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: firstname.lastname@example.org.
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.
culture dishes (175 mm2) were required to obtain sufficient pp120 for amino acid
After reduction and S-carboxymethylation, the immobilized protein on the
membrane filter was digested with 0.2 pmol of Achromobacter protease I (ly-
sylendopeptidase) (Wako), essentially as described previously (13). Generated
peptides were separated by high-performance liquid chromatography with a
Wakosil-II AR octyldecyl silane column (2.0 by 150 mm) (Wako). Amino acid
sequencing of the peptides was performed with a gas-phase sequencer (model
PPSQ-10; Shimazu). The resultant phenylthiohydantoin derivatives were identi-
fied by isocratic high-performance liquid chromatography as described previ-
Cloning of SHPS-1 cDNAs. Eight amino acid sequences obtained from the 12
pp120 peptides isolated were used to design degenerate oligonucleotide primers
for PCR. DNA fragments were amplified by PCR with all possible combinations
of degenerate primers and rat brain cDNA (Clontech) as a template. PCR was
performed for three cycles of denaturation at 94?C for 1 min, annealing at 50?C
for 1.5 min, and extension at 72?C for 2 min, followed by 37 cycles of 94?C for 1
min, 55?C for 1.5 min, and 72?C for 2 min. A 168-bp PCR fragment (nt 289 to 456
of the cDNA sequence) was obtained with a sense primer [5?-TT(T/C)ATIG
GIGGIGA(A/G)CA(T/C)TT] derived from the peptide AP-11 and an antisense
primer [5?-TTIAT(C/T)TCIGT(G/A)TCIGG(C/T)TC] derived from the peptide
AP-6. This fragment was chosen as a screening probe because it contained two
amino acid sequences in the same coding frame as determined by sequence
analysis. A rat 3Y1 cell ZAP-II cDNA library (kindly provided by H. Nojima) was
then screened as previously described (28), and the nucleotide sequence of the
longest cDNA clone was determined in both directions by dideoxy termination
methods with a Sequencing Pro kit (TOYOBO). Screening of a mouse brain
ZAP-II cDNA library (Stratagene) and a human brain ?gt10 cDNA library
(Clontech) with32P-labeled rat SHPS-1 cDNA as a probe yielded a 3.0-kb mouse
full-length SHPS-1 cDNA and a 1.6-kb human partial SHPS-1 cDNA, both of
which were isolated and sequenced.
Northern (RNA) blot analysis. A rat multiple-tissue Northern blot (Clontech)
containing 2 ?g of poly(A)?RNA was hybridized consecutively with a32P-
labeled 3.7-kb cDNA fragment of rat SHPS-1 cDNA and a32P-labeled mouse
?-actin cDNA probe.
Transfection of SHPS-1 cDNA. Full-length rat SHPS-1 cDNA was inserted
into the EcoRI site in the pSR? expression vector (pSR?-SHPS-1). To construct
a pSR? expression vector encoding MYC epitope-tagged SHPS-1 (pSR?-SHPS-
1-MYC), we performed PCR with a pBluescript vector (Stratagene) containing
full-length rat SHPS-1 cDNA as a template, T3 primer as the sense primer, and
the antisense primer 5?-GTGAATTCTCAGAGGTCTTCTTCCGATATCAGC
TTCTGTTC, which results in deletion of the natural termination codon of
SHPS-1 cDNA and addition of another sequence encoding the amino acids
EQKLISEEDL followed by a termination codon and an EcoRI site. The ampli-
fied DNA fragment was digested with EcoRI and inserted into the EcoRI site of
pSR?, yielding pSR?-SHPS-1-MYC.
Semiconfluent SR-3Y1 cells cultured in DMEM supplemented with 10% FBS
were transfected with 10 ?g of pSR?-SHPS-1 with the use of Lipofectamine
(Gibco). Cells were harvested 2 days after transfection and lysed as described
above for immunoblot analysis with antibodies to SHPS-1.
CHO-IR cells (?5 ? 105cells per 10-cm-diameter dish) were transfected with
both 10 ?g of pSR?-SHPS-1-MYC and 1 ?g of pHyg, which contains the
hygromycin B phosphotransferase gene, by the calcium phosphate precipitation
method (33). The cells were cultured in Ham’s F-12 medium containing hygro-
mycin B (200 ?g/ml) (Wako) and 10% FBS, and colonies were isolated 14 to 21
days after transfection. Several cell lines expressing MYC epitope-tagged
SHPS-1 (CHO-IR-SHPS-1) were identified by immunoblotting cell lysates with
polyclonal antibody to SHPS-1 as described above.
Stimulation of cells with mitogens and cell adhesion. CHO-IR cells or CHO-
IR-SHPS-1 cells cultured in Ham’s F-12 medium containing 10% FBS were
deprived of serum for 16 h and then stimulated with 100 nM insulin for 5 min.
Confluent Rat-1-IR cells cultured in DMEM supplemented with 10% FBS were
also deprived of serum and stimulated with mitogens. After stimulation, the
culture medium was aspirated and the cells were immediately washed with
ice-cold PBS and frozen in liquid nitrogen. Cell lysates were prepared and
subjected to immunoprecipitation and immunoblot analysis as described above.
For adhesion experiments, CHO-IR cells were treated with PBS containing 1
mM Ca2?and 0.5 mM Mg2?or with PBS without Ca2?and Mg2?for 10 min as
described previously (24), after which lysates were prepared. Confluent CHO-IR
cells were also detached by treatment with 0.05% trypsin and 0.02% EDTA,
washed three times with serum-free Ham’s F-12 medium, plated on polystyrene
dishes which were coated with fibronectin (Falcon) or uncoated, and incubated
at 37?C for 1 h in serum-free Ham’s F-12 medium as described previously (35).
Bound cells were then lysed and subjected to immunoprecipitation and immu-
In vitro phosphorylation of the cytoplasmic portion of SHPS-1 by IRs and its
binding to SH2 domains of SHP-2. IRs were partially purified from lysates of
insulin-stimulated CHO-IR cells with WGA-agarose as described previously
(14). The GST fusion protein containing the cytoplasmic region of SHPS-1,
purified as described above (?1 ?g), was incubated for 30 min at 25?C with
partially purified IRs immobilized on WGA-agarose beads in 50 ?l of a solution
containing 50 mM HEPES-NaOH (pH 7.6), 3 mM MnCl2, 10 mM MgCl2, 1 mM
dithiothreitol, and 10 ?M ATP. The supernatant of the reaction mixture was
separated from WGA-agarose beads by centrifugation at 10,000 ? g for 5 min.
Thereafter, the supernatant was incubated with 10 ?l of CNBr-activated Sepha-
rose beads coupled with a GST protein or a GST fusion protein containing SH2
domains of SHP-2 (?0.5 ?g of protein coupled to 1 ?l of beads) for 4 h at 4?C.
The beads were washed three times with 1 ml of WG buffer, and the proteins
bound to GST protein beads were separated by SDS-PAGE and subjected to
immunoblot analysis with PY-20.
Nucleotide sequence accession number. The nucleotide sequence data re-
ported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide
sequence databases under accession no. D85183.
The pp120 protein that binds to SHP-1 in SR-3Y1 cells is a
membrane glycoprotein. As has been shown previously (29), an
?120-kDa tyrosine-phosphorylated protein, pp120, was coim-
munoprecipitated with tyrosine-phosphorylated SHP-1 in SR-
3Y1-P cells (Fig. 1A, lane 2). The pp120 protein did not form
a complex with an SHP-1 protein lacking SH2 domains in
SR-3Y1-C cells (Fig. 1A, lane 3). On incubation of SR-3Y1 cell
lysates with a GST fusion protein containing either full-length
SHP-1 (GST–SHP-1) or SHP-1 lacking SH2 domains (GST–
SHP-1?SH2), a 120-kDa tyrosine-phosphorylated protein
bound to GST–SHP-1 but not to GST alone or GST–SHP-
1?SH2 (Fig. 1A, lanes 4 to 6). These data suggest that SHP-1
forms a complex with pp120 both in vivo and in vitro, presum-
ably through its SH2 domains.
