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INTRODUCTION |
Angiogenesis is critical for normal embryogenesis, growth, and
tissue repair. Pathological angiogenesis, however, can support tumor
growth and possibly lead to metastatic spread (reviewed in Ref. 1).
Furthermore, neoangiogenesis in ischemic ocular retinal diseases, such
as diabetic retinopathy and possibly age-related macular degeneration,
can culminate in blindness. Tumor angiogenesis, growth, and metastasis
(2) and pathological ocular neovascularization (3) have been correlated
with increased local expression of vascular endothelial growth factor
(reviewed in Ref. 4).
Two high affinity VEGF1
receptors, Flt-1 (VEGFR-1) and KDR (Flk-1/VEGFR-2), have been
identified (5-7). These receptors can be divided into three structural
regions: seven extracellular Ig-like domains that contain the growth
factor binding sites, a single polypeptide chain hydrophobic
transmembrane sequence, and intracellular cytoplasmic domains that
confer the tyrosine kinase activity required for signal transduction.
In addition, Flt-1 pre-mRNA is alternatively spliced to produce not
only the full-length membrane-spanning form but also a soluble
truncated version retaining the N-terminal six Ig-like extracellular
domains containing the ligand binding sites that can heterodimerize
with the corresponding region of KDR and antagonize the activity of VEGF in vitro (8) and in vivo (9).
KDR binds with high affinity not only to VEGF (6, 10, 11), also denoted
VEGF-A, but also to other recently identified homologous family
members, such as VEGF-D (12), and to the lymphathic endothelial cell
mitogen VEGF-C (12). The HIV transcription factor, tat, has also been
reported to bind to KDR and stimulate receptor autophosphorylation
(13). In addition, VEGF and placenta growth factor (10, 11), VEGF-B and
VEGF-D homologues bind with high affinity to Flt-1 (12). Although KDR
is able to mediate VEGF-stimulated vascular endothelial cell
mitogenesis, the functional role of full-length Flt-1 is not yet clear
because it does not appear to transduce VEGF mitogenic signaling in
these cells (14). In addition, although both the KDR and Flt-1
homozygous knockouts are each embryonically lethal, mice devoid of KDR
contain few, if any, vascular endothelial cells, whereas those without
Flt-1 have endothelial cell-lined vessels exhibiting a somewhat
disorganized structure (15). KDR, which has not been shown to be
alternatively spliced, was found to be an important mediator of VEGF
function in endothelial cells through activation of the intracellular
tyrosine kinase (14). Neuropilin-1, recently found to be an
isoform-specific VEGF receptor, does not have intrinsic tyrosine kinase
activity and is not sufficient to mediate either a mitogenic or a
chemotactic signal in response to VEGF binding but might enhance
VEGF165 binding to KDR (16).
KDR can be autophosphorylated on at least four tyrosine residues
located within the cytoplasmic domains of the protein (17). Two of
these residues, tyrosines 1054 and 1059, are located in the activation
loop of the tyrosine kinase domains. The other two tyrosine
phosphorylation sites at amino acid positions 951 and 996 are located
in a poorly conserved region, known as the tyrosine kinase insert loop,
that is characteristic of type III receptor tyrosine kinases (18).
Phosphorylation of the corresponding tyrosines in PDGF
-receptor
provide docking sites for downstream signal transduction proteins
(18).
We have cloned the KDR tyrosine kinase from a human umbilical vein
endothelial cell cDNA library, and we report a functionally important discrepancy in the tyrosine kinase active site with the
published sequence (6). Using the active kinase, we found that
phosphorylation of the activation loop tyrosine residues at positions
1054 and 1059 is required for activation of the catalytic activity of
the KDR kinase. Finally, we demonstrated that antagonists of this
tyrosine kinase can preferentially inhibit either the unactivated or
activated form of the enzyme.
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EXPERIMENTAL PROCEDURES |
Materials--
[
-33P]ATP (3000 Ci/mmol) and the
ECL kit were from Amersham Pharmacia Biotech. ATP and poly(Glu, Tyr)
4:1 (pEY) were from Sigma. All DNA modifying enzymes were from Promega
unless otherwise stated. Staurosporine was from Calbiochem, and
3-(4-dimethylamino-benzylidene)-1,3-dihydro-indol-2-one was from
Salor (Tokyo, Japan) or prepared as follows: equimolar quantities of
oxindole (Aldrich, 318 mg, 2.4 mmol) and p-N,N-dimethylamino benzaldehyde (Aldrich, 350 mg, 2.4 mmol) were mixed in MeOH (5 ml).
Piperidine was added (95 µl, 1 mmol), and the reaction was stirred at
reflux for 3.5 h. The reaction was cooled to room temperature, and
the orange solid was filtered and dried. 1H NMR and mass
spectroscopy (M + 1)+265 data were consistent with the
desired structure.
