Inhibition of Spontaneous Receptor Phosphorylation by Residues in
a Putative
-Helix in the KIT Intracellular Juxtamembrane Region*
Yongsheng
Ma
,
Matthew E.
Cunningham§,
Xiaomei
Wang
,
Indraneel
Ghosh¶,
Lynn
Regan¶, and
B. Jack
Longley
§
From the Departments of
Dermatology and
§ Pathology, College of Physicians and Surgeons, Columbia
University, New York, New York 10032 and the ¶ Department of
Molecular Biophysics and Biochemistry, Yale University, New Haven,
Connecticut 06520
 |
ABSTRACT |
KIT receptor kinase activity is repressed, prior
to stem cell factor binding, by unknown structural constraints. Using
site-directed mutagenesis, we examined the role of KIT intracellular
juxtamembrane residues Met-552 through Ile-563 in controlling receptor
autophosphorylation. Alanine substitution for Tyr-553, Trp-557,
Val-559, or Val-560, all sitting along the hydrophobic side of an
amphipathic
-helix (Tyr-553-Ile-563) predicted by the Chou-Fasman
algorithm, resulted in substantially increased spontaneous receptor
phosphorylation, revealing inhibitory roles for these residues. Alanine
substitution for other residues, most of which are on the hydrophilic
side of the helix, caused no or slightly increased basal receptor
phosphorylation. Converting Tyr-553 or Trp-557 to phenylalanine
generated slight or no elevation, respectively, in basal KIT
phosphorylation, indicating that the phenyl ring of Tyr-553 and the
hydrophobicity of Trp-557 are critical for the inhibition. Although
alanine substitution for Lys-558 had no effect on receptor
phosphorylation, its substitution with proline produced high
spontaneous receptor phosphorylation, suggesting that the predicted
-helical conformation is involved in the inhibition. A synthetic
peptide comprising Tyr-553 through Ile-563 showed circular dichroism
spectra characteristic of
-helix, supporting the structural
prediction. Thus, the KIT intracellular juxtamembrane region contains
important residues which, in a putative
-helical conformation, exert
inhibitory control on the kinase activity of ligand-unoccupied receptor.
 |
INTRODUCTION |
KIT, encoded by the protooncogene c-KIT (1, 2), is the
receptor tyrosine kinase for stem cell factor
(SCF)1 (3). KIT and the
receptors for colony-stimulating factor 1 and platelet-derived growth
factor define the receptor tyrosine kinase type III subfamily (1, 2,
4). These receptors have in common five immunoglobulin-like motifs in
the extracellular domain and a bipartite kinase in the cytoplasmic
portion. The current model for activation of receptor tyrosine kinases
(4), which involves ligand binding-induced receptor dimerization and autophosphorylation, is well exemplified in the case of KIT (5, 6).
Molecular lesions that impair the kinase activity of KIT can lead to a
variety of developmental disorders (7), while mutations that
constitutively activate KIT (8-10) are associated with the
pathogenesis of mastocytosis (10-12) and gastrointestinal stromal
tumors (9). These activating mutations can transform cells in
vitro and confer aggressive behavior to the cells in vivo (9, 13).
Prior to SCF binding, the kinase activity of KIT is kept in a repressed
state. The structural basis for this repression is unknown. A number of
in-frame deletion mutations in the c-KIT intracellular
juxtamembrane coding region have recently been identified in
situ in gastrointestinal stromal tumors and in mastocytomas and
shown to cause SCF-independent receptor activation (9, 10). While these
findings imply that this region is involved in negative control of the
receptor kinase activity in the absence of SCF stimulation, the amino
acid(s) that play inhibitory roles are not known. This is because
deletion mutations are likely to result in conformational changes that
are not specifically related to the eliminated residues but are
necessary to compensate for the gap left by the ablation.
In this study we examined the role of a series of residues in the KIT
intracellular juxtamembrane region in controlling receptor autophosphorylation. Our results reveal important residues in this
region that exert inhibitory effects on the receptor kinase activity in
the SCF naive state and demonstrate conformational requirements for
these residues in repressing autoactivation of the receptor kinase.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Recombinant human SCF, mouse monoclonal and
rabbit polyclonal anti-human KIT antibody (Ab), and wild-type human
full-length KIT cDNA were provided by Amgen (Thousand Oaks, CA).
Mouse anti-phosphotyrosine (Tyr(P)) monoclonal Ab was purchased from
Upstate Biotechnology (Lake Placid, NY).
cDNA Construction and Transfection--
Single residue
substitutions were generated in human KIT cDNA in the pcDNA3
mammalian expression vector (Invitrogen, Carlsbad, CA) using the
Quikchange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
COS cells (90% confluent in 10-cm plate) were transfected with 5 µg
of plasmid using 15 µl of LipofectAMINETM (Life
Technologies, Gaithersburg, MD) in serum-free medium for 5 h. An
equal volume of medium with 20% bovine calf serum was then added, and
cells were incubated overnight, followed by 24-h culture in regular
medium prior to receptor phosphorylation experiments.
