From the Laboratory of Molecular Biophysics,
Department of Biochemistry, and Oxford Centre for Molecular Sciences,
University of Oxford, The Rex Richards Building, South Parks Road,
Oxford OX1 3QU, United Kingdom and § Centre de Recherche de
Biochemie Macromoleculaire, CNRS, Montpellier, 1919 Route de Mende,
34293 Montpellier Cedex, France
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ABSTRACT |
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We have prepared phosphorylated
cyclin-dependent protein kinase 2 (CDK2) for
crystallization using the CDK-activating kinase 1 (CAK1) from
Saccharomyces cerevisiae and have grown crystals using
microseeding techniques. Phosphorylation of monomeric human CDK2 by
CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A
complex. Phosphorylated CDK2 exhibits histone H1 kinase activity
corresponding to approximately 0.3% of that observed with the fully
activated phosphorylated CDK2-cyclin A complex. Fluorescence
measurements have shown that Thr160 phosphorylation
increases the affinity of CDK2 for both histone substrate and ATP and
decreases its affinity for ADP. By contrast, phosphorylation of CDK2
has a negligible effect on the affinity for cyclin A. The crystal
structures of the ATP-bound forms of phosphorylated CDK2 and
unphosphorylated CDK2 have been solved at 2.1-Å resolution. The
structures are similar, with the major difference occurring in the
activation segment, which is disordered in phosphorylated CDK2. The
greater mobility of the activation segment in phosphorylated CDK2 and
the absence of spontaneous crystallization suggest that phosphorylated
CDK2 may adopt several different mobile states. The majority of
these states are likely to correspond to inactive conformations, but a
small fraction of phosphorylated CDK2 may be in an active conformation
and hence explain the basal activity observed.
Progression through the eukaryotic cell cycle is critically
dependent upon the activity of a family of serine/threonine kinases, the cyclin-dependent protein kinases
(CDKs).1 CDKs are regulated
by both transcriptional and post-translational mechanisms that promote
the correct timing and order of events for cell growth and cell
division (1). Monomeric, nonphosphorylated CDKs have no detectable
kinase activity. Activation requires two components: (i) the
association of a positive regulatory cyclin subunit (2, 3) and (ii)
activating phosphorylation of the kinase on a threonine residue
(Thr160 in CDK2) located in a surface loop termed the
activation segment (4-8).
In metazoans, a CDK-cyclin pair, CDK7-cyclin H, that requires a third
protein, MAT-1, exhibits CDK-activating kinase (CAK) activity (9-16).
CDK7 and cyclin H are also components of the multimeric basal
transcription factor complex, TFIIH (17, 18). In Saccharomyces
cerevisiae, CAK activity is associated with a monomeric protein
called CAK1 or CIV1 (CAK
in
vivo) (19-21). CAK1 shows greatest
sequence similarity to the CDK family but does not have a cyclin
partner. Dephosphorylation of the threonine in the activation segment
is catalyzed by the kinase-associated phosphatase (KAP) (22, 23). For
CDK2, KAP will only accept monomeric phosphorylated CDK2 as a substrate
and not the binary complex of phosphorylated CDK2-cyclin A, suggesting
that in vivo KAP dephosphorylates Thr160 only
after the cyclin subunit is dissociated or degraded. Cyclin proteolysis
is mediated by the ubiquitin-dependent pathway (24-27). CDK-cyclin heterodimers may also be regulated by reversible
phosphorylation at other sites leading to inhibition (reviewed in Ref.
1) and by association with protein inhibitor molecules (CKIs) such as p27kip1 (reviewed in Ref. 28).
Several groups have addressed the question of whether the monomeric CDK
or the CDK-cyclin complex is the better substrate for phosphorylation
by CAK. The results suggest both a CDK and species dependence. Studies
using CAK purified from starfish oocytes (9) concluded that this enzyme
can only phosphorylate CDC2 (CDK1) when it is bound to a cyclin.
However, CAK purified from Xenopus laevis egg extract can
phosphorylate bacterially expressed human CDK2 (10) or GST-CDK2 (11) in
the absence of cyclin. Fisher and Morgan working with CAK purified from
both human and murine cells have shown that CAK activates complexes of
CDK2 and CDC2 with various cyclins and also phosphorylates CDK2, but
not CDC2, in the absence of cyclin (12). Studies with S. cerevisiae CAK1 have indicated that CDK2 and CDC28 phosphorylation
can occur with or without cyclin (19-21).
The requirement for CDKs to be both phosphorylated and associated with
a cyclin subunit for full activation indicates that more significant
changes need to occur than those promoted by phosphorylation alone.
Other kinases such as cAMP-dependent protein kinase (29,
30), lymphocyte kinase, Lck (31), insulin receptor tyrosine kinase
(32), and mitogen-activated protein kinase (33) are activated solely by
phosphorylation without the need for association with another protein
subunit. Kinase activity associated with monomeric phosphorylated CDKs
has been reported for CDK7, where dual phosphorylation on the two sites
in the activation segment (Ser170 and Thr176 in
X. laevis CDK7) is sufficient to generate CAK activity to one-third of its maximal value in vitro (34). Monomeric
phosphorylated CDK2 has been reported to be inactive (12).
Crystallographic studies on CDK2 have provided a detailed understanding
of the basis of CDK2 activation by cyclin A binding and phosphorylation
(35-37). CDK2 adopts a characteristic protein kinase fold composed of
a mostly Upon formation of the CDK2-cyclin A complex, there are no changes in
the structure of cyclin A (38), but there are substantial changes in
CDK2 that create the ATP triphosphate recognition site (36).
CDK2-cyclin A exhibits about 0.2% of the activity of the fully
activated phosphorylated binary complex. In the structure of
phosphorylated CDK2-cyclin A, further structural rearrangements occur
centered within the activation segment (37). Thr160, now
phosphorylated, turns in to contact three arginine residues: one
(Arg50) from the C-helix PSTAIRE motif within the
N-terminal domain, a second (Arg126) that is adjacent to
the catalytic aspartate (Asp127), and a third
(Arg150) from the start of the activation segment. The
phosphothreonine group acts as an organizing center, and a major
outcome of the realigned phosphorylated CDK2-cyclin A structure appears
to be the formation of the protein-substrate recognition site that is likely to depend most sensitively on the activation segment orientation.
In order to elucidate the structure and biological properties of
phosphorylated CDK2 in the absence of cyclin, we have prepared phosphorylated CDK2 by the action of CAK1 on CDK2. We have analyzed the
role of cyclin in the recognition of CDK2 by CAK1; the relative affinities of CDK2 and phosphorylated CDK2 for cyclin A, ATP, and
substrate; and the structural consequences of CDK2 phosphorylation. The
crystallographic results demonstrate disorder in the activation segment
of phosphorylated CDK2 compared with this region of nonphosphorylated CDK2. We propose that this disorder can explain the differences observed in ATP and substrate binding between phosphorylated and nonphosphorylated CDK2 and the low level of phosphorylated CDK2 histone
H1 kinase activity.