To determine the subcellular localization of pp120, we pre-
pared cytosolic and solubilized membrane fractions of SR-
3Y1-P cells. Each fraction was subjected to immunoprecipita-
tion with antibodies to SHP-1 and immunoblot analysis with
the PY-20 monoclonal antibody to phosphotyrosine. Src, which
has been known to associate with the cell membrane, was
recovered in the membrane fraction (Fig. 1B, lanes 5 and 6).
The pp120 protein was recovered exclusively in the membrane
fraction (Fig. 1B, lanes 1 and 2). The same result was obtained
when subcellular fractions prepared from SR-3Y1 cells were
incubated with GST–SHP-1 and the bound proteins were sub-
jected to analysis with PY-20 (Fig. 1B, lanes 3 and 4).
The diffuse nature of the pp120 band on gel electrophoresis
suggested that pp120 might be a glycosylated protein. When
pp120 bound to GST–SHP-1 was eluted and then incubated
with various lectins coupled to agarose beads, the amount of
pp120 recovered was greatest with ConA- or WGA-coupled
beads, with lesser amounts binding to LCA- or RCA-coupled
beads (Fig. 1C). In addition, incubation of SR-3Y1 cells with
tunicamycin (5 ?g/ml) for 48 h reduced the apparent molecular
size of pp120 from 120 to ?60 kDa (Fig. 1D). These results
thus suggest that pp120 may be a membrane-associated glyco-
SHP-2 binds to pp120 in vitro. Because SHP-2 is structurally
similar to SHP-1, it is possible that SHP-2 also binds to pp120
in SR-3Y1 cells. A 120-kDa tyrosine-phosphorylated protein
present in the solubilized membrane fraction of SR-3Y1 cells
bound to GST–SHP-2 as well as to GST–SHP-1 (Fig. 2, lanes
1 and 2). In addition, pp120 bound to a GST fusion protein
containing the SH2 domains of SHP-2 (GST–SHP-2-SH2) but
not to GST–SHP-2 fusion protein lacking the SH2 domains
(GST–SHP-2?SH2) (Fig. 2, lanes 3 and 4), suggesting that the
SH2 domains of SHP-2 may mediate the association of SHP-2
Affinity purification and amino acid sequence analysis of
pp120. Given that pp120 from SR-3Y1 cells was shown to bind
to both GST-SHP-1 and ConA, we attempted to purify this
protein from SR-3Y1 cell membranes by taking advantage of
these properties. SR-3Y1 cells from 100 culture dishes (175
mm2) were scraped into a hypotonic solution. After addition of
VOL. 16, 1996SHPS-1, A TARGET FOR SH2-DOMAIN-CONTAINING PTPase 6889
500 mM NaCl, which markedly reduced the amount of proteins
associated with membranes, the cells were homogenized and
separated into cytosolic and membrane fractions. The mem-
brane fraction was solubilized in a buffer containing 1% Triton
X-100 and then incubated with GST–SHP-1 immobilized on
CNBr-activated Sepharose beads. Of several conditions tested
for elution of pp120 from the GST–SHP-1 beads, we chose a
solution containing 0.1% SDS and 0.1% Triton X-100 at 25?C
because it resulted in efficient and the most specific elution of
pp120. We have also used immobilized GST–SHP-2 to purify
pp120; however, the results were inferior to those obtained
with GST–SHP-1 because GST–SHP-2 binds many more pro-
teins than GST–SHP-1, as revealed by silver staining (data not
shown). The proteins eluted from GST–SHP-1 beads were
then incubated with agarose beads conjugated with ConA.
After extensive washing of the beads, bound proteins were
eluted with ?-methyl-D-mannoside. The proteins were then
precipitated with trichloroacetic acid, separated on a 7.5%
polyacrylamide gel, transferred to a polyvinylidene difluoride
membrane filter, and stained with Ponceau S. A major band
corresponding to pp120 and several minor bands were de-
tected (Fig. 3A).
We repeated the purification procedure a total of 10 times to
obtain sufficient protein for amino acid sequence analysis. We
finally accumulated ?5 ?g of purified pp120, as estimated by
the intensity of the band stained with Ponceau S. The purified
protein was then subjected to internal amino acid sequencing.
More than 20 peptide fractions were obtained after digestion
of pp120 with Achromobacter protease I (Fig. 3B), and nine
single and three mixed sequences were determined (Table 1).
Searching the Swiss Prot database with the BLAST program
FIG. 2. Binding of SHP-2 to pp120 in vitro. The solubilized membrane frac-
tion of SR-3Y1 cells was incubated with the indicated GST fusion proteins
immobilized on beads. The bound proteins were then subjected to SDS-PAGE
and immunoblot analysis with PY-20.
FIG. 1. Characterization of the SHP-1-associated pp120 protein in SR-3Y1
cells as a membrane glycoprotein. (A) Lysates of SR-3Y1-P (lanes 1 and 2) or of
SR-3Y1-C(lane 3) cells were subjected to immunoprecipitation with preimmune
serum or polyclonal antibody to SHP-1 as indicated below the lanes. In addition,
SR-3Y1 cell lysates were incubated with GST protein (lane 4) or GST–SHP-1
(lane 5) or GST–SHP-1?SH2 (lane 6) fusion protein immobilized on glutathi-
one-Sepharose beads. The immunoprecipitates or the proteins bound to the
beads were then fractionated by SDS-PAGE and subjected to immunoblot anal-
ysis with the PY-20 monoclonal antibody to phosphotyrosine. The pp120 band and
the positions of molecular size standards are indicated. (B) Solubilized mem-
brane (M) and cytosolic (C) fractions of SR-3Y1-P cells were subjected to immu-
noprecipitation with polyclonal antibody to SHP-1 (lanes 1 and 2, respectively).
The corresponding fractions of SR-3Y1 cells were incubated with GST–SHP-1
beads (lanes 3 and 4, respectively). The precipitates and bead-bound proteins
were then subjected to SDS-PAGE and immunoblot analysis with PY-20. Solu-
bilized membrane and cytosolic fractions of SR-3Y1-P cells were also immuno-
blotted with monoclonal antibody to v-Src (lanes 5 and 6, respectively). (C) A
solubilized membrane fraction of SR-3Y1 cells was incubated with GST–SHP-1
immobilized on glutathione-Sepharose beads. The beads were washed and the
bound proteins were eluted with SDS elution buffer as described in Materials and
Methods. The eluted proteins were then incubated with agarose beads conju-
gated with ConA, WGA, LCA, or RCA. The proteins bound to the lectin-
coupled beads were separated by SDS-PAGE and subjected to immunoblot
analysis with PY-20. (D) SR-3Y1 cells were incubated in the absence or presence
of tunicamycin (5 ?g/ml) for 48 h at 37?C. Solubilized membrane fractions were
prepared and incubated with GST–SHP-1 immobilized on beads. The bound
proteins were subjected to SDS-PAGE and immunoblot analysis with PY-20.
6890FUJIOKA ET AL.MOL. CELL. BIOL.
revealed that none of the peptide sequences was identical to
listed sequences, indicating that pp120 is a previously unchar-
acterized protein. Because it is a potential target for SH2-
domain-containing PTPases, we designated pp120 SHPS-1, for
SHP substrate 1.