Immunoprecipitation and Immunoblotting--
For tyrosine
phosphorylation assay, cells expressing either wild-type or mutant KITs
were serum-starved for 18 h before incubation with, or without,
SCF at 200 ng/ml for 10 min at 37 °C. Cells were harvested in lysis
buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.5),
150 mM NaCl, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 1 mM sodium orthovanadate.
Centrifugation-clarified cell lysates were immunoprecipitated for
1.5 h at 4 °C with mouse anti-KIT Ab and protein A-agarose. Immunoprecipitates were washed with lysis buffer and heat-eluted in
sodium dodecyl sulfate (SDS) sample buffer. Samples were fractionated by 7.5% SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membrane, blocked by 5% bovine serum albumin
in TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
and 0.1% Tween 20), and probed with mouse anti-Tyr(P) Ab for 1 h,
followed by washing with TBST. Membranes were incubated in TBST with
horseradish peroxidase-linked secondary Ab for 45 min and washed, and
antigen-Ab complexes were detected using the ECL System (Amersham
Pharmacia Biotech). Anti-Tyr(P) blots were stripped in 100 mM
-mercaptoethanol, 2% SDS, and 62.5 mM
Tris-HCl (pH 6.7) at 50 °C for 30 min and then reprobed with rabbit
anti-KIT Ab.
Secondary Structure Prediction--
The amino acid sequence of
the KIT intracellular juxtamembrane region (Thr-544 through Lys-581)
was analyzed by the Chou-Fasman algorithm (14) using MacVector program
(Oxford Molecular Group) for prediction of secondary structures.
Homology modeling of this region's structure was tried, but
experimentally defined three-dimensional structures of proteins in the
Brookhaven Protein Data Bank showed too low homology to be used as
templates for modeling.
Circular Dichroism Spectroscopy--
An 11-residue peptide
(YEVQWKVVEEI) corresponding to residues 553 to 563 in the KIT
intracellular juxtamembrane region was synthesized by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University
and evaluated by circular dichroism (CD) spectroscopy. CD spectra of
the peptide were recorded at 25 °C in a 0.2-cm path length cell on a
62DS Circular Dichroism Spectrometer (Aviv Instruments) running Aviv
software. Spectra were recorded as an average of three scans either in
the presence or absence of trifluoroethanol in a pH 7.0 buffer
containing 10 mM sodium phosphate and 20 mM
NaCl with a step size of 1 nm and a 10-s equilibration time from 260 nm
to 196 nm. Peptide concentrations were determined by amino acid
analysis of a stock solution. The
-helical content was calculated
based on the mean residue ellipticity at 222 nm (15).
 |
RESULTS AND DISCUSSION |
A Number of Juxtamembrane Residues Are Necessary for Inhibition of
Spontaneous KIT Phosphorylation--
To examine which amino acid(s) in
the KIT intracellular juxtamembrane region may play inhibitory roles in
control of the receptor kinase activity, we generated a series of
mutant KITs with single alanine substitutions for residues Met-552
through Ile-563. This region covers most of the mutations identified in
gastrointestinal stromal tumors (9) and in mastocytomas (10) and
includes a putative
-helix (Tyr-553 through Ile-563) predicted by
the Chou-Fasman algorithm (14). We assessed tyrosine phosphorylation of
the mutated receptors in COS cells because these cells express neither
KIT nor SCF (16).
Compared with wild-type receptor which showed a low basal level of
spontaneous tyrosine phosphorylation in the absence of SCF stimulation,
five mutants displayed elevated spontaneous tyrosine phosphorylation
(Fig. 1A). Substantially
increased phosphorylation (>10-fold) resulted from removing the side
chain of Tyr-553, Trp-557, Val-559, or Val-560, and slightly increased
phosphorylation (~2-fold) was caused by eliminating the side chain of
Glu-554 (density-assessed after normalizing the difference in protein
expression shown in Fig. 1B). In the presence of SCF, these
mutants became more phosphorylated, but the increases varied; the
higher the basal level, the less the increase. Of the other six mutants
with no enhanced spontaneous phosphorylation, four (M552A, Q556A,
K558A, and I563A) underwent SCF-induced phosphorylation in a manner
comparable to the wild-type receptor, but two (V555A and E561A) showed
mild and severe impairments in ligand-induced phosphorylation,
respectively. These results reveal five residues in this region that
exert inhibitory effects on spontaneous receptor phosphorylation:
Tyr-553, Trp-557, Val-559, Val-560, and to a lesser extent Glu-554.

