MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 1998, American Society for Microbiology
Apr. 1998, p. 2089–2099Vol. 18, No. 4
SHP-1 Binds and Negatively Modulates the c-Kit Receptor by
Interaction with Tyrosine 569 in the c-Kit
MAYA KOZLOWSKI,1* LOUISE LAROSE,2FAI LEE,1DUC MINGH LE,1ROBERT ROTTAPEL,3
AND KATHERINE A. SIMINOVITCH4
Health Canada Life Sciences and the University of Ottawa, Ottawa,1Polypeptide Hormone Laboratory, McGill
University, Montreal,2and Departments of Medicine, Immunology and Medical Genetics and Microbiology,
University of Toronto, the Wellesley Hospital Research Institute, Wellesley Hospital,3and the
Samuel Lunenfeld Research Institute, Mount Sinai Hospital,4Toronto, Canada
Received 17 July 1997/Returned for modification 1 September 1997/Accepted 22 December 1997
The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad
spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the
cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which
associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion
proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains
of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit
juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be
mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with
mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides
encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site
for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this
interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine
substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding
and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit
tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second
c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again
showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to
tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate
as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling
of c-Kit engagement to proliferation.
The pivotal role of the SH2 domain-containing SHP-1
(PTP1C, HCP, or SHPTP1) tyrosine phosphatase in the regu-
lation of hemopoietic cell growth and development is now well
recognized (1). In contrast to the structurally similar, ubiqui-
tously expressed SHP-2 (Syp or PTP1D) tyrosine phosphatase
and its Drosophila csw homolog (10, 37), SHP-1 appears to
exert primarily inhibitory effects on the signaling cascades in
which it participates (34). SHP-1 has been shown, for example,
to suppress the growth-promoting properties of the activated
interleukin 3 (IL-3), erythropoietin, and colony-stimulating
factor 1 receptors, an effect mediated either directly by recep-
tor dephosphorylation or indirectly by dephosphorylation of
receptor-associated protein tyrosine kinases (PTKs) (4, 19, 55,
56). SHP-1 has also been implicated in downregulation of the
signaling pathways evoked by engagement of the B- and T-
lymphocyte antigen receptors (5, 32, 33), antigen receptor co-
modulators such as CD22, Fc?RIIB, and CD5, and cytosolic
signaling molecules such as Vav and Grb2/Sos1 which are
involved in Ras activation (6, 8, 20). The capacity of SHP-1 to
negatively modulate this broad spectrum of signaling effectors
is consistent with the enormous overexpansion of multiple
hemopoietic cell populations manifested by motheaten (me)
and viable motheaten (mev) mice, animals now known to be
homozygous for loss-of-function mutations in the SHP-1 gene
(21, 46). The presence of two SH2 domains in SHP-1, as well
as the possibility for altering its C-terminal SH2 domain by
alternative splicing of a 39-amino-acid segment (46), provides
a structural explanation for the diverse range of molecular
interactions in which this phosphotyrosine phosphatase (PTP)
appears to participate. Thus, while the precise structural basis
for and physiologic effects of SHP-1 interactions with the mol-
ecules with which it has been shown to associate require fur-
ther investigation, the current data concerning SHP-1 func-
tions identify this PTP as a critical player in the modulation of
hemopoietic cell growth and function.
In addition to the regulation of cell proliferation, SHP-1 has
also been implicated in the control of signaling cascades cou-
pling growth factor receptors to hemopoietic cell differentia-
tion. This role for SHP-1 has become particularly well appre-
ciated in the context of data derived from studies of SHP-1
interactions with the transmembrane PTK receptor encoded
by the c-Kit proto-oncogene. The latter receptor subserves
pivotal functions in promoting the development, survival, and
proliferation of hemopoietic stem cells, neural crest-derived
cells, and germ cells, a role well illustrated by the depletion of
erythroid precursors and mast cells and associated macrocytic
anemia, sterility, and hypopigmentation manifested by c-Kit-
deficient mice bearing the W mutation (7, 9, 11, 29, 31). The
* Corresponding author. Mailing address: Life Sciences Division,
Tunney’s Pasture, Ottawa K1A 0L2, Canada. Phone: (613) 941-6594.
Fax: (613) 941-8933. E-mail: Maya_Kozlowski@hc-sc.gc.ca.
signaling events induced by c-Kit engagement to its cognate
ligand, stem cell factor (SCF), have been extensively investi-
gated and shown to involve the initial induction of c-Kit auto-
phosphorylation and consequent association of the activated
receptor with phosphatidylinositol 3-kinase, phospholipase
C?-1, megakaryocyte-associated tyrosine kinase (MATK), and
a number of other cytosolic signaling effectors that act in the
downstream delivery of the ligand-binding signal (16, 24, 40–
42). While these data primarily identify biochemical events
which promote c-Kit signaling, the demonstration that SHP-1
not only associates with but also tyrosine dephosphorylates
activated c-Kit receptors strongly suggests that c-Kit represents
another growth factor receptor subject to negative regulation
by SHP-1 (54). This contention is supported by recent data
showing that bone marrow progenitor cells from me mice hy-
perproliferate in response to SCF as well as the finding that
defects in c-Kit signaling associated with the Wvmutation are
ameliorated in Wvmice carrying the me or mevmutation (25,
35). Together, these data strongly suggest that SHP-1 interac-
tion with c-Kit is relevant to the modulation of c-Kit functions
in vivo. Furthermore, in view of the oncogenic potential puta-
tively conferred by alterations in c-Kit activity (12, 15, 18, 22,
47, 48) as well as data revealing lymphoma frequency to be
increased in mice heterozygous for the me or mevmutation (1)
and thereby implying a tumor suppressor role for SHP-1, the
inhibition of c-Kit signaling by SHP-1 may also represent a
molecular event relevant to hemopoietic cell transformation.
Although the cumulative data indicate a significant role for
SHP-1 in modulating c-Kit signaling capacity, the structural
basis for SHP-1 interaction with c-Kit is not well defined. Thus,
although this interaction has been ascribed to the binding of
the SHP-1 N-terminal and, to a lesser extent, C-terminal SH2
domains to a phosphorylated tyrosine site(s) on c-Kit (54), the
precise phosphotyrosine residues within c-Kit which mediate
this association are unknown. Accordingly, to further elucidate
the molecular basis for SHP-1 effects on c-Kit, we have used a
series of glutathione S-transferase (GST) fusion proteins that
span the tyrosine-containing segments of the c-Kit cytosolic
domain to investigate the site on the c-Kit receptor with which
SHP-1 associates. Analysis of SHP-1 interactions with phos-
phorylated forms of these fusion proteins as well as with phos-
phorylated peptides encompassing the phosphotyrosine sites
within selected regions of c-Kit has allowed the identification
of Tyr569within the c-Kit juxtamembrane region as the binding
site for SHP-1. SHP-1 association with this single phosphoty-
rosine residue was confirmed biochemically, by data from com-
petitive binding studies using wild-type tyrosine-phosphory-
lated as well as tyrosine3phenylalanine-mutated peptides. By
contrast, the structurally similar SHP-2 tyrosine phosphatase,
which also associates with activated c-Kit, was found to bind
selectively to Tyr567, the tyrosine residue immediately up-
stream of Tyr569. Importantly, phenylalanine substitution of
tyrosine 569 or 567 not only disrupted c-Kit interactions with
SHP-1 and SHP-2, respectively, but also was associated with
expression of hyperproliferative responses to SCF stimulation.
These findings therefore confirm the capacity of SHP-1 to
downregulate c-Kit signaling and indicate that the inhibitory
effect of SHP-1 on c-Kit is realized through SHP-1 binding with
tyrosine 569 in the c-Kit juxtamembrane domain. The data also
implicate SHP-2 binding to tyrosine residue 567 in the down-
regulation of c-Kit signaling and thus suggest that the physio-
logic outcome of c-Kit receptor engagement can be tempered
by the negative influence of each of these PTPs.
MATERIALS AND METHODS
Reagents. Polyclonal anti-SHP-1 antibodies were generated in rabbits immu-
nized with GST–SHP-1 SH2 domain fusion proteins as previously described (21).
The polyclonal anti-c-Kit antibody was purchased from Santa Cruz Biotechnol-
ogy Inc. (Santa Cruz, Calif.), monoclonal antiphosphotyrosine 4G10 and anti-
GST antibodies were obtained from Upstate Biotechnology Inc. (Lake Placid,
N.Y.), and polyclonal anti-SHP-2 antibodies (generated against SHP-2 SH2
fusion proteins) were kindly provided by Gen-Sheng Feng (Indiana University
Medical Center) (10). Murine recombinant SCF was obtained from Genzyme
(Cambridge, Mass.), recombinant human SCF and human and murine IL-3 were
purchased from Life Technologies (Burlington, Ontario, Canada), and con-
canavalin A (ConA) was obtained from Sigma Chemical Co. (St. Louis, Mo.).
