From the Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook, New York 11794-8661
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ABSTRACT |
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c-Abl is a non-receptor tyrosine kinase that is
involved in a variety of signaling pathways. Activated forms of c-Abl
are associated with some forms of human leukemia. Presently, no high resolution structure of the tyrosine kinase domain of Abl is available. We have developed a structural homology model of the catalytic domain
of Abl based on the crystal structure of the insulin receptor tyrosine
kinase. Using this model as a guide, we selected residues near the
active site predicted to play a role in peptide/protein substrate
recognition. We expressed and purified 15 mutant forms of Abl with
single amino acid substitutions at these positions and tested their
peptide substrate specificity. We report here the identification of
seven residues involved in recognition of the P The c-abl proto-oncogene encodes a multidomain
non-receptor tyrosine kinase that is expressed ubiquitously in human
tissues (reviewed in Refs. 1-4). Mutant forms of c-abl are
found in patients with Philadelphia chromosome-positive chronic
myelogenous leukemia and acute lymphocytic leukemia (1-4). In these
diseases, a chromosomal translocation event produces a chimeric
oncogene consisting of 5'-sequences of bcr fused to
abl. The BCR-Abl fusion protein has elevated tyrosine kinase
activity relative to c-Abl, and the tyrosine kinase activity of the
BCR-Abl fusion protein is necessary for disease progression. Similarly,
tyrosine kinase activity is necessary for transformation of fibroblasts
or hematopoietic cells by BCR-Abl (5, 6).
In addition to its tyrosine kinase catalytic domain, c-Abl has a short
amino-terminal unique domain followed by SH3 and SH2 domains (1-4).
This domain organization is found in many non-receptor tyrosine
kinases. Abl also possesses a large carboxyl-terminal region that
includes a DNA-binding domain, an F-actin-binding domain, a nuclear
localization signal, and a proline-rich region implicated in mediating
protein-protein interactions. Studies aimed at understanding the normal
physiological role of c-Abl have shown the enzyme to be involved in
signal transduction, cytoskeletal rearrangement, RNA polymerase II
activation, DNA repair, and cell cycle control (1-4). c-Abl has been
shown to physically associate with at least seven unique proteins,
including p53 and the nuclear Rb protein (4). Mice with targeted
disruptions in the c-abl gene have high neonatal mortality
rates and are more susceptible to infection, suggesting a role for
c-abl in B-lymphocyte development (7).
At least eight in vivo substrates for Abl have been
identified (4). The amino acid sequences surrounding the
phosphorylation sites for two of these proteins, RNA polymerase II (8)
and c-Crk (9), have been described. These sequences do not share a
common primary sequence motif, suggesting that Abl may have a broad
range of substrate specificity. Studies using synthetic peptides have
been used to examine the substrate specificity of Abl and to define any
primary sequence determinants for substrate recognition (10, 11). These
studies suggest that, although Abl does not have an absolute consensus
sequence for phosphorylation, the best in vitro peptide
substrates for Abl contain the sequence Ile-Tyr-Ala-Xaa-Pro, where Xaa
is any amino acid. These studies indicate that the P The molecular basis of peptide/protein substrate recognition for
tyrosine kinases is not well understood. Presently, a single high
resolution crystal structure of a tyrosine kinase in complex with
peptide substrate is available: the tyrosine kinase domain of the
insulin receptor (IRK)1
complexed with a peptide substrate (12). This structure reveals interactions between enzyme and substrate that govern substrate specificity. Two adjacent hydrophobic pockets on the surface of the
C-terminal lobe of IRK accommodate Met side chains C-terminal to the
phosphorylated tyrosine on the peptide substrate. The crystal structure
of the activated IRK-peptide complex provides a structural basis for
understanding the primary signaling specificity of IRK and serves as a
general model for tyrosine kinase substrate recognition.
In this paper, we have developed a molecular homology model of the
kinase catalytic domain of Abl (Abl-CAT) to help identify amino acids
that may be important in substrate recognition. A similar approach was
used to propose a molecular model of the Bruton tyrosine kinase and to
provide a structural basis for understanding mutations in this enzyme
associated with the disease X-linked agammaglobulinemia (13). Our
molecular homology model is based on the crystal structure of the
ternary complex of IRK with peptide substrate and AMP-PNP bound (12).
