From the Department of Molecular, Cellular and
Developmental Biology, and the ** Department of Chemistry and
Biochemistry and the Interdepartmental Program in Biochemistry and
Molecular Biology, University of California, Santa Barbara, California
93106,
Howard Hughes Medical Institute, Cellular Biochemistry
and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New
York, New York 10021, and the ¶ Interdepartmental
Graduate Program in Biochemistry and Molecular Biology, University of
California, Santa Barbara, California 93106
Received for publication, August 11, 2000, and in revised form, September 29, 2000
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ABSTRACT |
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The activation of most protein kinases requires
phosphorylation at a conserved site within a structurally defined
segment termed the activation loop. A classic example is the regulation of the cell cycle control enzyme, CDK2/cyclin A, in which
catalytic activation depends on phosphorylation at
Thr160 in CDK2. The structural consequences of
phosphorylation have been revealed by x-ray crystallographic studies on
CDK2/cyclin A and include changes in conformation, mainly of the
activation loop. Here, we describe the kinetic basis for activation by
phosphorylation in CDK2/cyclin A. Phosphorylation results in a
100,000-fold increase in catalytic efficiency and an approximate
1,000-fold increase in the overall turnover rate. The effects of
phosphorylation on the individual steps in the catalytic reaction
pathway were determined using solvent viscosometric techniques. It was
found that the increase in catalytic power arises mainly from a
3,000-fold increase in the rate of the phosphoryl group transfer step
with a more moderate increase in substrate binding affinity. In
contrast, the rate of phosphoryl group transfer in the ATPase pathway
was unaffected by phosphorylation, demonstrating that phosphorylation at Thr160 does not serve to stabilize ATP in the ATPase
reaction. Thus, we hypothesize that the role of phosphorylation in the
kinase reaction may be to specifically stabilize the peptide
phosphoacceptor group.
Cellular proliferation is controlled by a family of protein
kinases in which the catalytic subunits are members of the
cyclin-dependent kinase
(CDK)1 family and the
regulatory subunits are cyclins. To date, nine distinct CDKs in
addition to eight different cyclins have been identified, in which
different CDK/cyclin combinations serve to regulate distinct points in
the mammalian cell division cycle. Although Cdc2 (CDK1)/cyclin B
controls the transition of cells from the G2 to M-phase,
the activities of CDK2/cyclin E and CDK2/cyclin A are critical
for G1/S-phase transition and progression through S-phase,
respectively (1). Since the critical role of the CDKs in cell cycle
control has been well established, understanding the details of their
regulation is now of fundamental importance.
The three-dimensional structures of several forms of CDK2 have been
solved by x-ray crystallography. Like all protein kinases, CDK2
displays a globular fold consisting of two lobes, a smaller N-terminal
lobe that is principally Activation of CDK2 requires binding to its regulatory subunit, cyclin,
in addition to phosphorylation at Thr160 (5). The
structural consequences of cyclin binding to CDK2 and phosphorylation
at Thr160 in CDK2 have been revealed by x-ray
crystallography. Interaction with cyclin A results in the repositioning
of an active site helix and the consequent alignment of key catalytic
residues in CDK2, including the invariant residues Lys33
and Glu51, which function to stabilize the Although abundant structural information regarding the activation of
CDK2 is available, it is not known how the associated alterations in
structure correlate with increased catalytic rate. In particular, it is
not known which steps along the catalytic reaction pathway are altered
in response to Thr160 phosphorylation to achieve kinase
activation. Thus, it has not been possible to make a correlation
between CDK structure and regulation. In this study, we describe the
catalytic reaction pathway for both the unphosphorylated and
Thr160-phosphorylated CDK2/cyclin A complexes and report
the kinetic basis for activation by phosphorylation. Our results show
that phosphorylation affects mainly the rate of chemistry, doing so by
specific stabilization of the protein phosphoacceptor group.
Protein Expression and Purification
Unphosphorylated CDK2/cyclin A ((non-p)CDK2/Cyclin A)--
Cdk2
was expressed as the full-length human CDK2 N-terminally fused to GST
in pGEX-2T. Cyclin A was expressed as a truncated fragment of bovine
cyclin A3, (corresponding to human cyclin A residues Val171
to the end) fused to a C-terminal hexahistidine tag in pET21d (9).