Protein A-Sepharose, glutathione S-Sepharose, and NHS-Sepharose were pur-
chased from Pharmacia (Baie d’Urfe ´, Quebec, Canada). All other chemicals
used for immunoprecipitation and immunoblotting analyses were obtained from
Cells and cell culture. The Mo7e megakaryocytic and FMA3 mastocytoma cell
lines were kindly provided by L. Pegorraro (University di Torino, Turin, Italy)
and T. Tsujimura (Osaka University, Osaka, Japan), respectively. The HEL
human erythroleukemia and EL4 thymoma cell lines were obtained from the
American Type Culture Collection. All cells were maintained in opti-MEM (Life
Technologies) medium supplemented with 100 ?g of penicillin-streptomycin per
ml and 10% fetal calf serum (FCS). The growth factor-dependent Mo7e cells
were also grown in opti-MEM supplemented with 100 ?g of penicillin strepto-
mycin per ml, 10% FCS, and 50 ng of recombinant human IL-3 per ml. For
stimulation of these lines, cells were cultured for 16 h in medium lacking FCS
and IL-3 and then treated for 10 min in the presence of 20 ?g of ConA per ml
(EL4 cells) or for 5 min with 100 ng of SCF per ml (all other cell lines). Ba/F3
cells expressing mutant and wild-type forms of c-Kit were derived as follows.
Tyr5673Phe-mutated forms of the c-Kit cDNA were first generated by PCR
mutagenesis (14), and these and the wild-type c-Kit cDNA were then cloned into
the LXSN retrovirus (26). Retroviral plasmids were calcium phosphate trans-
fected into the retroviral packaging cell line BOSC-23 (36), and supernatants
containing greater than 106infectious units/ml were recovered 48 h after retro-
viral transfection and used to infect the IL-3-dependent pro-B-cell line Ba/F3. At
72 h following retroviral infection, infected Ba/F3 cell populations were stained
with the c-Kit-specific monoclonal antibody ACK2 (28) and sorted for equivalent
c-Kit expression by fluorescence-activated cell sorting (FACS) (FACstar; Becton
Dickinson). For proliferation assays, 104Ba/F3 c-Kit-transfected cells were
stored overnight in RPMI plus 0.5% FCS, suspended at a density of 2.5 ? 105
cells/ml in culture medium, and cultured in 0.2-ml microtiter plates in the pres-
ence of various concentrations of SCF (0 to 400 ng/ml). After 48 h of culture in
SCF, the cells were pulsed for 6 h with 1 mCi of [3H]thymidine (Dupont,
Wilmington, Del.). Cells were then harvested onto microplate filters, and radio-
activity was measured by scintillation counting (Top Count; Canberra, Downers
Grove, Ill.). Data are presented as the means ? standard deviations (SD) of
counts per minute. Alternatively, Ba/F3 cells and Ba/F3 c-Kit-transfected cells
were suspended in opti-MEM supplemented with 10% FCS and G418 (for c-Kit
transfectants) and plated at 104cells/well in 96-well microtiter plates. IL-3 at a
final concentration of 5 ng/100 ?l was added on day 0 and every other day
thereafter, and cell proliferation was evaluated every 48 h by a Cell Titer 96
nonradioactive assay (Promega, Madison, Wis.) based on conversion of a tetra-
zolium salt to formazen. Proliferation was quantitated by enzyme-linked immu-
nosorbent assay at 570 nm with a microtiter cell reader (Thermomax; Molecular
Generation of GST–c-Kit proteins. GST–c-Kit fusion proteins were generated
by subcloning PCR-amplified murine c-Kit sequences into pGEX-2TK (Pharma-
cia). The amplified fragments (see Fig. 3A) subcloned into this expression plas-
mid included the c-Kit juxtamembrane region (residues 545 to 571), the kinase
insert (residues 684 to 761), and the C-tail region (residues 917 to 975). The first
methionine codon was designated residue 1. Point mutations for tyrosine con-
version to phenylalanine, tyrosine deletion, or other amino acid substitutions
were generated by overlap extension with PCR and oligonucleotide primers
encoding the desired mutation.
To generate GST fusion proteins containing the SHP-1 SH2 domains, the
N-terminal (SH2-N, residues 1 to 100), C-terminal (SH2-C, residues 109 to 205),
and N- plus C-terminal (SH2-NC, residues 1 to 213) SH2 domains of SHP-1 were
PCR amplified from the murine SHP-1 cDNA (the methionine encoded by the
first ATG codon is considered residue 1). To generate a GST fusion protein
representing the SHP-1 C-terminal SH2 domain splice variant (SH2-C?), cDNA
was prepared from 500 ng of RNA isolated from murine splenocytes by using an
RNA extraction kit from Qiagen (Chatsworth, Calif.) and subjected to PCR
amplification with a primer pair corresponding to SHP-1 nucleotides 348 to 362
and 613 to 628, which encompass the SH2-C? region, together with a Tth7 DNA
polymerase kit (Boehringer Mannheim) and reaction conditions of 1 min at
94°C, 1 min at 65°C, and 1 min at 72°C for 35 cycles. Following BamHI and
EcoRI digestion, the reverse transcription-PCR product was subcloned into
pGEX-2T, and the insert sequence was confirmed by direct sequence analysis.
Bacterial cells transformed by pGEX2T-SH2 expression plasmids were in-
duced with 2 mM isopropyl-1-thio-?-D-galactopyranoside (IPTG), and the fusion
proteins were purified with glutathione-conjugated Sepharose beads (Pharma-
2090KOZLOWSKI ET AL.MOL. CELL. BIOL.
cia). To derive tyrosine-phosphorylated GST–c-Kit fusion proteins, the various
pGEX–c-Kit constructs were transformed into TKX1 (Pharmacia), and the ty-
rosine-phosphorylated fusion proteins were purified from bacterial cells follow-
ing sequential incubations with IPTG and indole acetic acid (IAA) (to induce the
expression of the tyrosine kinase gene cloned into TKX1 bacteria). Fusion
protein tyrosine phosphorylation was confirmed by immunoblotting with 4G10
monoclonal antiphosphotyrosine antibodies (Upstate Biotechnology).
Immunoprecipitation and Western immunoblotting. Protein lysates were pre-
pared by resuspending 108resting or SCF (100 ng/ml)-treated HEL, Mo7e,
FMA3, EL4, and Ba/F3-Kit cells in 1 ml of lysis buffer (phosphate-buffered saline
containing 1% Triton X-100, 1% Tween 20, 1 mM sodium orthovanadate, 1 ?g
of leupeptin per ml, 1 ?g of aprotinin per ml, and 0.001 mM dithiothreitol) (21).
Lysate proteins (500 ?g) were electrophoresed through sodium dodecyl sulfate
(SDS)–8 or 10% polyacrylamide gels and electroblotted onto nitrocellulose, and
the blots were then incubated overnight at 4°C in 10 mM Tris (pH 8.0)–150 mM
NaCl–0.05% Tween 20 (TBST) containing 5% skim milk. Proteins were detected
by incubating blots for 2 h at room temperature with primary antibodies in TBST
followed by125I-protein A (Dupont). Blots were then washed with TBST and
exposed to Kodak XAR film at ?70°C. For immunoprecipitations, cell lysates
were clarified by centrifugation for 20 min at 10,000 ? g at 4°C, and 1,800 ?g of
cell lysate protein was incubated for 2 h at 4°C with selected antibodies and then
with 100 ?l of protein A-Sepharose (Pharmacia) for 30 min at 4°C. The immune
complexes were collected by centrifugation, washed three times with lysis buffer,
boiled for 5 min in SDS sample buffer, and then subjected to electrophoresis and
immunoblotting as described above.
In vitro binding assays. To evaluate SHP-1 or c-Kit binding to GST–c-Kit or
GST-SHP1 fusion proteins, protein lysates prepared from 1,800 ?g of EL4 or
SCF-treated HEL cells were incubated at 4°C for 2 h with 5 ?g of fusion protein
immobilized on glutathione-Sepharose beads. After several washes in lysis buffer,
complexes were resuspended in sample buffer, boiled, and analyzed by SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with ei-
ther anti-SHP-1 or anti-c-Kit antibodies and125I-protein A.