Using the model as a guide, we have targeted seven residues in Abl-CAT
for amino acid substitutions to examine effects on substrate
specificity. Site-directed mutants of Abl-CAT were engineered and
tested with a series of peptide substrates to monitor changes in
specificity. Kinetic analyses of these mutants with the peptide
substrates show distinct changes in substrate preferences.
Homology Model--
Amino acids 362-625 of v-Abl were used to
generate primary sequence alignments using the CLUSTALW alignment
algorithm (14) and were imported into Swiss-PDB Viewer (15). The
primary model of the Abl catalytic domain was prepared using the
spatial coordinates of Mutagenesis, Expression, and Purification--
Our experiments
were carried out on the isolated catalytic domain of v-Abl, expressed
in Escherichia coli as described previously (19). The
sequence numbering used is from the gag-Abl fusion protein of the
Abelson murine leukemia virus (20). Mutagenesis of the Abl catalytic
domain was carried out using a QuikChange mutagenesis kit (Stratagene).
Mutagenesis primers complementary to wild-type template were designed
with single, double, or triple nucleotide substitutions. The DNA
sequences encoding the entire catalytic domains of the mutants were
confirmed by DNA sequencing on an ABI373 automated DNA sequencer.
Wild-type and mutant proteins were expressed as glutathione
S-transferase fusion proteins in E. coli strain NB42 and purified using glutathione-agarose
(19). All proteins expressed to similar levels, were of the expected size, and purified to >98% homogeneity.
Peptides--
Synthetic peptides were prepared by solid-phase
synthesis using standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry on an Applied
Biosystems automated 431A peptide synthesizer. Peptides were purified
using semi-preparative reversed-phase high performance liquid
chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to confirm the identity of
the final products.
Kinase Assays--
Tyrosine kinase activity assays were carried
out using two methods. In both cases, the intact glutathione
S-transferase-Abl fusion proteins were used; we showed
previously that the specific activity and substrate specificity of
glutathione S-transferase-Abl are similar to those of
full-length Abl and that velocity versus [enzyme] plots
are linear over the concentrations used here (19). For initial
comparisons of several substrates, phosphocellulose binding assays were
used to measure incorporation of [32P]ATP into peptide
substrates (19, 21). These reactions were carried out in triplicate at
saturating concentrations of ATP (250 µM) and
Mg2+ (10 mM). The reactions were carried out in
volumes of 25 µl, using peptide concentrations of 50 µM
or 1.5 mM and 1 µg of enzyme. Picomoles of phosphate
incorporated into peptides were measured after 20 min as described
(19).
A continuous spectrophotometric assay (22) was used to measure initial
rates of phosphorylation and to determine kinetic constants for some
peptides. Reactions were carried out in volumes of 100 µl using 5 µg of purified enzyme. Saturating concentrations of ATP (500 µM) and Mg2+ (10 mM) were used,
and peptide concentrations ranged from 50 µM to 2 mM. Data were sampled every 30 s to determine rates of phosphorylation. Initial rates (<10% of the reaction) were measured in triplicate. The initial rate values were averaged, and
Vmax and Km were determined
by fitting to the hyperbolic velocity versus [substrate]
curves using the program MacCurve Fit. For some combinations of
peptides and mutants, initial experiments established that their
Km values were in the millimolar range (>2
mM). In addition, we observed that high concentrations of
these peptides were inhibitory. Thus, concentrations over 2 mM were not employed, and we were unable to determine
Km values accurately using initial rate kinetics.
For these peptides, the complete time courses for phosphorylation were
measured using peptide concentrations less than Km.
In these cases, we analyzed the data graphically as described (23) to
determine Vmax/Km.