Cloning of SHPS-1 cDNA. The amino acid sequences ob-
tained from purified SHPS-1 were used to design degenerate
oligonucleotide primers for the PCR. Screening of a rat 3Y1
cell ZAP-II cDNA library with a 168-bp32P-labeled PCR DNA
fragment as a probe yielded a full-length SHPS-1 cDNA. The
cloned SHPS-1 cDNA comprised 3,728 nt and contained a
single open reading frame encoding a protein of 509 amino
acids (nt 46 to 1572) (Fig. 4A). The first ATG codon (nt 46 to
48) matched the Kozak consensus sequence (19) for a trans-
lation initiation site and was presumed to be the translation
initiation codon. The 3? noncoding region of the cDNA con-
tains a typical polyadenylation signal (AATAAA) followed by
a poly(A) tail. The predicted amino acid sequence revealed
that residues 344 to 368 are highly hydrophobic, indicating that
SHPS-1 is a transmembrane protein, as expected. In addition,
the NH2-terminal 28 amino acids are also hydrophobic and
likely constitute a signal peptide (57). The entire putative ex-
tracellular region of SHPS-1 contains three homologous Ig-like
domains, indicating that SHPS-1 is a member of the Ig super-
family. Three types of Ig superfamily domain, V, C1, and C2,
have been differentiated (58). BLAST sequence analysis of the
Swiss Prot database revealed that the NH2-terminal Ig-like
domain of SHPS-1 is homologous to an Ig V domain, whereas
the second and third SHPS-1 domains resemble an Ig C1 do-
main. The extracellular region of SHPS-1 also contains 15 poten-
tial N-linked glycosylation sites (NXS or NXT, where X is any
amino acid), consistent with the observation that SHPS-1 is
highly glycosylated. In the cytoplasmic region of SHPS-1, four
tyrosine residues followed by XX(L/V/I) sequences (Y408ADL,
Y432ASIE, Y449ADL, and Y473ASV) represent potential ty-
rosine phosphorylation sites. Several putative serine-threonine
phosphorylation sites are also present in this region. The se-
quences downstream of the potential tyrosine phosphorylation
sites of SHPS-1 correspond well to binding sites for SHP-2 and
SHP-1 (7, 16, 45, 46, 48). BLAST analysis showed that the
sequences surrounding Tyr-408 and Tyr-449 of SHPS-1 are
similar to the sequence surrounding Tyr-1172 of IRS-1 (Fig.
4B). In addition, the sequences surrounding Tyr-432 and Tyr-
473 in SHPS-1 resemble that surrounding Tyr-1222 of IRS-1.
Furthermore, the sequences surrounding Tyr-408 and Tyr-449
of SHPS-1 resemble each other, as do those surrounding Tyr-
432 and Tyr-473 (Fig. 4B).
FIG. 3. Purification and protease digestion of pp120. (A) The pp120 protein was isolated from 100 culture dishes of confluent SR-3Y1 cells by a two-step affinity
purification protocol as described in Materials and Methods. The purified protein was subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane,
and stained with Ponceau S. The band corresponding to pp120 is indicated. (B) Purified pp120 was digested with Achromobacter protease I and the resulting peptides
were separated by reversed-phase high-performance liquid chromatography. Elution of the peptides was monitored by A205. The amino acid sequences of the peptides
in peaks AP-1 to AP-12 were determined by phenylthiohydantoin derivative analysis.
TABLE 1. Amino acid sequences of peptides derived
from purified pp120a
aPeptides generated by digestion of purified pp120 with Achromobacter pro-
tease I were subjected to NH2-terminal sequence analysis. Designations of pep-
tide fragments correspond to the absorbance peaks in Fig. 3B. Where two
sequences are shown for the same peak, the residues could not be assigned
unambiguously to the major or minor sequence because of their equal yields.
Residues in lowercase letters represent assignments of less than full confidence.
Positions for which no assignment was possible are indicated by X. The NH2-
terminal K in parentheses was deduced from the substrate specificity of the
VOL. 16, 1996 SHPS-1, A TARGET FOR SH2-DOMAIN-CONTAINING PTPase6891
In addition to the rat SHPS-1 cDNA, we also cloned mouse
and human SHPS-1 cDNAs. The four putative SH2-domain
binding sites, consisting of a tyrosine residue and the following
three amino acids, are completely conserved, and the overall
cytoplasmic region is highly conserved among the putative
SHPS-1 proteins from these three species (Fig. 4C). Detailed
characterization of the human and mouse SHPS-1 genes will
be given elsewhere. No sequences corresponding to known
catalytic domains were detected in the cytoplasmic region of
Northern blot analysis revealed that the rat SHPS-1 mRNA
is ?4.2 kb and was present in all tissues examined, being most
abundant in brain, lung, and spleen tissues (Fig. 5).
SHPS-1 is a substrate for the IR kinase and forms a com-
plex with SHP-2 in response to insulin. We have generated a
polyclonal antibody to a GST fusion protein containing the
cytoplasmic region of rat SHPS-1 (?SHPS-1). This antibody
recognized SHPS-1 purified from SR-3Y1 cell membranes
(Fig. 6A, lane 1). A 120-kDa protein was also detected in the
immunoblot analysis with this antibody of cell lysates prepared
FIG. 4. (A) Nucleotide sequence and deduced amino acid sequence of rat SHPS-1 cDNA. The putative signal peptide (solid underline), the transmembrane region
(boldface underline), the AATAAA box close to the polyadenylated 3? end of the cDNA (dots), the Ig-like domains (broken lines); (the Ig V-like domain, [small broken
line] and the Ig C1-like domains [large broken lines]), cysteine residues that potentially form disulfide bonds in the Ig-like domains (boxes), the putative binding sites
for SH2 domains of PTPases (boldface with underlines and small dots), and potential sites of N-linked glycosylation (boldface) are indicated. Potential serine-threonine
phosphorylation sites include Ser-381, Thr-437, and Thr-463 for protein kinase C [(S/T)X(R/K)] and Thr-407, Thr-448, and Ser-469 for casein kinase II [(S/T)XX(E/D)].
The numbers to the right of each row refer to nucleotide position (upper sequence) or to amino acid position (lower sequence) in the predicted mature SHPS-1 protein.
(B) Alignment of the amino acid sequences containing the putative SH2-domain binding sites of rat SHPS-1 with those of rat IRS-1 (47) and with each other. The
tyrosine residue in each sequence is indicated in parentheses. Residues shared by sequences (single-letter codes between two sequences) and similar amino acids (plus
signs) are indicated. The alignments were performed by using the BLAST program and the Swiss Prot database. (C) Alignment of the deduced amino acid sequences
of the putative cytoplasmic portions of rat, mouse, and human SHPS-1. Residues that are identical in all species (boxed), the putative binding sites for SH2 domains
of PTPases (boldface), and gaps introduced for optimal alignment (hyphens) are indicated.
6892 FUJIOKA ET AL.MOL. CELL. BIOL.
from 3Y1 cells and SR-3Y1 cells (Fig. 6A, lanes 2 and 3). The
120-kDa protein was immunoprecipitated with ?SHPS-1 poly-
clonal antibody but not with preimmune serum from 3Y1 cell
lysates (Fig. 6A, lanes 4 and 5). The immunoprecipitation of
the 120-kDa protein was significantly inhibited by incubation
of antibody with a GST fusion protein containing the cytoplas-
mic region of SHPS-1 (Fig. 6A, lane 6). Thus, these data
indicate that the polyclonal antibody ?SHPS-1 specifically rec-
ognized rat 120-kDa SHPS-1. We have found that ?SHPS-1
immunoprecipitated less SHPS-1 from SR-3Y1 cell lysates
than from 3Y1 cells (Fig. 6A, lane 7). Since ?SHPS-1 poly-
clonal antibody was generated against a GST fusion protein
containing the cytoplasmic region of SHPS-1, it is possible that
?SHPS-1 polyclonal antibody may not be able to immunopre-
cipitate the tyrosine-phosphorylated form of SHPS-1 or the
SHPS-1 complexed with SHP-2 in SR-3Y1 cells. Since we pu-
rified SHPS-1 from SR-3Y1 cells, we next transfected SR-3Y1
cells with cDNA of SHPS-1. The transfection of the SR-3Y1
cells yielded a polypeptide of ?120 kDa that was specifically
recognized by ?SHPS-1 polyclonal antibody (Fig. 6B, lane 2).
Thus, these results indicate that our cloned SHPS-1 cDNA
encodes SHPS-1 originally purified from SR-3Y1 cells.
We have shown that insulin stimulates both the tyrosine
phosphorylation of an ?115-kDa membrane glycoprotein,
pp115, and the subsequent association of SHP-2 with pp115 in
CHO-IR cells (32). In addition, the extent of tyrosine phos-
phorylation of pp115 is markedly increased in CHO-IR cells
overexpressing a catalytically inactive SHP-2, and the forma-
tion of a more stable complex of pp115 with the mutant protein
than with wild-type SHP-2 was observed (27, 32). We have
subsequently generated a monoclonal antibody, 4C6, which
specifically recognizes pp115 of CHO cells and can be used for
both immunoprecipitation and immunoblotting of pp115 (32).
