From the Department of Molecular, Cellular and Developmental
Biology, Interdepartmental Program in Biochemistry and Molecular
Biology, University of California, Santa Barbara, California 93106 and
the Graduate Program in Biomedical Sciences,
University of California, San Diego, California 92093
Received for publication, September 6, 2000
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ABSTRACT |
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The mitogen-activated protein (MAP) kinases are
characterized by their requirement for dual phosphorylation at a
conserved threonine and tyrosine residue for catalytic activation. The
structural consequences of dual-phosphorylation in the MAP kinase ERK2
(extracellular signal-regulated
kinase 2) include active site closure, alignment of key
catalytic residues that interact with ATP, and remodeling of the
activation loop. In this study, we report the specific effects of dual
phosphorylation on the individual catalytic reaction steps in ERK2.
Dual phosphorylation leads to an increase in overall catalytic
efficiency and turnover rate of approximately 600,000- and 50,000-fold,
respectively. Solvent viscosometric studies reveal moderate decreases
in the equilibrium dissociation constants (Kd) for
both ATP and myelin basic protein. However, the majority of the overall
rate enhancement is due to an increase in the rate of the phosphoryl
group transfer step by approximately 60,000-fold. By comparison, the
rate of the same step in the ATPase reaction is enhanced only
2000-fold. This suggests that optimizing the position of the invariant
residues Lys52 and Glu69, which stabilize the
phosphates of ATP, accounts for only part of the enhanced rate of
phosphoryl group transfer in the kinase reaction. Thus, significant
stabilization of the protein phosphoacceptor group must also occur. Our
results demonstrate similarities between the activation mechanisms of
ERK2 and the cell cycle control enzyme, Cdk2
(cyclin-dependent kinase 2). Rather
than dual phosphorylation, however, activation of the latter is
controlled by cyclin binding followed by phosphorylation at
Thr160.
Members of the family of protein kinases referred to as the
MAP1 kinases are critical
components of the biochemical processes that define the essence of
life, the ability of cells to sense their external environment and
respond. The prototype member of the MAP kinase family that mediates
signaling by all polypeptide mitogens is the extracellular
signal-regulated kinase, ERK2. Like all MAP
kinases, ERK2 participates in a three-tier protein phosphorylation cascade that, in response to growth factor receptor signaling, is
activated by dual phosphorylation catalyzed by an upstream activating
kinase, MEK1/MEK2. Among the targets of ERK2 are downstream kinases
involved in cellular growth control as well as nuclear transcription
factors. Thus, ERK2 provides an essential link in transducing the
diverse signals from transmembrane growth factor receptors into gene
regulatory events (for review see Refs. 1 and 2).
The x-ray crystallographic structure of ERK2 (357 amino acids) reveals
a conserved catalytic core (residues 22-311) flanked by N- and
C-terminal extensions that lie on the surface of the molecule. Like all
protein kinases, the catalytic core is globular, consisting of an N-
and C-terminal lobe, whose interface defines the active site cleft.
Within the active site, the adenine ring of ATP is deeply buried, with
the All protein kinases display near their active sites an "activation
loop," the conformation of which must be optimized for high catalytic
activity (4). For most, but not all, protein kinases, this
activity is dependent upon phosphorylation at a single conserved
residue located at the C-terminal end of the activation loop (4). In
many cases, phosphorylation at this site is catalyzed by a heterologous
kinase and constitutes a physiological mechanism for kinase regulation.
For example, phosphorylation of the cyclin-dependent
kinases at the conserved activation loop threonine (Thr160
in Cdk2) by a cdk activating kinase
(CAK) is critical for regulation of cell cycle progression (5).
Similarly, the MAP kinases are regulated by phosphorylation by their
upstream activators, the MAPK/ERK kinases (MEKs), in response to
extracellular signaling (2).
The hallmark of the MAP kinases is their unique requirement for dual
phosphorylation at a conserved threonine and tyrosine residue belonging
to the consensus sequence TXY for catalytic activation (6). In ERK2, these sites are Thr183 and
Tyr185. The phosphorylation of both of these residues is
catalyzed by the dual-specific upstream kinases MEK1 or MEK2 (2). The
structural role of both phosphorylations in catalytic activation has
been revealed by comparison of the crystallographic structures of
(non-p)ERK2 (3) and (pp)ERK2 (7). The major alteration upon dual
phosphorylation is a closure of the active site cleft, which
results in optimal alignment of the key catalytic residues that contact
the phosphate groups of ATP. In addition, there is significant
remodeling of the activation loop as well as the P+1
surface pocket,2 the latter
of which is necessary to accept the P+1 proline residue
essential for the recognition of all ERK substrates.
