MOLECULAR AND CELLULAR BIOLOGY, Dec. 1996, p. 6887–6899
Copyright ? 1996, American Society for Microbiology
Vol. 16, No. 12
A Novel Membrane Glycoprotein, SHPS-1, That Binds the
SH2-Domain-Containing Protein Tyrosine Phosphatase
SHP-2 in Response to Mitogens and Cell Adhesion
YOHSUKE FUJIOKA,1TAKASHI MATOZAKI,1* TETSUYA NOGUCHI,1AKIHIRO IWAMATSU,2
TAKUJI YAMAO,1NOBUAKI TAKAHASHI,2MASAHIRO TSUDA,1TOSHIYUKI TAKADA,1
AND MASATO KASUGA1
Second Department of Internal Medicine, Kobe University School of Medicine, Chuo-ku, Kobe 650,1
and Kirin Brewery Co. Ltd. Central Laboratories for Key Technology,
Kanazawa-ku, Yokohama, Kanagawa 236,2Japan
Received 19 June 1996/Returned for modification 29 July 1996/Accepted 11 September 1996
Protein tyrosine phosphatases (PTPases), such as SHP-1 and SHP-2, that contain Src homology 2 (SH2)
domains play important roles in growth factor and cytokine signal transduction pathways. A protein of ?115
to 120 kDa that interacts with SHP-1 and SHP-2 was purified from v-src-transformed rat fibroblasts (SR-3Y1
cells), and the corresponding cDNA was cloned. The predicted amino acid sequence of the encoded protein,
termed SHPS-1 (SHP substrate 1), suggests that it is a glycosylated receptor-like protein with three immu-
noglobulin-like domains in its extracellular region and four YXX(L/V/I) motifs, potential tyrosine phosphor-
ylation and SH2-domain binding sites, in its cytoplasmic region. Various mitogens, including serum, insulin,
and lysophosphatidic acid, or cell adhesion induced tyrosine phosphorylation of SHPS-1 and its subsequent
association with SHP-2 in cultured cells. Thus, SHPS-1 may be a direct substrate for both tyrosine kinases,
such as the insulin receptor kinase or Src, and a specific docking protein for SH2-domain-containing PTPases.
In addition, we suggest that SHPS-1 may be a potential substrate for SHP-2 and may function in both growth
factor- and cell adhesion-induced cell signaling.
SHP-2 (also named SH-PTP2, PTP1D, Syp, PTP2C, SH-
PTP3, and SAP-2) (1–3, 9, 10, 28, 56), is a non-transmembrane
protein tyrosine phosphatase (PTPase) that contains two Src
homology 2 (SH2) domains (17) and is thought to participate
in intracellular signaling in response to various growth factors,
such as platelet-derived growth factor (PDGF), epidermal
growth factor (EGF), and insulin (26, 27). SHP-2 binds to tyro-
sine-phosphorylated PDGF receptors in response to PDGF (9,
15, 22) and also to tyrosine-phosphorylated insulin receptor
substrate 1 (IRS-1) in response to insulin (20). In addition, it
has been demonstrated that expression of a catalytically inac-
tive SHP-2 inhibited the activation of RAS and mitogen-acti-
vated protein (MAP) kinase in response to insulin in a domi-
nant negative manner (33). The expression of a catalytically
inactive SHP-2 also inhibited the activation of MAP kinase in
response to insulin (31, 61) or fibroblast growth factor (50),
indicating that SHP-2 mediates growth factor stimulation of
the RAS-MAP kinase cascade that leads to DNA synthesis
(59). It has also been suggested that SHP-2 may play an im-
portant role in EGF-stimulated MAP kinase activation and
mitogenesis (4, 62). However, the mechanism by which SHP-2
mediates RAS-MAP kinase cascade activation in response to
insulin or other mitogens is largely unknown. Furthermore, the
site at which SHP-2 may act in the RAS-MAP kinase cascade
is still controversial. In contrast to our previous observation for
Chinese hamster ovary (CHO) cells that overexpress human
insulin receptors (IRs) (CHO-IR cells) (33), Sawada et al. have
reported that expression of a catalytically inactive SHP-2 in-
hibited the activation of MEK and Raf-1 kinase in response to
insulin and had no detectable effect on insulin-induced activa-
tion of RAS in NIH 3T3 cells overexpressing human IRs (39).