When lysates of unstimulated CHO-IR cells were subjected to
immunoprecipitation with 4C6 monoclonal antibody, the pre-
cipitated 115-kDa protein was detectable by immunoblot anal-
ysis with ?SHPS-1 polyclonal antibody to rat SHPS-1 (Fig. 7A,
lane 2). Lysates prepared from unstimulated CHO-SHP-2-C/S
cells, in which a catalytically inactive SHP-2 forms a stable
complex with tyrosine-phosphorylated pp115 (27, 32), were
also immunoprecipitated with normal mouse IgG or 4C6
monoclonal antibody to pp115 (Fig. 7B, lanes 1 and 2). The
resulting supernatants from the first immunoprecipitation were
then subjected to a second round of immunoprecipitation with
polyclonal antibody to SHP-2. When the second immunopre-
cipitate was in turn subjected to immunoblot analysis with
?SHPS-1 polyclonal antibody, the amount of pp115 complexed
with SHP-2 was significantly decreased by the first immuno-
precipitation with 4C6 monoclonal antibody (Fig. 7B, lanes 1
and 2). These results suggest that polyclonal antibody to
SHPS-1 recognizes pp115 which is immunoprecipitated with
4C6 monoclonal antibody from CHO-IR cells.
FIG. 5. Tissue distribution of rat mRNA. Poly(A)?RNAs (2 ?g) from var-
ious rat tissues were subjected to Northern blot analysis with32P-labeled full-
length rat SHPS-1 cDNA (upper panel) and, subsequently,32P-labeled mouse
?-actin cDNA (lower panel) as probes. The positions of molecular size standards
are shown on the left.
VOL. 16, 1996 SHPS-1, A TARGET FOR SH2-DOMAIN-CONTAINING PTPase6893
Lysates of CHO-IR cells expressing wild-type SHP-2 (CHO-
SHP-2-WT cells) or a catalytically inactive SHP-2 (CHO-SHP-
2-C/S cells) were subjected to immunoprecipitation with anti-
body to SHP-2 followed by immunoblot analysis with
polyclonal antibody to SHPS-1. The amount of SHP-2 immu-
noprecipitated from CHO-SHP-2-C/S cells was approximately
twice as great as that from CHO-SHP-2-WT cells (Fig. 7C,
lanes 1 and 2), because the level of expression of SHP-2 in
CHO-SHP-2-C/S cells was higher than that of CHO-SHP-
2-WT cells (33). However, SHPS-1 was shown to associate to a
much greater extent with SHP-2 in CHO-SHP-2-C/S cells than
in CHO-SHP-2-WT cells (Fig. 7C, lanes 1 and 2). Incubation
of CHO-IR cells that overexpress SHPS-1 containing a MYC
epitope tag (SHPS-1-MYC) with 100 nM insulin for 5 min
induced both tyrosine phosphorylation of SHPS-1-MYC and
the association of SHP-2 with SHPS-1-MYC (Fig. 7D, left
panel); similar amounts of SHPS-1-MYC were immunopre-
cipitated with the 9E10 monoclonal antibody to MYC tag
under the two conditions (Fig. 7D, right panel). Together,
these results strongly suggest that pp115 previously identified
in CHO cells (27, 32) may be SHPS-1.
When lysates prepared from insulin-stimulated or -unstimu-
lated CHO-IR cells were immunoblotted with PY-20, insulin
stimulation of CHO-IR cells resulted in a marginal but signif-
icant tyrosine phosphorylation of ?115-kDa protein (Fig. 8A,
lane 2) that corresponded to the tyrosine-phosphorylated
pp115 (Fig. 8A, lane 4). The extent of insulin-dependent ty-
rosine phosphorylation of pp115 was much weaker than that of
IRS-1, a major substrate for IRs, in the immunoblot analysis
with PY-20 of cell lysates.
The partially purified, activated IRs tyrosine phosphorylated
the GST fusion protein containing the cytoplasmic portion of
SHPS-1 but not GST protein alone (data not shown), suggest-
ing that SHPS-1 may be a direct substrate for the IR kinase in
vitro. Furthermore, when tyrosine-phosphorylated GST fusion
protein containing the cytoplasmic portion of SHPS-1 was in-
cubated with either GST alone or GST-SH2 domains of
SHP-2, the tyrosine-phosphorylated fusion protein bound to
GST-SH2 domains of SHP-2 but not to GST (Fig. 8B, lanes 1
and 2). This result suggests the possibility that SHP-2 may
directly bind to tyrosine-phosphorylated SHPS-1 through SH2
domains of SHP-2.
Mitogens and cell adhesion induce tyrosine phosphorylation
of SHPS-1 and its association with SHP-2. We next tested
whether other mitogens in addition to insulin also induce ty-
rosine phosphorylation of SHPS-1 and its subsequent associa-
tion with SHP-2 in Rat-1 fibroblasts that overexpress human
IRs (Rat-1-IR cells). The reason we used Rat-1 cells in this
experiment is that Rat-1-IR cells can respond to various mito-
gens such as serum, lysophosphatidic acid (LPA), and insulin
(5). Even in unstimulated cells, both tyrosine phosphorylation
of SHPS-1 and association of SHP-2 with SHPS-1 were appar-
ent (Fig. 9A). Incubation of serum-starved Rat-1-IR cells with
insulin, serum, or LPA, all of which induce MAP kinase acti-
vation in these cells (5), increased the extent of both tyrosine
phosphorylation of SHPS-1 and association of SHPS-1 with
SHP-2 (Fig. 9A). The ?180-kDa tyrosine-phosphorylated pro-
tein coimmunoprecipitated with SHP-2 from insulin-stimu-
lated Rat-1-IR cells was found to be IRS-1 (Fig. 9A, left panel)
when the same filter was reprobed with antibody to IRS-1
(data not shown), while the ?180-kDa unknown protein that
reacted with polyclonal antibody to SHPS-1 was coimmuno-
precipitated with SHP-2 from serum-stimulated Rat-1-IR cells
(Fig. 9A, right panel).
We hypothesized that the tyrosine phosphorylation of
SHPS-1 and its association with SHP-2 in unstimulated cells
might be attributable to the effects of cell adhesion. Since 4C6
monoclonal antibody was able to effectively immunoprecipi-
tate SHPS-1 which is tyrosine phosphorylated and complexed
with SHP-2, we used CHO-IR cells for following experiments.
When CHO-IR cells were incubated for 10 min with PBS free
of both Ca2?and Mg2?, the cells became round but remained
tightly bound to the polystyrene plates. This treatment of cells
was reported previously to reduce cell-substrate adhesion and
the extent of tyrosine phosphorylation of cellular proteins (24).
This treatment of CHO-IR cells also induced a marked de-
crease in the extent of tyrosine phosphorylation of the
pp125FAKfocal adhesion kinase (40) (data not shown), indi-
cating that it may reduce the number of focal contacts of
CHO-IR cells. The extent of tyrosine phosphorylation of
SHPS-1 and the amount of SHP-2 bound to SHPS-1 were both
markedly reduced in CHO-IR cells exposed to Ca2?- and
Mg2?-free PBS compared with those of control CHO-IR cells
(Fig. 9B, left panel, lanes 1 and 2); similar amounts of
pp115SHPS-1were immunoprecipitated with the 4C6 monoclo-
nal antibody under the two conditions (Fig. 9B, right panel,
lanes 1 and 2). Adherence of cells to cell matrix proteins such
as fibronectin increases the numbers of focal contacts and the
extent of tyrosine phosphorylation of various proteins associ-
ated with such contacts (40). We detached CHO-IR cells from
culture dishes by treatment with 0.05% trypsin and 0.02%
EDTA and then plated the cells in polystyrene dishes which
were coated with fibronectin or uncoated. Adherence of
CHO-IR cells to fibronectin induced a marked increase in both
the extent of tyrosine phosphorylation of SHPS-1 and the
amount of SHP-2 bound to SHPS-1 (Fig. 9B, left panel, lanes
3 and 4).