Escherichia coli BL21 (DE3) transformed with either GST-CDK2 or cyclin A-His6 were grown at 37 °C in LB to an
A600 of 0.6-0.8. Expression of GST-CDK2
was then induced with IPTG (0.4 mM) at room temperature for
18 h, whereas expression of cyclin A-His6 was induced
with IPTG (0.2 mM) at room temperature for 3 h. Cell pellets from 1 liter of each culture were separately resuspended in 10 ml of lysis buffer (20 mM MOPS, pH 7.4 50 mM
NaCl, 1 mM EDTA), mixed, and colysed in the presence of 1 mM dithiothreitol, 1 µg/ml each of leupeptin, pepstatin,
aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 100 mM MgCl2, 1 mM benzamidine, and 0.2 mg/ml lysozyme for 20 min on ice, sonicated briefly (five 20-s
bursts), and then centrifuged at 15,000 rpm (Sorvall SS-34) for
30 min. The supernatant was batch-incubated with 0.5 ml
glutathione-agarose (Sigma)/liter of GST-CDK2 culture for 1 h at 4 °C. The resin was washed with buffer A (20 mM
MOPS, 0.1 EDTA, 25 mM NaCl, 1 mM
dithiothreitol, pH 7.4), and bound protein was eluted in a column using
buffer A containing 15 mM glutathione (Sigma). The eluted
protein (2-5 mg/ml) was cleaved with thrombin (Sigma, 100 units/ml) in
a total volume of 2 ml at room temperature overnight. The cleaved
material was digested with Dnase I (Sigma, 0.2 µg/ml) for 15 min at
room temperature and then loaded onto a 1-ml Uno Q column (Bio-Rad Biologics). Protein was eluted by a gradient of increasing ionic strength (0.02-0.3 M NaCl/40 ml). The elution profile was
monitored by absorbance at A280, peak fractions
were analyzed by SDS-PAGE, and those containing protein subunits
corresponding to CDK2 and cyclin A were pooled, dialyzed against buffer
A, and chromatographed again on Uno Q anion exchange. Fractions
corresponding to the major peak at A280 were
analyzed for purity by SDS-PAGE followed by assays for ATPase activity
and kinase activity toward Peptide 1.
CAK1--
The cDNA clone encoding the Saccharomyces
cerevisae CAK1 protein fused to an N-terminal hexahistidine tag
was kindly provided by D. Morgan (University of California, San
Francisco). CAK1 was subcloned into pGEX-2T, and overexpression
of GST-CAK1 protein was carried out in E. coli BL21 (DE3)
according to Kaldis et al. (10). Ten liters of bacterial
culture were grown to an A600 of 0.6 and induced
with IPTG (0.4 mM) at room temperature for 20 h, the
cells were harvested, and an extract was prepared as described for
(non-p)CDK2/cyclin A with the exception that MgCl2 was
present at 5 mM. GST-CAK1 was enriched by
glutathione-agarose chromatography (0.5 ml packed resin/liter of
culture) as described for (non-p)CDK2/cyclin A.
[T160-P]CDK2/Cyclin A--
GST-CDK2 and cyclin
A-His6 were separately purified. GST-CDK2 was purified by
glutathione-agarose chromatography and subjected to thrombin cleavage.
Cyclin A-His6 was purified by chromatography over
Ni2+-NTA-resin according to Brown et al. (9)
except that 100 mM MgCl2 was included during
lysis as well as throughout purification. Cdk2 (0.8 mg) was
phosphorylated at Thr160 by incubation with GST-CAK1, ATP
(1 mM), and MgCl2 (10 mM) overnight at room temperature based on a protocol used by Brown et al.
(11). Pyruvate kinase (7.5 units/ml) and phosphoenolpyruvate (1 mM) were included to remove product ADP and to regenerate
ATP. After phosphorylation, [p-Thr160]CDK2 was
combined with cyclin A (4 mg), incubated on ice for 30 min, purified on
Superose 12, and then twice subjected to chromatography over Uno Q as
described for (non-p)CDK2/cyclin A. From 0.8 mg of CDK2, ~100 µg of
highly purified [p-Thr160]CDK2/cyclin A was
obtained. [p-Thr160]CDK2/cyclin A expressed in Sf9
cells was purified according to Russo (12).
Activation of (non-p)CDK2/Cyclin A--
(non-p)CDK2/cyclin A was
tested for its ability to undergo activation by CAK. (non-p)CDK2/cyclin
A (0.5 µM) was incubated with a concentrated extract of
glutathione-agarose-enriched GST-CAK1 in the presence of 1 mM ATP, 10 mM MgCl2, 20 mM MOPS, 1 mM phosphoenolpyruvate, 7.5 units/ml
pyruvate kinase, 50 mM KCl, 1 mM
dithiothreitol, and 0.1 mM EDTA, pH 7.4, (100 µl
total volume) for 14 h at room temperature. Ten µl of the
reaction mixture was then diluted into 90 µl of buffer containing 1 mM ATP and coupling reagents (15 units/ml lactate
dehydrogenase, 7.5 units/ml pyruvate kinase, 1 mM
phosphoenolpyruvate, and 130 µM NADH), and peptide
kinase activity was measured after the addition of 100 µM
Peptide 1 in a coupled spectrophotometric assay (see below). The
kinase activity was taken as the change in absorbance at
A340 after correction for ATPase activity, which
was determined by measurement in the absence of peptide.