Homology Model--
The overall topology of the Abl structural
homology model reflects the typical bilobal structure shared by all
eukaryotic protein kinases (24) with a five-stranded Design of Peptide Substrates--
Previous studies of the in
vitro substrate specificity of Abl have been carried out in this
laboratory and by others (for review, see Ref. 27). We designed two
groups of peptide substrates to examine any changes in specificity at
the P
We chose to use a different series of peptides to examine P Mutations That Affect P+3 Peptide Recognition--
Experiments
with synthetic peptides and peptide libraries have demonstrated that
Abl prefers proline at the P+3 residue of a substrate. In the crystal
structure of IRK, Leu-1219 is part of a binding pocket that surrounds
the P+3 methionine side chain of the peptide substrate (12). In the Abl
homology model, the residue homologous to Leu-1219 is Tyr-569. Tyr-569
is partially solvent-exposed in our model and could adopt the same role
as Leu-1219 in IRK (Fig. 2). Three
separate mutant forms of Abl-CAT were engineered with amino acid
substitutions at Tyr-569: Y569L, Y569A, and Y569W. Alanine was chosen
as a substitute for Tyr-569 to examine the effects of minimizing the
amino acid side chain length. In c-Src, the residue corresponding to
Tyr-569 is a leucine (28), and the specificity of c-Src at the P+3
position differs from that of Abl (10). For this reason, we chose to
introduce leucine as a substitute for Tyr-569. We also mutated Tyr-569
to Trp because the Abl homology model suggests that a large side chain
at this position might cause the putative P+3 substrate-binding pocket
to be too narrow to accommodate amino acids with large side chains such
as proline.
Initial screens of Abl mutants were carried out using peptide
concentrations of 1.5 mM and measuring
[32P]ATP incorporation after 20 min to assess any changes
in substrate phosphorylation (Fig. 3).
Experiments using low (50 µM) peptide concentrations
showed no changes in the rank order of specificity compared with those
using higher peptide concentrations (data not shown). These initial
activity measurements indicated that the Y569W and Y569L mutants had an
altered specificity (Fig. 3); in particular, recognition of Pro at the
P+3 position was greatly reduced. These changes in specificity were
characterized further by more detailed kinetic analyses (Table
II). The kinetic measurements show that
the Y569W mutant phosphorylated the
P+1Ala/P+3Met peptide best, with a
Vmax/Km value of 5.6. The
P+1Met/P+3Pro peptide, the best for the wild
type with a Vmax/Km value of
10.7, was phosphorylated less efficiently by this mutant, with a
Vmax/Km value of 1.3 (Table
II). The phosphorylation of other peptides by the Y569W mutant was
similar to the wild type (Fig. 3 and Table II). The difference in
specificity observed in the Y569W mutant therefore arises from a
decrease in Vmax/Km for the
P+3Pro peptide specifically rather than an overall decrease in the phosphorylation of all peptides. Kinetic measurements also showed a decrease in the phosphorylation of the
P+1Met/P+3Pro peptide by Y569L, with a
Vmax/Km value of 4.4 (wild-type Vmax/Km = 10.7)
(Table II). The Y569A mutant did not display any changes in
phosphorylation of the peptides tested compared with wild-type Abl
(Fig. 3), and we did not carry out kinetic analysis of this mutant.
Mutations That Affect P+1 Peptide Recognition--
Two residues in
Abl-CAT, Phe-521 and Ile-523, were identified as residues that might
contribute to P+1 substrate specificity (Fig. 2). Ile-523 was chosen
based on the homology model. A solvent-accessible surface
representation suggests that Ile-523 is partially solvent-exposed and
forms the edge of a groove into which a substrate amino acid side chain
may fit. Two substitutions were made for Ile-523: I523V and I523A.
These amino acid substitutions were chosen to examine the effect of
shortening the side chain on accommodating P+1 residues. Phe-521 was
selected to test the possibility that residues near the activation loop
contribute to specificity; based on homology to other protein kinases,
the activation loop in Abl is predicted to span residues 500-521. We
examined the effects of smaller (F521A) and larger (F521W) side chains
at this position. Initial measurements of activity were used to assess
changes in specificity (Fig. 4) using a
panel of peptides that vary at the P+1 position. Initial rate
measurements were used to characterize the changes in specificity shown
in the I523V and F521A mutants (Table
III). The I523A and F521W mutants did not
display any changes in specificity and were indistinguishable from
wild-type enzyme in our initial comparisons (Fig. 4). For this reason,
we did not pursue kinetic characterization of these mutants.
The specificity of the I523V mutant shows a change at the P+1 position.
Phosphorylation of the P+1Ala/P+3Met peptide by
the wild type (Vmax/Km = 6.3)
and I523V (Vmax/Km = 10.0)
was comparable (Table III). However, the I523V mutant phosphorylated the P+1Ile/P+3Met peptide ~3.5 times better
than the wild type (Table III). Initial rate measurements on the F521A
mutant showed generally higher
Vmax/Km values for the
P+1Ala/P+3Met, P+1Nle/P+3Met, and
P+1Ile/P+3Met peptides when compared with
wild-type values (Table III). Like wild-type enzyme, the F521A mutant
still phosphorylated the P+1Ala/P+3Met peptide
best, with Vmax/Km = 13.0, compared with the wild type, which has a
Vmax/Km value of 6.3 for the
same peptide.