Given the structural consequences of dual phosphorylation revealed by
x-ray crystallography, it is not known how such changes in structure
specifically affect the kinetics of individual reaction steps along the
catalytic reaction pathway. Thus, a correlation between structure and
mechanism, and therefore regulation, cannot be made. In this study, the
catalytic reaction pathway for nonphosphorylated ERK2 was determined
and compared with that of the fully active, dual-phosphorylated enzyme.
The results provide a quantitative understanding of the mechanistic
basis for catalytic activation by dual phosphorylation. Finally, our
studies reveal functional similarities between ERK2 and Cdk2 in terms
of their mechanisms of activation.
Materials--
All chemicals (KCl, EDTA, MgCl2,
MOPS, sucrose, dithiothreitol) were purchased from Fisher, except for
ATP (Sigma), and [ MBP Preparation--
Myelin basic protein (MBP) was purified
from a bovine brain acetone powder (Sigma B0508) by acid extraction
followed by cation exchange chromatography as described previously
(8).
ERK2 Preparation--
Expression and purification of the
recombinant rat ERK2 N-terminally fused to a hexahistidine tag was
carried out as described previously (9). Briefly, ERK2 overexpressed in
Escherichia coli was purified by Ni2+-NTA
affinity chromatography followed by FPLC anion exchange chromatography on Uno Q (Bio-Rad). Select fractions from peak 1 (see "Results") were used for all experiments.
(pp)ERK2 was generated as described previously (8). (non-p)ERK2
purified by Ni2+-NTA affinity chromatography was subject to
phosphorylation by recombinant MEK1 in vitro, and the
dual-phosphorylated material was further purified by FPLC anion
exchange chromatography on Uno Q (Bio-Rad). ERK2 was confirmed to be
dual-phosphorylated by electrospray mass spectrometry. Both (non-p)ERK2
and (pp)ERK2, purified as described above, were essentially homogeneous
based on analysis by SDS-polyacrylamide gel electrophoresis. The
concentration of ERK2 was determined spectrophotometrically based on an
extinction coefficient ( Kinase Assays and Data Analysis--
Kinase activity was
monitored by a radioisotope assay in which the rate of incorporation of
32P from [
Steady-state kinetic parameters were determined by nonlinear least
squares analysis of initial velocity data obtained from several
concentrations of MBP at several fixed concentrations of ATP. The
following equation (Equation 1) for a two-substrate sequential reaction
was globally fit to the data using the program Scientist (Micromath
Inc.),
Solvent Viscosometric Studies--
Steady-state assays, as
described above, were carried out in buffer containing sucrose ranging
from 0 to 40%, to give relative solvent viscosities ranging between 1 and 4.2. Relative solvent viscosity was determined as described
previously (8).
ATPase Assays--
ATPase activity of (non-p)ERK2 was determined
in a radioisotope assay in which the rate of 32P production
was monitored. Reactions were performed in phosphorylation buffer in a
total volume of 20 µl at 23 °C. Typically, reactions containing 3 µM (non-p)ERK2 were initiated by the addition of [
ATPase activity of (pp)ERK2 was determined using a coupled
spectrophotometric assay (11). The coupling reagents and their concentrations were as follows: 15 units/ml lactate
dehydrogenase, 7.5 units/ml pyruvate kinase, 1 mM
P-enolpyruvate, and 130 µM NADH. All reactions were
performed in phosphorylation buffer in a total volume of 75 µl at
23 °C. Reactions were initiated by the addition of 1 µM (pp)ERK to the reaction mix containing ATP at various
concentrations. Progress of the reaction was monitored by a continuous
decrease in absorbance at 340 nm in a Shimadzu UV1601
spectrophotometer. Initial velocities in µM/min were
calculated based on an extinction coefficient for NADH of 6220 cm
Experiments to determine solvent viscosity effects on
kcat and
kcat/Km for ATPase reactions
were carried out under the following conditions: (non-p)ERK2, 2 mM and 800 µM ATP; (pp)ERK2, 4 mM
and 90 µM ATP. Sucrose was varied between 0 and 40%.