Peptide competition. The following peptides encompassing tyrosine sites
within the juxtamembrane region of c-Kit were synthesized in the tyrosine-
phosphorylated and unphosphorylated states (Ottawa Regional Cancer Center):
peptide 1 (VLTpYKpYLQKPMK) and peptide 2 (VLTpYKYLQKPMK), which
correspond to residues 541 to 552; peptide 3 (KPMpYEVQWKVVE), repre-
senting residues 549 to 560; and peptide 4 (GNNpYVpYIDPTQK), peptide 5
(GNNpYVYIDPTQK), and peptide 6 (GNNYVpYIDPTQK), which encompass
residues 564 to 575 of the c-Kit juxtamembranous region. For competition
studies, EL4 cell lysates (1,800 ?g) were incubated with 5 ?g of GST–c-Kit-
juxtamembrane fusion protein in the presence of either 10 ?M each peptide,
various amounts of peptide 4, or 10 ?M peptide 4 preincubated with antiphos-
photyrosine antibodies. Protein complexes were washed four times and then
subjected to SDS-PAGE followed by immunoblotting with anti-SHP-1 antibody.
Direct binding of SHP-1 to synthetic phosphopeptides. Synthetic phosphopep-
tides 3 and 4 were coupled to NHS-Sepharose beads (Pharmacia) as recom-
mended by the manufacturer, and the immobilized peptides were then individ-
ually mixed with EL4 cell lysate. Following four washes, the proteins were
resolved by SDS-PAGE and blotted with anti-SHP-1 antibodies.
SHP-1 binding to the c-Kit receptor is enhanced following
SCF stimulation. To extend previous data indicating the ca-
pacity of SHP-1 to associate with c-Kit in SCF-treated Mo7e
cells (54), binding of SHP-1 to c-Kit was investigated in four
cell lines known to express high levels of c-Kit receptor and to
proliferate in response to SCF stimulation. These included
Mo7e cells, HEL cells, Ba/F3 pro-B cells engineered to express
c-Kit receptors (Ba/F3-Kit), and FMA3 cells (a cell line which
contains a constitutively active form of c-Kit). As shown in Fig.
1A, anti-SHP-1 immunoblotting analysis of anti-c-Kit immu-
noprecipitates derived from each of the four lines under study
revealed coimmunoprecipitation of SHP-1 with c-Kit in both
resting and stimulated cells and confirmed previous data sug-
gesting that the association of these molecules is increased in
conjunction with SCF stimulation (Fig. 1A). The c-Kit protein
was also detected in anti-SHP-1 immunoprecipitates from
FIG. 1. Increases in association and tyrosine phosphorylation of SHP-1 and c-Kit following SCF stimulation. Cell lysates were prepared from unstimulated (?) and
SCF (100 ng/ml)-treated Mo7e, FMA3, and HEL cells and from Ba/F3 cells stably expressing the full-length wild-type c-Kit cDNA (Ba/F3-Kit). (A) Protein lysates
(1,800 ?g) prepared from unstimulated or SCF-stimulated Mo7e, Ba/F3-Kit FMA3, and HEL cells were immunoprecipitated (Ip) with anti-c-Kit antibody, and the
immune complexes were subjected to SDS-PAGE, immunoblotting with anti-SHP-1 antibody, and visualization with125I-protein A. Ba/F3 total cell lysate protein (500
?g) was included as a control for SHP-1 expression. (B) Cell lysate proteins (1,800 ?g) prepared from unstimulated or SCF-stimulated Mo7e and HEL cells were
immunoprecipitated with anti-SHP-1 antibody, and the immunoprecipitated and cell lysate proteins (500 ?g) were resolved by SDS-PAGE and immunoblotted with
anti-c-Kit antibody. (C) SHP-1 immunoprecipitates prepared from unstimulated or SCF-treated Mo7e and HEL cells were analyzed by SDS-PAGE and immuno-
blotting with antiphosphotyrosine antibody. (D) Cell lysate proteins (500 ?g) as well as c-Kit immunoprecipitates from unstimulated or SCF-stimulated HEL cells (1,800
?g of lysate protein) were analyzed by SDS-PAGE and antiphosphotyrosine immunoblotting. For each panel, mobilities of molecular mass (MW) standards are shown
on the right and positions of SHP-1 and c-Kit are indicated by arrows.
VOL. 18, 1998 SHP-1 BINDS TO Tyr569WITHIN THE c-Kit CYTOSOLIC DOMAIN2091
Mo7e and HEL cells, and again the interaction of these mol-
ecules was increased following SCF treatment (Fig. 1B). To
determine whether c-Kit engagement in these cells is associ-
ated with tyrosine phosphorylation of not only c-Kit but also
SHP-1, these proteins were individually immunoprecipitated
from resting and SCF-stimulated Mo7e and HEL cells, and
their phosphorylation status was assessed by antiphosphoty-
rosine immunoblotting analysis. As shown in Fig. 1C and D,
respectively, SHP-1 and c-Kit are both tyrosine phosphorylated
at low levels constitutively, an observation which may reflect a
relatively high level of basal tyrosine phosphorylation in the
lines under study. However, the phosphorylation status of each
protein was also found to be markedly enhanced following Kit
engagement by SCF. These findings suggest that SHP-1 asso-
ciates with the c-Kit receptor and that this interaction is in-
creased by receptor activation and tyrosine phosphorylation.
SHP-1–c-Kit interaction involves association of the 71-kDa
SHP-1 isoform with the 145-kDa c-Kit isoform. Previous stud-
ies of SHP-1 expression have revealed the existence of two
SHP-1 species of ?67 and 71 kDa, which are differentially
expressed among various hemopoietic and epithelial cell types
(21, 49). Although both of these SHP-1 species appear to be
expressed in the cell lines used in this study, c-Kit immuno-
precipitates from these cells appeared to contain primarily the
high-molecular-weight species (Fig. 1A). This species, which is
generated by alternative splicing of the SHP-1 gene, has been
shown to be distinguished from the smaller SHP-1 isoform by
the presence of a 39-amino-acid insert in the SHP-1 C-terminal
SH2 domain (46). To investigate the potential role for the
71-kDa SHP-1 species in c-Kit binding, the SHP-1 region en-
compassing the C-terminal SH2 domain of this splice variant
(SH2-C?) was amplified by reverse transcription-PCR from
murine splenocytes and subcloned into pGEX-2T, and the
capacities of bacterial GST–SH2-C? fusion proteins as well as
GST fusion proteins containing the SHP-1 N-terminal, C-ter-
minal, or N- and C-terminal SH2 domains (Fig. 2A) to bind
c-Kit derived from SCF-stimulated HEL cells were then as-
sessed. As shown in Fig. 2B and consistent with previous data
concerning the binding of SHP-1 to c-Kit (54), the results of
this analysis revealed the interaction of c-Kit with the N-ter-
minal, but not the C-terminal, SHP-1 SH2 domain. However,
in contrast to the GST–SH2-C proteins, fusion proteins carry-
ing the larger SH2-C? C-terminal variant precipitated c-Kit to
a degree similar to that achieved with either the GST–N-
terminal or GST–N- plus C-terminal SHP-1 SH2 domain fu-
sion proteins. These observations are consistent with the im-
munoblotting data indicating that c-Kit associates with the
71-kDa SHP-1 isoform and suggest that both the N-terminal
and C-terminal variant (SH2-C?) SH2 domains play a role in
mediating SHP-1 binding to c-Kit.
SHP-1 associates with the juxtamembrane region of the
activated c-Kit receptor. As an initial step toward defining the
phosphotyrosine site(s) on c-Kit that interacts with SHP-1, a
series of GST fusion proteins carrying various portions of the
c-Kit cytosolic region were derived. As illustrated in Fig. 3A,
these fusion proteins contained the c-Kit juxtamembrane
(GST-JUX), kinase insert (GST-KI), and C-terminal tail
(GST-TAIL) regions, encompassing residues 544 to 574, 685 to
762, and 915 to 975, respectively.
To derive tyrosine-phosphorylated versions of each protein,
the relevant recombinant plasmids were transformed into a
TKXI bacterial strain expressing tyrosine kinase activity, and
their expression and phosphorylation status were then assessed
following IPTG and IAA induction (Fig. 3A). The phosphor-
FIG. 2. The Kit receptor preferentially associates with the SHP-1 SH2-N and SH2-C? domains. (A) Schematic diagram showing the structures of the 71- and 67-kDa
splice variants of SHP-1 and the SHP-1 SH2 domain sequences present in the GST–SHP-1 fusion proteins used for in vitro binding assays. The shaded region represents
a 39-amino-acid segment present in the C-terminal SH2 domain (SH2-C?) of the 71-kDa, but not the 67-kDa, SHP-1 species. (B and C) Cell lysates (1,800 ?g) prepared
from 108SCF-stimulated HEL cells were incubated for 2 h at 4°C with 5 ?g of purified GST–SHP-1 fusion protein immobilized on glutathione-Sepharose beads.