Protein Expression and Purification
Human CDK2 was expressed in Sf9 insect cells using a
recombinant baculovirus encoding CDK2 (Autographica californica
CDK2) following slight modifications to the published method (39). Briefly, Sf9 cells, adapted to Sf900-II serum-free medium
(Life Technologies, Inc.), were maintained in shake flasks at 28 °C. Cells (2 × 10 9) were infected with A. californica CDK2 at a multiplicity of infection of 10. At 48 h postinfection, the virus-infected cells were harvested and lysed in
hypotonic lysis buffer (25 mM NaCl, 1 mM EDTA,
1 µg/ml pepstatin, 1 µg/ml leupeptin, 0.1 mM
benzamidine, 10 mM Tris-HCl, pH 7.4). After homogenization,
the lysate was clarified by centrifugation (100,000 × g for 1 h at 4 °C) and loaded onto DEAE-Sepharose
(1.6 × 13 cm; Amersham Pharmacia Biotech), preequilibrated in
hypotonic lysis buffer. The NaCl concentration of the flow-through pool
was raised to 50 mM prior to loading onto an SP-Sepharose
column (1.6 × 30 cm, Amersham Pharmacia Biotech). CDK2 eluted in
the flow-through as two distinct peaks that were pooled separately and
then further purified using an ATP-agarose column (11-atom spacer,
ribose hydroxyl-linked, 1 × 5 cm; Sigma) preequilibrated in 25 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 10% glycerol, 10 mM HEPES, pH 7.4. CDK2
was eluted with an increasing NaCl gradient. The two CDK2 populations
eluting from the SP-Sepharose column behaved equivalently on the
ATP-agarose column, and both pools were successfully phosphorylated by CAK1.
Human CDK2 was also prepared as a GST fusion protein using a pGEX-KG
plasmid transformed into Escherichia coli strain JM101. 1-Liter cultures were induced with 0.1 mM
isopropyl-1-thio- A C-terminal fragment (residues 171-432) of bovine cyclin A, cyclin
A3, possessing a hexahistidine tag, was expressed and purified as
described (38).
A pGEX clone containing the CAK1 cDNA insert was transformed into
E. coli strain B834. Typically 1-2 liters of bacterial
culture were grown to an A600 of 0.7 at 37 °C
and then shifted to 16 °C for 2 h before induction with 0.4 mM isopropyl-1-thio- His-tagged CAK1 was overexpressed in S. cerevisiae strain
MD4/4C ( CDK2 phosphorylated on Thr160 was prepared by adding CAK1
activity (either as GST fusion or a histidine-tagged version) at a final CAK1:CDK2 ratio of between 1:20 and 1:2 (w/w) in the presence of
50 mM Tris-HCl, pH 7.5, 10 mM magnesium
chloride, 1 mM ATP for 0.5 h at 25 °C. As reported
by others (19, 20), we also found that CAK1 gave a burst of
phosphorylation followed by a fall in activity within minutes (data not
shown). In order to obtain complete phosphorylation of CDK2, 3-4
aliquots of CAK1 and ATP were added. With GST-CAK1, ratios of 1:2 were
required to achieve complete phosphorylation as judged by band shift on SDS-PAGE. Phosphorylated CDK2 was purified for analysis and
crystallization trials by gel filtration (Superdex 75; Amersham
Pharmacia Biotech), a step that removed high molecular weight
contaminants and glutathione. A glutathione-Sepharose column (Amersham
Pharmacia Biotech) was then used to remove GST-CAK1. With
histidine-tagged CAK1, lower amounts of CAK1 to CDK2 were used (1:20),
and ion exchange (MonoQ; Amersham Pharmacia Biotech) was employed to
resolve phosphorylated CDK2 from unmodified CDK2 and His-tagged CAK1.
The reaction mixture was dialyzed against 25 mM Tris-HCl,
pH 8.5, containing 1 mM EDTA and 1 mM
monothiolglycerol before loading onto the ion exchanger equilibrated in
the same buffer. Pure phosphorylated CDK2, as judged by SDS-PAGE,
eluted at approximately 75 mM salt upon gradient elution.
Binary complexes of CDK2-cyclin A3 and phosphorylated CDK2-cyclin A3
were formed by adding cyclin A3 in slight molar excess to the CDK2
subunit and purified by Superdex 75 gel filtration chromatography.
Human p27kip1 carrying a C-terminal His6 tag was
expressed in E. coli cells (0.1 mM
isopropyl-1-thio- Kinase Assays
Phosphorylation of CDK2 was monitored by gel shift on SDS-PAGE
using 15% acrylamide and also by incorporation of
32P-labeled phosphate. Initially GST-CAK1 was titrated, at
concentrations from 0.75 to 7.5 µM (50-500 ng in 10 µl), against 30 µM (1 µg in 10 µl) monomeric CDK2
as substrate. Kinase reactions were performed in 10 µl of buffer
containing 50 mM Tris-HCl, pH 7.5, 10 mM
magnesium chloride, 1.0 mM ATP for 30 min at 30 °C.
Radioactive analytical kinase reactions were performed using 1.5 µM GST-CAK1 (100 ng in 10 µl) in 50 mM
Tris-HCl, pH 7.5, 10 mM magnesium chloride, 0.1 mM ATP containing 1 µCi of Dephosphorylation of Phosphorylated CDK2 by KAP
15 µM purified phosphorylated CDK2 (0.5 µg in 10 µl) was incubated with 25 µM KAP (0.6 µg in 10 µl)
in a buffer containing 50 mM Tris-HCl, pH 7.5, in the
presence or absence of 1 mM sodium orthovanadate. The
reaction was stopped after 1 h at 25 °C by the addition of SDS
sample buffer. Samples were analyzed by SDS-PAGE (15% acrylamide).
Analysis of CDK/Nucleotide Interactions
2'(3')-O-(N-methylanthraniloyl)
(Mant)-derivated nucleotide analogues were synthesized as described by
Hiratsuka (40) and purified according to John et al. (41).
Cyclin A was overexpressed in E. coli and purified to
homogeneity as described by Lorca et al. (42). Fluorescence
measurements were performed at 25 °C using a Spex II fluorolog
spectrofluorometer, with spectral band passes of 2 and 8 nm, for
excitation and emission, respectively. The intrinsic tryptophan
fluorescence of CDK2 (0.2 µM of protein) was measured in
a fluorescence buffer containing 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5% glycerol, and 2 mM EDTA. Proteins
were incubated for 30 min in fluorescence buffer before starting the
experiments, and all measurements were corrected as already described
(43). The binding of ATP and ADP was monitored by the quenching of the intrinsic tryptophan fluorescence of CDK2 at 340 nm upon excitation at
295 nm. A fixed concentration of CDK2 (0.2 µM) was
titrated by increasing the concentration of ATP or ADP, respectively.