It has been suggested that Corkscrew (the putative Drosophila
homolog of SHP-2) may be required upstream for Ras1 acti-
vation or that it functions in conjunction with Ras1 during
Sevenless receptor tyrosine kinase signaling (12). Thus, the
identification of a phosphorylated substrate of SHP-2 is essen-
tial for understanding the SHP-2-mediated signaling pathway.
It has recently been shown that insulin induces tyrosine
phosphorylation of an ?115-kDa membrane glycoprotein,
pp115, and subsequent association of SHP-2 with pp115 in
CHO-IR cells (32). The extent of tyrosine phosphorylation of
pp115 was greatly increased, relative to that in CHO-IR cells,
in CHO-IR cells that also overexpress catalytically inactive
SHP-2 (27, 32). The lack of PTPase activity of the mutant
SHP-2 may result in its forming a stable complex with tyrosine-
phosphorylated pp115, suggesting that pp115 may be a physi-
ological substrate for SHP-2 and that it may mediate SHP-2
signaling to downstream components. It has also been reported
that transient expression of a catalytically inactive SHP-2 in-
duces hyperphosphorylation of a 120-kDa protein which binds
to the fusion protein containing SH2 domains of SHP-2 in NIH
3T3 cells overexpressing human IRs (31). SHP-2 has also been
demonstrated to bind to a tyrosine-phosphorylated 115-kDa
protein in response to insulin (63) or EGF (62), while this
115-kDa protein did not bind to a fusion protein containing
SH2 domains of SHP-2 (63).
It has also been demonstrated that SHP-1, another SH2-
domain-containing PTPase (1, 30, 37, 43, 64), formed a com-
plex with an ?120-kDa tyrosine-phosphorylated protein,
pp120, when SHP-1 was overexpressed in v-src-transformed rat
fibroblasts (SR-3Y1 cells) (29). SHP-1 is expressed predomi-
nantly in hematopoietic cells and is thought to play an inhibi-
tory role in cytokine-stimulated proliferation of these cells (16,
* Corresponding author. Mailing address: Second Department of
Internal Medicine, Kobe University School of Medicine, Kusunoki-
cho, Chuo-ku, Kobe 650, Japan. Phone: 81-78-341-7451, ext. 5522. Fax:
81-78-382-2080. Electronic mail address: email@example.com.
26, 44, 51, 65). Although the physiological substrate of SHP-1
remains to be identified, pp120 is a potential candidate.
We have now purified pp120 from SR-3Y1 cell membranes
and cloned its cDNA. Because pp120 is a potential substrate
for SHP-1 and SHP-2, we named this protein SHPS-1 (SHP
substrate 1). SHPS-1 possesses immunoglobulin (Ig)-like do-
mains in its putative extracellular region and potential binding
sites for the SH2 domains of SHP-1 and SHP-2 in its cytoplas-
mic region. In addition, we show that SHP-2 binds to tyrosine-
phosphorylated SHPS-1 in intact cells in response to mitogens
and cell adhesion.
MATERIALS AND METHODS
Cells and antibodies. 3Y1 rat fibroblast cells and SR-3Y1 cells were obtained
from the Japanese Cancer Research Resources Bank. SR-3Y1 cells that overex-
press either SHP-1 (SR-3Y1-P cells) or SHP-1 lacking SH2 domains (SR-3Y1-C
cells) were generated as previously described (29). CHO-IR cells that overex-
press either wild-type SHP-2 (CHO-SHP-2-WT cells) or catalytically inactive
SHP-2 (CHO-SHP-2-C/S cells) were generated as previously described (33).
To generate polyclonal antibodies to SHPS-1, we first prepared a cDNA
fragment encoding the putative cytoplasmic region of SHPS-1 by PCR amplifi-
cation with a sense primer (5?-TTGGATCCAAGAAAGCCAAGGGCTCAAC
TTCT; nucleotides [nt] 1246 to 1269), an antisense primer (5?-AAGAATTCTC
ACTTCCTCTGGACTTGGACACT; nt 1552 to 1575), and the full-length rat
SHPS-1 cDNA as a template, as described previously (33). The amplified PCR
fragment was inserted in frame into the BamHI and EcoRI site of pGEX-2T
(Pharmacia), and the glutathione-S-transferase (GST) fusion protein containing
the cytoplasmic region of SHPS-1 was expressed in Escherichia coli and purified.