Complexes as well as lysate alone were fractionated by SDS-PAGE and subjected to immunoblotting with anti-c-Kit (B) or anti-GST (C) antibodies. Mobilities of
molecular mass (MW) standards are shown on the right.
2092KOZLOWSKI ET AL.MOL. CELL. BIOL.
ylated fusion proteins were then individually immobilized on
gluthathione-Sepharose beads and incubated with lysates pre-
pared from EL4 thymoma cells, and their binding to SHP-1
was examined by anti-SHP-1 immunoblotting analysis. As
shown in Fig. 3B, the results of this analysis revealed the
precipitation of SHP-1 by the GST-JUX protein. By contrast,
SHP-1 was not precipitated by either the GST-KI or GST-
TAIL fusion protein (Fig. 3B), even when the experiment was
performed with amounts of the latter two fusion proteins three
times greater than the amount of GST-JUX protein required
to precipitate SHP-1 (data not shown). The precipitation of
SHP-1 by GST-JUX was enhanced by the use of larger
amounts of fusion protein and, as is consistent with the con-
tention that c-Kit selectively associates with the 71-kDa SHP-1
isoform, involved only the larger of the two SHP-1 species
expressed in EL4 cells (Fig. 3B). These data strongly suggest
that SHP-1 interactions with c-Kit are mediated by the inter-
actions of this PTP with the c-Kit juxtamembrane region.
Identification of Tyr569as the binding site for SHP-1 on
c-Kit. As the 30-amino-acid juxtamembrane region of c-Kit
contains five tyrosine residues (located at positions 544, 546,
552, 567, and 569), the specific tyrosine(s) involved in SHP-1
binding to c-Kit was next investigated by examining SHP-1
interactions with GST-JUX fusion proteins in which these ty-
rosine residues had been individually deleted or phenylalanine
substituted. Analysis of tyrosine-phosphorylated versions of
these fusion proteins for the capacity to precipitate SHP-1
from EL4 cell lysates revealed GST-JUX binding to SHP-1 to
be abrogated by deletion of Tyr569but unaffected by a
Tyr3Phe substitution at position 552 or by deletion of Tyr544
or Tyr567(Fig. 4). By contrast, SHP-1 was not precipitated by
unphosphorylated versions of these GST-JUX proteins. To
extend these observations, a series of synthetic phosphotyrosyl
peptides representing sequences encompassing the tyrosine-
containing regions within the c-Kit juxtamembrane region
were assessed for their capacities to compete with phosphor-
ylated GST-JUX fusion protein for SHP-1 binding and thereby
interfere with GST-JUX-mediated precipitation of SHP-1
from EL4 lysates. As shown in Fig. 5, peptides containing
phosphorylated Tyr544(peptide 2), Tyr544and Tyr546(peptide
1), or Tyr552(peptide 3) did not interfere with the precipitation
of SHP-1 from the stimulated cells by the GST-JUX fusion
proteins. By contrast, SHP-1 precipitation by phosphorylated
GST-JUX protein was abrogated in the context of competition
with phosphopeptide 4, a synthetic peptide containing phos-
phorylated Tyr567and Tyr569. Inhibition of GST-JUX binding
to SHP-1 by phosphopeptide 4 was found to be dose depen-
dent and was reduced by preincubation of peptide 4 with an-
tiphosphotyrosine antibody (Fig. 5B). Direct binding of SHP-1
to immobilized peptides 3 and 4 was also examined, and as
shown in Fig. 5C, the results of this analysis revealed that
phosphopeptide 4, but not 3, efficiently bound SHP-1. Because
phosphopeptide 4 contains two phosphotyrosine residues
(Tyr567and Tyr569) which might serve as SHP-1 binding sites,
an analysis of whether one or both of these residues are in-
volved in coupling c-Kit to SHP-1 was performed by further
FIG. 3. Association of SHP-1 with the c-Kit juxtamembrane region. (A) Top, schematic diagram showing domain structure of the c-Kit cytosolic region, including
the juxtamembrane (JUX), kinase insert (KI), and carboxy-tail (C-TAIL) domains and two kinase domains. Numbers indicate the positions of the amino acid residues
bordering each domain. TM, transmembrane. Bottom, pGEX2-T–Kit constructs encoding GST–c-Kit–JUX, –KI, or –C-TAIL fusion proteins were used for transfor-
mation into TKX1 cells, and the bacterial cells were treated sequentially with IPTG and IAA. Tyrosine-phosphorylated GST fusion proteins were then purified by
affinity chromatography with glutathione-Sepharose beads, fractionated by SDS-PAGE, and visualized by Coomassie blue staining (left) or anti-pTyr immunoblotting
analysis (right). (B) Top, cell lysates (1,800 ng) prepared from ConA (20 ?g/ml)-treated EL4 cells were either immunoprecipitated with anti-SHP-1 antibody (IP:
SHP-1) or incubated for 2 h with 5 ?g of tyrosine-phosphorylated GST or GST-JUX, -KI or –C-TAIL fusion proteins immobilized on glutathione-Sepharose beads.
Complexes and lysate protein alone were fractionated by SDS-PAGE and subjected to immunoblotting analysis with anti-SHP-1 antibody. Bottom, cell lysates (1,800
?g) prepared from ConA-treated EL4 cells were incubated with various amounts (3, 6, 9, or 12 ?l) of glutathione-Sepharose–GST-JUX fusion protein, and the
complexes were then subjected to SDS-PAGE and anti-SHP-1 immunoblotting analysis. Mobilities of molecular mass (MW) standards are shown on the right, and the
position of SHP-1 is indicated on the left.
VOL. 18, 1998SHP-1 BINDS TO Tyr569WITHIN THE c-Kit CYTOSOLIC DOMAIN 2093
competition analyses with two phosphopeptides (5 and 6)
which contained either one or the other of these two residues
in a phosphorylated state. As shown in Fig. 5D, of these two
latter phosphopeptides, only peptide 6, in which Tyr569but not
Tyr567was phosphorylated, inhibited GST-JUX binding to
SHP-1. These observations provide strong evidence that SHP-1
binding to the phosphorylated tyrosine at position 569 is re-
sponsible for the association of SHP-1 with the c-Kit jux-
Data from studies of another cytosolic PTP, SHP-2, have
indicated that this enzyme also interacts via its SH2 domains
with activated c-Kit in hemopoietic cells (45). The binding of
SHP-2 to c-Kit has been predicted to be mediated through a
tyrosine residue located in the c-Kit juxtamembrane region, in
this instance Tyr567(44). Accordingly, to further address the
specific role for c-Kit juxtamembrane tyrosine residues in me-
diating PTP binding to c-Kit, the respective capacities of
SHP-1 and SHP-2 to bind Tyr567and Tyr569were investigated
by using a second set of TXK1-phosphorylated GST-JUX fu-
sion proteins containing Phe substitutions at one or both of
these tyrosine sites. As illustrated by the anti-SHP-1 immuno-
blotting analysis shown in Fig. 6A, the GST-JUX fusion pro-
teins in which Tyr569alone or Tyr569together with Tyr567was
replaced with Phe failed to precipitate SHP-1 from EL4 ly-
sates. By contrast, GST-JUX proteins containing only a
Tyr5673Phe substitution did precipitate this phosphatase, al-
beit to a lesser extent than the wild-type JUX protein. SHP-2
was also precipitated by wild-type GST-JUX and not by the
Tyr567Tyr569double mutant GST-JUX proteins (Fig. 6B).
However in contrast to SHP-1, the SHP-2 PTP was also pre-
cipitated by the Tyr5693Phe mutant fusion protein and was
barely detectable in the precipitates derived by using GST-
JUX proteins containing the Tyr5673Phe substitution. While
alanine replacement of the valine residue at position 568 had
no effect on GST-JUX binding to either SHP-1 or SHP-2, both
of these interactions were abrogated by mutation of the hydro-
phobic residue at codon 570, a residue which occupies the first
and third sites carboxy terminal to Tyr567and Tyr569, respec-
tively. Taken together, these data identify Tyr569as the pivotal
binding site on c-Kit for SHP-1 and identify Tyr567as the site
through which c-Kit interacts with SHP-2. In addition, these
data indicate that the interactions of both SHP-1 and SHP-2
with c-Kit depend on the isoleucine residue at position 570.