The binding of Mant-nucleotides to CDK2 was monitored by the
enhancement of the Mant-group fluorescence at 450 nm upon excitation at
340 nm (41). Titration curve fitting was accomplished using Grafit software (Erithacus Software Ltd.) using a quadratic equation (44). All
the results correspond to the average of four separate experiments with
an S.D. value lower than 10% for intrinsic and extrinsic fluorescence titrations.
Analysis of CDK/Cyclin Interactions
Fluorescence Experiments--
The affinity between CDK2 or
phosphorylated CDK2 and cyclin A was measured by the direct
fluorescence titration of the CDK2-Mant-ATP complex with different
cyclin A concentrations. Mant-ATP-CDK2 complexes were incubated for 15 min in the presence of different cyclin concentrations before starting
the experiment. CDK2/cyclin interactions were also monitored using the
enhancement of Mant-ATP fluorescence at 430 nm. The Mant-ATP-CDK2
complexes were kept at a constant concentration of 0.2 µM, and the fluorescence emission was monitored at 430 nm
(excitation at 350 nm) as a function of the cyclin concentration. The
Kd was calculated by fitting the data to a standard
quadratic equation (44). For displacement experiments, CDK2-Mant-ATP
complexes at a concentration of 0.2 µM were incubated in
the presence of a 200-fold excess of ATP and with or without added
cyclin A (200 nM). Dissociation of the fluorescently
labeled nucleotides was monitored according to the quenching of Mant
fluorescence at 430 nm upon excitation at 350 nm. Data were fitted as
already described (44).
BIAcore Analysis--
GST-CDK2 was used for BIAcore analysis
because neither free cyclin A3 nor CDK2 could be immobilized in an
active form. GST-CDK2 was phosphorylated by His-tagged CAK1, as
described above, and the fusion protein was purified on Superdex 200 (Amersham Pharmacia Biotech). Phosphorylated GST-CDK2 showed minimal
mobility shifts on SDS-PAGE. Phosphorylation of the CDK2 fusion protein
was confirmed using Phosphorylated CDK2 Crystallization
Purified phosphorylated CDK2 was buffer-exchanged into 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM monothioglycerol and concentrated
to 10 mg/ml using a Centricon 10 concentrator (Amicon). Since initial
crystallization trials with phosphorylated CDK2 were not successful,
sitting drops (2 µl) were prepared by mixing equal volumes of protein
and well solution (12-16% PEG 4000, 0.1 M Tris-HCl, pH
7.5, 50 mM ammonium acetate) and allowed to equilibrate for
3 days before streak/microseeding with microcrystalline preparations of
unmodified CDK2 using a 50-µm diameter steel wire for the seed
transfer. Typically, crystals appeared within 2 days and continued to
grow for 1 week. Maximum crystal dimensions were 200 × 100 × 75 µm. CDK2 was crystallized using the hanging drop method by
mixing equal volumes (1 µl) of protein solution (at a concentration
of 10 mg/ml in 15 mM NaCl, 10 mM HEPES, pH 7.4) and well solution (10-15% PEG 3350, 50 mM ammonium
acetate, 0.1 M HEPES, pH 7.4). Crystals appeared overnight
and continued to grow for 5 days. Crystal complexes of phosphorylated
CDK2 and ATP and of CDK2 and ATP were prepared by soaking crystals in
the presence of 2.5 mM ATP and 5 mM
MgCl2.
Data Collection and Processing
Data for the Phosphorylated CDK2-ATP complex were collected on
beamline BW7B at DESY (Hamburg) operating at a wavelength of 1.044 Å using a 30-cm Mar Research image plate, oscillations of 1.0°, and
exposure time of 120 s. The CDK2-ATP complex data set was
collected on beamline 9.5 at the SRS operating at a wavelength of 1.20 Å using a 30-cm Mar Research image plate, oscillations of 1.0°, and
an exposure time of 450 s/frame. In each case, data were collected at
100 K after crystals had been transferred briefly to cryoprotectant
(mother liquor containing 20% glycerol) and then mounted using a nylon
loop. Images were integrated with the DENZO package and subsequently
scaled and merged using SCALEPACK (45). Statistics of the data sets
used are given in Table I.
Structure Solution and Refinement
Identical protocols were used for the refinement of
phosphorylated CDK2-ATP and unphosphorylated CDK2-ATP structures. The starting model for refinement was the structure of CDK2 in complex with
a purine-based inhibitor refined at 1.3-Å
resolution.2 This model
included protein residues 1-35 and 44-298. Residues 36-43, prior to
the C-helix, are disordered and have not been built in any reported
monomeric CDK2 structure. At first, rigid body refinement of this model
against the two data sets was performed. As the resolution of the data
included was increased from 3.0 to 2.1 Å, an increasing number of
rigid bodies was used, so that initially the whole molecule was treated
as a single rigid body, and finally five amino acid segments were
allowed to refine independently. Refinement of the models was then
pursued with alternating cycles of interactive model building (46) and
maximum likelihood refinement using the program REFMAC (47). In all
cases where manual intervention was required, the structural changes
indicated were similar for both structures. No electron density trace
could be seen for the tip of the activation segment (residues 153-164)
of the phosphorylated CDK2-ATP structure, and neither omit refinement
nor refinement of this region in alternative conformations could
produce such a trace. The initial coordinates of MgATP were taken from
the structure of CDK2-ATP determined by DeBondt et al. (35),
and optimal geometry for this ligand was taken from the default PROTIN dictionary. Toward the end of the refinement, water molecules were
added using ARP (48). Statistics of the final models are given in Table
I.
Monomeric CDK2 is a Substrate for CAK1--
Phosphorylation of
CDK2 on Thr160 results in increased electrophoretic
mobility of CDK2 on SDS-PAGE (Fig.
1a) (9, 12). We used this band
shift to follow CDK2 phosphorylation by S. cerevisiae CAK1.
The GST-CAK1 preparation contained several contaminants (see Fig.
1a, lane 2, and "Experimental
Procedures") but provided a good source of CAK activity, and no
further purification steps were introduced at this stage. CDK2
phosphorylation was also monitored by incorporation of 32P
from
As an additional test for CAK1 specificity, purified phosphorylated
CDK2 was treated with the CDK-associated phosphatase, KAP. This enzyme
specifically dephosphorylates monomeric phosphorylated CDK2 at
Thr160 but shows no activity toward phosphorylated
CDK2-cyclin A (24). Incubation of monomeric phosphorylated CDK2 with
purified recombinant KAP resulted in a gel shift to a position
characteristic of unmodified CDK2 (Fig.
2).
Mass spectroscopic analysis of phosphorylated CDK2 was used as a final
test for CAK1 specificity. The results of this analysis (not shown)
confirmed that CDK2 incubated with CAK1 is only phosphorylated on
residue Thr160.