Kinetic Assays and Data Analysis--
The phosphorylation of
Peptide 1 was monitored by a radioisotope assay in which the direct
incorporation of 32P from [
Steady-state kinetic parameters for the phosphorylation of Peptide 1 were determined from initial velocity data obtained from a matrix of
several fixed ATP concentrations at varied peptide substrate
concentrations. Equation 1 was globally fit to the data using
the program Scientist (Micromath Inc.).
ATPase activity was monitored using a coupled spectrophotometric assay
in which the regeneration of ATP from ADP and phosphoenolpyruvate catalyzed by pyruvate kinase is coupled to the reduction of pyruvate by
NADH to form lactate and NAD+, the latter of which is
catalyzed by lactate dehydrogenase (13). The concentrations of the
coupling reagents were as follows: 15 units/ml lactate dehydrogenase,
7.5 units/ml pyruvate kinase, 1 mM
phosphoenolpyruvate, and 130 µM NADH. Reactions were
performed in phosphorylation buffer with coupling reagents in a total
volume of 100 µl at 23 °C. Progress of the reaction over time was
monitored as a linear decrease in absorbance at 340 nm in a Shimadzu
UV1601 spectrophotometer. The micromolar change in product
concentration was calculated based on an extinction coefficient for
NADH of 6220 cm Solvent Viscosity Studies--
Steady-state assays were carried
out as described above in buffer containing varied sucrose or fructose.
Relative solvent viscosity ( Purification and Steady-state Kinetic
Analysis--
[p-Thr160]CDK2/cyclin A was produced by
overexpression in either E. coli or Sf9 insect cells.
Steady-state kinetic parameters for the phosphorylation of a peptide
substrate displaying the canonical recognition motif
XT/SPXK/R (Peptide 1; PKTPKKAKKL) were
determined by Michaelis-Menten analysis using a two-substrate model for
sequential binding (see "Materials and Methods"). The data are
shown in Fig. 1 in double reciprocal
form. The regression yields a turnover number
(kcat) of 7 ± 0.7 s
(non-p)CDK2/cyclin A was expressed and purified from E. coli. SDS-PAGE analysis of fractions corresponding to the major
protein peak eluting from the final chromatographic step is shown in
Fig. 2. Each fraction was assayed for
ATPase activity, which was found to correspond exactly to the profile
of CDK2/cyclin A protein. However, analysis of the same fractions for
peptide kinase activity revealed that, while the early fractions of the
protein peak displayed low activity, the later fractions exhibited
substantially higher kinase activity (Fig. 2, B and
C). The basis for the high kinase activity in these later
fractions is not known. For all studies on [un-P]CDK2/cyclin A,
fractions free of the contaminating high kinase activity species were
used (fractions 31-34, Fig. 2). In these fractions, peptide
kinase activity corresponded exactly to the levels of CDK2/cyclin A
protein and steady-state kinetic parameters, measured for both fraction
31 and fractions 31-34 pooled, were identical (not shown).
(non-p)CDK2/cyclin A in these fractions displayed low but measurable
kinase activity. The kinase activity of (non-p)CDK2/cyclin A] was
linear for up to 2 h, demonstrating that autoactivation does not
occur within this time frame. To test the possibility that the low
level of activity may be attributable to partial denaturation,
(non-p)CDK2/cyclin A was tested for activation by phosphorylation (see
"Materials and Methods"). Incubation of (non-p)CDK2/cyclin A with
MgATP and CAK resulted in a turnover rate (~9
s
It was found that the maximal ATPase rate (10 min Solvent Viscosity Studies--
The Michaelis-Menten parameters
described above are composed of microscopic rate constants combined in
a manner dependent upon the order of substrate addition. The
steady-state data for both [p-Thr160]CDK2/cyclin A and
(non-p)CDK2/cyclin A are consistent with both random and compulsorily
ordered mechanisms. For both enzymes, however, if the kinetic mechanism
is ordered, it is necessarily ordered with ATP binding first; this is
true because the crystal structures of both
[p-Thr160]CDK2/cyclin A and (non-p)CDK2/cyclin A were
obtained with bound ATP alone. Furthermore, both enzymes display
substantial ATPase activity in the absence of peptide or protein substrate.
Under saturating conditions of ATP, the catalytic mechanism of
CDK2/cyclin A can therefore be described by Scheme
1. In this model, the catalytic
efficiency is given by
(kcat/Km(peptide) = k2 × k3/(k
A similar solvent viscosometric analysis was conducted on
(non-p)CDK2/cyclin A. Although saturation with Peptide 1 could not be
achieved because of the high Km value for this
substrate, subsaturating substrate concentrations equal to 0.36, 1.8, and 3.6 times the Km(peptide) revealed
no viscosity effect on initial rates (Fig. 4B).