FIG. 6. Transient expression of SHPS-1. (A) The pp120 protein was purified
from SR-3Y1 cell membranes as described in the legend to Fig. 3A. Purified
pp120 (lane 1) or cell lysates prepared from 3Y1 cells (lane 2) and SR-3Y1 cells
(lane 3) were subjected to immunoblot analysis with polyclonal antibody to
SHPS-1. Cell lysates prepared from 3Y1 cells (lanes 4 to 6) or SR-3Y1 cells (lane
7) were subjected to immunoprecipitation (IP) with preimmune serum and
?SHPS-1 polyclonal antibody as indicated below the lanes. Immunoprecipitation
was carried out in the presence of 1 ?g of GST fusion protein containing the
cytosolic domain of SHPS-1 (GST–SHPS-1-cyto) (lane 6). The immunoprecipi-
tates were then fractionated by SDS-PAGE and subjected to immunoblot anal-
ysis with ?SHPS-1. (B) Cell lysates prepared from nontransfected SR-3Y1 cells
(lane 1) or SR-3Y1 cells transfected with a pSR? vector containing the full-
length rat SHPS-1 cDNA (pSR?-SHPS-1) (lane 2), as well as purified pp120
protein from SR-3Y1 cells (lane 3), were subjected to immunoblot analysis with
polyclonal antibody to SHPS-1.
6894FUJIOKA ET AL.MOL. CELL. BIOL.
The biochemical characterization and molecular cloning of
SHPS-1 have demonstrated that it is a transmembrane glyco-
protein that contains three Ig-like domains in the extracellular
region and four putative binding sites for SH2-domain-con-
taining PTPases in the cytoplasmic region. In SR-3Y1 cells,
SHPS-1 is presumably tyrosine phosphorylated by the v-src-
encoded kinase and forms a complex with overexpressed
SHP-1 through the SH2 domains of SHP-1. It has recently
been shown that the extent of tyrosine phosphorylation of
pp115, a membrane glycoprotein, was greatly increased, rela-
tive to that in CHO-IR cells, in CHO-IR cells that also over-
express catalytically inactive SHP-2 (26, 27, 32). We have also
demonstrated that insulin stimulates tyrosine phosphorylation
of pp115 and subsequent association of SHP-2 with pp115 (32).
Although we purified pp120 and cloned its cDNA from rat
SR-3Y1 cells, the biochemical characteristics of SHPS-1 and
pp115 seem to be almost identical; the two are similar in
molecular size, membrane glycoproteins, and tyrosine-phos-
phorylated proteins bound to the SH2 domains of SHP-2.
Furthermore, the anti-SHPS-1 polyclonal antibody that was
raised against the GST-cytosolic domain of SHPS-1 recognized
pp115 immunoprecipitated with 4C6 monoclonal antibody to
pp115 from CHO-IR cells. This monoclonal antibody has re-
cently been generated against the partially purified pp115 from
CHO-IR cells expressing catalytically inactive mutant SHP-2
(32). In addition, when the lysates of CHO-SHP-2-WT cells or
CHO-SHP-2-C/S cells were subjected to immunoprecipitation
FIG. 7. The pp115 of CHO cells may be SHPS-1. (A) Lysates prepared from CHO-IR cells were subjected to immunoprecipitation (IP) with normal mouse IgG
(NMG) (lane 1) or 4C6 monoclonal antibody to pp115 (lane 2). The immunoprecipitates were in turn subjected to immunoblot analysis with polyclonal antibody to
SHPS-1. (B) Lysates prepared from unstimulated CHO-SHP-2-C/S cells were immunoprecipitated with normal mouse IgG (lane 1) or 4C6 monoclonal antibody to
pp115 (lane 2). The resulting supernatants from the first immunoprecipitation in lanes 1 and 2 were then subjected to a second round of immunoprecipitation with
polyclonal antibody to SHP-2. The immunoprecipitates were in turn subjected to immunoblot analysis with polyclonal antibodies to SHPS-1 (top) and SHP-2 (bottom).
(C) Lysates prepared from CHO-SHP-2-WT (lane 1) or CHO-SHP-2-C/S (lane 2) cells were subjected to immunoprecipitation with polyclonal antibody to SHP-2. The
immunoprecipitates were in turn subjected to immunoblot analysis with polyclonal antibodies to SHPS-1 (top) and SHP-2 (bottom). (D) CHO-IR cells that overexpress
SHPS-1 tagged with a MYC epitope (SHPS-1-MYC) (lanes 1 and 2) were incubated in the absence (?) or presence (?) of 100 nM insulin for 5 min as indicated. Cell
lysates were then prepared and subjected to immunoprecipitation with antibody to MYC and immunoblot analysis with PY-20 (left panel, top) and antibody to SHP-2
(left panel, bottom). The upper portion of the blot was reprobed with polyclonal antibody to SHPS-1 (right).
VOL. 16, 1996 SHPS-1, A TARGET FOR SH2-DOMAIN-CONTAINING PTPase6895
with antibody to SHP-2 followed by immunoblot analysis with
polyclonal antibody to SHPS-1, SHPS-1 was shown to associate
to a much greater extent with SHP-2 in CHO-SHP-2-C/S cells
than in CHO-SHP-2-WT cells. When MYC-tagged SHPS-1
was overexpressed in CHO-IR cells, insulin induced both ty-
rosine phosphorylation of MYC-tagged SHPS-1 and the sub-
sequent binding of SHP-2 to MYC-tagged SHPS-1. Together,
these results strongly suggest that SHPS-1 and the pp115 that
was previously identified in CHO cells (32) are likely to be the
same. WGA-purified activated IRs phosphorylated the recom-
binant cytoplasmic portion of SHPS-1 in vitro. Thus, SHPS-1
appears to be a direct substrate for tyrosine kinases such as Src
and the IR kinase.
Milarski and Saltiel have demonstrated that transient ex-
pression of a catalytically inactive SHP-2 induces hyperphos-
phorylation of 120-kDa protein which binds to SH2 domains of
SHP-2 in NIH 3T3 cells overexpressing human IRs (31). How-
ever, this tyrosine-phosphorylated 120-kDa protein could not
be detected by immunoprecipitation with an SHP-2 antibody
(31). Yamauchi and Pessin have reported that SHP-2 binds to
tyrosine-phosphorylated 115-kDa protein in response to insu-
lin (63) or EGF (62). Yamauchi et al. have also demonstrated
that the extent of tyrosine phosphorylation of a 115-kDa pro-
tein was greatly increased, relative to that in CHO-IR cells, in
CHO-IR cells that overexpress catalytically inactive SHP-2
(63). However, they reported that they did not observe any
precipitation of a 115-kDa protein from cell extracts with the
SH2 domains of SHP-2 fusion protein (63). Thus, there appear
to exist different types of tyrosine-phosphorylated 115- to 120-
kDa proteins which interact with SHP-2, and further efforts
will be required to characterize each of these proteins.