CDK2 Phosphorylated on Thr160 Possesses Partial Histone
H1 Kinase Activity--
CDK2 histone H1 kinase activity in the
presence and absence of cyclin A3 was assayed before and after
phosphorylation by CAK1 (Fig.
3a). Both unmodified CDK2 and
GST-CAK1 showed no histone H1 kinase activity (Fig. 3a,
lanes 3 and 2, respectively). CDK2 phosphorylated by CAK1 exhibited partial histone H1 kinase activity (Fig. 3a, lane 1). This activity was
comparable with that of the unmodified CDK2-cyclin A3 complex (Fig.
3a, lane 5). Phosphorylated CDK2
incubated with cyclin A3 showed maximal histone H1 kinase activity
(Fig. 3a, lane 4). PhosphorImager
analysis indicated that the phosphorylated CDK2 and the unmodified
CDK2-cyclin A binary complex had 0.3 and 0.2%, respectively, of the
activity seen in the fully activated phosphorylated CDK2-cyclin A3
binary complex (data not shown). We investigated the possibility that small amounts of insect cyclin may have co-purified with the CDK2 and
provided an apparent activity for the highly purified monomeric phosphorylated CDK2. As little as 1% contamination would be
significant, since the fully activated phosphorylated CDK2-cyclin A
complex is more active by at least 2 orders of magnitude. Experiments were repeated with GST-CAK1 and CDK2 expressed in E. coli
cells. Fig. 3b shows that the bacterially expressed,
monomeric CDK2 phosphorylated by CAK1 also exhibits partial, but
significant, histone H1 kinase activity.
CDKs can be inhibited by members of the CKI family (28).
p27kip1 inhibited the histone H1 kinase activity observed with
both the fully active phosphorylated binary complex and the partially
active unphosphorylated complex (Fig. 3c). The partial
histone H1 kinase activity of phosphorylated monomeric CDK2 was also
inhibited (Fig. 3d).
Fluorescence experiments were performed to determine the dissociation
constants between various forms of CDK2 and its substrate histone H1.
The binding of histone H1 to CDK2-cyclin A induces a 25% quenching of
the intrinsic fluorescence of the complex (Fig. 3e). This
quenching was used to quantify the interaction between CDK2-cyclin A
and histone H1. Titrations were performed using a fixed concentration
of kinase with increasing concentrations of substrate.
Kd values of 1 and 0.7 µM were
calculated for CDK2-cyclin A and phosphorylated CDK2-cyclin A,
respectively. Monomeric phosphorylated CDK2 was found to bind histone
H1 with very low affinity, with a calculated Kd of
about 100 µM. Histone H1 binding to monomeric CDK2 was
not detected. The results are summarized in Table
II.
ATP Binding to CDK2--
Binding of ATP to phosphorylated CDK2 was
first monitored by following the quenching of intrinsic tryptophan
fluorescence. ATP binding to phosphorylated CDK2 results in quenching
of the intrinsic fluorescence by 48%. This value is similar to that
reported for unphosphorylated CDK2 (44). The titration curves are shown in Fig. 4a, where a fixed
concentration of CDK2 (0.2 µM) phosphorylated or not on
Thr160 was titrated with an increasing concentration of ATP
or ADP. The curve fitting carried out using the results of the
phosphorylated CDK2 experiment calculates an apparent dissociation
constant of 120 nM for ATP. This value is 2-fold lower than
that determined for unphosphorylated CDK2 (254 nM). In
contrast, as shown in Fig. 4a, CDK2 phosphorylation
decreases by 4.5-fold the CDK2 affinity for ADP. Curve fitting yields
apparent Kd values of 1.4 and 6.7 µM
for ADP association with CDK2 and phosphorylated CDK2, respectively.
The fluorescence of Mant-ATP was also used to quantify the binding of
ATP to phosphorylated CDK2. We have demonstrated that the binding of
Mant-ATP to CDK2 overexpressed in E. coli cells results in a
10-fold increase in the Mant-group fluorescence (44). This experiment
was repeated using as the source of CDK2 the enzyme expressed in
recombinant baculovirus-infected insect cells. The binding of Mant-ATP
to either unphosphorylated CDK2 or phosphorylated CDK2 results in an
increase in the fluorescence of the Mant-group (Fig. 4b).
These changes reveal that the highly hydrophobic character and the
conformation of the CDK2 active site are not significantly modified by
Thr160 phosphorylation. A fixed concentration of Mant-ATP
or Mant-ADP (0.2 µM) was titrated by increasing the CDK2
concentration (Fig. 4b). The curve fitting yielded
Kd values of 256 and 170 nM for Mant-ATP
and 1.5 and 5.4 µM for Mant-ADP against unphosphorylated CDK2 and phosphorylated CDK2, respectively. Mant-ATP bound to CDKs can
be fully displaced by an excess of unlabeled nucleotide, resulting in a
5-fold decrease in the fluorescence of the Mant-group. As previously
published, the dissociation rate of Mant-ATP from CDK2 using a 200-fold
excess of ATP occurs according to a single exponential with a rate
constant of 5.4 × 10 Cyclin Binding Is Unaffected by Thr160 Phosphorylation
of CDK2--
Cyclin A binding to CDK2 and phosphorylated CDK2 was
quantified by using the fluorescence of Mant-ATP previously bound to the kinase. The binding of cyclin A to CDK2 enhanced the bound Mant-ATP
fluorescence 3-fold (Fig. 5). The
titration curves followed a monophasic pattern, and their fitting
yielded Kd values of 52 nM for
CDK2-cyclin A and 48 nM for phosphorylated CDK2-cyclin A
with a 1:1 (CDK/cyclin) stoichiometry in both cases (Fig. 5). These
results show that phosphorylation on Thr160 does not modify
the affinity of CDK2 for cyclin A and that phosphorylation is not
required, as previously proposed (44), for the formation of a
CDK2-cyclin A complex. The enhancement of Mant-ATP fluorescence is
similar (3-fold) for cyclin A binding to CDK2 or phosphorylated CDK2,
and the magnitude of the enhancement suggests that substantial conformational changes in the nucleotide binding site accompany cyclin
binding.
Surface plasmon resonance analysis (BIAcore) was also used to compare
the cyclin binding properties of CDK2 and phosphorylated CDK2. Because
of the difficulty in immobilizing CDK2 or cyclin A3 in an active form
using the standard amine coupling chemistry, a GST-CDK2 protein
expressed in E. coli cells was used. The CDK2 fusion protein
was captured by immobilized anti-GST immunoglobulin. Analysis of the
sensorgram curves (results not shown) gave a Kd of
0.7 nM for the GST/CDK2-cyclin A3 interaction and a value
of 0.5 nM for GST-phosphorylated CDK2-cyclin A3. Although
these values are probably significantly affected by the use of dimeric
GST fusion proteins (see Ref. 49 and "Discussion") they also
indicate no significant difference in apparent affinity of
phosphorylated CDK2 or of unmodified CDK2 for cyclin A3.