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Fig. 1.
Effect of alanine substitution on KIT
phosphorylation. A, anti-Tyr(P) (pTyr) blot
of immunoprecipitated wild-type and mutant KITs expressed in COS cells
treated (+), or not ( ), with SCF (200 ng/ml, 37 °C, 10 min) after
18 h of serum starvation shows that basal receptor phosphorylation
is highly increased with mutations at Tyr-553, Trp-557, Val-559, or
Val-560 (lanes 5, 13, 17,
and 19) and slightly increased by mutating Glu-554
(lane 7) in contrast to wild-type KIT (lane 1).
Substituting Val-555 or Glu-561 impairs SCF-induced phosphorylation
(lanes 10 and 22) compared with
wild-type KIT (lane 2). B, reprobing the
anti-Tyr(P) blot with anti-KIT Ab (after stripping) shows that all the
mutants (lanes 3-24) are expressed as two
protein products of 145 kDa and 125 kDa corresponding to wild-type KIT
(lanes 1 and 2). WT, wild-type KIT.
Molecular mass markers are indicated in kDa on the
left.
|
|
The Phenyl Ring of Tyr-553 Is Critical for Inhibition--
To look
more closely at the inhibitory role of Tyr-553, we mutated this
tyrosine residue to phenylalanine. Removal of the hydroxyl group of
Tyr-553 resulted in a slight increase (~2-fold) in basal receptor
phosphorylation in comparison with wild-type receptor (Fig.
2). This result, together with the
substantially increased spontaneous receptor phosphorylation caused by
Y553A substitution (Fig. 1, lane 5), indicates that the
phenyl ring of Tyr-553 exerts a major inhibitory effect, while its
hydroxyl group plays a relatively minor inhibitory role. Whether this
hydroxyl group interacts directly with another structural element or it is subject to phosphorylation is not known and awaits further study.

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Fig. 2.
Effect of phenylalanine substitution on KIT
phosphorylation. A, anti-Tyr(P) blot of
immunoprecipitated wild-type and mutant KITs expressed in COS cells
treated (+), or not ( ), with SCF (200 ng/ml, 37 °C, 10 min) after
18 h of serum starvation shows that substitution for Tyr-553
results in slightly increased basal receptor phosphorylation
(lane 3) compared with wild-type KIT (lane 1),
and substitution for Trp-557 has no effect on receptor phosphorylation
(lanes 7 and 8) compared with
wild-type KIT (lanes 5 and 6).
B, reprobing the anti-Tyr(P) blot with ant-KIT Ab (after
stripping) shows that the mutants are expressed as two isoforms of 145 kDa and 125 kDa (lanes 3 and 4 and
lanes 7 and 8) corresponding to
wild-type KIT (lanes 1 and 2 and
lanes 5 and 6). WT,
wild-type KIT. Molecular mass markers are indicated in kDa on the
left.
|
|
The Hydrophobicity of Trp-557 Is Important for
Inhibition--
Since the side chain of tryptophan allows both
hydrophobic and hydrophilic interactions, the inhibitory effect of
Trp-557, as indicated by the high spontaneous phosphorylation of the
W557A mutant (Fig. 1, lane 13), could be mediated through
either type of interaction. To test whether the amphipathic property of
tryptophan is important for the inhibition, we converted Trp-557 to
phenylalanine. With a hydrophobic phenyl ring at position 557, the
mutant receptor showed no alteration in phosphorylation compared with
wild-type receptor (Fig. 2). This result therefore suggests that it is
the hydrophobic character of Trp-557 that contributes to repression of
spontaneous KIT phosphorylation.
A Predicted
-Helical Conformation Is Involved in
Inhibition--
The spontaneous phosphorylation pattern displayed by
this series of mutant receptors is consistent with the corresponding residues being contained in an amphipathic
-helix (Tyr-553 through Ile-563) predicted by the Chou-Fasman algorithm (14). Specifically, residues (Tyr-553, Trp-557, Val-559, and Val-560) that exert large inhibitory effects on spontaneous receptor phosphorylation all sit
along the hydrophobic side of the predicted helical cylinder (analyzed
by plotting the sequential residues along a helix wheel). In contrast,
Glu-554, which plays a small inhibitory role, as well as Lys-558 and
Glu-561, which are not involved in the repression, all lie on the
hydrophilic side of the helix. We did not assess Glu-562 but it is
predicted to have at most a minor inhibitory role because of its
placement on the hydrophilic side of the helix. In addition, these data
demarcate the longitudinal boundaries critical for the inhibition in
that Met-552 (adjacent to the beginning of the helix) and Ile-563 (at
the end of the helix) are not necessary for the inhibition, even though
they both possess large hydrophobic side chains.