Site-directed Mutagenesis--
The cDNA encoding human KDR
was cloned from a human umbilical vein endothelial cell cDNA
library as described (8). The 5' end of the cytoplasmic region of KDR
was modified by polymerase chain reaction to facilitate subsequent
cloning steps by introduction of two silent mutations into the coding
sequence to generate a KpnI (ACC 65 I) site at residues
Gly800-Tyr801-Leu802 and to
eliminate the endogenous BamHI site at Asp807.
The cytoplasmic region of KDR was then cloned as a GST fusion into
pBluebac 4 (Invitrogen). Using this construct as the template, site-directed mutagenesis was performed with QuikChangeTM
site-directed mutagenesis kit (Stratagene), according to the protocol
of the manufacturer. Point mutations were introduced with the
oligonucleotides 5'-GGT GCC TTT GGC CAA GAG ATT GAA GCA GAT GC-3' and
5'-GC ATC TGC TTC AAT CTC TTG GCC AAA GGC ACC-3' for the V848E mutant,
5'-G GCC CGG GAT ATT TTT AAA GAT CCA GAT TAT GTC AG-3' and 5'-CT GAC
ATA ATC TGG ATC TTT AAA AAT ATC CCG GGC C-3' for the Y1054F mutant,
5'-CGG GAT ATT TAT AAA GAT CCA GAT TTT GTC AGA AAA GGA G-3' and 5'-C
TCC TTT TCT GAC AAA ATC TGG ATC TTT ATA AAT ATC CCG-3' for the Y1059F
mutant, and 5'-GCC CGG GAT ATT TTT AAA GAT CCA GAT TTT GTC AGA AAA
GG-3' and 5'-CC TTT TCT GAC AAA ATC TGG ATC TTT AAA AAT ATC CCG GGC-3'
for the Y1054F/Y1059F double mutant. The resultant changes were
confirmed by DNA sequencing using an ABI 377 sequencer.
Expression and Purification of the GST-fused KDR Cytoplasmic
Domain in Insect Cells--
The above cDNA constructs encoding
either the wild-type or mutant cytoplasmic domain of KDR fused with GST
were transfected into insect Sf21 cells with
Bac-N-BlueTM transfection kit (Invitrogen) according to the
protocol of the manufacturer. Recombinant virus preparation and protein
expression were done as described by Kendall and Thomas (8). Cell
pellets were lysed in Buffer A (50 mM Tris-HCl, pH 7.5; 0.5 M NaCl; 5 mM dithiothreitol; 1 mM
EDTA; 10% glycerol; 10 µg/ml each leupeptin, pepstatin A, and
aprotinin; and 1 mM phenylmethylsuflonyl fluoride) containing 0.5% Triton X-100, and the lysate was spun at 100,000 × g for 1 h at 4 °C. The resulting supernatant was
batch adsorbed to glutathione-Sepharose 4B resin (Amersham Pharmacia
Biotech) that was then transferred to a column and washed with 10 volumes of the above buffer followed by 10 volumes of Buffer A/0.05%
Triton X-100 and eluted with 10 mM reduced glutathione in
Buffer A/0.05% Triton X-100, pH 7.8. Protein concentration was
measured with the Bio-Rad protein assay kit, and peak protein fractions
were pooled and dialyzed against Buffer A containing 0.05% Triton
X-100 and 50% glycerol. The purity of the kinases was assessed by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie
Brilliant Blue R-250 staining. The resulting protein preparations were
generally 80-95% pure.
Phosphorylation Assays--
Phosphorylation assays were carried
out with 5 nM enzyme in kinase buffer (20 mM
Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM
dithiothreitol, 100 mM NaCl, and 500 µg/ml bovine serum
albumin) for 15 min at 25 °C in a total volume of 50 µl. Reactions
were stopped by the addition of an equal volume of 30% trichloroacetic
acid/0.1 M sodium pyrophosphate. Precipitates were formed
by incubation at 4 °C for 15 min and collected on Millipore filter
plates (MAFC NOB 10) by filtration. Optiphase Supermix (Wallac) was
added (50 µl), and the plates were counted using a Wallac 1450 Microbeta plate counter. Raw cpms were converted and expressed as nmol
of phosphate/min/mg of protein and analyzed using the Grafit software package (19).
Autophosphorylation Assay--
Twelve ng of either
KDRcyt or KDRcytY1054F/Y1059F were incubated in
reaction buffer with either increasing concentrations of ATP or 1 mM ATP in a total volume of 20 µl. The reaction was stopped by the addition of an equal volume of 2× SDS-PAGE sample buffer (Bio-Rad) and boiled for 5 min. Reaction products were separated
by 7.5% SDS-PAGE and analyzed by a Western blot probed with the
anti-phosphotyrosine antibody PY20 (Transduction Laboratories), visualized using the ECL detection kit, and quantified by scanning densitometry (Molecular Dynamics).