Female rabbits were injected with the GST fusion protein, and polyclonal anti-
bodies were affinity purified with CNBr-activated Sepharose beads (Pharmacia)
coupled to the GST–SHPS-1 protein as described previously (33).
Monoclonal antibody to pp115 of CHO cells, 4C6, was generated by injecting
mice with pp115 partially purified from CHO-IR cells as described elsewhere
(32). This monoclonal antibody reacts well with pp115 of CHO cells but poorly
with the corresponding protein of other species, such as rats or mice.
Rabbit polyclonal antibodies to SHP-1 were generated against a synthetic
peptide corresponding to the COOH-terminal region of SHP-1 as described
previously (52). Rabbit polyclonal antibodies to SHP-2 were generated against a
GST fusion protein containing the COOH-terminal region of SHP-2 as described
previously (33). The PY-20 monoclonal antibody to phosphotyrosine was ob-
tained from Transduction Laboratories. Monoclonal antibody 9E10, to MYC
epitope tag, was purified from the ascites of mice injected with MYC1-9E10
hybridoma cells that were obtained from American Type Culture Collection.
Monoclonal antibody to v-Src (Ab-1) was obtained from Oncogene Science.
Immunoprecipitation and immunoblot analysis. SR-3Y1 cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS). Confluent cells (10-cm-diameter plates) were washed with
phosphate-buffered saline (PBS) and immediately frozen in liquid nitrogen. The
cells were then lysed on ice in 1 ml of ice-cold lysis buffer (20 mM Tris-HCl [pH
7.6], 140 mM NaCl, 2.6 mM CaCl2, 1 mM MgCl2, 1% Nonidet P-40, 10%
[vol/vol] glycerol) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1
mM sodium vanadate. The lysates were centrifuged at 10,000 ? g for 15 min at
4?C, and the resulting supernatants were subjected to immunoprecipitation and
immunoblot analysis. Supernatants were incubated for 4 h at 4?C with various
antibodies bound to protein G-Sepharose beads (2 ?g of antibody on 20 ?l of
beads) (Pharmacia) in the presence or absence of the immune antigen. For in
vitro binding experiments, the supernatants prepared from SR-3Y1 cells were
also incubated with various GST fusion proteins immobilized on glutathione-
Sepharose beads (Pharmacia). The beads were then washed twice with 1 ml of WG
buffer (50 mM N-2-hydroxyethylpiperazine-N?-2-ethanesulfonic acid [HEPES]-
NaOH [pH 7.6], 150 mM NaCl, 0.1% Triton X-100) and resuspended in sodium
dodecyl sulfate (SDS) sample buffer. Gel electrophoresis and immunoblot anal-
ysis with PY-20 or other antibodies and an enhanced chemiluminescence detec-
tion kit (Amersham) were performed as described previously (29, 33).
SR-3Y1 cells were also incubated in the absence or presence of tunicamycin (5
?g/ml) (Sigma) for 48 h at 37?C. Solubilized membrane fractions were prepared
as described below and incubated with GST–SHP-1 fusion protein immobilized
on beads. The bound proteins were subjected to SDS-polyacrylamide gel elec-
trophoresis (SDS-PAGE) and immunoblot analysis with PY-20.
Expression and purification of recombinant PTPases. Recombinant full-
length SHP-1 was generated with the use of the GST fusion protein system. PCR
amplification was performed with a sense primer (5?-ATTGAATTCTGTCCCG
TGGGTGGTTTCAC; nt 256 to 279), an antisense primer (5?-ACCGAATTCT
CACTTCCTCTTGAGGGAACC; nt 2029 to 2049), and the full-length SHP-1
cDNA as a template (53). In addition, an SHP-1 cDNA lacking the sequence
encoding the SH2 domains was constructed by PCR amplification of the portion
of the full-length cDNA coding for the PTPase domain and the COOH-terminal
region; full-length cDNA served as the template, the sense primer was 5?-TTA
GAATTCACCATGAACTTGCACCAGCGTCTGGAA (nt 1033 to 1053), and
the antisense primer was 5?-TATGAATTCTCACTTCCTCTTGAGGGAACC
(nt 2029 to 2049). The amplified PCR fragments were digested with BamHI and
EcoRI and inserted in frame into the BamHI and EcoRI site of pGEX-2T. The
GST fusion protein containing full-length SHP-1 (GST–SHP-1) or that contain-
ing SHP-1 lacking SH2 domains (GST–SHP-1?SH2) was expressed in E. coli
(10-ml culture) as described previously (53), and the cells were immobilized on
glutathione-Sepharose beads, which were then subjected to in vitro binding
experiments. Incubation of cell lysate supernatants with the beads was performed
under the same conditions as for immunoprecipitation.
