(Received for publication, March 8, 1995; and in revised form, April 27, 1995)
From the
Immunoprecipitation of the cytosolic Src homology 2
domain-containing protein-tyrosine phosphatase, SHPTP2, from
insulin-stimulated 3T3L1 adipocytes or Chinese hamster ovary cells
expressing the human insulin receptor resulted in the
coimmunoprecipitation of a diffuse tyrosine-phosphorylated band in the
115-kDa protein region on SDS-polyacrylamide gels. Although
platelet-derived growth factor induced the tyrosine phosphorylation of
the platelet-derived growth factor receptor and SHPTP2, there was no
significant increase in the coimmunoprecipitation of
tyrosine-phosphorylated pp115 with SHPTP2. SHPTP2 was also associated
with tyrosine-phosphorylated insulin receptor substrate-1, but this
only accounted for <2% of the total immunoreactive SHPTP2 protein.
Similarly, only a small fraction of the total amount of
tyrosine-phosphorylated insulin receptor substrate-1 (<4%) was
associated with SHPTP2. Expression and immunoprecipitation of a Myc
epitope-tagged wild-type SHPTP2 (Myc-WT-SHPTP2) and a catalytically
inactive point mutant of SHPTP2 (Myc-C/S-SHPTP2) also demonstrated an
insulin-dependent association of SHPTP2 with tyrosine-phosphorylated
pp115. Furthermore, expression of the catalytically inactive SHPTP2
mutant resulted in a marked enhancement in the amount of
coimmunoprecipitated tyrosine-phosphorylated pp115 compared with the
expression of wild-type SHPTP2. These data indicate that the
insulin-stimulated tyrosine-phosphorylated 115-kDa protein is the
predominant in vivo SHPTP2-binding protein and that pp115 may
function as a physiological substrate for the SHPTP2 protein-tyrosine
phosphatase. Similar to many growth factor receptors, the insulin receptor is
a ligand-stimulated transmembrane tyrosine-specific protein kinase that
phosphorylates itself as well as intracellular substrates on specific
tyrosine residues(1, 2) . The two best characterized
substrates for the insulin receptor kinase have been identified as a
185-kDa protein termed IRS1 The SHPTP2 phosphatase (also
termed PTP1D, SHPTP3, PTP2C, PTPL1, or Syp) is one member of a family
of cytosolic protein tyrosine-specific phosphatases that contain two
amino-terminal SH2 domains and a carboxyl-terminal catalytic
domain(12, 13, 14, 15, 16, 17) .
These SH2 domains target SHPTP2 to the tyrosine-phosphorylated
epidermal growth factor receptor, the platelet-derived growth factor
(PDGF) receptor, and
IRS1(13, 18, 19, 20) . In addition
to mediating the targeting of SHPTP2, the SH2 domains also appear to
regulate SHPTP2 catalytic activity. Several studies have demonstrated
that the unoccupied SH2 domains maintain the SHPTP2 phosphatase
activity in a repressed state, whereas the binding of
tyrosine-phosphorylated proteins or peptides results in a marked
activation of protein-tyrosine phosphatase
activity(22, 23, 24, 25, 26, 27) .
Furthermore, the phosphorylation of SHPTP2 on tyrosine 542 in response
to PDGF may also contribute to the activation of its phosphatase
activity(28, 29) . In general, tyrosine
phosphatases function as the inactivating arms of kinase activation
pathways; however, they may also function as positive effectors for
downstream signaling. In the case of T cell receptor signaling, the
CD45 protein-tyrosine phosphatase dephosphorylates the inhibitory
carboxyl-terminal tyrosine phosphorylation site on Fyn and/or
Lck(30) . The subsequent relief of this kinase inhibition is an
essential requirement for T cell receptor-mediated biological responses (31, 32) . Similarly, SHPTP2 is the mammalian
homologue of the Drosophila SH2 domain-containing
protein-tyrosine phosphatase, termed corkscrew(33) .