Kinase Activation--
KDRcyt (150 nM)
was incubated with 0-4 mM ATP or with just 1 mM ATP in kinase buffer for 10 min at 37 °C. Activation
was stopped by diluting 10-fold with enzyme dilution buffer (50 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 100 mM NaCl, 100 µg/ml bovine serum albumin) and cooling
quickly on ice. Activated KDRcyt (1.5 nM) was
incubated with 10 µM/5 µCi of
[
-33P]ATP and 0.3 mg/ml pEY in kinase buffer for 5 min
at 37 °C. Reactions were stopped and processed as described
above. The mutant enzymes were activated by incubating 150-400
nM enzyme with 1 mM ATP, and activity was
tested as described for the wild-type enzyme.
Enzyme Kinetics--
Kinetic constants were determined by
arranging a two-dimensional array across a 96-well microtiter plate:
increasing ATP on the x axis and increasing pEY in the y direction.
Reactions were done as described above. Kinetic analysis was done using
the following equations (20).
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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These equations describe the ternary complex, competitive
inhibition, and noncompetitive inhibition, respectively, where v is the measured velocity; KA and
KB are the Michaelis constants for substrates A
and B, respectively; and KiA and
KiB are dissociations constants for
substrates A and B from EA and EB, respectively. Kinetic constants were
determined from a nonlinear least squares best fit of the data
(19).
Inhibitor IC50 and Ki
Determinations--
Unactivated (nonphosphorylated)
KDRcyt (10 nM) was incubated with 25 µM/5 µCi of [
-33P]ATP, 0.3 mg/ml pEY,
and inhibitors diluted in Me2SO (final Me2SO concentration of 5%) in kinase buffer for 15 min at 22 °C.
KDRcyt enzyme, activated as described above, was diluted to
a final concentration of 1.5 nM in kinase buffer containing
25 µM/10 µCi of [
-33P]ATP and 0.3 mg/ml pEY and then incubated in the presence of increasing
concentrations of inhibitor for 15 min at 22 °C and processed as
described above.
Molecular Modeling--
Homology models of the unactivated
(nonphosphorylated) and the activated (phosphorylated) forms of the KDR
kinase were based on the published coordinates of the FGF receptor 1 (FGFR1) (21) and insulin receptor (22) kinases, respectively. The
sequences of the FGFR1, insulin receptor kinase (IRK) and KDR were
aligned by hand based on conserved structural elements, and the
three-dimensional models were generated using the program LOOK (23).
Docking of the ligands into these models was done manually, based on
the crystallographically established binding of staurosporine in PKA (24) and SU4984 in FGFR1 (25). The enzyme-ligand complex was then
minimized using the CHARMM force field as implemented in Quanta97 (26).
The ligand and the side chains of any residue within 5.0 Å were
allowed to move, and the remainder of the enzyme was fixed. The models
were considered to be minimized when the rms force was <0.01
kcal/mol/Å.
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RESULTS |
Identification of the Sequence of Enzymatically Active KDR
Kinase--
The amino acid sequences deduced from three independent
clones of human KDR from a human umbilical vein endothelial cell
cDNA library each contain a valine residue at position 848 rather
than the glutamic acid residue listed in the published sequence (Ref. 6; GenBankTM accession number L04947). This residue is
located in the N-terminal portion of the kinase domain just C-terminal
to the glycine-rich loop. Molecular modeling of the KDR kinase sequence
into the known crystal structure of the homologous FGFR1 tyrosine
kinase (21) positions the side chain of amino acid 848 in the ATP
binding site in proximity of the adenine ring of ATP (data not shown). Valine is the most prevalent amino acid found at this position in both
tyrosine and serine/threonine kinases (27), and it could stabilize the
binding of ATP through hydrophobic interactions with the adenine ring.
Modeling of the glutamic acid residue in this position generates a
structure in which the negatively charged carboxyl group could
interfere with ATP binding and suggests that the kinase might be less
active. Although not obvious from the modeled structure because this
carboxyl group could become protonated, purified KDRcyt-
Glu848 is unable to autophosphorylate in the presence of 1 mM ATP (Fig. 1). The mutant
protein also cannot catalyze phosphorylation of an exogenous peptide
substrate but can itself be phosphorylated by a truncated version of
the wild-type kinase (data not shown). In contrast,
KDRcytVal848 isolated in a >99%
tyrosine-dephosphorylated form is a functional kinase (Fig. 1), as
demonstrated by autophosphorylation in the presence of 1 mM
ATP/10 mM MgCl2 and the ability to
phosphorylate pEY. All subsequent characterization was carried out
using the active cytoplasmic region of the KDR kinase containing
Val848, denoted KDRcyt.