Mutations That Affect P
Abl-CAT residues Gly-556 and Ser-558 were chosen because these residues
are solvent-accessible in the homology model, in close proximity to
Leu-444 (Fig. 2). We produced G556A, G556V, S558A, and S558N mutants.
The G556A and S558A mutants were indistinguishable from the wild type
in our initial screens (Fig. 5). The G556V mutant, however, has a
reduced preference for Ile at the P
We also chose to mutate Trp-525 of Abl based on our modeling studies.
To identify residues in Abl that may make contacts with the P Indirect Effects--
We examined the possibility that mutations
designed to affect recognition of one position in the substrate might
have effects at other positions as well. Mutants with predicted changes
in P+3 recognition were tested with five peptides containing Met, Ile,
Ala, Glu, or Nle at the P+1 position (Fig. 3). Whereas the pattern of
peptide phosphorylation by Y569W and Y569A closely resembled that of
wild-type Abl, Y569L showed some differences from the wild type (Fig.
3). We examined these differences more closely by kinetic analysis of
P+1Ala/P+3Met and
P+1Nle/P+3Met (Table II). These measurements
showed a decrease in the phosphorylation of
P+1Ala/P+3Met, with
Vmax/Km = 2.4 (wild-type
Vmax/Km = 6.3). Thus, in the
Y569L mutant, a change in the P+3 region has effects on P+1 recognition
as well. We also screened the P+3 mutants with the following peptides
varying at P
We tested for indirect effects using the P+1 and P
We screened the following four P Although Abl is capable of phosphorylating a wide range of peptide
and protein substrates, the best peptide substrates for Abl contain the
sequence Ile-Tyr-Ala-Xaa-Pro, as shown in peptide library studies (10,
11). Ile at the P The mutant forms of Abl described here fall into three classes with
respect to substrate specificity. 1) Two mutants (Y569W and Y569L) have
altered substrate specificity. These mutants no longer prefer proline
at the P+3 position in peptide substrates. 2) Many of the mutants
(e.g. I523V, W525H, and L444E) showed no change in the major
determinants for substrate recognition, but differed from the wild type
in their phosphorylation of other peptide substrates. For example,
mutations aimed at altering recognition of the P The Y569W mutation in Abl has the most dramatic effect on substrate
specificity of the mutants we report here. Wild-type Abl phosphorylates
a peptide substrate with Pro at the P+3 position best. The Y569W mutant
phosphorylates P+1Met/P+3Pro ~10 times less
efficiently than does wild-type Abl (Table II). All other amino acid
side chains tested at the P+3 position of the substrate were
phosphorylated at the same level as in the wild type (Fig. 3). Our
structural model suggests that this change in specificity arises from a
steric clash between the side chain of Trp-569 in the mutant and the
side chain of proline in the substrate. This is not the case with the
other peptide substrates tested that have smaller amino acid side
chains at the P+3 position. Proline at the P+3 position plays a role in
substrate recognition in vivo in at least one case: Abl
phosphorylates c-Crk at Tyr-221 within the sequence Tyr-Ala-Gln-Pro
(9). Phosphorylation by Abl is believed to modulate the protein binding
and transforming activity of Crk (29). Preliminary experiments indicate
that, in contrast to wild-type Abl, the Y569W mutant has no activity
toward Crk in
vitro.3
Mutations predicted to affect substrate recognition at the P+1 position
(I523V) or at the P There are residues in the three-dimensional structures of Src family
kinases that appear to correspond to residues identified in our study
(30, 31). The substrate specificity of Src differs from that of Abl at
the P+3 position (10). Src prefers a phenylalanine at the P+3
position in a peptide substrate, whereas Abl prefers proline. The
residue homologous to tyrosine 569, a residue involved in P+3
specificity in Abl (Fig. 3), is a leucine (Leu-472) in Src. This
sequence difference may account for the differences seen in substrate
specificity; an L472Y mutant of Src might phosphorylate P+3Pro-containing peptides more efficiently. A tryptophan
substitution at this position could prevent large side chain amino
acids from binding in this region, as we observed for Abl.
