cytoplasm as an
inactive complex with an I
B inhibitory protein. This class of
protein includes I
B-
, I
B-
, and I
B-
, which all contain ankyrin repeats necessary for complexation with NF-
B (for a review, see Ref. 3). In the case of I
B-
, which is the most carefully studied member of this class, stimulation of cells with agents that
activate NF-
B-dependent gene transcription results in a phosphorylation of I
B-
at Ser-32 and Ser-36 (4). This is critical
for subsequent ubiquitination and proteolysis of I
B-
, which then
leaves NF-
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-
B and results in an I
B-
that is neither phosphorylated, ubiquitinated, nor proteolytically digested (7). Analogous serines have
been identified in both I
B-
and I
B-
, and phosphorylation at
these residues appears to regulate the proteolytic degradation of these
proteins by a mechanism similar to that of I
B-
(8, 9). Because of
its important role in the activation of NF-
B, the inhibition of this
signal-inducible phosphorylation of I
B is an important target for
novel anti-inflammatory agents.
A high molecular mass (500-900 kDa) multisubunit I
B kinase
(IKK)1 that phosphorylates at
Ser-32 and Ser-36 of I
B-
has been isolated from HeLa cells
(10-12). The IKK also phosphorylates at Ser-19 and Ser-23 in the N
terminus of I
B-
(13). The kinase activity is greatly enhanced if
the cells are first treated with tumor necrosis factor-
, which
appears to activate a kinase cascade leading to the phosphorylation of
the IKK (11, 13).
Recently, two catalytic subunits (termed IKK-
and IKK-
) of IKK
have been identified, cloned, and shown to be widely expressed in human
tissues (12-16). IKK-
and IKK-
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 I
B-
was accomplished by both
antisense inhibition of IKK-
and the use of dominant negative, catalytically inactive mutants of IKK-
and IKK-
(12, 15, 16).
Both approaches abrogated cytokine-induced activation of NF-
B. The
signal-induced activation of IKK appears to proceed through
phosphorylation of the IKK-
and/or IKK-
subunits by a
mitogen-activated protein kinase kinase (such as mitogen-activated protein kinase/ERK kinase kinase-1 or NF-
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
I
B
.
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EXPERIMENTAL PROCEDURES |
Materials--
GST-I
B
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-
was purchased
from R&D Systems, and [
-33P]ATP (1000 Ci/mmol) was
purchased from Amersham Pharmacia Biotech.
Peptide Synthesis--
An N-terminal peptide corresponding to
amino acids 26-42 of I
B-
(LDDRHDSGLDSMKDEEY) was prepared along
with a C-terminal peptide corresponding to amino acids 279-303 of
I
B-
(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-
-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-
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 I
B-
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 I
B-
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 I
B-
--
The
I
B-
substrate used was a GST-I
B
fusion protein. Enzymatic
assays were performed by adding IKK at 30 °C to solutions of the
GST-I
B
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 I
B-
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-I
B
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 [
-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.
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RESULTS |
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 I
B-
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 KI
B
and
KI
B
are the dissociation constants for I
B-
in the absence and presence, respectively, of ATP in the active site;
KATP and
KATP are the
dissociation constants for ATP in the absence and presence,
respectively, of I
B-
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 KI
B
, KATP, and
,
respectively. A value of
<1 demonstrates that the binding of one
substrate increases the affinity for the second substrate (17).

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Fig. 1.
Initial velocity patterns for IKK with
varying levels of ATP and I B- . Hanes plot of the initial
rate of IKK-catalyzed phosphorylation of I B- 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.
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Peptide Analogs of I
B-
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
I
B-
(termed the N-terminal peptide) or amino acids 279-303 of
I
B-
(a C-terminal peptide) were tested as inhibitors of the the
IKK-catalyzed phosphorylation of I
B-
. 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 I
B-
. 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.