Extrapolation to zero and infinite peptide substrate concentrations
showed that neither the catalytic efficiency nor turnover rate,
respectively, was sensitive to solvent viscosity. The release rates for
both substrate peptide (k The majority of protein kinases require phosphorylation at a
conserved site within their activation loops for full catalytic activation (3). This site in CDK2/cyclin A is Thr160. Its
phosphorylation by CAK is of interest for at least two reasons: 1) it
has been established that phosphorylation at this site is a critical
regulatory mechanism for control of CDK2/cyclin A activity and
progression through the cell cycle (5); 2) the structural basis for
activation of CDK2 by both cyclin binding and phosphorylation is known
(6-8), and the structure of [p-Thr160]CDK2/cyclin A
bound to a synthetic peptide substrate has been solved (18). Given the
abundant structural data, the consequences of phosphorylation at
Thr160 on the specific steps for substrate binding and
catalytic turnover have not been investigated. Thus, a correlation
between atomic structure and the mechanism of regulation is not known.
In this study, we have characterized the kinetic reaction pathway for the phosphorylation of a model peptide substrate by unphosphorylated CDK2/cyclin A as well as CDK2/cyclin A phosphorylated at
Thr160 by CAK. The information reveals the kinetic basis
for activation by phosphorylation in terms of the individual reaction steps.
We have employed a synthetic peptide (Peptide 1) as a model substrate
in which the primary structure (PKTPKKAKKL) is patterned after a region
of similar sequence found in histone H1 protein (19). The catalytic
efficiency of Peptide 1 for [p-Thr160]CDK2/cyclin A is
one of the highest reported for protein kinases (kcat/Km The dramatic increase in catalytic efficiency is in part attributable
to a moderate but significant increase in peptide binding affinity. The
crystal structure of [p-Thr160]CDK2/cyclin A bound to
AMPPNP and a peptide substrate (HHASPRK) (18) reveals the structural
basis for interaction with peptide and protein substrates.
Phosphorylation of CDK2/cyclin A at Thr160 results in
displacement of the activation loop by 5.3-7.1 Å and rotation of the
carbonyl oxygen of Val163 out of the so called
"P+1 binding pocket" (7, 8). This movement is necessary
to accommodate binding of the essential proline residue found in the
P+1 position (P0 is the phosphorylation site)
in all CDK2/cyclin substrates (18). In addition, the substrate lysine
residue at position P+3 forms a hydrogen bond with the
phosphate group in [p-Thr160]CDK2/cyclin A (18). Overall,
phosphorylation of CDK2/cyclin A at Thr160 increases the
binding affinity of Peptide 1 by ~50-fold.
Nonetheless, the large increase in both catalytic efficiency and
turnover rate with respect to the kinase pathway is attributable mostly
to an approximate 3000-fold increase in the rate of the phosphoryl
group transfer step. In contrast, phosphorylation has no effect on the
rate of ATP hydrolysis. Solvent viscosometric studies on the ATPase
reaction of both [p-Thr160]CDK2/cyclin A and
(non-p)CDK2/cyclin A show that phosphorylation affects neither the
overall turnover rate nor the rate of the phosphoryl group transfer
step (Table I).
The large rate enhancement of chemical reactions afforded by enzymes is
classically accounted for by specific stabilization of the transition
state with respect to the ground state Michaelis complex (20). Thus,
the 3000-fold increase in the rate of phosphoryl group transfer may be
attributed to optimization of the alignment of either ATP or peptide,
or both, in response to phosphorylation. Phosphorylation does not,
however, optimize the alignment of ATP for phosphotransfer in the
ATPase reaction, because no rate enhancement of this reaction step was
observed; this is consistent with the observation that phosphorylation
does not affect the positioning of the invariant Lys33,
Glu51, and Asp145 catalytic triad, which serves
to stabilize the phosphates of ATP. Instead, the alignment of these
residues is achieved by cyclin binding (2).
These results raise the possibility that phosphorylation may serve
specifically to align the peptide or protein portion of the
transition-state structure for phosphotransfer in the kinase reaction.
The dramatically slower rate of phosphotransfer to peptide in
(non-p)CDK2/cyclin A compared with [p-Thr160]CDK2/cyclin
A may be explained by an unfavorable orientation of the peptide
substrate hydroxyl group as a consequence of the steric interference
from the carbonyl oxygen of Val163, which is relieved by
phosphorylation at Thr160. We are currently conducting
studies to test our hypothesis that the alignment of ATP
versus that of the peptide/protein substrate may be
separately controlled by cyclin binding versus
phosphorylation at Thr160, respectively.
Substrate turnover by [p-Thr160]CDK2/cyclin A is
partially (65%) limited by product release (k4 = 11 s Studies addressing the role of autophosphorylation in the activation of
cAMP-dependent protein kinase (PKA) have been carried out
employing a nonphosphorylatable mutant as a model of the
unphosphorylated enzyme (21). Although the site of phosphorylation in
PKA (Thr197) is homologous to residue Thr160 in
CDK2/cyclin A, clear differences exist between these enzymes in their
kinetic mechanisms of activation. In particular, phosphorylated wild-type PKA, in comparison with PKA(T197A), displays no change in
substrate binding affinity, a small increase in the overall rate of
substrate turnover (<10-fold), and a moderate increase in the rate of
the phosphoryl group transfer step (<200-fold) (21) (cf.