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Fig. 1.
Tyrosine phosphorylation of
KDRcyt mutants. Twelve or 120 ng of purified
KDRcytGlu848 (E848) and
KDRcytVal848 (V848) were
incubated either with (+) or without ( ) 1 mM ATP at
37 °C for10 min. The reaction products were separated by SDS-PAGE
and analyzed by Western blots probed with either an
anti-phosphotyrosine (left panel) or a KDR-specific
(right panel) antibody.
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KDR Tyrosine Kinase Activation--
The ability of
KDRcyt to autophosphorylate and the effect of
phosphorylation on catalytic activation of the kinase enyzmatic activity were analyzed. KDRcyt was incubated with 10 mM MgCl2 in the presence of increasing
concentrations of ATP. Fig. 2 shows the
quantitative densitometric scan of an anti-phosphotyrosine Western blot
(Fig. 2, inset) measuring the ATP-dependent
increase in tyrosine phosphorylation of the recombinant
KDRcyt enzyme. A plot of phosphorylation versus
ATP concentration (Fig. 2) yields an apparent
KmATP of 0.29 mM for
autophosphorylation.

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Fig. 2.
Autophosphorylation of
KDRcyt. KDRcyt was phosphorylated with
increasing ATP concentrations for 10 min at 37 °C. The reaction
products were analyzed by a Western blot probed with an
anti-phosphotyrosine antibody (inset) and plotted as
relative activity quantified by densitometric scanning
versus ATP concentration.
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To determine the effect of autophosphorylation on the activation of the
kinase catalytic activity, KDRcyt was first preincubated with increasing concentrations of ATP in the presence of
MgCl2, the reaction mixture was diluted, and ATP was
adjusted to a final concentration of 10 µM. Phosphate
incorporation into the exogenous polypeptide substrate pEY was
measured. Without prior incubation with ATP, the level of
KDRcyt autophosphorylation at 10 µM ATP was
very low (Fig. 2), and any additional activation during the second
reaction was minimal. The concentration of ATP required for
half-maximal activation of the catalytic activity of the kinase was
0.52 mM, as shown in Fig.
3.

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Fig. 3.
ATP-dependent activation of
KDRcyt catalytic activity. KDRcyt was
activated by incubation in the presence of increasing concentrations of
ATP at 37 °C for 10 min. The enzyme was then diluted, adjusting the
ATP concentration to 10 µM; pEY was added to a final
concentration of 0.30 mg/ml; and the reactions were incubated for 5 min
at 22 °C. 33P incorporation into pEY substrate was
measured and plotted versus the ATP concentration used in
the initial activation reaction.
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To further study the mechanism of activation of the KDR kinase, one or
both of the tyrosines 1054 and 1059, known autophosphorylation sites in
the activation loop (17), were changed by site-directed mutagenesis to
phenylalanine residues. Activation of the purified mutant and wild-type
enzymes was carried out with 1 mM ATP, and the resulting
activities were measured using the exogenous pEY substrate. As shown in
Fig. 4, the basal activities of these
wild-type and mutant kinases were essentially equivalent. In contrast,
prephosphorylated wild-type enzyme exhibited an 18-fold increase in
tyrosine kinase activity, whereas the KDRcytY1054F and
KDRcytY1059F single mutants were each activated only
6-fold. The double mutant KDRcytY1054F/Y1059F showed less
than a 2-fold increase in activity demonstrating the importance of both
of these tyrosine residues for the activation of KDR tyrosine kinase
activity. The increased activities resulting from preinculation in 1 mM ATP were not the result of instability of the enzyme in
the absence of ATP at 37 °C because the activity of
KDRcyt without the incubation step was equivalent to the
activity of the enzyme incubated at 37 °C without ATP (data not
shown). Despite substitution of the two activation loop tyrosines by
phenylalanine residues, the double mutant still autophosphorylated
(Fig. 5), consistent with the presence of
additional previously identified KDR phosphotyrosine residues (17).

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Fig. 4.
Activation of wild-type and tyrosine to
phenylalanine mutants of KDRcyt. Wild-type and mutant
enzymes were incubated either with (open bar) or without
(filled bar) 1 mM ATP for 10 min at 37 °C.
Each enzyme was then diluted 100-fold, the ATP concentration was
adjusted to 10 µM containing 10 µCi of
[ -33P]ATP, and pEY was added to a final concentration
of 0.30 mg/ml. The reaction was maintained at 37 °C for 5 min, and
33P incorporation into pEY was measured.
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Fig. 5.