The Phosphorylated CDK2 Structure Reveals Activation Segment
Disorder--
Initial crystallization trials with phosphorylated CDK2
were not successful. However, conditions similar to those required to
produce CDK2 crystals were able to sustain crystal growth in microseeding experiments with nuclei obtained from nonphosphorylated CDK2 crystals. We have determined structures for phosphorylated CDK2
(results not shown), the ATP-phosphorylated CDK2 complex, and
unphosphorylated CDK2 in complex with ATP at 2.1-Å resolution. Our own
results and those of others have shown that the structure and mobility
of monomeric crystalline CDK2 can depend critically on the nature of
the ligand bound to the ATP binding site. In order to compare as
closely as possible phosphorylated and unphosphorylated CDK2, we
determined the structures of the ATP complexes of both forms of the
enzyme, using crystals of comparable size, identical crystal soaking
conditions, and identical structure refinement protocols. Statistics
relating to the quality of the two data sets and of the two refined
models are presented in Table I.
The structure of CDK2, first described by DeBondt et al.
(35), is that of a minimal protein kinase catalytic core, consisting of
an N-terminal domain formed principally from Phosphorylated CDK2 has been prepared by treatment of purified,
nonphosphorylated CDK2 expressed in baculovirus-infected insect cells
with CAK1 expressed either in E. coli or in S. cerevisiae. Purified phosphorylated CDK2 exhibited histone H1
kinase activity representing about 0.3% of the activity measured for
the fully active phosphorylated CDK2-cyclin A and comparable with that
shown by the unmodified CDK2-cyclin A binary complex. The possibility that activity was the result of association of the CDK2 with endogenous insect cell cyclins was excluded by repeating the experiments using
CDK2 and GST-CAK1 expressed in E. coli cells. Fisher and Morgan (12) could not detect histone H1 kinase activity associated with
monomeric phosphorylated CDK2 prepared by treating CDK2 with reconstituted CAK (CDK7-cyclin H), but the activity reported here is
below the sensitivity of the curves shown in that paper.
CDK2 has been isolated from synchronized mammalian cell lines in
multiple high and low molecular weight complexes by gel filtration analysis (50). Monomeric CDK2 was identified as a mixture of fast and
slow migrating forms as analyzed by SDS-PAGE, suggesting that monomeric
phosphorylated CDK2 is present in mammalian cells. These results,
together with the results described in this paper suggest that
monomeric CDK2 phosphorylated on Thr160 may be biologically
relevant, and as previously proposed (12), there may be alternative
assembly pathways to generate a fully active CDK2-cyclin binary
complex. We have found that the cyclin-dependent kinase
inhibitor p27kip1 does inhibit phosphorylated CDK2 as well as
the activity observed with both the fully activated
phosphorylated binary complex and the partially active unphosphorylated
complex. The concentrations of p27kip1 required to
inhibit monomeric phosphorylated CDK2 are consistent with the
observation that p27kip1 has a much lower affinity for
monomeric CDKs compared with CDK-cyclin complexes (Ref. 51; and see
Ref. 52). The biological significance of the low levels of kinase
activity associated with monomeric phosphorylated CDK2 remains to be determined.
The phosphorylated CDK2 structure reveals that Thr160
phosphorylation is insufficient to fully activate the kinase and
provides an explanation for the very low levels of kinase activity
observed. The major CDK2 structural changes required for activation are promoted by cyclin association. Protein kinases show significant variation in their dependence on phosphorylation for activity. Significant structural changes that lead to activation in some protein
kinases (e.g. the insulin receptor tyrosine kinase (32)) can
be promoted solely by phosphorylation, whereas other protein kinases
(e.g. phosphorylase kinase (53)) require no phosphorylation on the activation segment.
Phosphorylated CDK2 is observed to have both higher affinities for ATP
and substrate histone H1 and a lower affinity for ADP than the
nonphosphorylated CDK2. In the unphosphorylated monomeric CDK2
structure, Thr160 is located close to the side chain of
Glu12, and the aromatic ring of Tyr159 stacks
against the planar peptide backbone around Gly16 (Fig.
6d). The glycine loop structure that favors this set of interactions is also promoted by additional interactions between backbone atoms of residues Glu12-Thr14 with
the ribose-triphosphate group of ATP. Phosphorylation of Thr160 by both steric and charge constraints precludes this
set of stabilizing interactions between residues in the glycine-rich
loop and the activation segment. In the phosphorylated CDK2 structure,
increased structural flexibility extends to residues
His161, Glu162, Val163, and
Val164 in the activation loop. Two structures of protein
kinases in complex with peptide substrates, insulin receptor kinase
(32) and phosphorylase kinase (54), show a defined conformation of the
protein substrate across the catalytic site. In particular, there is an
antiparallel In contrast, phosphorylation of CDK2 does not significantly alter its
affinity for cyclin A. Using fluorescence methods, CDK2 and
phosphorylated CDK2 showed high affinity for cyclin A with measured
Kd values of 52 and 48 nM for
phosphorylated CDK2-cyclin A and CDK2-cyclin A association,
respectively. Repeating the measurements using BIAcore analysis gave
Kd values of 0.7 nM for phosphorylated
CDK2-cyclin A and 0.5 nM for CDK2-cyclin A. These results
confirmed that the two CDK2 species have comparable affinity for cyclin
A. The low values observed for Kd in the BIAcore
experiments may be an artifact produced by the use of GST-CDK2. Ladbury
et al. (49) have shown that avidity effects that result from
the dimerization of GST fusion proteins could be responsible for
overestimates of affinities measured by surface plasmon resonance.
We have shown that CAK1 phosphorylates monomeric CDK2 more effectively
than the binary CDK2-cyclin A complex. This observation is consistent
with structural results. Although the structure of CAK1 is not yet
known, it is anticipated to have the kinase fold reported for the 14 protein kinase structures determined to date by x-ray crystallographic
analysis. Different protein kinases may demand slightly different
substrate conformations, as seems to be the case for the cAPK-peptide
inhibitor complex (55) and for CDKs that recognize Ser/Thr-Pro motifs.
However, the structural conservation of the catalytic sites in the
active kinase conformations suggests, as discussed above, that the
protein substrate needs to be able to adopt an approximately extended conformation for recognition.