Expression of Tyr5693Phe- or Tyr5673Phe-mutated c-Kit
receptors enhances SCF-induced proliferation of Ba/F3-Kit
cells. To investigate whether the interaction between SHP-1
and the c-Kit Tyr569or Tyr567residue is responsible for the
previously demonstrated inhibitory influence of SHP-1 on c-
Kit signaling, Ba/F3 cells lacking endogenous c-Kit were in-
fected with retroviral vectors carrying cDNAs for either the
full-length wild-type (Ba/F3-Kit), Tyr5693Phe-mutated (Ba/
F3-Kit Y569F), or Y5673Phe-mutated (Ba/F3-Kit Y567F) c-Kit
receptor. FACS (Fig. 7C, bottom panel) and Western analysis
(data not shown) revealed comparable levels of c-Kit expres-
sion in these transfectants. However, as shown in Fig. 7C (top
panel), the cells expressing the Tyr5693Phe mutant c-Kit pro-
tein exhibited a markedly enhanced proliferative response to
SCF relative to that detected in cells expressing wild-type c-Kit
receptors. As is consistent with the identification of Tyr569on
c-Kit as a required residue for c-Kit binding to SHP-1, SHP-1
and c-Kit were not coimmunoprecipitated from the SCF-
treated Ba/F3-Kit Y569F cells but were co-immunoprecipitated
from stimulated Ba/F3-Kit and Ba/F3-Kit Y567F cells (Fig.
7A). By contrast, the capacity of SHP-2 to associate with acti-
vated c-Kit in the Ba/F3–c-Kit transfectants was unaffected in
the context of Tyr569mutation but was markedly reduced by
mutation of the tyrosine residue at position 567 (Fig. 7B). Most
significantly, as shown in Fig. 7C, expression of c-Kit proteins
carrying Phe substitutions at either Tyr567or Tyr569was asso-
ciated with markedly enhanced proliferation of the Ba/F3-Kit
cells in response to various amounts of SCF. These increases in
proliferation were specifically related to interactions between
SCF and the mutant Kit receptors, as proliferative responses to
IL-3 in Ba/F3-Kit Y567F and Ba/F3-Kit Y569F cells were com-
parable to those detected in Ba/F3-Kit cells (Fig. 7D). These
observations indicate a critical role for Tyr569and Tyr567in
mediating the association of c-Kit with SHP-1 and SHP-2,
respectively, in vivo and indicate that SHP-2 as well as SHP-1
can negatively modulate c-Kit signaling by interacting with
these specific tyrosine residues.
Recent data derived from biochemical and genetic studies of
SHP-1 interactions with the c-Kit PTK receptor have revealed
that SHP-1 negatively regulates c-Kit signaling and thereby
FIG. 4. Deletion of c-Kit Y569abrogates binding of SHP-1 to the c-Kit juxtamembrane region. Tyrosine-phosphorylated or unphosphorylated GST–c-Kit–JUX
fusion proteins containing either wild-type (JUX) or mutated versions of the c-Kit juxtamembrane domain were immobilized on glutathione-Sepharose beads and
incubated with cell lysates (1,800 ?g) prepared from ConA-treated EL4 cells. Complexes and lysate protein (500 ?g) were then resolved by SDS-PAGE and
immunoblotted with anti-SHP-1 antibody. Sites of the JUX domain mutations are indicated above the lanes and include deletions of tyrosine residues at position 544,
567, or 569 and, in the left panel only, replacement of Tyr552with phenylalanine (Y552F). For each panel mobilities of molecular mass (MW) standards are shown on
the right, and the position of SHP-1 is indicated on the left.
2094 KOZLOWSKI ET AL.MOL. CELL. BIOL.
mitigates the signaling events linking c-Kit engagement to he-
mopoietic cell proliferation and differentiation (25, 35). We
have investigated the structural basis for SHP-1 binding to and
consequent inhibitory influence on c-Kit and report here that
SHP-1 binds to a specific tyrosine-containing peptide sequence
within the juxtamembrane region of c-Kit. Our data indicate
that both SHP-1 SH2 domains participate in its interaction
with c-Kit and identify the tyrosine residue at position 569
FIG. 5. Identification of Tyr569as the c-Kit binding site for SHP-1. (A) Phosphopeptides (12-mers) spanning the five tyrosine sites contained in the c-Kit
juxtamembrane region were synthesized with tyrosines in phosphorylated or unphosphorylated states (upper diagram), and the individual phosphopeptides (10 ?M)
were then incubated with cell lysates (1,800 ?g) from EL4 cells in the presence of 5 ?g of glutathione-Sepharose–GST-JUX fusion protein. Complexes were washed
four times, and the complexes and lysate protein were then subjected to SDS-PAGE followed by anti-SHP-1 immunoblotting analysis. (B) Lysates prepared from
ConA-stimulated EL4 cells were incubated with glutathione-Sepharose–GST-JUX fusion proteins (5 ?g) in the presence of various amounts (1, 5, or 10 ?M) of
phosphopeptide 4 and with 10 ?M phosphopeptide 4 preincubated with anti-pTyr antibody (anti-pY). Following washing, complexes and lysate protein (500 ?g) were
subjected to SDS-PAGE and anti-SHP-1 immunoblotting analysis. (C) c-Kit phosphopeptides 3 and 4 were individually coupled to NHS-Sepharose beads and then
incubated with ConA-treated EL4 cell lysates. Following washing, the complexes and lysate protein were resolved by SDS-PAGE and subjected to anti-SHP-1
immunoblotting analysis. (D) Glutathione-Sepharose–GST-JUX fusion protein (5 ?g) was incubated with lysates from ConA-treated EL4 cells in the absence or
presence of 10 ?M phosphopeptide 4, 5, or 6, and the complexes and lysate proteins were subjected to SDS-PAGE and anti-SHP-1 immunoblotting analysis. In each
panel, mobilities of molecular mass (MW) standards are shown on the right and the position of SHP-1 is indicated on the left.
VOL. 18, 1998 SHP-1 BINDS TO Tyr569WITHIN THE c-Kit CYTOSOLIC DOMAIN 2095
within the phosphorylated c-Kit juxtamembrane region as the
major SHP-1 binding site on c-Kit. Mutation of Tyr569not only
abrogates SHP-1 interaction with c-Kit but also results in en-
hanced c-Kit signaling in response to SCF stimulation. There-
fore, binding of SHP-1 to Tyr569appears to be critical to the
capacity of SHP-1 to both associate with c-Kit and negatively
modulate the signaling pathways coupling the activated recep-
tor to cellular responses.
The data reported here confirm previous observations indi-
cating that tyrosine-phosphorylated c-Kit interacts with the
SHP-1 N-terminal, but not C-terminal, SH2 domain (54).
Binding of the alternatively spliced C-terminal SHP-1 SH2
domain (SH2-C?) to phosphorylated c-Kit in vitro was also
demonstrated in the current study and appeared to be equiv-
alent to the interaction detected between c-Kit and the SHP-1
N- and C-terminal SH2 domains together (Fig. 2B). Thus,
c-Kit association with SHP-1 can be mediated through either
the SH2-N, SH2-C?, or tandem SH2-N and -C (or -C?) do-
mains. These data provide the first direct evidence that the
ligand binding properties of the SHP-1 C and C? SH2 domains
and, by extension, the two SHP-1 isoforms may be distinct, a
conclusion consistent with structural data on SH2 domains
indicating that the 39-amino-acid SH2 domain segment distin-
guishing the SHP-1 isoforms maps within a region forming
EF? strands implicated in defining SH2 domain peptide-bind-
ing specificity (2, 50). Along similar lines, the SHP-1 SH2-N
and SH2-C domains also diverge in terms of their binding
specificities, SHP-1 association with the activated erythropoi-
etin, CD22, and IL-3? receptors being mediated through the
N-terminal SHP-1 SH2 domain (23, 55, 56), while its interac-
tion with the natural killer inhibitory and Fc?RII? receptors is
mediated by the C-terminal SH2 domain (3, 6). Such diver-
gence in terms of the peptide-binding specificities of its indi-
vidual SH2 domains gives SHP-1 the potential to associate with
and modulate a broad array of signaling effectors and, accord-
ingly, to assume pivotal roles in regulating many facets of
hemopoietic and epithelial cell behavior.