Autophosphorylation of the wild-type and
Y1054F/Y1059F double mutant of KDRcyt. Wild-type and
mutant enzymes were incubated either with (+) or without ( ) 1 mM ATP for 10 min at 37 °C, and reactions were stopped
by the addition of sample buffer and boiled. The reaction products were
separated by SDS-PAGE, analyzed on a Western blot probed with an
anti-phosphotyrosine antibody, and visualized by autoradiography.
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Kinetic Mechanism--
The reaction mechanism of the activated
kinase was investigated by a two substrate kinetic analysis varying the
concentrations of both ATP and pEY. Double reciprocal plots of
1/v versus 1/[ATP] and 1/[pEY], displayed in
Fig. 6, show an intersecting line pattern indicative of a sequential mechanism in which a ternary complex composed of the enzyme and both substrates forms before any products are released (20). These data rule out a ping-pong mechanism, indicated
by a parallel line pattern in this graphical analysis, in which one of
the substrates binds, is converted to a product by modification of the
enzyme or a co-enzyme, and is released prior to binding the second
substrate.

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Fig. 6.
Kinetic mechanism of KDRcyt and
KDRcytY1054F/Y1059F. A two substrate analysis of the
mechanism of KDRcyt is shown. Enzyme reactions were carried
out with wild-type (A and B) or mutant
(C and D) enzymes for 5 min at 37 °C either
varying ATP concentrations at a series of fixed pEY concentrations
(0.063 ( ), 0.125 ( ), 0.25 ( ), and 0.50 ( ) mg/ml)
(A) or varying pEY concentrations at several fixed ATP
concentrations (0.05 ( ), 0.10 ( ), 0.20 ( ), and 0.30 ( )
mM) (B). In the case of the mutant enzyme,
reactions were carried out either varying the concentrations of ATP at
fixed pEY concentrations (0.19 ( ), 0.38 ( ), 0.75 ( ), and 1.5 ( ) mg/ml) (C) or varying the concentrations of pEY at
fixed ATP concentrations (0.12 ( ), 0.25 ( ), 0.50 ( ), and 1.0 ( ) mM) (D). Incorporation of 33P
was measured, and the values were fit to the equation for ternary
complex formation (Equation 1) and plotted as 1/v
versus 1/[S].
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The kinetic constants, determined by nonlinear curve fitting (19), are
displayed in Table I. The Michaelis
constants KmATP and
KmpEY are similar to the dissociation
constants KiATP (KiA) and
KipEY
(KiB) calculated from Equation 1,
indicating that each substrate binds independently. This is consistent
with a rapid equilibrium model in which association and dissociation of
each substrate is rapid compared with catalysis and binding is random.
Patterns of product inhibition can be used to distinguish reaction
mechanisms (20). The results of inhibitor studies using the product ADP
and the dead-end substrate AMP-PCP are plotted in Fig.
7. Both inhibitors show a competitive
pattern versus ATP and are noncompetitive with the peptide
substrate, further supporting a random substrate addition mechanism. A
product inhibitor of the peptide substrate, which is not available,
would be required to rule out an ordered mechanism in which ATP binds first (20). The reaction mechanism of the wild-type unactivated form of
the kinase could not be determined because autophosphorylation and
consequently enzyme activation would occur during the reaction at high
ATP concentrations, as shown in Fig. 2.
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Table I
Kinetic constants of activated KDRcyt and KDRcyt
Y1054F/Y1059F
Kinetic constants were calculated from the equation for ternary complex
formation (Equation 1).
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Fig. 7.
Inhibition of activated KDRcyt by
ADP and AMP-PCP. Enzyme reactions were done with activated
KDRcyt for 5 min at 37 °C, varying ATP concentrations at
1 mg/ml pEY (A and C) or varying pEY
concentrations at 0.3 mM ATP. Inhibition of these reactions
by either 0 ( ), 0.5 ( ), 1.0 ( ), 2.0 ( ), and 4.0 ( )
mM AMP-PCP (A and B) or 0 ( ), 0.06 ( ), 0.12 ( ), 0.24 ( ), and 0.48 ( ) mM ADP
(C and D) was monitored. Incorporation of
33P into pEY was measured and the data were fit to the
equations for competitive (Equation 2) and noncompetitive (Equation 3)
inhibition and plotted as 1/v versus
1/[S].
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A two substrate analysis was done for the
KDRcytY1054F/Y1059F double mutant in an effort to model the
unactivated kinase (Fig. 6). The KmATP
and KmpEY values and
KiATP and
KipEY were marginally different (Table
I), consistent with some synergy between substrates (28). Similar to
the wild-type enzyme, ADP and AMP-PCP are both competitive with respect
to ATP and are noncompetitive with the peptide substrate (data not
shown). As shown in Table I, the Km values for ATP
and pEY in the double mutant are increased by 6.9- and 2.7-fold,
respectively, compared with the wild-type enzyme, with essentially no
change in the turnover numbers.