The substrate specificities of tyrosine kinase catalytic domains are
important in maintaining the fidelity of cellular signal transduction
pathways. This is best illustrated in the case of the RET receptor. A
naturally occurring mutation in the kinase domain of this receptor
changes a methionine residue to a threonine residue in a region
homologous to the region of Abl shown here to be involved in substrate
recognition of the P+1 residue (32, 33). This change affects the
substrate specificity of the enzyme at the P+1 position, changing the
preference from methionine at that position in the substrate to alanine
(10). This mutant form of the RET receptor is implicated in multiple
endocrine neoplasia type 2A (32, 33).
Mutations throughout the Abl protein have been reported previously (4).
Many of these mutations affect the regulation of the enzyme in
vivo. One such mutation, which is sufficient to activate c-Abl
enzymatic activity in vivo, is found in the catalytic domain. This mutation changes a tyrosine to phenylalanine within the
ATP-binding fold of the enzyme (34). Mutations that affect substrate
recognition by the catalytic domain, however, have not been reported
previously. Our studies on Abl have highlighted seven residues as
playing important roles in peptide substrate recognition. We also show
that a single amino acid change of Tyr-569 to tryptophan can affect the
substrate specificity dramatically. The results raise the possibility
of altering tyrosine kinase substrate specificity in vivo by
protein engineering.
1, P+1, and P+3
positions of bound peptide substrate. Mutations in these residues cause
distinct changes in substrate specificity. The results suggest features
of Abl substrate recognition that may be relevant to related tyrosine kinases.
INTRODUCTION
Top
Abstract
Introduction
References
1 (Ile) and the
P+3 (Pro) positions are most important for substrate recognition.
EXPERIMENTAL PROCEDURES
-carbons from the crystal structure of the
ternary form of IRK with peptide substrate and AMP-PNP bound (12). Gaps
in the sequence alignment were ligated manually, minimizing large
steric clashes. Energy minimization and loop insertions were carried out using the Swiss-Model automated modeling system (16). Additional rounds of energy minimization were carried out using Sculpt for Power
Macintosh (17). The stereochemical quality of the model was checked
using PROCHECK Version 3.3 (18), which reports no distorted main-chain
bonds, five distorted main-chain angles, and no distorted planar
groups. The distorted main-chain angles were outside of the predicted
substrate-binding region and do not interfere with our interpretation
of the model. Solvent-accessible surface area was calculated and
visualized using Web Lab Viewer (Molecular Simulations Inc.) with a
1.4-Å probe. Figs. 1A and 2 were prepared using Strata
Studio Pro (Strata Inc., St. George, UT).
RESULTS
-sheet and a
single
-helix in the amino-terminal lobe, responsible for MgATP
binding, and a highly helical carboxyl-terminal lobe (Fig.
1). Based on results for other protein
kinases, the C-terminal lobe is predicted to make most of the contacts
with peptide/protein substrates. The total root mean square deviation
of the polypeptide backbone between the Abl model and the IRK structure
is 1.8 Å. The crystal structure of the activated insulin receptor
catalytic domain with peptide bound (12) served as a model for the
orientation and structure a peptide may adopt in the Abl active site.
Comparison of the C-terminal peptide-binding domains of IRK and Abl
indicated that the greatest differences are in the regions responsible
for binding the P+3 amino acid side chain. The P
1 region differs only
slightly from that of IRK. The activation loop (amino acids 500-521;
see Fig. 1) is extremely flexible when examined using molecular
mechanics (Sculpt, Interactive Simulations, Inc.), and the structure of
the loop in our model is one of many possible conformations.
Additionally, the structure of IRK used for our model is multiply
phosphorylated on the activation loop (12). Abl contains a single
tyrosine within the activation loop, Tyr-513, which is phosphorylated
in vivo and in vitro and is believed to be
involved in enzyme activation (25, 26). Although we believe that our
model represents the activated form of Abl, the activation loop
tyrosine is modeled in its unphosphorylated state.
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Fig. 1.
A, ribbon diagram of the Abl tyrosine
kinase catalytic domain molecular homology model. B,
sequence alignment of the v-Abl and human insulin receptor (IRK)
tyrosine kinase catalytic domains. The sequence shown for Abl is the
portion modeled in A. This portion of Abl is the same as the
region expressed in E. coli for the mutagenesis experiments.