In the CDK2-cyclin A complex, residues 159-162 around
Thr160 adopt a turn of 310 helix (36). A
conformational change would be required for this region to adapt to the
activating kinase catalytic site if an extended conformation is
required for recognition, although Thr160 itself is
accessible. In unmodified CDK2, although the preferred conformation of
the activation segment buries Thr160, the region exhibits
considerable mobility (Fig. 6c). This mobility would allow
displacement of Thr160 from its buried position and provide
scope for the activation segment to adapt its conformation to the CAK1
catalytic site. Just such an adaptation occurs when the mobile
N-terminal region of glycogen phosphorylase b adapts to the
catalytic site of phosphorylase kinase (54). p27kip1 binding to
the binary complex promotes phosphorylation of Thr160 by
CAK1. This result is in contrast to the ability of p27kip1 when
bound to CDK2-cyclin E complexes to inhibit phosphorylation of
Thr160 by CAK (56). Although there is no structural
information about the unphosphorylated ternary complex of CDK2-cyclin
A-p27kip1 our results suggest that the mobility of the
activation loop is enhanced in this complex. In the phosphorylated
CDK2-cyclin A-p27kip1 complex (57), the p27kip1
inhibitor contacts both cyclin A and CDK2, causing significant conformational changes in CDK2 that result in displacement of the The notion of increased mobility in the activation segment in
phosphorylated CDK2 compared with phosphorylated CDK2-cyclin A may also
explain the catalytic activity of KAP. It has been shown that KAP will
not dephosphorylate the binary phosphorylated CDK2-cyclin A complex but
only monomeric phosphorylated CDK2 (24). In the phosphorylated
CDK2-cyclin A structure, the phospho-Thr160 is buried, and
the activation segment is well localized so that it would be difficult
for the phosphatase to have access to the phosphothreonine. In
monomeric phosphorylated CDK2, the activation segment is mobile and
available for modification by KAP.
In summary, the mobility of the activation segment in phosphorylated
CDK2 observed in the crystal structure analysis provides an explanation
for the effects observed in vitro with purified enzymes. We
have shown that phosphorylated CDK2 has basal activity, that monomeric
CDK2 is a better substrate than CDK2-cyclin A for CAK1, and that
phosphorylated CDK2 and unmodified CDK2 have similar affinities for
cyclin A. The significance of these results for the in vivo
situation remains to be evaluated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet N-terminal domain that contains one helix, the
C-helix, and a predominantly
-helical C-terminal domain. The
ATP-binding site is situated at the domain-domain interface. In the
structure of inactive, monomeric CDK2, residues at the ATP-binding site
are wrongly disposed and are unable to promote the correct alignment of
the triphosphate moiety for catalysis, although the inactive monomer
can bind ATP (35). The inactive conformation arises mainly from the
organization of two key elements of structure. These are the C-helix,
which contains the PSTAIRE motif (single letter amino acid code,
residues 45-51), and the activation segment, which includes residues
that lie between the conserved DFG and APE motifs (residues 145-147
and 170-172, respectively). The position of the C-helix in the
inactive CDK2 monomer results in the loss of the stabilizing
interaction between Glu51 (the E of the PSTAIRE sequence)
and Lys33 that is important for correct localization of the
ATP triphosphate. The activation segment conformation buries
Thr160 away from solvent facing in toward the conserved
glycine-rich loop.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-D-galactopyranoside and incubated for
16 h at 25 °C. Harvested cells were lysed by sonication of a
freeze/thawed sample to which lysozyme (0.1 mg/ml) had been added.
Following clarification, the lysate was applied to a 5-ml
glutathione-Sepharose column (Amersham Pharmacia Biotech) preequilibrated in Hepes-buffered saline (10 mM Hepes, pH
7.4, 134 mM NaCl, 2 mM EDTA), and after
washing, the GST fusion protein eluted with freshly prepared
Hepes-buffered saline containing 20 mM glutathione. Yields
of soluble fusion were greater than 100 mg/liter. Following thrombin
cleavage (Sigma; 16 h at 1:1000 (w/w) at 25 °C), CDK2 was
purified by gel filtration (Superdex 75; Amersham Pharmacia
Biotech) and glutathione-Sepharose (Amersham Pharmacia Biotech).
-D-galactopyranoside and
incubation for a further 28 h at 16 °C. Harvested cells were lysed, and GST-CAK1 was purified using glutathione-Sepharose as described above. Yields of soluble fusion protein were 2 mg/liter. The
partially purified preparation showed three bands around the expected
position of the fusion protein on gels. Thrombin digests demonstrated
that the middle band was GST-CAK1. Since the preparation was an
effective source of CAK activity, further purification was not carried
out. Partially purified fusion was found to be stable for at least 2 weeks when stored on ice.
leu2 his3 ura2 trp1) as described (20). The cell lysate was
loaded onto a nickel-nitrilotriacetic acid resin (Qiagen) and purified
according to the manufacturer's instructions. The His-tagged CAK1
showed similar enzymatic properties to those of the fusion protein.
-D-galactopyranoside, 3-4 h at
37 °C) and purified in one step using nickel-nitrilotriacetic acid
resin. Purified recombinant KAP was a kind gift of Dr. Neil Hanlon.
-32P-labeled
ATP (Amersham Pharmacia Biotech) and either 15 µM
CDK2-cyclin A3 complex (1 µg in 10 µl) or 15 µM
monomeric CDK2 (0.5 µg in 10 µl) as substrate. The reactions
proceeded for 30 min at 30 °C before termination with SDS sample
buffer. Following SDS-PAGE (15% acrylamide), the stained and dried
gels were subjected to autoradiography. Histone H1 kinase activity was
examined using the same reaction conditions to which 300 µM histone H1 (Boehringer Mannheim) was added. Inhibition
of kinase activity by p27kip1 was measured by including 35 µM p27kip1 (1.0 µg in 10 µl) in the
radioactive kinase assays carried out under the same reaction
conditions as described above. PhosphorImager analyses (Molecular
Dynamics, Inc., Sunnyvale, CA) were carried out on the fixed, stained,
and dried gels.
-32P-labeled ATP (Amersham Pharmacia
Biotech). Samples of GST-CDK2 and GST-phosphorylated CDK2 were captured
on the sensor chip (CM5) using immobilized anti-GST immunoglobulin
(Amersham Pharmacia Biotech). Cyclin A3, at concentrations of 5-100
µg/ml, was passed over the chip at a flow rate of 5 µl/min. Data
analysis was performed with the BIAevaluation software (version 2.1, Amersham Pharmacia Biotech).
Statistics of the data sets used and of the refined structures
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-labeled ATP (Fig. 1b). Radiolabeled phosphorylated
CDK2 co-migrated with the faster migrating CDK2 band seen on
Coomassie-stained gels. Comparative studies showed that monomeric human
CDK2 is a better substrate for CAK1 than the human CDK2-bovine cyclin A3 complex as judged by both incorporation of 32P (Fig.
1b) and mobility shift (Fig. 1c). Cyclin A3
migrates as a faster band than CDK2 or phosphorylated CDK2 (Fig.
1c, lane 4). (Bovine cyclin A3
corresponds to human cyclin A residues 171-432.) The addition of the
CKI, p27kip1 to monomeric CDK2 had no effect on the
phosphorylation of Thr160 by GST-CAK1. However,
p27kip1 addition to the binary complex enhanced phosphorylation
by CAK1 to levels observed with monomeric CDK2 (Fig. 1d). In
these experiments, there was no evidence of radiolabeling of CAK1 by
either phosphorylated CDK2 or phosphorylated CDK2-cyclin A3 (Fig.