Table I). The differences in activation of PKA versus CDK2/cyclin A may relate to their different physiological mechanisms of
regulation. For example, reversible phosphorylation of the CDKs by CAK
is critical to their function in cell cycle control (5), whereas the
role of autophosphorylation in the regulation of PKA remains unclear
(22, 23). Instead, the regulation of PKA is achieved mainly by
association with either the RI or RII regulatory subunits. Comparison
of CDK2/cyclin A with PKA (21) and the v-fps tyrosine kinase
(24) does, however, reveal an apparent common theme, that
phosphorylation of the activation loop in all cases serves mainly to
enhance the rate of phosphotransfer with less influence on the affinity
of substrate binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheet and a larger C-terminal lobe that is
principally
-helix. The bilobal interface constitutes the active
site cleft into which the adenine ring of substrate ATP is deeply
buried. The ATP
-phosphate is directed toward the mouth of the
active site where peptide and protein substrates bind and where
phosphoryl group transfer occurs (for a review see Ref. 2). Located
near the mouth of the active site is a conserved loop structure termed
the activation loop (residues 146-166). This loop structure is present
in all protein kinases (3), and phosphorylation at a conserved site
within the activation loop is necessary for full activation of most
protein kinases. In CDK2, this site is Thr160,
phosphorylation of which is catalyzed by a heterologous kinase, CAK
(cdk-activating kinase) (4, 5).
- and
-phosphates of ATP, and Asp145, which chelates an
essential Mg2+ ion also serving to stabilize ATP.
Furthermore, the active site cavity of CDK2 is exposed upon cyclin
binding by repositioning of the activation loop by over 15 Å(6, 7).
Subsequent phosphorylation of the CDK2/cyclin A complex at
Thr160 (in CDK2) is associated with less dramatic changes
in structure that are localized primarily to the activation loop (8).
Nonetheless, the latter modification is associated with a dramatic
increase in catalytic power.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP into
the peptide was measured. Reactions were carried out in phosphorylation
buffer (20 mM MOPS, 50 mM KCl, 10 mM MgCl2(free),1 mM
dithiothreitol, 0.1 mM EDTA, pH 7.4) containing
enzyme and varying amounts of one substrate with the other held fixed.
Assays on [p-Thr160]CDK2/cyclin A contained 0.5 mg/ml
bovine serum albumin. In the case of (non-p)CDK2/cyclin A, an ATP
regeneration system consisting of phosphoenolpyruvate (1 mM) and pyruvate kinase (7.5 units/ml) was employed. The
decay in [
-32P]ATP specific radioactivity over time
follows first-order kinetics with rate constant k = [rate of ADP
formation]/[ATP].2 It was
assumed that the rate of ADP formation was effectively equal to the
rate of ATP hydrolysis and that ADP generated from the kinase reaction
was insignificant. The rate of ADP formation at a given concentration
of ATP and enzyme was therefore calculated by solving for the
Michaelis-Menten equation using Km and
kcat values of 90 µM and 10 min
1, respectively, for the ATPase reaction,
which were determined in a spectrophotometric assay (see below; data
not shown). The calculated rate constant corresponding to each ATP
concentration was used to simulate a decay profile using the equation,
y = yo × e
kt,
where y is the ATP-specific activity at any time,
t, yo is the original ATP-specific
activity, and k is the rate constant for decay, as defined
above. The average ATP-specific radioactivity at any time,
t, was obtained by determining the ATP-specific activity time integral for the entire reaction time course, dividing by the
total time. Calculations were performed using the program Scientist (Micromath Inc., Salt Lake City, UT). Reactions were initiated by the addition of [
-32P]ATP (300-500
cpm/pmol) and allowed to proceed at 23 °C for 6 to 30 min, at which
time reactions were terminated with 25% acetic acid. Reaction samples
(20 µl) were spotted and subjected to ascending chromatography on
phosphocellulose paper (Whatman P81) in 20 mM H3PO4, pH 2. In this system, ATP and
Pi migrate with the solvent front; the phosphorylated
peptide product remains at the origin. Papers were dried, and
radioactivity corresponding to the phosphopeptide spots were excised
and quantified by Cerenkov counting.
where v is the initial velocity, V is the
maximal initial velocity, A and B are the
concentrations of 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)
1
M
1 at 340 nm. Steady-state
kinetic parameters were determined by nonlinear least-squares analysis
using the Michaelis-Menten equation, which was fit to the velocity data.
rel) was determined from the
following equation:
rel = t/to ·
/
o, where
rel is the solvent viscosity relative to buffer
containing no viscosogen, t is the solvent transit time
measured by an Ostwald capillary viscometer, and
is the solvent
specific gravity. The superscript "o" denotes the absence of viscosogen.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 and Km values for ATP
and peptide equal to 55 ± 5 µM and 8 ± 0.7 µM, respectively. The optimized steady-state parameter values yield a catalytic efficiency for peptide that is among the
highest reported for protein kinases
(kcat/Km(peptide) = 0.9 µM
1
s
1). The kinetic properties of
[p-Thr160]CDK2/cyclin A produced in Sf9 cells (12)
were also determined and were found to be similar
(kcat = 4.5 ± 1 s
1,
kcat/Km(peptide) = 0.4 µM
1
s
1).