In vitro binding studies with a phosphotyrosyl peptide library
have suggested that the SH2 domains of SHP-2 bind to phos-
phopeptides that contain the sequence motif pYXXL/V/I (pY,
phosphorylated tyrosine) (45). In fact, SHP-2 was subsequently
shown to bind to pY1009TAV of the PDGF ? receptor (15, 22)
and to pY1172IDL or pY1222ASI of IRS-1 (36, 48) in a ligand-
dependent manner. The cytoplasmic region of SHPS-1 con-
tains four YXX(L/V/I) sequences, Y408ADL, Y432ASIE,
Y449ADL, and Y473ASV. Thus, the SH2 domains of SHP-2
may bind to one or more phosphorylated tyrosine residues in
the cytoplasmic region of SHPS-1. The sequences surrounding
Tyr-408 and Tyr-449 of SHPS-1 are similar to that surrounding
Tyr-1172 of IRS-1, whereas the sequences surrounding Tyr-
432 and Tyr-473 of SHPS-1 are homologous to that surround-
ing Tyr-1222 of IRS-1. The NH2-terminal and COOH-terminal
SH2 domains of SHP-2 are thought to bind to Y1172IDL and
Y1222ASI of IRS-1, respectively, in response to insulin stim-
ulation (8, 36). Thus, it is possible that one SHP-2 molecule
may bind to the two NH2-terminal SH2 binding sites
(Y408ADL and Y432ASI) of SHPS-1 and another SHP-2 mol-
ecule may bind to the two COOH-terminal SH2 binding sites
(Y449ADL and Y473ASV) of SHPS-1. The SH2 domains of
SHP-1 have been suggested to bind to phosphopeptides that
contain the motif pYXXL, which is very close to that of SHP-2
(7, 46). Thus, SHP-1 may bind to tyrosine-phosphorylated
SHPS-1 when SHP-1 is overexpressed in SR-3Y1 cells. How-
ever, since the physiological functions of these two SHP pro-
teins were quite different (26), these two proteins may not bind
to the same phosphorylated tyrosine residue(s) of SHPS-1.
We have recently shown that both the extent of tyrosine
phosphorylation of pp115SHPS-1and the association of SHP-2
with pp115SHPS-1are maximal at 1 to 5 min after insulin stim-
ulation of CHO-IR cells (32). In contrast, the extent of tyrosine
phosphorylation of pp115SHPS-1continued to increase for up to
30 min after stimulation of CHO-SHP-2-C/S cells, presumably
because of a lack of PTPase activity of mutant SHP-2. In
addition, we have shown that recombinant SHP-2 effectively
dephosphorylates tyrosine-phosphorylated SHPS-1, which was
prepared from SR-3Y1 cell membranes, in vitro (11). Thus, we
FIG. 8. Insulin and IR kinase tyrosine phosphorylates SHPS-1. (A) CHO-IR cells were incubated with (lanes 2 and 4) or without (lanes 1 and 3) 100 nM insulin
for 5 min. Cell lysates were then prepared and subjected to immunoblot analysis with PY-20 (lanes 1 and 2). The cell lysates were also subjected to immunoprecipitation
(IP) with 4C6 monoclonal antibody and immunoblot analysis with PY-20 (lanes 3 and 4). (B) The GST fusion protein containing the cytoplasmic portion of SHPS-1
(GST–SHPS-1-cyto) was incubated in the presence of 10 ?M ATP with IRs purified with WGA-agarose from insulin-stimulated CHO-IR cells as described in Materials
and Methods. Phosphorylated GST fusion protein containing the cytoplasmic portion of SHPS-1 was then incubated with CNBr-activated Sepharose beads coupled with
GST alone (lane 1) or GST-SH2 domains of SHP-2 fusion proteins (lane 2). The bound proteins were then subjected to SDS-PAGE and were detected by
immunoblotting with PY-20.
6896FUJIOKA ET AL.MOL. CELL. BIOL.
propose a model that SHP-2 may dephosphorylate one or
more phosphotyrosine residues of SHPS-1 after interaction of
the two proteins in response to insulin stimulation. However,
further efforts will be required to clarify whether SHPS-1 is a
physiological substrate for SHP-2 in a growth factor-mediated
signal transduction pathway.
We have shown that stimulation with serum or LPA also
induced tyrosine phosphorylation of SHPS-1 and its subse-
quent association with SHPS-1. LPA, the receptor for which is
coupled to a G protein, stimulates the RAS-MAP kinase path-
way (18, 55) and also induces the tyrosine phosphorylation of
several proteins, including pp125FAK(42) and SHC (54). Thus,
SHPS-1 appears to be a new target protein for tyrosine phos-
phorylation induced by LPA, although the mechanism by
which LPA achieves this effect is not known. We further
showed that cell adhesion to fibronectin induces tyrosine phos-
phorylation of SHPS-1 and the binding of SHP-2 to tyrosine-
phosphorylated SHPS-1, effects presumably mediated by an
integrin-coupled pathway (23). Engagement of cell surface in-
tegrins rapidly stimulates the tyrosine phosphorylation of sev-
eral intracellular proteins, including paxillin, tensin, FAK, and
p130CAS(35, 40). These proteins are also tyrosine phosphory-
lated in v-src-transformed cells (35, 40), suggesting the possi-
bility that Src family kinases or FAK might mediate the cell
adhesion-induced tyrosine phosphorylation of SHPS-1. Both
integrin-mediated cell adhesion and LPA activate RAS and
MAP kinase in a manner independent of protein kinase C (6,
41). Because SHP-2 may mediate RAS and MAP kinase acti-
vation in response to various growth factors, the association of
SHP-2 with tyrosine-phosphorylated SHPS-1 may contribute
to LPA- or integrin-induced RAS and MAP kinase activation.
Rat SHPS-1 mRNA was present in all tissues examined, with
the greatest abundance in brain, lung, and spleen tissues. Hu-
man SHPS-1 mRNA was also detected in the thymus, leuko-
cytes, and the intestine, in addition to the rat tissues tested;
again, human SHPS-1 mRNA was most abundant in the brain
(60). The tissue distribution of SHPS-1 mRNA resembles that
of SHP-2 mRNA rather than that of SHP-1 mRNA, the latter
being mostly restricted to hematopoietic tissues (26, 64). Both
SHPS-1 and SHP-2 (49) are highly expressed in the brain,
suggesting the possible role of SHPS-1 in SHP-2-mediated
signal transduction in the brain. In contrast, the level of ex-
pression of SHPS-1 in skeletal muscle is relatively low, as
shown in Fig. 5, while the level of expression of SHP-2 in the
same tissue in fact is high (10, 49). This indicates that SHPS-1
may not be important for the function of SHP-2, and an alter-
native target for SHP-2 may exist in the skeletal muscle. SHP-2
has previously been shown to be expressed in the mouse em-
bryo (9). SHPS-1 mRNA was also detected together with
SHP-2 mRNA in early-stage mouse embryos, in which SHP-1
mRNA was virtually undetectable (60), indicating that SHP-2
and SHPS-1 might function simultaneously during embryogen-
We discovered that the human SHPS-1 cDNA appears to be
the same as the CCA53 cDNA, which was recently cloned by
direct screening of a human brain cDNA library with an oli-
gonucleotide containing CCA repeats (25). However, the func-
tion of CCA53 cDNA-encoded protein was not identified. Ex-
pansion of trinucleotide repeats is responsible for several
hereditary neurological disorders, including myotonic dystro-
FIG. 9. Effects of mitogens and cell adhesion on the tyrosine phosphorylation
of SHPS-1 and its subsequent association with SHP-2. (A) Serum-starved Rat-
1-IR cells were incubated for 5 min in the absence (?) or presence of 100 nM
insulin, 20% serum, or 1 ?M LPA. Cell lysates were prepared and subjected to
immunoprecipitation with polyclonal antibody to SHP-2 followed by immunoblot
analysis with PY-20 (top left panel) or antibody to SHPS-1 (top right panel). The
lower portions of the same blots were also probed with antibody to SHP-2. (B)
CHO-IR cells were incubated for 10 min at 37?C with PBS containing (lane 1) or
not containing (lane 2) Ca2?and Mg2?. Alternatively, CHO-IR cells were plated
on a control polystyrene dish (lane 3) or a fibronectin-coated dish (lane 4) and
incubated for 1 h at 37?C. Cell lysates were prepared and then subjected to
immunoprecipitation with 4C6 monoclonal antibodies to pp115SHPS-1followed
by immunoblot analysis with PY-20 (top left panel) or to SHP-2 (bottom left
panel). The upper portions of the same filters were reprobed with polyclonal
antibody to SHPS-1 (right panel).
VOL. 16, 1996 SHPS-1, A TARGET FOR SH2-DOMAIN-CONTAINING PTPase6897
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.
1. Adachi, M., E. H. Fischer, J. Ihle, K. Imai, F. Jirik, B. Neel, T. Pawson, S.-H.
Shen, M. Thomas, A. Ullrich, and Z. Zhao. 1996. Mammalian SH2-contain-
ing protein tyrosine phosphatases. Cell 85:15.