Purification of (non-p)ERK2--
(non-p)ERK2 was expressed and
purified from E. coli. Purification using anion exchange
chromatography revealed two peaks of protein corresponding to ERK2,
both of which displayed identical activities and activation properties.
These observations are consistent with those reported previously during
the crystallization of ERK2, in which only peak 1 formed crystals (9).
Accordingly, we have used this fraction of ERK2 for all of our studies.
The activity of each fraction from peak 1 was determined. When assayed
for kinase activity, it was found that the latter fractions of the peak
exhibited substantially higher kinase activity than those eluting
earlier (Fig. 1). The high specific
activity associated with the later fractions may be attributable to
enzyme that had undergone autophosphorylation during induction. In all
our studies, however, fractions free of the high kinase activity
(fractions 42-43, Fig. 1) were used. In these fractions,
the profile of kinase activity corresponded to the ERK2 protein
concentration exactly, and when subjected to phosphorylation by MEK1
the enzyme could be activated to a form that displayed high catalytic
activity (kcat/Km = 1 µM Steady-state Kinetic Analysis--
The steady-state kinetic
parameters for the phosphorylation of myelin basic protein by
(non-p)ERK2 were determined. MBP phosphorylation was linear for at
least 2 h, indicating that autoactivation of (non-p)ERK2 did not
occur within this time frame. A data set of initial reaction velocities
obtained under conditions of varied MBP concentrations at several fixed
concentrations of ATP was analyzed. Fig.
2 shows the experimental data and the
best-fit regression curves in double reciprocal form. The regression
analysis yielded values of kcat = 2 × 10 Solvent Viscosometric Analysis--
The Michaelis-Menten
parameters described above are a composite of microscopic rate
constants combined in a manner dependent upon the order of substrate
addition. The steady-state data for (non-p)ERK2 are consistent with
both randomly and compulsorily ordered mechanisms. However, if the
kinetic mechanism of this enzyme is ordered, it is necessarily ordered
with ATP binding first. This is true because the crystal structure of
(non-p)ERK2 has been obtained with bound ATP alone (3). In addition,
(non-p)ERK2 displays measurable ATPase activity in the absence of MBP
(see below).
Under saturating conditions of ATP, the catalytic mechanism of
(non-p)ERK2 can therefore be described by Scheme
1. In this scheme, catalytic efficiency
is given by
[kcat/Km(MBP) = k2·k3/(k
The viscosity effect (designated by superscripted
The viscosity effect on
kcat/Km for ATP was also
determined.3 No viscosity
effect on this parameter was observed. Thus, the lack of a viscosity
effect on both
kcat/Km(MBP) and
kcat/Km(ATP), as
well as on kcat, supports a kinetic scheme in
which both substrates exist in rapid equilibrium with the ternary
Michaelis complex, whose breakdown to form products is entirely limited
by phosphoryl group transfer. In such a model, the true affinities
(Kds) of both substrates to form the ternary complex
are given by their Km values. Thus, our steady-state
data reveal that dual phosphorylation enhances the affinity of ATP
binding by approximately 12-fold, whereas the affinity for MBP is
enhanced ATPase Activity--
An important consequence of
dual-phosphorylation is the optimization of the alignment of the
invariant residues, Lys52, which coordinates to the Mechanism of Activation by Dual Phosphorylation--
We previously
characterized the kinetic reaction pathway of the dual-phosphorylated
form of ERK2 (8). In comparison to (pp)ERK2, (non-p)ERK2 displays
kcat/Km(MBP) and
kcat values that are decreased by approximately
600,000- and 50,000-fold, respectively (Table I). Yet, the
Km values for MBP and ATP are each increased by only
10-fold. Therefore, the exceedingly low activity of (non-p)ERK2 is due
to the dramatically decreased rates of substrate capture and turnover
and not the inability to saturate the enzyme with substrate under
steady-state conditions.