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Fig. 1.
Steady-state kinetic analysis of
phosphorylation by [p-Thr160]CDK2/cyclin A. Initial
velocities for the phosphorylation of Peptide 1 were determined as
described under "Materials and Methods." A model describing
two-substrate sequential binding was fit to the untransformed data
using global nonlinear regression analysis. The experimental data and
the fit were then transformed to the double reciprocal form for
display. The concentration of enzyme was 2 nM. ATP
concentrations were fixed at 20, 50, 100, 200, and 500 µM
(from top to bottom). Kinetic parameters are
reported in Table I.
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Fig. 2.
Anion exchange chromatography of
(non-p)CDK2/cyclin A. (non-p)CDK2/cyclin A was produce by
overexpression in E. coli and purified as described under
"Materials and Methods." A, A280
of eluted protein and buffer conductivity from Uno Q anion exchange
chromatography are shown. B, SDS-PAGE analysis of
protein-containing fractions. (non-p)CDK2 and cyclin A migrate with the
same relative mobility. Molecular mass markers are 97, 66, 55, 42/40, 31, 21.5, and 14.4 kDa from top to bottom.
C, peptide kinase activity. Five µl of each fraction was
assayed in the presence of 100 µM peptide and 1 mM [ -32P]ATP (~300 dpm/pmol) for 10 min
at 23 °C, as described under "Materials and Methods." Although
two peaks of CDK2/cyclin A are observed in panel A, the
steady-state kinetic parameters measured for both fraction 31 and the
pool of fractions 31-34 were identical (not shown). The pool was thus
used for all studies.
1) corresponding to that of
[p-Thr160]CDK2/cyclin A obtained after extensive
purification (data not shown). These data indicate that all of the
(non-p)CDK2/cyclin A was activable and was therefore native. In
addition, the maximal ATPase rates of (non-p)CDK2/cyclin A produced in
E. coli (10 min
1) compared with
insect cells (~14 min
1) were similar, as
were their peptide kinase activities.
1) was ~20-fold higher than the maximal
rate of the peptide kinase reaction (0.5 min
1). This finding presented two problems
for kinetic analysis. First, (non-p)CDK2/cyclin A binds ADP with a
Kd value of ~1 µM (14). Thus, under
initial velocity conditions with respect to the kinase reaction,
significant product inhibition would be expected to occur. Second, the
fraction of ATP depleted at low initial ATP concentrations would be
large even at small fractions of peptide phosphorylation, precluding
kinetic measurement under initial velocity conditions. To circumvent
these difficulties, an ATP-regenerating system was employed. In this
system, ADP is efficiently removed from the reaction mix and the
concentration of ATP remains constant. However, under these conditions,
the [
-32P]ATP-specific radioactivity decays over the
reaction time course as ATP is regenerated. This decay was accounted
for in all analyses of peptide phosphorylation (see "Materials and
Methods"). The corrected initial velocity data for the
phosphorylation of Peptide 1 by (non-p)CDK2/cyclin A are shown in Fig.
3, and the optimized steady-state kinetic
parameter values are reported in Table I. The turnover rate for (non-P)CDK2/cyclin A is 843-fold lower than that
of [p-Thr160]CDK2/cyclin A, whereas the
Km(peptide) is 137-fold higher. Thus,
phosphorylation of CDK2/cyclin A results in an ~100,000-fold increase
in catalytic efficiency.
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Fig. 3.
Steady-state kinetic analysis of
phosphorylation by (non-p)CDK2/cyclin A. Initial velocities for
the phosphorylation of Peptide 1 were determined as described under
"Materials and Methods," and kinetic parameters were obtained as
described in the legend to Fig. 1. The concentration of enzyme was 1 µM. Peptide concentrations were fixed at 0.5, 1, 2, 4, and 6 mM (from top to bottom), and
ATP was varied. Kinetic parameters are reported in Table I.