Numbering for Abl is from the gag-Abl fusion protein sequence (20).
Residues mutated in this study are shaded.
Asterisks indicate positions that are identical in Abl and
insulin receptor kinases; colons show positions with
conservative substitutions.
1 and P+1/P+3 positions for engineered mutant forms of Abl
(Table I). (Amino acids in peptide
substrates are designated by their position relative to the
phosphorylated tyrosine. For example, in the sequence
Ile-Tyr-Ala-Ser-Pro, Ile is at the P
1 position, Ala is at
the P+1 position, and Pro is at the P+3 position.) Peptides used to
examine P+1 and P+3 specificity share the sequence
Ser-Arg-Gly-Asp-Tyr-Xaa1-Thr- Xaa2 -Gln-Ile-Gly, where either Xaa1 or Xaa2 is
varied. These peptides are based on a peptide sequence derived from a
phosphorylation site of insulin receptor substrate-1 used previously in
our laboratory to examine the substrate specificity of wild-type Abl at
the P+1, P+2, and P+3 positions (19). In these earlier studies, we
found that amino acids at the P+2 position do not strongly influence substrate recognition. Moreover, in the ternary structure of IRK, specificity in peptide binding is achieved through interactions with
the P+1 and P+3 residues (12). For these reasons, we did not examine
the effects of residue changes at the P+2 position in this study.
Amino acid sequences and designations of peptides used in this study
1
specificity (Table I). This is because, in the context of the insulin
receptor substrate-1 peptides, we did not observe a strong dependence
on the amino acid at the P
1 position for Abl
phosphorylation.2 Peptides
designed to examine specificity at the P
1 position share the sequence
Leu-Ile-Glu-Asp-Ala-Xaa-Tyr-Ala-Ala-Arg-Gly, where Xaa is varied. This
sequence is based on the autophosphorylation site of Src and has been
previously used in our laboratory to examine P
1 specificity in
wild-type Abl (11). Amino acids for the substituted position were
chosen to explore the effect of size, charge, and hydrophobic
character. Because the two groups of peptides are dissimilar in
sequence, we did not attempt to draw conclusions about the relative
importance of P
1 versus P+1/P+3 recognition for each mutant.
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Fig. 2.
Three pairs of models showing amino acids
targeted for mutagenesis. A and B show
residues involved in P 1 substrate specificity. C and
D show residues involved in P+1 specificity. E
and F show the residue involved in P+3 specificity.
A, C, and E, molecular surface
representations of the Abl homology model showing residues targeted for
mutagenesis in red-orange. B, D, and
F, tube schematic of the Abl homology model showing residues
targeted for mutagenesis in ball and stick
representation.
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Fig. 3.
Initial comparisons of wild-type and mutant
forms of Abl (Y569W, Y569L, and Y569A) with P+1/P+3 peptide
variants. Wild-type Abl and the three mutant forms of Abl were
tested with a panel of eight peptides. The incorporation of
[32P]phosphate into peptides was determined after a
20-min reaction using the phosphocellulose paper assay.
Kinetic measurements for wild-type Abl and two mutant forms of Abl
(Y569W and Y569L)
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Fig. 4.
Initial comparisons of wild-type and mutant
forms of Abl (F521A, F521W, I523V, and I523A) with P+1 peptide
variants. Each of the four mutant forms of Abl was tested with the
panel of eight peptides. The incorporation of
[32P]phosphate into peptides was determined after a
20-min reaction using the phosphocellulose paper assay.
Kinetic measurements for wild-type Abl and two mutant forms of Abl
(I523V and F521A)
1 Peptide Recognition--
Isoleucine at
the P
1 position is a strong determinant for substrate recognition in
wild-type Abl (10, 11). We produced Abl mutants with amino acid
substitutions at residues predicted to be near the P
1-binding region
of the enzyme. Abl-CAT residue Leu-444 was chosen as a target for
mutagenesis based the role of the corresponding residue in IRK,
Lys-1085. In IRK, Lys-1085 extends from the
D helix and makes a
water-mediated hydrogen bond with the P
1 residue (Asp) of the
substrate peptide (12). In our model, Leu-444 extends from the
structurally homologous helix and is partially solvent-exposed. We
chose to change Leu-444 to lysine to examine the possibility of a
change in substrate specificity to that of IRK (which prefers Glu at
P
1) (10). An L444E mutation was made to examine the possibility that
introduction of an acidic residue at this position may favor the
binding of a basic amino acid at the P
1 position of peptide
substrate. The L444K and L444E mutants retained the overall preference
for Ile at the P
1 position, although they differed from the wild type in phosphorylation of other substrates (Fig.