The extreme rate enhancements caused by dual phosphorylation are
the most dramatic of any protein kinase for which the kinetic basis for
activation by phosphorylation has been investigated. The large
increases in catalytic efficiency and turnover rate are attributable to
an approximate 60,000-fold increase in the rate of phosphotransfer,
12-fold higher binding affinity for ATP, and a minimum 100-fold higher
affinity for MBP. The ability to bind MBP more tightly correlates to a
remodeling of the P+1 surface pocket that functions to bind
the essential proline residue found in all ERK2 substrates; this is
achieved in part by hydrogen bonding between the phosphoryl group
oxygens of PO3-Tyr185 and the side chains of
Arg189 and Arg192 (7). Although our data
provides only a lower limit value on the absolute enhancement in
substrate binding affinity, it should be noted that tighter binding of
the substrate to (pp)ERK2 will not result in higher catalytic
efficiency, because the rate of MBP binding to (pp)ERK2 already occurs
at the diffusion-controlled limit (8). Thus, the catalytic efficiency
of the dual-phosphorylated enzyme depends only on the rate of substrate encounter.
Dual phosphorylation results in a rotation of the N- and C-terminal
lobes, which closes the active site cleft and optimizes the alignment
of the essential catalytic residues, Lys52 and
Glu69 (7). This results in a 12-fold tighter binding of ATP
to the enzyme, and a 2000-fold increase in the rate of phosphoryl group transfer with respect to the ATPase reaction. Nonetheless, this increase in phosphotransfer rate is approximately 30-fold down from
that seen in the kinase reaction, demonstrating that stabilization of
the phosphate moieties of ATP accounts for only a portion of the
overall enhanced rate of chemistry with respect to the phosphorylation of protein substrates.
Correlation of the ATPase and kinase activities of ERK2 reveals
similarities in its mechanism of activation to that of Cdk2/cyclin A. For example, activation of both enzymes involves two steps. In
Cdk2/cyclin A, these steps are cyclin binding followed by
phosphorylation at Thr160 (5), whereas in ERK2,
phosphorylation at Tyr185 followed by phosphorylation at
Thr183 is required (6, 17). In Cdk2/cyclin A, the rate of
phosphotransfer in the ATPase reaction is unchanged by phosphorylation
at Thr160 (18). This is consistent with crystallographic
information which shows that the role of phosphorylation at
Thr160 is not to align the conserved lysine
(Lys33) and glutamate (Glu51) side chains, thus
stabilizing ATP. Instead, alignment of these residues in Cdk2 is
achieved by the binding of cyclin. However, the rate of the same step
in the kinase reaction of Cdk2/cyclin A is enhanced nearly 3000-fold
(18). Thus, although cyclin binding serves to stabilize the ATP
portion of the transition state for phosphoryl group transfer,
phosphorylation at Thr160 serves to stabilize the protein
phosphoacceptor group. Thr160 in Cdk2 and
Thr183 in ERK2 occupy structurally analogous roles (4);
both coordinate to a positively charged triad of arginine side chains
that bridge the N- and C-terminal lobes of the catalytic cores. Thus,
it is possible that phosphorylation at Thr183 in ERK2 may
function analogously to that at Thr160 in Cdk2/cyclin A,
whereas phosphorylation at Tyr185 in ERK2 may play a role
analogous to cyclin binding.
In summary, we have demonstrated that dual phosphorylation of ERK2
results in 10-100-fold greater rate enhancements compared with other
protein kinases in which activation is dependent on phosphorylation at
only a single site. Most of the increase in catalytic power in ERK2 is
attributable to an increased rate of phosphotransfer, which is
coordinately accomplished by two independent mechanisms: 1)
stabilization of the phosphate moieties of ATP via alignment of
Lys52 and Glu69 and 2) stabilization of the
protein phosphoacceptor group. In Cdk2, we propose that these may be
separately achieved by cyclin binding and phosphorylation at
Thr160, respectively (18). We hypothesize that a
similar mechanism of activation may be functionally conserved in ERK2,
except that stabilization of the ATP and protein phosphoacceptor groups
may instead be controlled separately by phosphorylation at
Tyr185 and Thr183.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-phosphate pointing toward the mouth of the active site where
protein substrates bind and where phosphotransfer occurs (3).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32-P]ATP (ICN).
280 = 44, 825 cm
1 M
1)
calculated from its primary amino acid sequence (10).
-32P]ATP into MBP was directly
measured. Reactions were carried out in phosphorylation buffer (20 mM MOPS, pH 7.4, 50 mM KCl, 0.1 mM
EDTA, 1 mM dithiothreitol, 10 mM
MgCl2(total)) containing 3 µM ERK2 and varied
concentrations of MBP. Reactions were initiated by the addition of
[
-32P]ATP (300-500 cpm/pmol) at varied concentrations
and allowed to proceed at 23 °C for 45 min, after which time they
were terminated with 25% acetic acid. The
32P-labeled MBP product was resolved from
unincorporated [
-32P]ATP by ascending chromatography
on P81 phosphocellulose paper (Whatman) as described previously (8).