Kinetic and thermodynamic constants for (non-p)CDK2/cyclin A and
[p-Thr160]CDK2/cyclin A
2 + k3)], while the turnover rate is given by
[kcat = k3 × k4/(k3 + k4)]. 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
non-diffusive steps (k3) (15-17). Initial
velocity data for peptide phosphorylation by
[p-Thr160]CDK2/cyclin A were obtained as a function of
peptide substrate concentration at several concentrations of sucrose
(Fig. 4A). At these sucrose
concentrations, the maximum
rate3 of ATP hydrolysis (12 min
1) in the absence of peptide was constant,
indicating that sucrose does not perturb the structure of the active
site. Equation 2,
(Eq. 2)
which describes the effect of relative solvent viscosity on
initial rate, was fit to the data, where E is the
concentration of enzyme, S is the concentration of Peptide
1,
is the relative solvent viscosity, and Kd is
the equilibrium dissociation constant for Peptide 1 binding to the
E·ATP complex
(k
2/k2). All other
constants are defined in Scheme 1. The derived values for the kinetic
constants are reported in Table I. Solvent viscosity studies performed
on [p-Thr160]CDK2/cyclin A produced in insect cells
revealed similar kinetic parameters
(Kd(peptide) = 20 µM,
k3 = 8 s
1,
k4 = 11 s
1).
View larger version (4K):
[in a new window]
Scheme 1.
View larger version (17K):
[in a new window]
Fig. 4.
Effect of relative solvent viscosity on
Michaelis-Menten parameters. A,
[p-Thr160]CDK2/cyclin A. Equation 2,
corresponding to Scheme 1, was fit to a matrix of initial velocity data
obtained with varied peptide substrate concentrations at several fixed
concentrations of sucrose (giving relative solvent viscosities of 1, 2, 2.5, and 4.2, from top to bottom). The optimized
microscopic rate constants are given in Table I. The concentration of
ATP was fixed at 1 mM. Identical results were obtained at 2 mM ATP. The concentration of enzyme was 2 nM.
B, (non-p)CDK2/cyclin A. Relative changes in initial rates
were determined as a function of increasing sucrose concentration at
three different subsaturating concentrations of peptide substrate (0.4 mM, solid line, open circles; 2 mM,
long dashes, open squares; 4 mM,
short dashes, open diamonds). The concentration
of ATP was 1 mM. Identical results were obtained at 2 mM ATP. The concentration of enzyme was 1 µM.
The "viscosity effect" on the reaction rate
(kobs ) is defined as the slope of
the line at a given substrate concentration and can vary
between the theoretical limits of 0 and 1. The viscosity effects on
catalytic efficiency
(kcat/Km
) and
turnover rate (kcat
) are obtained
by extrapolation to zero and infinite substrate concentration,
respectively. kcat/Km
and
kcat
relate to the individual
rate constants in Scheme 1 as follows:
kcat/Km
= k3/(k
2 + k3); kcat
= k3/(k3 + k4)(25). A value for
kcat/Km
and
kcat
equal to zero implies that
k
2
k3 and
k3
k4,
respectively.
2) and product
(k4) can therefore be assumed to exceed the rate
of chemistry (k3) by at least 10-fold (Table I).
Thus, phosphoryl group transfer in (non-p)CDK2/cyclin A is rate
determining. A viscosometric analysis was also carried out on the
ATPase reaction. No viscosity effect on the turnover rate was observed,
suggesting that phosphoryl group transfer in the ATPase reaction is
also rate determining (Table I). The lack of a viscosity effect on the
steady-state kinetic parameters for both the kinase and ATPase reactions indicates that sucrose perturbs only the rates of diffusion.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 µM
1
s
1) and is similar to that of histone
H1 protein (not shown). The lack of a viscosity effect on
kcat/Km(peptide)
for both [un-P]CDK2/cyclin A and [p-Thr160]CDK2/cyclin
A demonstrates that during steady-state turnover Peptide 1 equilibrates
rapidly with the active site of both enzymes. Thus, catalytic
efficiency for the kinase activity in both cases is a function of only
the affinity of peptide binding and the rate of phosphoryl group
transfer (kcat/Km
k3/Kd). Phosphorylation at
Thr160 increases catalytic efficiency by ~100,000-fold,
while the turnover rate is enhanced nearly 1000-fold.
1). However, our studies do not discern
whether the slow step in this event is the release of ADP or
phosphopeptide. If the dissociation rate of the phosphopeptide product
is similar to, or greater than, that of the peptide substrate
(k
2
220 s
1), then
it is the release of ADP that is slow. In addition, we cannot assess
the effect that phosphorylation at Thr160 has on the
product dissociation rates, as the lack of a viscosity effect on
kcat in (non-p)CDK2/cyclin A permits only a
lower limit value on the net rate of product release to be estimated
(k4
0.083 s
1).
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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.
§ These authors made equal contributions to this work.
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 11, 2000, DOI 10.1074/jbc.M007337200
2
dATP32/dt = V · ATP32/(ATP32 + Km · (1 + ATP/Km)) = k · ATP32 and
dATP/dt = V · ATP/(ATP + Km · (1 + ATP32/Km)) = k
· ATP, where k
V/(ATP + Km) and therefore: k = (
dATP/dt)/ATP = (dADP/dt)/ATP.