In the current study, the tyrosine residue at position 569 in
the c-Kit juxtamembrane region was identified as the critical
site for SHP-1 association with activated c-Kit receptors. This
tyrosine, in turn, is flanked by Tyr and Val residues at the ?2
and ?1 positions, respectively, followed by Ile, Asp, and Pro at
the ?1, ?2, and ?3 positions, respectively. By contrast, pre-
vious studies of the binding motifs for SHP-1 interactions with
the CD22 and Fc?RIIB receptors on B lymphocytes and KIR,
the p58 receptor on natural killer cells, have identified the
sequence Val/IleX[pTyr]XXLeu as a consensus motif for as-
sociation of SHP-1 with each of these receptors. Engagement
of the latter receptors and their coincident recruitment of
SHP-1 inhibit activation through the B-cell antigen (in the
cases of Fc?RIIB and CD22) or CD16 (in the case of p58)
receptors, and accordingly, this conserved motif has been des-
ignated the immunoreceptor tyrosine-based inhibitory motif
(6, 30). The SHP-1 N-terminal SH2 domain has also been
shown to select the peptide sequence [pTyr]-hydrophobic-X-
hydrophobic from a degenerate phosphopeptide library (43).
This finding is consistent with the identification here of
[pTyr]IsoAspPro as the SHP-1 binding site on c-Kit as well as
data identifying [pTyr]ThrIsoLeu as the SHP-1 binding site on
the erythropoietin receptor (19). Together these data indicate
the capacity for SHP-1 to recognize phosphotyrosines in a
multiplicity of amino acid contexts, a property not solely at-
tributable to structural differences between the SHP-1 N- and
C-terminal SH2 domains, as receptors such as the erythropoi-
etin and CD22 receptors both bind SHP-1 via its N-terminal
SH2 domain despite the differences in their SHP-1 binding site
In contrast to SHP-1, the SHP-2 PTP was shown to bind
c-Kit by interacting with a tyrosine residue (Tyr567) within the
sequence [pTyr]ValTyrIle, a motif which matches the peptide
sequence ([pTyr]Val/IleX Val/Ile) that the SHP-2 N-terminal
SH2 domain preferentially selects from a degenerate peptide
library (44). Importantly, phenylalanine replacement of this
tyrosine in GST–c-Kit–JUX fusion proteins was associated not
only with disruption of SHP-2 binding, but also with some
reduction in SHP-1 binding to the phosphorylated fusion pro-
tein (Fig. 6). By contrast, Phe replacement of Tyr569had no
effect on the capacity of either GST–c-Kit–JUX fusion proteins
(Fig. 6B) or c-Kit receptors expressed in Ba/F3 cells (Fig. 7B)
to associate with SHP-2. Thus, unlike Tyr569, which is required
FIG. 6. Definition of SHP-1 and SHP-2 binding sites within the c-Kit juxtamembrane region. (A) GST-c–Kit–JUX fusion proteins containing either the wild-type
(JUX) or mutated versions of the c-Kit juxtamembrane region were expressed in TKXI cells, and the tyrosine-phosphorylated proteins were then immobilized on
gluathione-Sepharose beads and incubated with lysates prepared from 108ConA-treated EL4 cells. Complexes and lysate proteins were then subjected to SDS-PAGE
and anti-SHP-1 immunoblotting analysis. The specific JUX domain mutations are indicated above the lanes and include phenylalanine replacement of tyrosine 567 and
569 individually (Y567F and Y569F, respectively) or together [DM (Y567, Y569F)], alanine replacement of valine 568, and alanine replacement of isoleucine 570. The
position of SHP-1 is indicated on the left. (B) The filter shown in panel A was stripped and reblotted with anti-SHP-2 antibody. The position of SHP-2 is indicated
on the left.
2096 KOZLOWSKI ET AL.MOL. CELL. BIOL.
for SHP-1 but not involved in SHP-2 interactions with c-Kit,
Tyr567may play both an essential role in SHP-2 binding and a
facilitory role in SHP-1 binding to phosphorylated c-Kit. In this
context, it is possible that SHP-1 and SHP-2 compete for bind-
ing to the latter site on the c-Kit juxtamembrane region.
The involvement of Tyr567in SHP-2 and, potentially, SHP-1
binding to c-Kit is of particular interest in view of previous data
showing that deletion of the comparable tyrosine and juxta-
posed valine residues (Tyr568and Val569) substantially en-
hances the mitogenic and transforming properties of the feline
c-Kit receptor and also represents one of the mutations which
distinguishes the wild-type receptor from the oncogenic coun-
terpart, v-Kit (13). Although a similar link between Tyr569
mutation and c-Kit transforming capacity has not been de-
scribed, the finding that SCF-induced proliferation of Ba/F3-
Kit cells is enhanced by either Tyr5693Phe or Tyr5673Phe
substitutions of the c-Kit receptors on these cells indicates that
SHP-1 and SHP-2 can independently exert negative regulatory
effects on c-Kit signaling and, by extension, that mutations of
c-Kit which reduce or abrogate its binding to SHP-1 or SHP-2
can engender enhanced mitogenic and potentially oncogenic
c-Kit activity. This apparent overlap in the effects of SHP-1 and
SHP-2 on c-Kit signaling suggests that in at least some cell
lineages c-Kit signaling may be unaffected by loss of function of
one of these PTPs and thus provides a molecular explanation
for the cell lineage-dependent effects of SHP-1 on c-Kit func-
tion observed in Wv/motheaten mice (25).
While our data provide evidence for the capacity of both
SHP-1 and SHP-2 to negatively regulate c-Kit signaling, the
mechanisms whereby this inhibitory influence is realized re-
main to be defined. For example, although c-Kit has been
identified as an SHP-1 substrate in vitro, it is unclear whether
SHP-1 or SHP-2 directly dephosphorylates c-Kit in vivo and/or
elicits dephosphorylation of the receptor indirectly by dephos-
phorylating and inhibiting cytosolic PTKs that act on c-Kit.
Downregulation of c-Kit signaling by these PTPs may also
reflect the dephosphorylation of signaling effectors involved in
downstream transduction of the ligand-binding activation sig-
nal, a paradigm recently demonstrated with respect to SHP-1
interactions with the erythropoietin receptor (17). In the latter
example, the negative influence of SHP-1 on receptor signaling
has been linked to SHP-1-mediated dephosphorylation of the
cytosolic JAK2 PTK (17, 19, 52). As JAK2 has also been shown
to associate with and modulate the activated c-Kit receptor
(51, 53), it is possible that SHP-1 dephosphorylation of JAK2
either impairs c-Kit phosphorylation following ligand engage-
ment or, by analogy with the erythropoietin receptor, inter-
feres with JAK2-mediated activation and recruitment of sig-
naling effectors required to evoke a cellular response. In
addition to these possibilities, previous data indicating that the
association of Src family tyrosine kinases with the c-Kit related
platelet-derived growth factor receptor is mediated through
Tyr579and Tyr581, sites which represent homologs of Tyr567
and Tyr569on c-Kit (27, 39), also suggest that SHP-1 and/or
SHP-2 inhibitory effects on c-Kit signaling may reflect the
capacity of these PTPs to compete with and displace Src PTKs.
FIG. 7. Mutations at the SHP-1 or SHP-2 binding sites on c-Kit enhance SCF-driven proliferation of Ba/F3-Kit cells. Cell lysates were prepared from unstimulated
(?) or SCF (100 ng/ml)-treated (?) Ba/F3 transfectants infected with a retroviral vector carrying the full-length wild-type (Ba/F3-Kit), Y5673F-mutated (Ba/F3-Kit
Y567F), or Y5693F-mutated (Ba/F3-Kit Y569F) c-Kit cDNA. (A and B) Lysate proteins (1,800 ?g) from the unstimulated and stimulated cells were immunopre-
cipitated (Ip) with anti-c-Kit antibody, and the immune complexes and lysate proteins were then subjected to SDS-PAGE and anti-SHP-1 (A) and anti-SHP-2 (B)
immunoblotting analysis. (C) Top, Ba/F3-Kit wild-type (Wt), Kit Y567F, and Kit Y569F cells were suspended at 2.5 ? 105cells/ml in culture medium in the presence
of various concentrations (0-400 ng/ml) of SCF. Cultures were harvested at 48 h following a 6-h pulse with 1 mCi of [3H]thymidine, and proliferation was measured
by beta scintillation counting. Results (means ? SD) represent averages for triplicate cultures and three independent experiments. Bottom, Ba/F3 c-Kit wild-type (WT),
Y567F, and Y569F cells (105) were stained with the ACK2 anti-Kit antibody and examined for expression of c-Kit by FACS analysis. (D) Ba/F3, Ba/F3-Kit wild-type
(Wt), Kit Y567F, and Kit Y569F cells were suspended at 104cells/ml in culture medium, and IL-3 (50 ng/ml) was added at day zero and every second day thereafter.