Radioactivity was quantified by Cerenkov counting.
where v is the initial velocity, V is the maximal initial
velocity, A and B are the fixed and varied substrates, respectively, Km is the Michaelis constant, and
KiA is the dissociation constant for A. kcat was determined by dividing the maximal
initial velocity by the enzyme concentration.
(Eq. 1)
32-P]ATP (500 cpm/pmol) at various concentrations,
allowed to proceed for 2 h at 23 °C, then terminated in 1 ml of
0.1 N HCl. To determine the amount of phosphate produced,
the stopped reactions were incubated with 200 µl of charcoal solution
(10% activated charcoal (Sigma C-6289), 10% acetic acid, 2.5 mM KH2PO4) for 1 h on ice and
then centrifuged at maximum speed in a microcentrifuge for 30 min. Radioactivity in the supernatant was quantified by counting 500 µl of
the supernatant using the Cerenkov method.
1
M
1at 340 nm. Measurement of
ATPase activity of (pp)ERK2 by spectrophotometric or by radioisotope
labeling methods gave identical results. Steady-state kinetic
parameters for both (non-p)ERK2 and (pp)ERK2 were determined from
nonlinear regression analysis of initial velocities as a function of
ATP concentration using the Michaelis-Menten equation.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1
s
1 and kcat = 10 s
1) (8).
View larger version (20K):
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Fig. 1.
Anion exchange chromatography of
(non-p)ERK2. ERK2 was purified by Ni2+ chelate
chromatography and then further purified by FPLC over UnoQ (Bio-Rad).
The Uno Q trace is shown. Solid circles ( ) correspond to
A280. Bars correspond to kinase
activity toward MBP, determined as follows: 5 µl of each fraction was
incubated with 100 µM MBP and 1 mM
[
-32P]ATP (300 cpm/pmol) in phosphorylation buffer (15 µl total volume) for 1 h, and total
32P-labeled MBP was produced was determined as
described under "Experimental Procedures." Fractions 42 or 43 were used for all assays. The dashed line (- - -
) indicates the salt gradient, which runs from 100 mM
(fraction 38) to 250 mM (fraction 52)
NaCl.
4 s
1,
Km(MBP) = 50 µM and
Km(ATP) = 700 µM. The
kcat value is down 50,000-fold from that of
(pp)ERK2, whereas the Km values for MBP and ATP are
each up by only 10-fold. The changes in the steady-state parameters
correspond to an overall catalytic efficiency toward MBP
(kcat/Km(MBP)), which is attenuated 600,000-fold.
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Fig. 2.
Steady-state kinetic analysis of MBP
phosphorylation. Initial velocities obtained at various MBP
concentrations and several fixed concentrations of ATP were determined
by a radioisotope assay as described under "Experimental
Procedures." A Michaelis-Menten model describing a two-substrate
sequential addition was globally fit to the data by nonlinear
regression analysis. The best-fit curves and experimental data are
shown in double reciprocal form. The optimized kinetic parameters
resulting from the regression analysis are reported in Table I. ATP was
fixed at 4000 ( ), 3000 (
), 2000 (
), 1000 (
), and 500 µM (+) (from bottom to top). The
concentration of (non-p)ERK2 was 3 µM.
2 + k3)], whereas the turnover rate is given by
[kcat = k3·k4/(k3 + k4)] (12). To solve for the microscopic constants, k2, k
2,
k3, and k4, we employed
steady-state solvent viscosometric techniques, which allow separation
of the diffusive (k2,
k
2, k4) from the
nondiffusive (k3) reaction steps (13-15).
Initial velocity data were obtained as a function of MBP concentration
at several fixed concentrations of sucrose, and the effect of solvent
viscosity on kcat or
kcat/Km(MBP) was
determined. Since it was not possible to carry out these experiments at
a single, saturating concentration of ATP, because of the high Km(ATP) value, viscosity data were
collected at several subsaturating ATP concentrations. The
viscosity effect on appkcat (Fig.