5 and Table IV). The L444K mutant has
Vmax/Km values of 8.0 for the
P
1His peptide and 21.0 for the P
1Ile
peptide, whereas wild-type Abl has
Vmax/Km values of 6.8 for
P
1His and 12.3 for P
1Ile (Table
IV). Thus, L444K shows enhanced
recognition of P
1Ile relative to wild-type Abl. The L444E
mutant demonstrated a specificity different from that of L444K. The
Vmax/Km values for the L444E
mutant are 14.5 for P
1His and 18.9 for
P
1Ile (Table IV); thus, there is a selective increase in
P
1His recognition relative to the wild type. Both L444K
and L444E have lower Vmax/Km values for P
1Glu than wild-type Abl. Wild-type enzyme has
a Vmax/Km value of 1.3 for
P
1Glu. The L444K mutant has a
Vmax/Km value of 0.5 for
P
1Glu, and the L444E mutant has a
Vmax/Km value of 0.07, a
19-fold reduction (Table IV).
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Fig. 5.
Initial comparisons of wild-type and mutant
forms of Abl (G556V, G556A, L444K, L444E, S558N, S558A, W525H, and
W525F) with P 1 peptide variants. Each of
the eight mutant forms of Abl shown here was tested with a panel of
five peptides: P
1Ala, P
1Gln,
P
1Ile, P
1Glu, and P
1His. The
incorporation of [32P]phosphate into peptides was
determined after a 20-min reaction using the phosphocellulose paper
assay.
Kinetic measurements for wild-type Abl and six mutant forms of Abl
(L444K, L444E, S558N, G556V, W525H, and W525F)
1 peptides (Fig. 5). Kinetic constants
for those enzyme/peptide combinations with significant changes from the
wild type are shown. To compare phosphorylation of a particular peptide
by mutant versus wild type, the
Vmax/Km value for the mutant was
divided by the value for the wild type. This ratio is given in the last
column.
1 position and an increased
preference for Leu at the P
1 position. For wild-type enzyme, the
Vmax/Km value for
P
1Ile is 12.3. For the G556V mutant, the
Vmax/Km value for
P
1Ile is 4.2 (Table IV). The S558N mutant showed subtle
changes in specificity. The Vmax/Km value for
P
1Ile, 12.0, is similar to the value of 12.3 for
wild-type Abl, and the
Vmax/Km value of this mutant
for P
1His, 5.4, is close to the value of 6.8 for
wild-type Abl (Table IV). The
Vmax/Km value of this mutant
for P
1Glu, 0.08, is substantially lower than the
corresponding value of 1.3 for wild-type Abl phosphorylating
P
1Glu (Table IV).
1 side
chain, we used the activated IRK structure to model Ile (in place of
Asp) at the P
1 position of the peptide substrate. The indole of
Trp-1175 of IRK packs against the side chain of one energetically
favorable rotamer of the modeled Ile. In our Abl model, the residue
homologous to Trp-1175, Trp-525, lies at the bottom of the putative
P
1-binding pocket (Fig. 2). Trp at this position is conserved in
protein-tyrosine kinases (28). We substituted the bulky residues Phe
and His for Trp-525 to minimize perturbations in the structure. The
W525F and W525H mutants preferred the P
1Ile peptide
overall and showed modest changes toward other substrates. For example,
the enzymes showed different abilities to phosphorylate the
P
1His and P
1Glu peptides (Table IV). W525H had a decreased selectivity for P
1His and an increased
selectivity for P
1Glu relative to wild-type Abl (Table
IV). These results suggest that Trp-525 of Abl is in the vicinity of
the P
1 position of bound substrate, although it appears not to play a
dominant role in substrate selection.
1: P
1His, P
1Glu, P
1Gln, P
1Ile, and P
1Ala.
These comparisons showed no differences between wild-type Abl and any
of the mutants (data not shown).
1 mutants as well.