Inhibition of Unactivated Versus Activated Enzyme--
The ability
of kinase antagonists to inhibit selectively either the activated or
unactivated form of the enzyme was tested. Two commercially available
inhibitors, staurosporine and the indolinone 3-(4-dimethylamino-benzylidene)-1,3-dihydro-indol-2-one (Fig. 8), were found to bind competitively with
ATP and noncompetitively with the polypeptide substrate (data not
shown). KDRcyt either was used without a prior activation
step or was activated with 1 mM ATP as described under
"Experimental Procedures." The reaction measuring the incorporation
of phosphate into the pEY substrate was done at a low ATP concentration
(25 µM), at which little, if any, autophosphorylation
(Fig. 2) and activation (Fig. 3) occurred in the time course of the
assay. Under these conditions, staurosporine exhibits a modest 6-fold
selectivity for the activated (Ki = 2.5 nM) compared with the unactivated (Ki = 16 nM) form of the enzyme (Table
II). In contrast, the indolinone is an
approximately 100-fold more potent inhibitor of the unactivated compared with the activated enzyme under these same assay conditions, consistent with a higher affinity for the unactivated form
(Ki = 0.04 µM) compared with the
activated form of the enzyme (Ki = 4 µM).

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Fig. 8.
KDR inhibitors. Structures of two
compounds found to be potent inhibitors of the KDR kinase:
A, 3-(4-dimethylamino-benzylidene)-1,3-dihydro-indol-2-one;
B, staurosporine.
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Table II
Inhibition of wild-type and Y1054F/Y1059F mutant KDRcyt
Ki values of staurosporine,
3-(4-dimethylamino-benzylidene)-1,3-dihydro-indol-2-one (indolinone),
and ADP were measured for the activated and mutant form of the enzyme.
The Ki for the unactivated form was calculated based
on the equation IC50 = (1 + ([S]/Km))Ki using the
experimentally determined IC50 value of the unactivated
wild-type enzyme at 25 µM ATP and the measured
KmATP value of the Y1054F/Y1059F mutant.
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The binding modes of staurosporine and the indolinone docked into the
ATP binding site of the KDR model are shown in Fig. 9. These models suggest that the
bidentate hydrogen bonds formed by N1 and N6 of ATP are mimicked by the
lactam moiety of the inhibitors. In addition to these hydrogen bonds,
there are several hydrophobic contacts between Leu840,
Val848, Lys868, Val899,
Val916, Val918, Cys919,
Gly922, Leu1035, Cys1045, and the
aromatic regions of the inhibitors (not shown). Comparisons of the KDR
ATP binding sites from the FGFR1- and IRK-derived models revealed that
although the hydrogen bonds and the hydrophobic pocket formed by
Val848, Lys868, Val899, and
Val916 are preserved in the unactivated FGFR1-like enzyme,
the position of Leu840 is much closer to the binding site
in the activated IRK-like conformation. Because staurosporine is flat
in the region of the binding site formed by Leu840, the
change in the position of this residue on activation does not generate
unfavorable steric interactions. In contrast, the dimethylamino-phenyl ring from the inhibitor
3-(4-dimethylamino-benzylidene)-1,3-dihydro-indol-2-one occupies and
extends beyond the same region of the binding site. In the more open
conformation of the unphosphorylated kinase, the phenyl moiety of the
indolinone can also be accommodated. However, in the active
phosphorylated form of the enzyme, this ring appears to be partially
occluded by the Leu840 side chain. Presumably, the energy
required for the enzyme conformational change that facilitates the
binding of this inhibitor lowers its affinity for this form of the
kinase. Consistent with this hypothesis, the crystal structure of
staurosporine in complex with PKA (24) shows inhibitor induction of a
more open kinase binding site conformation.

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Fig. 9.
The conformations of staurosporine and
3-(4-dimethylamino-benzylidene)-1,3-dihydro-indol-2-one modeled into
the ATP binding site of KDR. The KDR polypeptide backbone model,
based on the nonphosphorylated FGFR1 crystallographic coordinates, is
shown in blue, with the glycine-rich flap including the
Leu840 side chain highlighted in red. The
shifted position of the glycine-rich flap including the Leu side chain,
modeled from the phosphorylated IRK coordinates, is shown in
yellow. The two models were aligned by superposition of the
energy-minimized position of staurosporine (white). It was
not possible to minimize the indolinone (green) in the IRK
based model because of unfavorable steric interactions.