To further test whether the predicted
-helical conformation is
involved in the inhibition, we tried to disrupt the helical structure
by introducing in this region proline residues that are sterically
incompatible with
-helical conformation. We selected Val-555 and
Lys-558 to be replaced by proline, since the alanine substitution assay
had demonstrated that their side chains have no inhibitory effects, and
they are located within the predicted helix. Therefore, any
significantly increased basal receptor phosphorylation resulting from
the proline substitution can be specifically ascribed to steric
constraints imposed by proline rather than to loss of inhibitory side
chain function. Substitution of proline for Lys-558 led to a very high
level of constitutive receptor phosphorylation, which was even higher
than SCF-induced wild-type receptor phosphorylation (Fig.
3). This result thus suggests that the
predicted
-helical conformation is involved in the repression of
spontaneous receptor phosphorylation. Substitution of proline for
Val-555 did not produce any increased basal receptor phosphorylation
but resulted in a decrease in SCF-induced phosphorylation (Fig. 3), as
did substitution of alanine for Val-555 (Fig. 1, lane 10).
The lack of autoactivation of the V555P mutant is most likely due to
the absence of the side chain of Val-555, which is critical for KIT
activation.

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Fig. 3.
Effect of proline substitution on KIT
phosphorylation. A, anti-Tyr(P) blot of
immunoprecipitated wild-type and mutant KITs expressed in COS cells
treated (+), or not ( ), with SCF (200 ng/ml, 37 °C, 10 min) after
18 h of serum starvation shows that substitution for Lys-558 leads
to a high level of constitutive receptor phosphorylation (lane
5), and substitution for Val-555 impairs SCF-induced
phosphorylation (lane 4), compared with wild-type KIT
(lanes 1 and 2), respectively.
B, reprobing the anti-Tyr(P) blot with anti-KIT Ab (after
stripping) shows that the mutants are expressed as two products of 145 kDa and 125 kDa (lanes 3-6), as is wild-type KIT
(lanes 1 and 2). WT,
wild-type KIT. Molecular mass markers are indicated in kDa on the
left.
|
|
The Tyr-553-Ile-563 Fragment Forms an
-Helix in
Solution--
To examine more closely the predicted
-helical
structure, we measured the CD spectra of an 11-residue peptide
synthesized corresponding to Tyr-553 through Ile-563. This peptide
displayed negative minima in ellipticity at 208 nm and 222 nm, which
are characteristic of
-helical structure (15). The overall helical content of this peptide was 11% in aqueous solution and increased to
76% in the presence of trifluoroethanol (Fig.
4), which stabilizes the
-helical
structure of peptides that have inherent propensity to be
-helical
but are marginally stable in water (17, 18). The fact that this peptide
can fold into an
-helix reinforces the prediction that an
-helical conformation of the juxtamembrane Tyr-553-Ile-563 region
is involved in repression of spontaneous receptor phosphorylation.

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Fig. 4.
CD spectra of the Tyr-553-Ile-563
peptide. The spectra of 0.1 mM solution of an
11-residue peptide synthesized corresponding to KIT Tyr-553 through
Ile-563 were recorded in 10 mM sodium phosphate (pH 7.0)
and 20 mM NaCl with (+), or without ( ), 70%
trifluoroethanol.
|
|
The features of the inhibitory site, as we have shown, suggest that it
interacts with another structural element for the inhibition. In light
of the domain-domain interactions for repressing the kinase activity of
the Src and Hck tyrosine kinases (19, 20), we speculate that the KIT
juxtamembrane inhibitory site interacts with an epitope of the adjacent
kinase domain and, in doing so, affects the kinase activity. Other
mechanisms may also contribute to inhibition of KIT autoactivation and
remain to be elucidated.
In summary, the present study identifies a number of residues in the
KIT intracellular juxtamembrane region that exist in a putative
-helical conformation and exert inhibitory effects on the kinase
activity of SCF-unoccupied receptor. These findings provide a
structural basis for understanding why multiple deletion and missense
mutations in this region, which have been identified in situ
in gastrointestinal stromal tumors and mastocytomas, are able to cause
constitutive activation of the receptor kinase (9, 10).
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants RO1 AR43356 and AR44535 (Columbia Skin Disease Research Core Center).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: Dept. of
Dermatology, Section of Dermatopathology, College of Physicians and
Surgeons, Columbia University, 630 West 168th St., Vanderbilt Clinic
15-221, New York, NY 10032. Tel.: 212-305-2155, Fax: 212-927-9704;
E-mail: jl691{at}columbia.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
SCF, stem cell
factor;
Tyr(P), phosphotyrosine;
Ab, antibody;
CD, circular
dichroism.
 |
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