Genetic epistasis experiments have also demonstrated that corkscrew is an essential gene for the appropriate development of
anterior/posterior structures during embryogenesis, functioning
downstream of the torso tyrosine kinase, a homologue of the
mammalian PDGF receptor, and upstream of polehole, a homologue
of the mammalian raf serine/threonine
kinase(34, 35, 36) . Recently, several
studies have suggested that SHPTP2 also plays an important positive
role in insulin signaling. Although increased levels of tyrosine
phosphatase activity would be expected to inhibit signaling events,
expression of the wild-type catalytically active SHPTP2 had no
significant effect on insulin-stimulated biological responsiveness (37, 38, 39) . Furthermore, microinjection of
SHPTP2-specific antibodies completely blocked insulin-stimulated DNA
synthesis in fibroblasts expressing high levels of the insulin
receptor(40) . In addition, expression of the SHPTP2 SH2
domains or a full-length catalytically inactive SHPTP2 mutant blocked
activation of the mitogen-activated protein kinase pathway as well as
c-fos transcription and DNA synthesis without significant
effect on the tyrosine phosphorylation state of IRS1 or
Shc(37, 38, 39, 40) . Even though
these data have provided strong evidence for a positive signaling role
of SHPTP2 in mediating insulin action, a physiological substrate has
not been identified. In this report, we have identified an
insulin-stimulated tyrosine-phosphorylated protein (pp115) that is the
major SHPTP2-binding protein and that may be an important substrate for
the SHPTP2 phosphatase.
Figure 1:
Insulin-stimulated SHPTP2 association
with a tyrosine-phosphorylated pp115 protein. CHO/IR cells (A)
and differentiated 3T3L1 adipocytes (B) were either left
untreated (control (C); lanes1 and 4) or treated with 100 nM insulin (I; lanes2 and 5) or 100 ng/ml PDGF (P; lanes3 and 6) for 10 min at 37
°C. Total detergent-lysed cell extracts were prepared and
immunoprecipitated with a carboxyl-terminal SHPTP2 polyclonal antibody
as described under ``Experimental Procedures.'' The resulting
immunoprecipitates were then subjected to Western blotting using either
a phosphotyrosine antibody (PY20; lanes 1-3) or an
amino-terminal SHPTP2 antibody (SHPTP2; lanes 4-6). IP, immunoprecipitation; IB,
immunoblot.
Figure 3:
Time dependence of insulin-stimulated
pp115 tyrosine phosphorylation and association with SHPTP2. CHO/IR
cells (A) and differentiated 3T3L1 adipocytes (B)
were either left untreated (lane1) or incubated with
100 nM insulin for 0.5 (lane2), 2 (lane3), 5 (lane4), 15 (lane5), 30 (lane6), and 60 (lane7) min at 37 °C. Total detergent-lysed cell extracts
were prepared and immunoprecipitated with a carboxyl-terminal SHPTP2
polyclonal antibody as described under ``Experimental
Procedures.'' The resulting immunoprecipitates were then subjected
to Western blotting using the PY20 phosphotyrosine antibody. IP, immunoprecipitation; IB,
immunoblot.
To ensure that the association of SHPTP2 with pp115 was not
restricted to CHO cells expressing high levels of the insulin receptor,
we also investigated the association of the pp115 protein with SHPTP2
in the insulin-responsive 3T3L1 adipocytes. In this cell type, insulin
also stimulated the coimmunoprecipitation of SHPTP2 with
tyrosine-phosphorylated IRS1 (Fig. 1B, lanes1 and 2). Similar to the CHO/IR cells, the
relative amount of tyrosine-phosphorylated IRS1 that associated with
SHPTP2 was small compared with the association of pp115 with SHPTP2 (Fig. 1B, lanes1 and 2).
PDGF treatment of 3T3L1 adipocytes resulted in the
coimmunoprecipitation of the tyrosine-phosphorylated PDGF receptor as
well as tyrosine-phosphorylated SHPTP2 without any significant increase
in the amount of the tyrosine-phosphorylated 115-kDa protein (Fig. 1B, lanes1 and 3).
Under these conditions, essentially identical amounts of SHPTP2 protein
were immunoprecipitated as determined by an anti-SHPTP2 immunoblot (Fig. 1B, lanes 4-6).