1b). Similar results were obtained using His-tagged CAK1
expressed in S. cerevisiae as a source of CAK activity
(results not shown). CDK2 phosphorylated by CAK1 was used to prepare
phosphorylated CDK2 crystals.
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Fig. 1.
a, phosphorylation of CDK2 by GST-CAK1.
Lane 1, molecular weight markers. Markers are at
200, 98, 66, 45, 30, 20, and 12 kDa. Lane 2,
GST-CAK1. Lane 3, monomeric CDK2. Different
concentrations of GST-CAK1 (7.5, 3.0, 1.5, 0.75, and 0.12 µM GST-CAK1 in lanes 4-8,
respectively) were co-incubated with 30 µM monomeric
CDK2. Proteins were visualized by Coomassie stain following 15%
SDS-PAGE. As shown, phosphorylated CDK2 has a higher electrophoretic
mobility by SDS-PAGE than unphosphorylated CDK2.
b,CAK1-catalyzed incorporation of 32P from
-labeled ATP into monomeric CDK2. CDK2 was phosphorylated by
coincubation with GST-CAK1 in the absence (lane
2) or presence (lane 4) of cyclin A3.
The GST-CAK1 (lane 1), CDK2 (lane
3), or CDK2-cyclin A3 (lane 5) samples
show no autophosphorylation activity. c, comparative study
of CDK2 and CDK2-cyclin A3 phosphorylation by CAK1. Monomeric CDK2 is a
more efficient substrate for CAK1-catalyzed phosphorylation than the
binary CDK2-cyclin A3 complex. Lane 1, molecular
weight markers; lane 2, GST-CAK1; lane
3, CDK2 co-incubated with CAK1; lane
4, CDK2-cyclin A3 complex with CAK1; lane
5, CDK2. All reactions were carried out under the conditions
described under "Experimental Procedures." d, effect of
p27kip1 upon phosphorylation of CDK2 by GST-CAK1 in the
presence and absence of cyclin A3. CDK2 (0.3 µM) was
incubated with GST-CAK1 (0.1 µM) in the presence and
absence of p27kip1 (50 nM) and cyclin A3 (0.3 µM), and the reaction was allowed to proceed as described
under "Experimental Procedures." Enhancement of phosphorylation of
the CDK2-cyclin A3 binary complex to levels observed for the monomeric
CDK2 is seen in the presence of p27kip1 (compare
lanes 1 and 2). In this experiment,
p27kip1 was also labeled and is apparent just below
the phosphorylated CDK2 band in lane 2. No
difference is seen when using monomeric CDK2 as a substrate (compare
lanes 3 and 4).
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Fig. 2.
CDK-associated phosphatase KAP
dephosphorylates phosphorylated CDK2. KAP specifically
dephosphorylates monomeric phosphorylated CDK2. CDK2, phosphorylated
CDK2, cyclin A3, KAP, and orthovanadate were co-incubated as indicated
and under conditions as described under "Experimental
Procedures."
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Fig. 3.
a, CDK2 phosphorylated on
Thr160 has partial histone H1 kinase activity. CDK2, cyclin
A3, GST-CAK1, and histone H1 were co-incubated in various combinations
as indicated in the presence of radiolabeled [32P]ATP and
under conditions as described under "Experimental Procedures."
Radiolabeled histone H1 was visualized following 15% SDS-PAGE and
autoradiography. b, CDK2 expressed in E. coli
cells is a substrate for CAK1. The experiment described in a
above was repeated using CDK2 synthesized in E. coli cells.
c, the histone kinase activity of phosphorylated CDK2-cyclin
A3 and CDK2-cyclin A3 are inhibited by p27kip1.
p27kip1 (0.5 µM) inhibits both phosphorylated and
nonphosphorylated binary complexes (0.3 µM) (compare
lanes 1 and 2 and lanes
3 and 4, respectively). d, the histone
kinase activity of phosphorylated CDK2 is inhibited by
p27kip1. The addition of increasing quantities of
p27kip1 (5 nM, 50 nM, 500 nM, and 5 µM) to 50 nM
phosphorylated monomeric CDK2 (lanes 2-5) revealed that 5 µM p27kip1 showed strong inhibitory activity.
e, binding of histone H1 to CDK2. A fixed (0.1 µM) concentration of CDK2 ( ), phosphorylated CDK2
(
), CDK2-cyclin A (
), and phosphorylated CDK2-cyclin A (
) were
titrated by increasing concentration of histone H1. The binding was
monitored by following the quenching of intrinsic fluorescence at 340 nM, and the data were fitted according to a quadratic
equation that assumes the presence of a single site for histone
H1.
Affinity constants of CDK2 and CDK2-cyclin A complexes for nucleotides
and fluorescent analogues and histone H1
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Fig. 4.
a, nucleotide binding to CDK2 and
phosphorylated CDK2. ATP or ADP binding to phosphorylated CDK2 was
monitored by following the quenching of intrinsic tryptophan
fluorescence at 340 nm upon excitation at 290 nm. A fixed concentration
of CDK2 (0.2 µM) (open symbols) and
phosphorylated CDK2 (closed symbols) were
titrated with increasing concentrations of ATP ( ,
) and ADP
(
,
). b, Mant-ATP and Mant-ADP binding to
CDK2 and phosphorylated CDK2. Mant-nucleotides binding to either CDK2
or phosphorylated CDK2 result in an increase in the fluorescence of the
Mant-group at 440 nm upon excitation at 350 nm. A fixed concentration
of Mant-nucleotide (0.2 µM) (ATP (
,
), ADP (
,
)) was titrated with increasing concentrations of CDK2
(open symbols), or phosphorylated CDK2
(closed symbols). c, Mant-ATP
displacement from CDK2 and phosphorylated CDK2. Mant-ATP displacement
from CDK2 (
) and phosphorylated CDK2 (
) by a 200-fold excess of
ATP was monitored using the decrease of the Mant fluorescence at 450 nm. The dissociation kinetics were fitted as
a single exponential to a rate constant of 5.2 × 10
3 s
1 and 3.1 × 10
3
s
1 for CDK2 and phosphorylated CDK2, respectively.
3 s
1 (44). When
this experiment was repeated with phosphorylated CDK2, the dissociation
rate constant of Mant-ATP was reduced to a value of 3.1 × 10
3 s
1 (Fig. 4c). The results
are summarized in Table II. These displacement experiments confirm that
CDK2 phosphorylation increases the enzyme's affinity for ATP.
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Fig. 5.