Proliferation was evaluated every 48 h by the Cell Titer assay and enzyme-linked immunosorbent assay at 570 nm. Results (means ? SD) represent averages of triplicate
cultures. OD, optical density.
VOL. 18, 1998 SHP-1 BINDS TO Tyr569WITHIN THE c-Kit CYTOSOLIC DOMAIN2097
Resolution of these issues should elucidate the molecular
mechanisms whereby c-Kit signaling is regulated and trans-
lated to particular biological outcomes.
This work was supported in part by grants from the Medical Re-
search Council of Canada and the National Cancer Institute of Canada
and by the Health Canada Bureau of Drug Research. Katherine A.
Siminovitch is a Senior Scientist and Robert Rottapel is a Research
Scholar of the Arthritis Society of Canada, and Louise Larose is a
recipient of a Medical Research Council of Canada/Canadian Re-
search Society scholarship.
1. Bignon, J. S., and K. A. Siminovitch. 1994. Identification of PTP1C mutation
as the genetic defect in motheaten and viable motheaten: a step toward
defining the roles of protein tyrosine phosphatases in the regulation of
hemopoietic cell differentiation and function. Clin. Immunol. Immuno-
2. Birge, R. B., and H. Hanafusa. 1993. Closing in on SH2 specificity. Science
3. Burshtyn, D. N., A. M. Scharenberg, N. Wagtmann, S. Raja Apopalan, K.
Berrada, T. Alan, K. Berrada, T. Yi, J-P. Kinet, and E. O. Long. 1996.
Recruitment of tyrosine phosphatase HCP by the killer cell inhibitory re-
ceptor. Immunity 4:77–85.
4. Chen, H. E., S. Chang, T. Trub, and B. G. Neel. 1996. Regulation of colony-
stimulating factor 1 receptor signaling by the SH2 domain-containing ty-
rosine phosphatase SHPTP1. Mol. Cell. Biol. 16:3685–3697.
5. Cyster, J. G., and L. G. Goodnow. 1995. Protein tyrosine phosphatase 1C
negatively regulates antigen receptor signaling in B lymphocytes and deter-
mines thresholds for negative selection. Immunity 2:13–24.
6. D’Ambrosio, D., K. L. Hippen, S. A. Minskoff, I. Mellman, G. Pani, K. A.
Siminovitch, and J. C. Cambier. 1995. Recruitment and activation of PTP1C
in negative regulation of antigen receptor signaling by Fc?RIIB1. Science
7. der Mujum, S. K., K. Brown, F.-H. Qui, and P. Besmer. 1988. c-Kit protein,
a transmembrane kinase: identification in tissues and characterization. Mol.
Cell. Biol. 8:4896–4903.
8. Doody, G. M., L. B. Justement, C. C. Delibrias, J. R. Mathews, J. Lin, M. L.
Thomas, and D. T. Fearon. 1995. A role in B cell activation for CD22 and the
protein tyrosine phosphatase SHP. Science 269:242–244.
9. Dutflinger, R., K. Manova, G. Berrozpe, T.-Y. Chu, V. DeLeon, I. Timokhina,
R. S. K. Chaganti, A. D. Zelentz, R. F. Bachvarova, and P. Besmer. 1995. The
Wsh and Ph mutations affect the c-Kit expression profile: c-Kit misexpres-
sion impairs melanogenesis in Wsh and Ph mutant mice. Proc. Natl. Acad.
Sci. USA 92:3754–3758.
10. Feng, G.-S., C. C. Hui, and T. Pawson. 1993. SH2-containing phosphoty-
rosine phosphatase as a target of protein-tyrosine kinases. Science 259:1607–
11. Geissler, E. N., M. A. Ryan, and E. D. Housman. 1988. The dominant-white
spotting (w) locus of the mouse encodes the c-Kit proto-oncogene. Cell
12. Gokkel, E., Z. Grossman, B. Ramot, Y. Yarden, G. Rechavi, and D. Givol.
1992. Structural organization of the murine c-kit proto-oncogene. Oncogene
13. Herbst, R., S. Munemitsu, and A. Ullrich. 1995. Oncogenic activation of v-kit
involves deletion of a putative tyrosine-substrate interaction site. Oncogene
14. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989.
Site-directed mutagenesis by overlap extension using the polymerase chain
reaction. Gene 77:51–59.
15. Ikeda, H., Y. Kanakura, T. Tamaki, A. Kuriu, H. Kitayama, J. Ishikawa, Y.
Kanayama, T. Yonezawa, S. Tarui, and J. D. Griffin. 1991. Expression and
functional role of the proto-oncogene c-Kit in acute myeloblastic leukemia
cells. Blood 78:2962–2968.
16. Jhun, B. H., B. Rivnay, D. Price, and H. Avraham. 1995. The MATK tyrosine
kinase interacts in a specific and SH2-dependent manner with c-Kit. J. Biol.
17. Jiao, H., K. Berrada, W. Yang, M. Tabrizi, L. C. Platanias, and T. Yi. 1996.
Direct association with and dephosphorylation of Jak2 kinase by the SH2-
domain-containing protein tyrosine phosphatase SHP-1. Mol. Cell. Biol.
18. Kitayama, H., Y. Kanakura, T. Furitsu, T. Tsujimura, K. Oritani, H. Ikede,
H. Sugahara, H. Mitsui, Y. Kanayama, and Y. Matsuzawa. 1995. Constitu-
tively activating mutations of c-Kit receptor tyrosine kinase confer factor-
independent growth and tumorigenicity of factor dependent hematopoietic
cell lines. Blood 85:790–798.
19. Klingmuller, U., U. Lorenz, L. Cantley, B. Neel, and H. Lodish. 1995. Spe-
cific recruitment of SH-PTP1 to the erythropoietin receptor causes inacti-
vation of JAK2 and termination of proliferative signals. Cell 80:729–738.
20. Kon-Kozlowski, M., G. Pani, T. Pawson, and K. A. Siminovitch. 1996. The
tyrosine phosphatase PTP1C associates with Vav, Grb2, and mSos1 in he-
matopoietic cells. J. Biol. Chem. 271:3856–3862.
21. Kozlowski, M., I. Mlinaric-Rascan, G.-S. Feng, R. Shen, T. Pawson, and
K. A. Siminovitch. 1993. Expression and catalytic activity of the tyrosine
phosphatase PTP1C is severely impaired in motheaten and viable motheaten
mice. J. Exp. Med. 178:2157–2163.
22. Kuriu, A., H. Ikeda, Y. Kanakura, J. D. Griffin, B. Druker, H. Yagura, H.
Kitayama, J. Ishikawa, J. Nishihura, Y. Kanayama, T. Yonezawa, and S.
Tarui. 1991. Proliferation of human leukemia cell line associated with the
tyrosine phosphorylation and activation of the proto-oncogene c-Kit product.
23. Law, C. L., S. P. Sidorenki, K. A., Chandran, Z. Zhao, S. H. Shen, E. H.
Fischer, and E. A. Clark. 1996. CD22 associates with protein tyrosine phos-
phatase 1C, Syk and phospholipase C-?1 upon B cell activation. J. Exp. Med.
24. Lev, S., D. Givol, and Y. Yarden. 1991. A specific combination of substrates
is involved in signal transduction by the Kit-encoded receptor. EMBO J.
25. Lorenz, U., A. D. Bergemann, H. N. Steinberg, J. G. Flanagan, X. Li, S. J.
Galli, and B. G. Neel. 1996. Genetic analysis reveals cell type-specific regu-
lation of receptor tyrosine kinase c-Kit by the protein tyrosine phosphatase
SHP1. J. Exp. Med. 184:1111–1126.
26. Miller, A. D., and G. J. Rosman. 1989. Improved retroviral vectors for gene
transfer and expression. BioTechniques 7:980–982.
27. Mori, S., L. Ronnstrand, K. Yokote, A. Engstrom, S. A., Courtneidge, L.
Claesson-Welsh, and C.-H. Heldin. 1993. Identification of two juxtamem-
brane autophosphorylation sites in the PDGF ?-receptor; involvement in the
interaction with Src family tyrosine kinases. EMBO J. 12:2257–2264.