3A) or
appkcat/Km(MBP)
(Fig. 3B) was determined at each ATP concentration by
plotting the fold decrease in the respective rate parameter as a
function of the relative solvent viscosity. The viscosity effect is
given by the slopes of the best-fit lines in Fig. 3, A and
B. The true viscosity effect on kcat
and kcat/Km(MBP) was determined by extrapolation to infinite ATP concentration. We saw
no significant viscosity effect on either rate parameter at any ATP
concentration tested, and we therefore conclude that both
kcat and
kcat/Km(MBP) are
insensitive to the relative solvent viscosity.
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Scheme 1.
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[in a new window]
Fig. 3.
Solvent viscosity effects on
kcat and
kcat/Km(MBP)
for the phosphorylation of MBP. The "viscosity effect" on a
given rate parameter is defined as the rate of change in reaction rate
with respect to the relative solvent viscosity and corresponds to the
slopes of the lines in panels A and B.
The true viscosity effects on kcat (panel
A) or kcat/Km(MBP)
(panel B) were determined from the apparent rate values
measured at three subsaturating concentrations of ATP (
, 0.2 mM; -----
-----, 1 mM;
, 2 mM) as a function of increasing relative
solvent viscosity, followed by extrapolation to infinite ATP
concentration. In panel A, MBP was either varied or fixed at
600 µM. In panel B, MBP was fixed at 20 µM. The viscosity effects on
kcat/Km
(km
) and
kcat
(kcat
) relate to the individual
rate constants in Scheme 1 as follows:
kcat/Km
= k3/(k
2 + k3); kcat
= k3/(k3 + k4 (16). A value for
kcat/Km
and kcat
approaching zero implies
that k
2
k3 and
k3
k4, respectively.
) on
kcat/Km(MBP) is
given by
[kcat/Km(MBP)
= k3/(k
2 + k3)], whereas the viscosity effect on
kcat is given by
[kcat
= k3/(k3 + k4)] (16). The kinetic constants derived from
the viscosity studies are shown in Table
I. The lack of viscosity effect on
kcat implies that the overall rate of substrate
turnover is limited by phosphoryl group transfer
(k3) and that the diffusion of either product
from the active site (k4) is not rate-limiting. The rate of phosphoryl group transfer can therefore be assigned a value
of 0.012 min
1, which is 60,000-fold lower
than that in (pp)ERK2.
Kinetic and thermodynamic parameters for (non-p)ERK2 versus (pp)ERK2
100-fold.
-
and
-phosphates of ATP, and Glu69, which stabilizes
Lys52, in (pp)ERK2 (7). To determine the relative
contribution of these interactions to the overall increase in the
phosphoryl group transfer step of the kinase reaction, we compared the
fold increase in catalytic parameters of the kinase reaction to those
of the ATPase reaction (Table I). The steady-state parameters and
individual rate constants for the ATPase reaction for both (non-p)ERK2
and (pp)ERK2 were determined by solvent viscosometric analysis (see "Experimental Procedures"). Similar to the kinase reaction, the major effect of dual phosphorylation was a large (2000-fold) increase in the phosphotransfer rate, whereas the observed increase in ATP
binding affinity was only 12-fold. However, the 2,000-fold increase in
the rate of phosphotransfer is significantly less than the 60,000-fold
rate enhancement of this step seen in the kinase reaction. This
finding suggests that the net stabilization of the
transition-state complex for phosphoryl group transfer to MBP occurs
only in part by stabilization of the ATP moiety and that significant
stabilization of the protein phosphoacceptor substrate moiety must also occur.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Larry Brunton, University of California, San Diego, Biomedical Sciences Program, for invaluable support and encouragement.
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FOOTNOTES |
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* 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. Tel.: 805-893-5336; Fax: 805-893-4724; E-mail: lew@lifesci.ucsb.edu.
Published, JBC Papers in Press, October 2, 2000, DOI 10.1074/jbc.M008137200
2 P0 is the substrate phosphorylation site.
3 Initial velocities were determined at 1 mM MBP, 175 µM ATP, and varied sucrose. Identical conditions, except in 300 µM ATP, gave proportionally higher rates, indicative of true kcat/Km conditions.
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ABBREVIATIONS |
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The abbreviations used are: MAP, mitogen-activated protein; ERK2, extracellular signal-regulated kinase 2; (non-p)ERK2, nonphosphorylated ERK2; (pp)ERK2, dual-phosphorylated ERK2; Cdk, cyclin-dependent kinase; MBP, myelin basic protein; MOPS, 4-morpholinepropanesulfonic acid; FPLC, fast protein liquid chromatography.
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