CDK2 and phosphorylated CDK2 binding to
cyclin A3. Binding titration of cyclin A to phosphorylated CDK2
was followed by Mant-ATP fluorescence enhancement. CDK2 ( ) and
phosphorylated CDK2 (
) previously saturated with Mant-ATP (1 µM) were titrated by increasing concentrations of cyclin
A. The binding of cyclin A to CDK2 enhances the bound Mant-ATP
fluorescence 3-fold at 440 nm upon excitation at 350 nm. The titration
curves followed a monophasic pattern, with Kd values
of 52 nM for CDK2-cyclin A and 48 nM for
phosphorylated CDK2-cyclin A. In both cases, the stoichiometry of
binding is 1:1 (CDK/cyclin). Cyclin binding is unaffected by
Thr160 phosphorylation of CDK2.
-sheet and a C-terminal
domain formed principally from
-helix. ATP binds in the cleft
between the two domains (Fig.
6a), forming contacts with
both N- and C-terminal lobes. The structure of unphosphorylated monomeric CDK2 differs from that of CDK2 in the active phosphorylated binary form in two major respects: the orientation of the C-helix ("PSTAIRE helix"), and the conformation of the "activation
segment" between the conserved sequence motifs DFG
(Asp145-Phe146-Gly147) and APE
(Ala170-Pro171-Glu172). The
incorrect orientation of the C-helix in the inactive form of CDK2
prevents the correct conformation of key catalytic residues, while an
inappropriate conformation of the activation segment precludes
productive association of a polypeptide substrate at the active site.
The refined structures of phosphorylated and nonphosphorylated CDK2 in
complex with ATP are remarkably similar (root mean square difference
for 290 C-
positions = 0.5 Å). For the part of the structure
that is well defined by electron density in both cases (all except
residues 36-43 and 153-164), no part of the structure had to be built
differently in the phosphorylated as compared with the
nonphosphorylated structure. Of the two regions excepted above, the
former (residues 36-43) could not be traced in either the
phosphorylated or the unphosphorylated CDK2-ATP complex and is known to
become well defined only in the binary complex of CDK2 with cyclin A
(36). The latter region (residues 153-164) can clearly be traced in
the nonphosphorylated structure of CDK2 in complex with ATP but has no
continuous electron density in the structure of phosphorylated CDK2 in
complex with ATP (Fig. 6b). The increased mobility of both
the activation segment and the glycine-rich loop in the
ATP-phosphorylated CDK2 structure as compared with the structure of
ATP-CDK2 is reflected in the main chain B-factor values for each of the
models in these regions. With the exception of these two regions, the B
factor values agree closely for the two models (Fig. 6c).
The relevance of this change in mobility to the activity of
phosphorylated monomeric CDK2 is discussed later.
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Fig. 6.
a, the fold of monomeric CDK2. The
structure is shown in a schematic representation with regions of
-sheet shown as arrows and
-helix shown as
ribbons. The N-terminal domain is colored principally
white, with the exception of the glycine-rich loop (colored
magenta), and the C-helix (PSTAIRE helix, colored
gold). The region of the N-terminal domain for which no
trace is visible (residues 36-43) is indicated by small
black spheres identifying residues 35 and 44. ATP
is shown in ball and stick
representation at the interface between the N- and C-terminal domains.
The C-terminal domain is colored pink, with the activation
segment (residues 145-172) highlighted in cyan.
b, comparison of electron density for the tip of the
activation segment. The upper stereo
pair shows electron density defining the conformation of
residues at the tip of the activation segment (residues 155-165) in
the ATP complex of unphosphorylated monomeric CDK2, while the
lower stereo pair shows the equivalent
electron density in phosphorylated monomeric CDK2. In this
figure the phosphate group attached to Thr160
has been omitted from the phosphorylated CDK2 structure for clarity.
The maps were calculated using (2Fo
Fc)
calc coefficients generated by
REFMAC and are contoured at a level of 0.2e
Å
3. c, B-factor plots for CDK2-ATP and phosphorylated
CDK2-ATP. The mean main chain B-factor of each residue along the
polypeptide chain is shown for unphosphorylated CDK2 (thin
lines) and phosphorylated CDK2 (thick
lines). The outstanding regions of difference include the
glycine loop (residues 8-18) and the tip of the activation segment
(residues 155-165). d, detail of the fold of the CDK2-ATP
complex. The interaction of Tyr159 and Thr160,
at the tip of the activation segment, with residues
Glu12-Tyr15 in the glycine-rich loop is shown.
The coloring scheme is the same as for
a.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet between the amino acids in the P+1 and P+3
positions of the protein substrate and those of the activation segment
immediately following the negatively charged residue that is
responsible for activation (Tyr1163 in insulin receptor
kinase and Glu182 in phosphorylase kinase). The structural
results show that the increased mobility of the activation segment in
phosphorylated CDK2 compared with the unmodified CDK2 structure results
in accessibility of the catalytic site for binding substrate and a
potential for the kinase to adopt the active conformation, albeit for a
short time and for a small proportion of the population, when in
solution. This structural mobility may contribute to the observed
higher affinity of phosphorylated CDK2 for ATP and histone H1 and its observed lower affinity for ADP than unmodified CDK2. Fluorescence studies support this structural interpretation and suggest mechanisms by which phosphorylation of Thr160 could enhance CDK2's
catalytic activity. It is assumed that, upon crystallization, the more
thermodynamically stable and more prevalent inactive conformation of
phosphorylated CDK2 has been selected.
1
and
2 strands and the glycine loop.
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ACKNOWLEDGEMENTS |
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We acknowledge with gratitude the help of the beamline scientists at X-ray Diffraction Elettra (Trieste); BW7B, DESY (Hamburg); and 9.5, SRS (Daresbury). We thank Carol Robinson and Paula Tito for the mass spectroscopic analysis of phosphorylated CDK2. We thank David Morgan for the gift of the baculoviral construct expressing human CDK2, Carl Mann for various constructs expressing CAK1, Neil Hanlon for KAP, and Tim Hunt for the cyclin A3 and GST-CDK2 constructs together with much stimulating discussion. At the Laboratory of Molecular Biophysics (Oxford), we thank Stephen Lee, Richard Bryan, Kathryn Measures, and Irene Taylor for assistance.
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FOOTNOTES |
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* This work was supported by the Medical Research Council, BBSRC, Royal Society, The Wellcome Trust, CNRS, ARC (contract 1244), and the Ligue Nationale Contre le Cancer.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.
The atomic coordinates for the phosphorylated CDK2-ATP complex structure (1b39), its associated structure factors (r1b39sf), the nonphosphorylated CDK2-ATP complex (1b38), and its associated structure factors (r1b38sf) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ To whom correspondence should be addressed.
2 M. E. M. Noble, A. M. Lawrie, P. Tunnah, L. N. Johnson, and J. A. Endicott, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CDK, cyclin-dependent kinase; CAK, CDK-activating kinase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; KAP, kinase-associated phosphatase; Mant, 2'(3')-O-(N-methylanthraniloyl).
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