28. Nishikawa, S., M. Kusakabe, K. Yoshinaga, M. Ogawa, S. Hayashi, T.
Kunisada, T. Era, T. Sakakura, and S. Nishikawa. 1991. In utero manipu-
lation of coat color formation by a monoclonal anti-c-kit antibody: two
distinct waves of c-kit-dependency during melanocyte development. EMBO
29. Nocka, K., S. Majumder, B. Chabot, P. Ray, M. Gervonne, A. Bernstein, and
P. Besmer. 1989. Expression of the c-Kit proto-oncogene in known cellular
targets of W mutations in normal and W mutant mice: evidence for an
impaired c-Kit kinase in mutant mice. Genes Dev. 3:816–826.
30. Olcese, L., P. Lang, F. Vely, A. Cambiaggi, D. Marguet, M. Blery, K. L.
Hippen, R. Biassoni, A. Moretta, L. Moretta, J. C. Cambier, and E. Vivier.
1996. Human and mouse killer cell inhibitory receptors recruit PTP1C and
PTP1D tyrosine phosphatases. J. Immunol. 156:4531–4534.
31. Onoue, H., K. Maeyama, S. Nomura, T. Kasugai, H. Teri, H. M. Kim, T.
Watamabe, and Y. Kitamura. 1993. Absence of immature mast cells in the
skin of Ws/Ws rats with a small deletion at the tyrosine kinase domain of the
c-Kit gene. J. Pathol. 193:1001–1007.
32. Pani, G., M. Kozlowski, J. C. Cambier, G. B. Mills, and K. A. Siminovitch.
1995. Identification of the tyrosine phosphatase PTP1C as a B cell antigen
receptor-associated protein involved in the regulation of B cell signaling. J.
Exp. Med. 181:2077–2084.
33. Pani, G., K. D. Fischer, I. Mlinaric-Rascan, and K. A. Siminovitch. 1996.
Signaling capacity of the T cell antigen receptor is negatively regulated by the
PTP1C tyrosine phosphatase. J. Exp. Med. 184:839–852.
34. Pani, G., and K. A. Siminovitch. 1997. Protein tyrosine phosphatase roles in
the regulation of lymphocyte signaling. Clin. Immunol. Immunopathol. 84:
35. Paulson, R. F., S. Vesely, K. A. Siminovitch, and A. Bernstein. 1996. Signal-
ing by the W/kit receptor tyrosine kinase is negatively regulated in vivo by the
protein tyrosine phosphatase Shp1. Nat. Genet. 13:309–315.
36. Pear, W. S., G. P. Nolan, M. L. Scott, and D. Baltimore. 1993. Production of
high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad.
Sci. USA 90:8392–8396.
37. Perkins, L. A., I. Larsen, and N. Perrimon. 1992. Corkscrew encodes a
putative protein tyrosine phosphatase that functions to transduce the termi-
nal signal from the receptor tyrosine kinase torso. Cell 70:225–236.
38. Piao, X., J. E. Curtis, S. Minkin, M. D. Minden, and A. Bernstein. 1994.
Expression of the Kit and KitA receptor isoforms in human acute myelog-
enous leukemia. Blood 83:476–481.
39. Qui, F., P. Ray, K. Brown, P. E. Barker, S. Jhanwar, F. H. Ruddle, and P.
Besmer. 1988. Primary structure of c-Kit: relationship with the CSF-1/PDGF
receptor kinase family—oncogenic activation of v-kit involves deletion of
extracellular domain and C-terminus. EMBO J. 7:1003–1011.
40. Reith, A. D., C. Ellis, D. L. Lynman, D. M. Anderson, D. E. Williams, A.
Bernstein, and T. Pawson. 1991. Signal transduction by normal isoforms and
W mutant variants of the Kit receptor tyrosine kinase. EMBO J. 10:2451–
41. Rottapel, R., M. Reedijk, D. E. Williams, S. D. Lynman, D. M. Anderson, T.
Pawson, and A. Bernstein. 1991. The Steel/W transduction pathway: Kit
autophosphorylation and its association with a unique subset of cytoplasmic
signaling proteins is induced by steel factor. Mol. Cell. Biol. 11:3043–3051.
42. Shearman, M. S., R. Herbst, J. Schlessinger, and A. Ullrich. 1993. Phos-
2098KOZLOWSKI ET AL.MOL. CELL. BIOL.
phatidylinosital 3?-kinase associates with p145c-Kit as part of a cell type Download full-text
characteristic multimeric signalling complex. EMBO J. 12:3817–3826.
43. Song yang, Z., S. E. Shoelson, M. Chandhuri, G. Gish, T. Pawson, W. G.
Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B.
Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C.
Cantley. 1994. Specific motifs recognized by the SH2 domains of Csk, 3BP2,
Fps/Fes, GRB-2, HCP, SHC, Syk, and Vav. Mol. Cell. Biol. 14:2777–2785.
44. Songyang, Z., S. E. Schoelson, H. McGlade, P. Oliver, T. Pawson, X. R.
Bustelo, M. Barbacid, H. Sabe, H. Hanafusa, T. Yi, R. Ren, D. Baltimore, S.
Ratnofsky, R. A. Feldman, and L. C. Cantley. 1994. SH2 domains recognize
specific phosphopeptide sequences. Cell 72:767–778.
45. Tauchi, T., G. S. Feng, M. S. Marshall, R. Shen, C. Mantel, T. Pawson, and
H. E. Broxmeyer. 1994. The ubiquitously expressed Syp phosphatase inter-
acts with c-Kit and Grb2 in hematopoietic cells. J. Biol. Chem. 269:25206–
46. Tsui, H. W., K. A. Siminovitch, L. de Souza, and F. W. L. Tsui. 1993.
Motheaten and viable motheaten mice have mutations in the hemopoietic
cell phosphatase gene. Nat. Genet. 4:124–129.
47. Tsujimura, T., T. Furitsu, M. Morimoto, K. Isozaki, S. Nomura, Y. Matsu-
zawa, Y. Kitamura, and Y. Kanakura. 1994. Ligand-independent activation
of c-Kit receptor tyrosine kinase in a murine mastocytoma cell line P-815
generated by a point mutation. Blood 83:2619–2626.
48. Tsujimura, T., M. Morimoto, K. Hashimoto, Y. Moriyama, H. Kitayama, Y.
Matsuzawa, Y. Kitamura, and Y. Kanakura. 1996. Constitutive activation of
c-Kit in FMA3 murine mastocytoma cells caused by deletion of seven amino
acids at the juxtamembrane domain. Blood 87:273–283.
49. Uchida, T., T. Matozaki, K. Matsuda, T. Suzuki, S. Mutozaki, O. Nakano, K.
Wada, Y. Konda, C. Sakamoto, and M. Kasugo. 1993. Phorbolester stimu-
lates the activity of protein tyrosine phosphatase containing SH2 domains
(PTP1C) in the HL-60 leukemia cells by increasing gene expression. J. Biol.
50. Waksman, G., S. E. Shoelson, N. Pant, D. Cowburn, and John Kurigan.
1993. Binding of a high affinity phosphotyrosol peptide to the Src SH2
domain: crystal structures of the complexed and peptide free forms. Cell
51. Weiler, S. R., S. Mou, C. S. DeBerry, J. R. Keller, F. W. Ruscetti, D. K.
Ferris, D. L. Longo, and D. Linnekin. 1996. JAK2 is associated with the c-Kit
proto-oncogene product and is phosphorylated in response to stem cell
factor. Blood 87:3688–3693.
52. Witthuhn, B. A., F. W. Quelle, O. Silvennoinen, T. Yi, B. Tang, O. Miura,
and J. N. Ihle. 1993. JAK2 associates with the erythropoietin receptor and is
tyrosine phosphorylated and activated following stimulation with erythropoi-
etin. Cell 74:227–236.
53. Wu, H., U. Klingmuller, P. Besmer, and H. F. Lodish. 1995. Interaction of
the erythropoietin and stem-cell-factor receptors. Science 337:242–246.
54. Yi, T., and J. N. Ihle. 1993. Association of hematopoietic cell phosphatase
with c-Kit after stimulation with c-Kit ligand. Mol. Cell. Biol. 13:3350–3358.
55. Yi, T., A. L. F. Mui, G. Krystal, and J. N. Ihle. 1993. Hemopoietic cell
phosphatase associates with the interleukin-3 (IL-3) receptor ? chain and
down-regulates IL-3-induced tyrosine phosphorylation and mitogenesis.
Mol. Cell. Biol. 13:7577–7586.
56. Yi, T., J. Zhang, O. Miura, and J. N. Ihle. 1995. Hematopoietic cell phos-
phatase associates with erythropoietin (Epo) receptor after Epo-induced
receptor tyrosine phosphorylation: identification of potential binding sites.
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