2. Adachi, M., M. Sekiya, T. Miyachi, K. Matsuno, Y. Hinoda, K. Imai, and A.
Yachi. 1993. Molecular cloning of a novel protein-tyrosine phosphatase SH-
PTP3 with sequence similarity to the src-homology region 2. FEBS Lett.
3. Ahmad, S., D. Banville, Z. Zhao, E. H. Fischer, and S.-H. Shen. 1993. A
widely expressed human protein-tyrosine phosphatase containing src homol-
ogy 2 domains. Proc. Natl. Acad. Sci. USA 90:2197–2201.
4. Bennett, A. M., S. F. Hausdorff, A. M. O’Reilly, R. M. Freeman, Jr., and
B. G. Neel. 1996. Multiple requirements for SHPTP2 in epidermal growth
factor-mediated cell cycle progression. Mol. Cell. Biol. 16:1189–1202.
5. Burgering, B. M. T., and P. J. Coffer. 1995. Protein kinase B (c-Akt) in
phsophatidylinositol-3-OH kinase signal transduction. Nature (London) 376:
6. Chen, Q., M. S. Kinch, T. H. Lin, K. Burridge, and R. L. Juliano. 1994.
Integrin-mediated cell adhesion activates mitogen-activated protein kinases.
J. Biol. Chem. 269:26602–26605.
7. Doody, G. M., L. B. Justement, C. C. Delibrias, R. J. Matthews, 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.
8. Eck, M. J., S. Pluskey, T. Trub, S. C. Harrison, and S. E. Shoelson. 1996.
Spacial constraints on the recognition of phosphoproteins by the tandem
SH2 domains of the phosphatase SH-PTP2. Nature (London) 379:277–280.
9. Feng, G.-S., C.-C. Hui, and T. Pawson. 1993. SH2-containing phosphoty-
rosine phosphatase as a target of protein tyrosine kinase. Science 259:1607–
10. Freeman, R. M., Jr., J. Plutzky, and B. G. Neel. 1992. Identification of a
human src homology 2-containing protein-tyrosine-phosphatase: putative
homolog of Drosophila corkscrew. Proc. Natl. Acad. Sci. USA 89:11239–
11. Fujioka, Y., T. Matozaki, and M. Kasuga. Unpublished data.
12. Herbst, R., P. M. Carroll, J. D. Allard, J. Schilling, T. Raabe, and M. A.
Simon. 1996. Daughter of sevenless is a substrate of the phosphotyrosine
phosphatase corkscrew and functions during sevenless signaling. Cell 85:
13. Iwamatsu, A. 1992. S-carboxymethylation of proteins transferred onto poly-
vinylidene difluoride membranes followed by in situ protease digestion and
amino acid microsequencing. Electrophoresis 13:142–147.
14. Kasuga, M., M. F. White, and C. R. Kahn. 1985. Phosphorylation of the
insulin receptor in cultured hepatoma cells and a solubilized system. Meth-
ods Enzymol. 109:609–621.
15. Kazlauskas, A., G.-S. Feng, T. Pawson, and M. Valius. 1993. The 64-kDa
protein that associates with the platelet-derived growth factor receptor ?
subunit via Tyr-1009 is the SH2-containing phosphotyrosine phosphatase
Syp. Proc. Natl. Acad. Sci. USA 90:6939–6942.
16. Klingmu ¨ller, U., U. Lorentz, L. C. Cantley, B. G. Neel, and H. F. Lodish.
1995. Specific requirement of SH-PTP1 to the erythropoietin receptor causes
inactivation of JAK2 and termination of proliferative signals. Cell 80:729–
17. Koch, C. A., D. Anderson, M. F. Moran, C. Ellis, and T. Pawson. 1991. SH2
and SH3 domains: elements that control interactions of cytoplasmic signal-
ing proteins. Science 252:668–674.
18. Koch, W. J., B. E. Hawes, L. F. Allen, and R. J. Lefkowitz. 1994. Direct
evidence that Gi-coupled receptor stimulation of mitogen-activated protein
kinase is mediated by G beta gamma activation of p21ras. Proc. Natl. Acad.
Sci. USA 91:12706–12710.
19. Kozak, M. 1987. An analysis of 5?-noncoding sequences from 699 vertebrate
messenger RNAs. Nucleic Acids Res. 15:8125–8148.
20. Kuhne ´, M. R., T. Pawson, G. E. Lienhard, and G.-S. Feng. 1993. The insulin
receptor substrate 1 associates with the SH2-containing phosphotyrosine
phosphatase Syp. J. Biol. Chem. 268:11479–11481.
21. La Spada, A. R., H. L. Paulson, and K. H. Fischbeck. 1994. Trinucleotide
repeat expansion in neurological disease. Ann. Neurol. 36:814–822.
22. Lechleider, R. L., S. Sugimoto, A. M. Bennett, A. S. Kashishian, J. A.
Cooper, S. E. Shoelson, C. T. Walsh, and B. G. Neel. 1993. Activation of the
SH2-containing phosphotyrosine phosphatase SH-PTP2 by its binding site,
phosphotyrosine 1009, on the human platelet-derived growth factor ?.
J. Biol. Chem. 268:21478–21481.
23. Loftus, J. C., J. W. Smith, and M. H. Ginsberg. 1994. Integrin-mediated cell
adhesion: the extracellular face. J. Biol. Chem. 269:25235–25238.
24. Maher, P. A. 1993. Activation of phosphotyrosine phosphatase activity by
reduction of cell-substrate adhesion. Proc. Natl. Acad. Sci. USA 90:11177–
25. Margolis, R. L., T. S. Breschel, S. H. Li, A. S. Kidwai, S. E. Antonarakis,
M. G. McInnis, and C. A. Ross. 1995. Characterization of cDNA clones
containing CCA trinucleotide repeats derived from human brain. Somatic
Cell Mol. Genet. 21:279–284.
26. Matozaki, T., and M. Kasuga. 1996. Roles of protein tyrosine phosphatases
in growth factor signaling. Cell. Signaling 8:13–19.
27. Matozaki, T., T. Noguchi, T. Suzuki, and M. Kasuga. 1995. Insulin receptor
signalling and protein-tyrosine phosphatases. Adv. Protein Phosphatases
28. Matozaki, T., T. Suzuki, T. Uchida, J. Inazawa, T. Ariyama, K. Matsuda, K.
Horita, H. Noguchi, H. Mizuno, C. Sakamoto, and M. Kasuga. 1994. Mo-
lecular cloning of a human transmembrane-type protein tyrosine phos-
phatase and its expression in gastrointestinal cancers. J. Biol. Chem. 269:
29. Matozaki, T., T. Uchida, Y. Fujioka, and M. Kasuga. 1994. Src kinase
tyrosine phosphorylates PTP1C, a protein tyrosine phosphatase containing
src homology-2 domains, that down-regulates cell proliferation. Biochem.
Biophys. Res. Commun. 204:874–881.
30. Matthews, R. J., D. B. Bowne, E. Flores, and M. L. Thomas. 1992. Charac-
terization of hematopoietic intracellular protein tyrosine phosphatases: de-
scription of a phosphatase containing an SH2 domain and another enriched
in proline-, glutamic acid-, serine-, and threonine-rich sequences. Mol. Cell.
31. Milarski, K. L., and A. R. Saltiel. 1994. Expression of catalytically inactive
Syp phosphatase in 3T3 cells blocks stimulation of mitogen-activated protein
kinase by insulin. J. Biol. Chem. 269:21239–21243.
32. Noguchi, T., T. Matozaki, Y. Fujioka, T. Yamao, M. Tsuda, T. Takada, and
M. Kasuga. Characterization of a 115-kDa protein that binds to SH-PTP2, a
protein-tyrosine phosphatase with SRC homology 2 domains, in Chinese
hamster ovary cells. J. Biol. Chem., in press.
33. Noguchi, T., T. Matozaki, K. Horita, Y. Fujioka, and M. Kasuga. 1994. Role
of SH-PTP2, a protein-tyrosine phosphatase with src homology-2 domains,
in insulin-stimulated Ras activation. Mol. Cell. Biol. 14:6674–6682.