GST–SHP-1 was also isolated from 0.5 to 2 liters of bacterial culture as
described previously (33) and bound to glutathione-Sepharose beads. The beads
were washed twice with 10 ml of NETN lysis solution (20 mM Tris-HCl [pH 8.0],
100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), and the bound proteins were
then eluted by incubation with 7 ml of a solution containing 50 mM Tris-HCl (pH
8.0) and 10 mM glutathione (Sigma) for 30 min at 4?C. Proteins eluted from the
beads were dialyzed overnight against 1 liter of a solution containing 25 mM
Tris-HCl (pH 7.5), 1 mM EDTA, and 1 mM dithiothreitol and then concentrated
with a Centriprep-30 filtration cell (Amicon).
Expression and purification of GST fusion proteins containing full-length
SHP-2 (GST–SHP-2), the SH2 domains of SHP-2 (GST–SHP-2-SH2), or SHP-2
lacking SH2 domains (GST–SHP-2?SH2) were performed as previously de-
Subcellular fractionation. Confluent cells (10-cm-diameter plate) were washed
with ice-cold PBS, frozen in liquid nitrogen, scraped into 1 ml of ice-cold hypo-
tonic lysis solution, which consisted of 20 mM HEPES-NaOH (pH 7.6), 5 mM
NaPPi, 5 mM ethylene glycol-bis(?-aminoethyl ether)-N,N,N?,N?-tetraacetic acid
(EGTA), and 1 mM MgCl2, containing aprotinin (10 ?g/ml), 1 mM PMSF, and
1 mM sodium vanadate, and homogenized with a Dounce homogenizer. The
homogenate was centrifuged at 100,000 ? g for 60 min, and the resulting super-
natant was referred to as the cytosolic fraction. The pellet was suspended in 1 ml
of membrane solubilization solution (20 mM Tris-HCl [pH 7.5], 1% Triton
X-100, 150 mM NaCl, 1 mM MgCl2) supplemented with 1 mM PMSF and 1 mM
sodium vanadate. The suspension was centrifuged at 100,000 ? g for 60 min, and
the resulting supernatant was referred to as the solubilized membrane fraction.
All procedures were performed at 4?C.
Affinity purification of pp120. Confluent SR-3Y1 cells from a total of 100
culture dishes (175 mm2) were washed with ice-cold PBS and immediately frozen
in liquid nitrogen. Subsequent procedures were performed at 4?C unless indi-
cated otherwise. The cells were scraped with a rubber policeman on ice into a
total volume of 150 ml of hypotonic lysis solution containing aprotinin (10
?g/ml), 1 mM PMSF, and 1 mM sodium vanadate. After addition of NaCl to a
final concentration of 500 mM, the cells were homogenized with a Dounce ho-
mogenizer (100 strokes). The homogenates were then centrifuged at 100,000 ?
g for 60 min, and the resulting pellets were resuspended in 30 ml of Triton lysis
solution (20 mM Tris-HCl [pH 7.5], 1% Triton X-100, 150 mM NaCl, 2 mM
EDTA, 10% glycerol) supplemented with 1 mM PMSF and 1 mM sodium
vanadate. The suspension was again homogenized with a Dounce homogenizer
(100 strokes) and centrifuged at 100,000 ? g for 60 min, and the resulting
supernatant was passed through a 0.8-?m-pore-size membrane filter (Amicon).