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DISCUSSION |
KDR is critical for VEGF-induced mitogenic and chemotactic
signaling in vascular endothelial cells. To study the regulation and
enzymatic activities of the tyrosine kinase portion of this receptor,
we cloned the cytoplasmic domain and expressed it as a GST fusion
protein. The GST domain not only provided a simple and rapid affinity
purification of the kinase but also dimerized the protein (data not
shown) in a manner that is presumably similar to that which is promoted
by binding of the dimeric VEGF ligand, thereby facilitating
transphosphorylation. Kinase domains from other transmembrane tyrosine
kinase receptors have been studied and found to be good models for
investigating the catalytic activity and activation of full-length
receptor kinases (29, 30).
Comparing the predicted amino acid sequence of the KDR cDNA clone
that we isolated to the published sequence (6) revealed a difference at
amino acid position 848. In our cDNA clone, the glutamic acid was
replaced by a valine residue. The equivalent valine residue is very
conserved in both the tyrosine and serine/threonine kinases families of
kinases and is reported to be a valine in the amino acid sequences
deduced from both of the published mouse KDR (flk-1) sequences (31, 32)
(GenBankTM accession numbers X59397 and X70842) and the
closely related flt-1 (GenBankTM accession number X51602)
and flt-4 (GenBankTM accession number X68203) receptor
cDNA sequences (5, 33). Molecular modeling of the KDR kinase domain
based on the FGFR1 coordinates (21) suggests that a negatively charged
glutamic acid side chain at this position could diminish ATP binding.
The purified GST-KDRcytGlu848 fusion protein
was soluble and could be phosphorylated by a truncated KDR "core"
kinase but was unable either to autophosphorylate or to catalyze
phosphate incorporation into an exogenous polypeptide substrate. The
lack of activity of GST-KDRcytGlu848 might be
attributable either to inability of the properly folded enzyme to bind
substrate or to improper folding of the kinase domains.
The cytoplasmic domains of KDR contain 19 tyrosine residues, 4 of which
are known to be autophosphorylation sites (17). Two of these sites (951 and 996) are located in a poorly conserved sequence known as the type
III receptor tyrosine kinase insert loop (18). Phosphorylation of
homologous tyrosine residues in PDGF
-receptor provide docking sites
for other downstream signal transduction molecules to form a signaling
complex (18). These tyrosine residues might have a similar function in
KDR. The remaining two tyrosine phosphorylation sites (1054 and 1059)
are located in the activation loop of the kinase. In other receptor
tyrosine kinases, phosphorylation of tyrosine residues in the
activation loop has been associated with an activation of kinase
activity (34-36).
In an effort to investigate the activation mechanism of KDR, we changed
the two tyrosines in the activation loop, individually and together, to
phenylalanine residues and compared their ability to activate to that
of the wild-type protein. The GST-fused
KDRcytVal848 wild-type kinase isolated in the
dephosphorylated state could undergo autophosphorylation with
concomitant 18-fold activation at physiologic magnesium/ATP
concentrations. Each of the kinases containing a single tyrosine to
phenylalanine mutation was also able to activate, but by only 6-fold.
However, the catalytic activity of the double mutant increased by less
than 2-fold following incubation with physiologic ATP concentrations.
The wild-type enzyme and three mutant enzymes had similar basal levels
of kinase activity in the absence of phosphorylation. Not only the
single but also the double mutants were capable of autophosphorylation
at other sites, presumably including the two other tyrosine
phosphorylation sites in the kinase insert domain. These data indicate
that phosphorylation of both activation loop tyrosines are required for
full activation of the kinase activity of KDR. This result is similar
to those obtained with the insulin (34), hepatocyte growth factor (35), and FGFR1 kinases (36), in which phosphorylation of the activation loop
tyrosines is required for the full activation of kinase activity.
The kinetics of the KDR reaction were studied in a series of
experiments with the preactivated wild-type enzyme and the double mutant. Results from a two substrate analysis are typical for a
reaction mechanism that proceeds through formation of a ternary complex, indicating a sequential mechanism (20) in which both substrates bind prior to release of products. Inhibitor profiles from
experiments with either the product ADP or the dead-end substrate AMP-PCP are consistent with either a steady state ordered mechanism with ATP bound first or a rapid equilibrium Bi Bi (reaction with two
substrates producing two products) random mechanism (20). A suitable
peptide substrate product inhibitor is not available that can be used
to distinguish between the obligatory initial binding of ATP and random
addition of substrates. The catalytic mechanism of the full-length
ligand-stimulated activated PDGF
-receptor kinase is also found to
be a sequential rather than ping-pong mechanism (37), and the epidermal
growth factor receptor (38) and insulin receptor kinase (39) reactions
are reported to occur by a sequential Bi Bi rapid equilibrium random
mechanism. In addition, sequential mechanisms are reported for other
tyrosine kinases, such as csk (40) and pp60 src (41), and for the
serine/threonine kinases, cAMP-dependent protein kinase
(42), and p38 kinase (43), the latter utilizing an ordered sequential
mechanism in which the peptide substrate binds first (43).