Figure 2:
Insulin concentration dependence of pp115
tyrosine phosphorylation and association with SHPTP2. CHO/IR cells (A) and differentiated 3T3L1 adipocytes (B) were
either left untreated (lane1) or incubated with 0.1 (lane2), 1 (lane3), 10 (lane4), and 100 (lane5) nM insulin for 10 min at 37 °C. Total detergent-lysed cell
extracts were prepared and immunoprecipitated with a carboxyl-terminal
SHPTP2 polyclonal antibody as described under ``Experimental
Procedures.'' The resulting immunoprecipitates were then subjected
to Western blotting using the PY20 phosphotyrosine antibody. IP, immunoprecipitation; IB,
immunoblot.
Figure 4:
Quantitation of the total amount of
tyrosine-phosphorylated IRS1 associated with SHPTP2. Differentiated
3T3L1 adipocytes were either left untreated (lanes1, 3, 5, and 7) or incubated with 100 nM insulin (lanes2, 4, 6, and 8) for 10 min at 37 °C. Total detergent-lysed cell
extracts were prepared and subjected to immunoprecipitation with the
carboxyl-terminal SHPTP2 polyclonal antibody (A) or the IRS1
polyclonal antiserum (B) as described under
``Experimental Procedures.'' The resulting supernatants from
the initial SHPTP2 immunoprecipitation were subjected to a second round
of IRS1 (lanes3 and 4) or SHPTP2 (lanes7 and 8) immunoprecipitation. The
immunoprecipitates were then Western-blotted with the PY20
phosphotyrosine antibody (lanes 1-4) or with the
amino-terminal SHPTP2 antibody (lanes 5-8). IP,
immunoprecipitation; IB,
immunoblot.
In
a complementary approach, we next determined the relative amount of the
total SHPTP2 pool associated with tyrosine-phosphorylated IRS1 (Fig. 4B). 3T3L1 adipocyte extracts from control and
insulin-stimulated cells were immunoprecipitated with the IRS1
antiserum. Phosphotyrosine Western blotting demonstrated the expected
presence of IRS1 following insulin stimulation (Fig. 4B, lanes 1 and 2). The
resulting supernatants from the initial IRS1 immunoprecipitation were
then subjected to a second round of IRS1 immunoprecipitation.
Phosphotyrosine Western blotting following the second IRS1
immunoprecipitation demonstrated the presence of
tyrosine-phosphorylated IRS1, but at a significantly reduced level (Fig. 4B, lanes 3 and 4). Laser
scanning densitometry of several experiments indicated that the initial
incubation with the IRS1 antiserum resulted in the immunoprecipitation
of
Figure 5:
Insulin-stimulated tyrosine-phosphorylated
pp115 does not associate with Grb2, Nck, or Crk. CHO/IR cells were
either left untreated (lanes1, 3, and 5) or incubated with 100 nM insulin (lanes2, 4, and 6) for 10 min at 37 °C. A, total detergent-lysed cell extracts were prepared and
immunoprecipitated with an SHPTP2 antibody (lanes1 and 2), a Crk antibody (lanes3 and 4), or a Nck antibody (lanes5 and 6) as described under ``Experimental Procedures.'' B, total detergent-lysed cell extracts were prepared and
immunoprecipitated with an SHPTP2 antibody (lanes1 and 2) or a Grb2 antibody (lanes3 and 4). The resulting immunoprecipitates were then subjected to
Western blotting using the PY20 phosphotyrosine antibody. IP,
immunoprecipitation; IB,
immunoblot.