3 No change in rate was observed at 1 versus 2 mM ATP.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CDK, cyclin-dependent kinase;
(non-P)CDK2/cyclin A, unphosphorylated CDK2/cyclin A;
[p-Thr160]CDK2/cyclin A, CDK2/cyclin A phosphorylated at Thr160 in CDK2;
PKA, cAMP-dependent protein kinase;
IPTG, isopropyl-1-thio--D-galactopyranoside;
MOPS, 4-morpholinepropanesulfonic acid;
PAGE, polyacrylamide gel
electrophoresis;
CAK, CDK-activating kinase;
GST, glutathione
S-transferase.
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REFERENCES |
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1. | Morgan, D. O. (1997) Annu. Rev. Cell Dev. Biol. 13, 261-291[CrossRef][Medline] [Order article via Infotrieve] |
2. | Pavletich, N. P. (1999) J. Mol. Biol. 287, 821-828[CrossRef][Medline] [Order article via Infotrieve] |
3. | Johnson, L. N., Noble, M. E., and Owen, D. J. (1996) Cell 85, 149-158[Medline] [Order article via Infotrieve] |
4. | Kaldis, P. (1999) Cell. Mol. Life Sci. 55, 284-296[CrossRef][Medline] [Order article via Infotrieve] |
5. | Morgan, D. O. (1995) Nature 374, 131-134[CrossRef][Medline] [Order article via Infotrieve] |
6. | De Bondt, H. L., Rosenblatt, J., Jancarik, J., Jones, H. D., Morgan, D. O., and Kim, S. H. (1993) Nature 363, 595-602[CrossRef][Medline] [Order article via Infotrieve] |
7. | Jeffrey, P. D., Russo, A. A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, N. P. (1995) Nature 376, 313-320[CrossRef][Medline] [Order article via Infotrieve] |
8. | Russo, A. A., Jeffrey, P. D., and Pavletich, N. P. (1996) Nat. Struct. Biol. 3, 696-700[Medline] [Order article via Infotrieve] |
9. | Brown, N. R., Noble, M. E., Endicott, J. A., Garman, E. F., Wakatsuki, S., Mitchell, E., Rasmussen, B., Hunt, T., and Johnson, L. N. (1995) Structure 3, 1235-1247[Medline] [Order article via Infotrieve] |
10. | Kaldis, P., Sutton, A., and Solomon, M. J. (1996) Cell 86, 553-564[Medline] [Order article via Infotrieve] |
11. |
Brown, N. R.,
Noble, M. E.,
Lawrie, A. M.,
Morris, M. C.,
Tunnah, P.,
Divita, G.,
Johnson, L. N.,
and Endicott, J. A.
(1999)
J. Biol. Chem.
274,
8746-8756 |
12. | Russo, A. A. (1997) Methods Enzymol. 283, 3-12[CrossRef][Medline] [Order article via Infotrieve] |
13. | Cook, P. F., Neville, M. E., Jr., Vrana, K. E., Hartl, F. T., and Roskoski, R., Jr. (1982) Biochemistry 21, 5794-5799[Medline] [Order article via Infotrieve] |
14. | Heitz, F., Morris, M. C., Fesquet, D., Cavadore, J. C., Doree, M., and Divita, G. (1997) Biochemistry 36, 4995-5003[CrossRef][Medline] [Order article via Infotrieve] |
15. | Brouwer, A. C., and Kirsch, J. F. (1982) Biochemistry 21, 1302-1307[Medline] [Order article via Infotrieve] |
16. | Blacklow, S. C., Raines, R. T., Lim, W. A., Zamore, P. D., and Knowles, J. R. (1988) Biochemistry 27, 683-733 |
17. | Adams, J. A., and Taylor, S. S. (1992) Biochemistry 31, 8516-8522[Medline] [Order article via Infotrieve] |
18. | Brown, N. R., Noble, M. E. M., Endicott, J. A., and Johnson, L. N. (1999) Nat. Cell Biol. 1, 438-443[CrossRef][Medline] [Order article via Infotrieve] |
19. | Felix, M. A., Labbe, J. C., Doree, M., Hunt, T., and Karsenti, E. (1990) Nature 346, 379-382[CrossRef][Medline] [Order article via Infotrieve] |
20. | Hackney, D. D. (1990) The Enzymes , Vol. 19 , pp. 1-36, Academic Press, New York |
21. | Adams, J. A., McGlone, M. L., Gibson, R., and Taylor, S. S. (1995) Biochemistry 34, 2447-2454[Medline] [Order article via Infotrieve] |
22. |
Cauthron, R. D.,
Carter, K. B.,
Liauw, S.,
and Steinberg, R. A.
(1998)
Mol. Cell. Biol.
18,
1416-1423 |
23. |
Cheng, X.,
Ma, Y.,
Moore, M.,
Hemmings, B. A.,
and Taylor, S. S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9849-9854 |
24. | Saylor, P., Hanna, E., and Adams, J. A. (1998) Biochemistry 37, 17875-17881[CrossRef][Medline] [Order article via Infotrieve] |
25. | Nakatani, H., and Dunford, H. B. (1979) J. Am. Chem. Soc. 83, 2662-2665 |