Measurement of kinetic rate constants for the nonactivated KDR kinase
is difficult because activation can occur at high ATP concentrations
during the course of the experiment. The double mutant provides a model
of the unactivated kinase that cannot be substantially activated by
autophosphorylation and appears to retain an unactivated ATP binding
site that resembles the wild-type enzyme, as reflected by its similar
intrinsic catalytic activity and binding of inhibitors. Therefore, the
mechanism of catalytic activation can be studied by comparing kinetic
constants of the wild-type and double tyrosine mutant enzymes.
Activation of wild-type KDR by autophosphorylation is accompanied by 7- and 3-fold decreases in KmATP and
KmpEY, respectively, with no alteration in kcat. These data suggest that activation of
KDR is a result of an increased affinity for both substrates and not a
change in the intrinsic catalytic activity of the enzyme, as is
reported for the full-length ligand-stimulated epidermal growth factor receptor kinase (38). In addition, ligand activation of PDGF
-receptor is reported to result in 4- and 2.3-fold decreases in the
Km values for ATP and peptide substrate,
respectively (37). However, Kovalenko et al. (44) found no
change in the Km for the peptide substrate but did
observe a 3-fold decrease in the KmATP
on activation.
Surprisingly, the largely unactivable double mutant could undergo
autophosphorylation at high ATP concentrations. The unactivated kinase
retains a basal level of activity and can still phosphorylate the
exogenous substrate, albeit at a much reduced rate compared with the
activated kinase at low ATP concentrations. However, the
kcat values of the wild-type and double mutant
enzymes are essentially the same, indicating that at saturating
concentrations of substrates, the same Vmax is
achievable. Phosphorylation of the activation loop tyrosines in KDR
leads to a lowering of the Km for both substrates,
which might ensure that the kinase is operating either at or near
saturating substrate concentrations. The resulting increased level of
phosphorylation could be required to outpace dephosphorylation by
phosphatases, thereby sustaining a phosphorylation signal long enough
to form a competent signal transduction complex. Autophosphorylation of
the intact PDGF
-receptor stimulated by PDGF containing a similar
activation loop mutation was also observed (45). Phosphorylation of the
activation loop tyrosine was reported to be important for the
activation of kinase activity but not for autophosphorylation.
Tyrosines in the juxtamembrane region that are known to be
phosphorylation sites are critical for autophosphorylation, leading to
the suggestion that multiple events are required for full activation of
PDGF
-receptor. By analogy, phosphorylation of other tyrosines
residues in KDR may be required for additional activation events
in vivo. Phosphorylation events mediated by other kinases
in vivo might also play a role in KDR activation.
Finally, we find that inhibitors that are competitive with ATP can
interact differently with the activated and unactivated forms of the
kinase. Staurosporine, a nonselective kinase inhibitor, has an
approximately 6-fold higher affinity for the activated than for the
unactivated wild-type kinase and double mutant. ADP, as expected, binds
with higher affinity to the activated form of the enzyme, mimicking ATP
binding. However, a more selective kinase inhibitor of the indolinone
structural class (46) binds with an approximately 40- and 100-fold
higher affinity to the double mutant and unactivated wild-type enzymes,
respectively, compared with the activated wild-type kinase. These
results indicate that conformational changes in the ATP binding site
probably occur upon activation consistent with molecular modeling in
which staurosporine appears to be better accommodated by a slightly
more open binding site in the active kinase, whereas the indolinone
would require a conformational change in the position of the
glycine-rich flap. The partial occlusion of the inhibitor binding site
by Leu-840 and the generation of unfavorable steric interactions is
likely to decrease the affinity of the indolinone for the activated
kinase. The double mutant and unactivated wild-type enzymes bind
inhibitors, including ADP, with similar affinities, suggesting that the
double mutant is a good surrogate for the unactivated wild-type enzyme.
Kovalenko et al. (44) found that a tyrphostin (AG1295)
displays a different mode of inhibition of activated and nonactivated PDGF
-receptor, indicating conformational changes in the ATP binding
site that alter inhibitor binding. Furthermore, they observed less than
a 2-fold decrease in the Ki of this inhibitor on
receptor kinase activation with ligand. The KDR inhibitor
3-(4-dimethylamino-benzylidene)-1,3-dihydro-indol-2-one, however, shows
no change in the mode of
inhibition2 but displays a
dramatic decrease in Ki for the activated kinase.
Inhibition of the unactivated form of the kinase might be
therapeutically sufficient. Such an inhibitor has the advantage of
competing with a form of the enzyme that has decreased affinity for ATP
and could prevent catalytic activation of the enzyme. These data
demonstrate that not only can inhibitors be found that are
kinase-selective, but also conformational selectivity within the same
molecule can be achieved.