34. Noguchi, T., T. Matozaki, and M. Kasuga. Unpublished data.
35. Nojima, Y., N. Morino, T. Mimura, K. Hamasaki, H. Furuya, R. Sakai, T.
Sato, K. Tachibana, C. Morimoto, Y. Yazaki, and H. Hirai. 1995. Integrin-
mediated cell adhesion promotes tyrosine phosphorylation of p130cas, a Src
homology 3-containing molecule having multiple Src homology 2-binding
motifs. J. Biol. Chem. 270:15398–15402.
6898FUJIOKA ET AL.MOL. CELL. BIOL.
36. Pluskey, S., T. J. Wandless, C. T. Walsh, and S. E. Shoelson. 1995. Potent
stimulation of SH-PTP2 phosphatase activity by simultaneous occupancy of
both SH2 domains. J. Biol. Chem. 270:2897–2900.
37. Plutzky, J., B. G. Neel, and R. D. Rosenberg. 1992. Isolation of a src homol-
ogy 2-containing tyrosine phosphatase. Proc. Natl. Acad. Sci. USA 89:1123–
38. Ross, C. A., M. G. McInnis, R. L. Margolis, and S.-H. Li. 1993. Genes with
triplet repeats: candidate mediators of neuropsychiatric disorders. Trends
39. Sawada, T., L. Kim, and A. R. Saltiel. 1995. Expression of a catalytically inert
Syp blocks activation of MAP kinase pathway downstream of p21ras. Bio-
chem. Biophys. Res. Commun. 214:737–743.
40. Schaller, M. D., and J. T. Parsons. 1993. Focal adhesion kinase: an integrin-
linked protein kinase. Trends Cell Biol. 3:258–262.
41. Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. van der Geer. 1994.
Integrin-mediated signal transduction linked to Ras pathway by GRB2 bind-
ing to focal adhesion kinase. Nature (London) 372:786–791.
42. Seufferlein, T. E., and E. Rozengurt. 1994. Lysophosphatidic acid stimulates
tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130. J. Biol.
43. Shen, S.-H., L. Bastien, B. I. Posner, and P. Chretien. 1991. A protein-
tyrosine phosphatase with sequence similarity to the SH2 domains of the
protein-tyrosine kinases. Nature (London) 352:736–739.
44. Shultz, L. D., P. A. Schweitzer, T. V. Rajan, T. Yi, J. N. Ihle, R. J. Matthews,
M. L. Thomas, and D. R. Beier. 1993. Mutations at the murine motheaten
locus are within the hematopoietic cell protein phosphatase (HCPH) gene.
45. Songyang, Z., S. E. Shoelson, M. Chaudhuri, 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. 1993. SH2 domains recognize specific phosphopeptide sequences.
46. Songyang, Z., S. E. Shoelson, J. McGlade, P. Olivier, 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. Specific motifs recog-
nized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk,
and vav. Mol. Cell. Biol. 14:2777–2785.
47. Sun, X. J., P. Rothenberg, C. R. Kahn, J. M. Backer, E. Araki, P. A. Wilden,
D. A. Cahill, B. J. Goldstein, and M. F. White. 1991. Structure of the insulin
receptor substrate IRS-1 defines a unique signal transduction protein. Na-
ture (London) 352:73–77.
48. Sun, X. J., L.-H. Wang, Y. Zhang, L. Yenush, M. G. Myers, Jr., E. Glasheen,
W. S. Lane, J. H. Pierce, and M. F. White. 1995. Role of IRS-2 in insulin and
cytokine signalling. Nature (London) 377:173–177.
49. Suzuki, T., T. Matozaki, A. Mizoguchi, and M. Kasuga. 1995. Localization
and subcellular distribution of SH-PTP2, a protein-tyrosine phosphatase
with src homology-2 domains, in rat brain. Biochem. Biophys. Res. Commun.
50. Tang, T. L., R. M. Freeman, Jr., A. M. O’Reilly, B. G. Neel, and S. Y. Sokol.
1995. The SH2-containing protein-tyrosine phosphatase SH-PTP2 is re-
quired upstream of MAP kinase for early Xenopus development. Cell 80:
51. Tsui, H. W., K. A. Siminovitch, L. deSouza, and F. W. L. Tsui. 1993. Moth-
eaten and viable motheaten mice have mutations in the haematopoietic cell
phosphatase gene. Nature Genet. 4:124–129.
52. Uchida, T., T. Matozaki, K. Matsuda, T. Suzuki, S. Matozaki, O. Nakano, K.
Wada, Y. Konda, C. Sakamoto, and M. Kasuga. 1993. Phorbol ester stimu-
lates the activity of a protein tyrosine phosphatase containing SH2domains
(PTP1C) in HL-60 leukemia cells by increasing gene expression. J. Biol.
53. Uchida, T., T. Matozaki, T. Noguchi, T. Yamao, K. Horita, T. Suzuki, Y.
Fujioka, C. Sakamoto, and M. Kasuga. 1994. Insulin stimulates the phos-
phorylation of Tyr538and the catalytic activity of PTP1C, a protein tyrosine
phosphatase with Src homology-2 domains. J. Biol. Chem. 269:12220–12228.
54. van Blesen, T., B. Hawes, D. K. Luttrell, K. M. Krueger, K. Touhara, E.
Porfirl, M. Sakauem, L. M. Luttrell, and R. J. Lefkowitz. 1995. Receptor-
tyrosine-kinase- and G??-mediated MAP kinase activation by a common
signalling pathway. Nature (London) 376:781–784.
55. van Corven, E. J., P. L. Hordijk, R. H. Medema, J. L. Bos, and W. H.
Moolenaar. 1993. Pertussis toxin-sensitive activation of p21ras by G protein-
coupled receptor agonists in fibroblasts. Proc. Natl. Acad. Sci. USA 90:1257–
56. Vogel, W., R. Lammers, J. Huang, and A. Ullrich. 1993. Activation of a
phosphotyrosine phosphatase by tyrosine phosphorylation. Science 259:
57. von Heijne, G. 1986. A new method for predicting signal sequence cleavage
sites. Nucleic Acids Res. 14:4683–4690.
58. Williams, A. F., and A. N. Barclay. 1988. The immunoglobulin superfamily-
domains for cell surface recognition. Annu. Rev. Immunol. 6:381–405.
59. Xiao, S., D. W. Rose, T. Sasaoka, H. Maegawa, T. R. Burke, Jr., P. P. Roller,
S. E. Shoelson, and J. M. Olefsky. 1994. Syp (SH-PTP2) is a positive medi-
ator of growth factor-stimulated mitogenic signal transduction. J. Biol.
60. Yamao, T., T. Matozaki, and M. Kasuga. Unpublished data.
61. Yamauchi, K., K. L. Milarski, A. R. Saltiel, and J. E. Pessin. 1995. Protein-
tyrosine-phosphatase SHPTP2 is a required positive effector for insulin
downstream signaling. Proc. Natl. Acad. Sci. USA 92:664–668.
62. Yamauchi, K., and J. E. Pessin. 1995. Epidermal growth factor-induced
association of the SHPTP2 protein tyrosine phosphatase with a 115-kDa
phosphotyrosine protein. J. Biol. Chem. 270:14871–14874.
63. Yamauchi, K., V. Ribon, A. R. Saltiel, and J. E. Pessin. 1995. Identification
of the major SHPTP2-binding protein that is tyrosine-phosphorylated in
response to insulin. J. Biol. Chem. 270:17716–17722.
64. Yi, T., J. L. Cleveland, and J. N. Ihle. 1992. Protein tyrosine phosphatase
containing SH2 domains: characterization, preferential expression in hema-
topoietic cells, and localization to human chromosome 12p12-13. Mol. Cell.
65. Yi, T., A. L.-F. Mui, G. Krystal, and J. N. Ihle. 1993. Hematopoietic 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.
VOL. 16, 1996 SHPS-1, A TARGET FOR SH2-DOMAIN-CONTAINING PTPase6899