We carried out initial comparisons of the P+1 mutants using four
peptides that vary at the P+3 position:
P+1Met/P+3Met, P+1Met/P+3Thr,
P+1Met/P+3Ala, and
P+1Met/P+3Pro (Fig. 4). In these experiments,
there was no observable difference between the substrate specificities
of wild-type Abl and the P+1 mutants toward the peptides varying at P+3
(Fig. 4). Similarly, we observed no differences between the P+1 mutants
and the wild type in recognition at the P
1 position (data not shown).
1 mutants of Abl for indirect
effects: L444K, L444E, S558N, and S558A. We tested the following P+1/P+3 peptides: P+1Met/P+3Met,
P+1Ala/P+3Met,
P+1Glu/P+3Met, P+1Met/P+3Ala, and
P+1Met/P+3Thr. All four of the mutants
displayed the same rank order of substrate preference as the wild type
in this experiment (P+1Ala/P+3Met > P+1Met/P+3Met > P+1Glu/P+3Met > P+1Met/P+3Ala
P+1Met/P+3Thr) (data not shown). We conclude from these studies on indirect effects that the sites on Abl for recognition of the P
1 and P+1/P+3 positions are distinct. On the
other hand, the P+1 and P+3 sites may have some overlap, as at least
one mutation (Y569L) had an effect on recognition of both positions.
DISCUSSION
1 position and Pro at the P+3 position are the most
important determinants of substrate specificity for Abl. Here, we have
identified residues in the catalytic domain of Abl involved in peptide
substrate binding and specificity. These residues are located primarily
in the C-terminal lobe of the catalytic domain, which has been
implicated previously in substrate binding for other protein
kinases (12, 24, 27).
1 position resulted
in enzymes that still preferred Ile at P
1, but that diverged from the
wild type when screened against peptides containing other amino acids
at P
1. In these cases, these residues may not be involved in direct
interactions with the P
1 residue of substrate. Instead, because of
their vicinity to the P
1 position, they may act indirectly,
stabilizing the local structure to interact favorably with Ile at the
P
1 position. 3) Some mutants (e.g. Y569A, I523A, and
G556A) showed no changes in specificity when assayed against a variety
of peptide substrates. These mutants were not characterized by kinetic analysis.
1 position (L444K, L444E, G556V, S558N, W525F, and
W525H) do so in a more subtle manner. These mutations do not change the
overall preference for Ile at P
1 or Ala at P+1; however, we observed
effects on specificity when we screened these mutants against peptides
containing other residues at P
1 or P+1. For example, the I523V mutant
still phosphorylated the P+1Ala/P+3Met peptide
best (of the peptides tested), but the Vmax/Km value for the
P+1Ile/P+3Met peptide was 3.5 times higher in
this mutant than in the wild type (Table III). The L444E mutant, while
still preferring Ile at the P
1 position, was 2.1 times more efficient
at phosphorylating the P
1His peptide than the wild type
and 19 times less efficient at phosphorylating the P
1Glu
peptide than the wild type (Table IV). There are at least two
explanations for these subtle effects on substrate specificity. (i) The
residues may not make direct contact with bound substrate, but might
instead be involved indirectly in maintaining the three-dimensional structure of Abl to favor certain amino acids in the substrate. (ii)
Additionally or alternatively, substrate specificity at P
1 or P+1 may
be achieved by a combination of residues, such that single amino acid
substitutions do not cause complete alterations in substrate
recognition. Indirect effects could explain why, for example, the L444K
mutant phosphorylates the P
1Ile peptide better than the
wild type and shows a decrease in the phosphorylation of
P
1Glu when compared with the wild type (Table IV).
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ACKNOWLEDGEMENT |
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We thank Professor Stevan Hubbard (New York University Medical School) for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA58530 (to W. T. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 516-444-3533;
Fax: 516-444-3432; E-mail: miller{at}physiology.pnb.sunysb.edu.
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ABBREVIATIONS |
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The abbreviations used are: IRK, insulin receptor tyrosine kinase domain; Abl-CAT, Abl catalytic domain; AMP-PNP, 5'-adenylylimidodiphosphate; Nle, norleucine.
2 D. A. Hinds, W. T. Miller, and S. E. Shoelson, unpublished observations.
3 J. H. Till and W. T. Miller, unpublished observations.
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REFERENCES |
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