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Fig. 2.
Inhibition by peptides of the IKK-catalyzed
phosphorylation of I B- . Dixon plot of the initial rate of
IKK-catalyzed phosphorylation of I B- using the following peptides
as inhibitors: closed circles, N-terminal peptide;
open circles, C-terminal peptide. [I B- ] = 51 nM; [ATP] = 2.3 µM; [IKK] = 39 µg/ml.
See "Experimental Procedures" for details.
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As represented in Fig. 3, the inhibition
of the IKK-catalyzed phosphorylation of I
B-
by the C-terminal
peptide was shown to be competitive with respect to I
B-
. 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 I
B-
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 I
B-
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).

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Fig. 3.
Competitive inhibition by the C-terminal
peptide with respect to I B- . Varying concentrations of
I B- 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.
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Scheme 2.
Equilibria in a random sequential
mechanism showing an inhibitor (I) that competes with
I B- but allows ATP to bind (17). Here, KI
and KI represent the dissociation constants of
the inhibitor in the absence and presence, respectively, of ATP; and
KATP and KATP
represent the dissociation constants of ATP in the absence and
presence, respectively, of inhibitor.
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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 I B- 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.
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Values for KI and
were estimated from the data
represented in Figs. 3 and 4 and from the values of
KATP, KI
B
, and
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
of 0.3 ± 0.5. This value, within experimental error, was the same as that obtained for
. Because the
value for
is less than 1, it demonstrates that the binding of the
peptide also increases the affinity for ATP.
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(Eq. 1)
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(Eq. 2)
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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
the
Vmax for phosphorylation of I
B-
under identical conditions (results not shown). An analog of the N-terminal peptide, which had residues corresponding to Ser-32 and Ser-36 of
I
B-
changed to alanines, was not phosphorylated under these conditions. However, this mutant peptide inhibited the IKK-catalyzed phosphorylation of I
B-
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.

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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.
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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).

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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.
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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).
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DISCUSSION |
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 I
B-
.
Indeed, this C-terminal peptide inhibited the IKK-catalyzed
phosphorylation of I
B-
. Recognition by the enzyme of the C
terminus of I
B-
is surprising because IKK has been shown to
phosphorylate at Ser-32 and Ser-36 within the N terminus of I
B-
(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-
B activation and
phosphorylation of I
B-
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
I
B-
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 I
B-
. 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 I
B-
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 I
B-
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 I
B-
phosphorylation. This dissociation constant for
the N-terminal peptide is also smaller than the Km
value of 140 µM because the C terminus of I
B-
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 I
B-
to the enzyme. Moreover,
this model may account for the specificity of phosphorylation of
I
B-
at Ser-32/Ser-36 because activation of the active site would
only occur when the C terminus of I
B-
is bound to the enzyme. In fact, when the C terminus of I
B-
is bound to the regulatory site,
the N terminus may then be optimally positioned to interact with the
now-activated active site.

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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.
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This finding that the IKK recognizes and is activated by the C terminus
of I
B-
also has important ramifications for our understanding of
both the regulation of the degradation of I
B-
and the activation
of NF-
B. Indeed, the presence and phosphorylation state of the C
terminus of I
B-
has been shown to play a role in the
signal-induced degradation of I
B-
(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
I
B-
(8, 25). Interestingly, a recent report has indicated that
recombinantly transferring the C terminus (along with the N terminus)
of I
B-
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 I
B-
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 I
B-
results from binding
to an allosteric site on the IKK-
and/or IKK-
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-
and IKK-
need to form a heterodimer
for maximal enzyme activity (15), an intriguing possibility is that the
binding of the C terminus of I
B-
to one of the catalytic subunits
(IKK-
, for instance) allosterically affects the other subunit
(IKK-
in this example) to accelerate the N-terminal phosphorylation
of a second I
B-
molecule. We are currently addressing these
questions through the use of purified IKK-
and IKK-
catalytic
subunits.