The filtrate was incubated with 100 ?l of CNBr-activated Sepharose beads
coupled with the GST–SHP-1 fusion protein (?1 ?g of protein coupled to 1 ?l
of beads) in two 15-ml tubes with gentle rotation for 4 to 12 h. The beads were
washed with Triton lysis solution in the 15-ml tubes, transferred to a 1.5-ml
Eppendorf tube, and washed with 1 ml of Triton wash buffer containing 20 mM
Tris-HCl (pH 7.5), 1% Triton X-100, 2 mM EDTA, and 500 mM NaCl, with
rotation, for 1 to 2 h at 25?C. The bound proteins were then eluted with 500 ?l
of SDS elution buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM EDTA,
0.1% Triton X-100, 0.1% SDS) at 25?C. After the addition of MnCl2to a final
concentration of 1 mM, the eluate was incubated for 2 h with 100 ?l of agarose
beads coupled with concanavalin A (ConA) (Seikagaku Kogyo Co.). The beads
were washed twice with 1 ml of Triton wash buffer, and the bound proteins were
eluted with 500 ?l of Tween buffer (10 mM Tris-HCl [pH 7.5], 0.1% Tween 20,
150 mM NaCl) containing 200 mM ?-methyl-D-mannoside (Wako). The eluted
proteins were then precipitated with 10% (wt/vol) trichloroacetic acid, washed
once with cold (?20?C) acetone, and dried with a Speed-Vac concentrator.
In a separate experiment, a solubilized membrane fraction of SR-3Y1 cells was
incubated with GST–SHP-1 immobilized on glutathione-Sepharose beads; there-
after, the beads were washed and the bound proteins were eluted with SDS
elution buffer as described above. The eluted proteins were then incubated with
agarose beads conjugated with wheat germ agglutinin (WGA), Lens culinaris
agglutinin (LCA), or Ricinus communis agglutinin (RCA) (Seikagaku Kogyo
Co.) under the same conditions used for ConA. The proteins bound to the
lectin-coupled beads were separated by SDS-PAGE and subjected to immuno-
blot analysis with PY-20.
Separation of pp120 by SDS-PAGE and internal amino acid sequencing.
Affinity-purified pp120 was dissolved in SDS sample buffer, subjected to SDS-
PAGE in a 7.5% gel, and transferred to a polyvinylidene difluoride filter (ProB-
lott; Applied Biosystems). Proteins were visualized by being stained with 0.1%
Ponceau S in 1% acetic acid, and the band corresponding to pp120 was excised
for amino acid sequence analysis. Confluent SR-3Y1 cells from a total of 1,000
6888 FUJIOKA ET AL.MOL. CELL. BIOL.
phy, fragile X syndrome, and Huntington’s disease (21, 38).
Given that human SHPS-1 mRNA is also abundant in the
brain (60), the expansion of the triplet repeats in the SHPS-1
gene may underlie some neurological disease.
It has previously been demonstrated that SHP-2 may regu-
late an upstream element necessary for RAS activation in
response to insulin (33), while the place of SHP-2 during the
activation of the RAS-MAP kinase cascade in response to
insulin is still controversial (12, 39). Although we have now
clarified the structure of SHPS-1, to which SHP-2 binds, it
remains unclear how SHPS-1 may couple SHP-2 to RAS acti-
vation. No known catalytic domain was detected in the cyto-
plasmic region of SHPS-1. In the preliminary experiment, nei-
ther Grb2, Shc, nor GAP bound to SHPS-1 in response to
insulin in CHO-IR cells (34). Thus, SHPS-1 may simply act as
a docking protein, similar to IRS-1, and induce translocation of
SHP-2 from the cytosol to the plasma membrane in response
to mitogens and cell adhesion. Approximately 2% of the total
SHP-2 was recovered in the SHPS-1 immunoprecipitates with
4C6 monoclonal antibody when CHO-IR cells were stimulated
with insulin (34). However, it is possible that the minor fraction
of SHP-2 may be enough to mediate its downstream signals.
After binding to SHPS-1, SHP-2 may dephosphorylate and
dissociate from SHPS-1, in the process possibly activating an
SOS-like guanine nucleotide exchange protein near the plasma
membrane by catalyzing its tyrosine dephosphorylation. In ad-
dition, it remains to be determined whether SHP-1 also binds
to SHPS-1 in response to cytokines in hematopoietic cells and
plays a role in cytokine signal transduction.
We thank K. Shii and R. Sakai for helpful advice regarding the
purification of pp120 and H. Nojima for kindly providing the rat 3Y1
cell cDNA library.
This work was supported by a grant-in-aid for cancer research, a
grant-in-aid for Scientific Research (A) from the Ministry of Educa-
tion, Science, and Culture of Japan, and a grant for diabetic research
from Ohtsuka Pharmaceutical Co. Ltd.
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