Figure 6:
Expression of phosphatase-negative SHPTP2
increases the extent of SHPTP2-associated pp115 tyrosine
phosphorylation. CHO/IR cells were transfected with the empty
expression vector CLDN (lanes1 and 2), CLDN
containing Myc-WT-SHPTP2 (lanes3 and 4),
and CLDN containing Myc-C/S-SHPTP2 (lanes5 and 6). Subsequently, the cells were incubated in the absence (lanes1, 3, and 5) or presence (lanes2, 4, and 6) of 100 nM insulin for 10 min at 37 °C. Total detergent-lysed cell
extracts were prepared and subjected to immunoprecipitation with either
the Myc epitope 9E10 monoclonal antibody (A and C),
or the carboxyl-terminal SHPTP2 polyclonal antibody (B). The
resulting immunoprecipitates were then Western-blotted with the
amino-terminal SHPTP2 antibody (A) or the PY20 phosphotyrosine
antibody (B and C). IP, immunoprecipitation; IB, immunoblot.
To determine the effect of SHPTP2 expression on the
coimmunoprecipitation and tyrosine phosphorylation of pp115, the
transfected CHO/IR cells were immunoprecipitated with an SHPTP2
antibody and subjected to phosphotyrosine immunoblotting (Fig. 6B). As previously observed, pp115 was
coimmunoprecipitated with SHPTP2 in cells transfected with the empty
expression vector (Fig. 6B, lanes1 and 2). Increased expression of wild-type SHPTP2
(Myc-WT-SHPTP2) had no significant effect on the extent of
SHPTP2-coimmunoprecipitated tyrosine-phosphorylated pp115 following
insulin stimulation (Fig. 6B, lanes3 and 4). However, expression of the phosphatase-negative
SHPTP2 mutant (Myc-C/S-SHPTP2) increased the basal
coimmunoprecipitation of pp115 with SHPTP2 (Fig. 6B, lane5) and markedly enhanced the detection of
tyrosine-phosphorylated pp115 following insulin stimulation (Fig. 6B, lane6). To confirm the
association of pp115 with the proteins encoded by the transfected
SHPTP2 cDNAs, we also immunoprecipitated the transfected SHPTP2
proteins with the 9E10 antibody (Fig. 6C). The
insulin-stimulated tyrosine phosphorylation of the pp115 protein was
also detected in the 9E10 immunoprecipitates from cells expressing the
wild-type SHPTP2 cDNA (Myc-WT-SHPTP2), but not from cells transfected
with the empty vector (Fig. 6C, lanes
1-4). In addition, a small amount of tyrosine-phosphorylated
IRS1 was coimmunoprecipitated with the 9E10 antibody in the
Myc-WT-SHPTP2-transfected cells following insulin stimulation, but not
in the mock-transfected cells. In contrast, a marked increase in the
amount of tyrosine-phosphorylated pp115 was detected in the 9E10
immunoprecipitates of extracts from cells expressing the catalytically
inactive SHPTP2 mutant (Myc-C/S-SHPTP2) (Fig. 6C, lanes5 and 6). Furthermore, both the basal
and insulin-stimulated tyrosine phosphorylation of the mutant SHPTP2
protein was observed (Fig. 6C, lanes5 and 6). These changes in tyrosine phosphorylation
occurred with no effect on the extent of coimmunoprecipitated
tyrosine-phosphorylated IRS1. It should be noted that both a pp120
protein and the insulin receptor Over the past several years, substantial progress has been
made in elucidating various intracellular signaling events mediating
positive protein-protein interactions by tyrosine kinase receptor
activation. On the other hand, the processes that mediate the
inactivation of these events through tyrosine dephosphorylation remain
less defined. The tyrosine phosphatase SHPTP2 is characteristic of a
family of related cytosolic enzymes that contain two amino-terminal SH2
domains(12, 13, 14, 15, 16, 17, 18, 19) .
This phosphatase is ubiquitously expressed and is regulated by its
association with a variety of tyrosine-phosphorylated receptors and
receptor substrates through engagement of its SH2 domains. For example,
SHPTP2 has been observed to associate with tyrosine-phosphorylated
IRS1, resulting in the stimulation of catalytic
activity(22, 23, 24) . Several laboratories
have also demonstrated that expression of a catalytically inactive
SHPTP2 protein inhibits the insulin stimulation of the
mitogen-activated protein kinase pathway, thereby blocking c-fos transcription and
mitogenesis(37, 38, 39, 40) . Thus,
SHPTP2 appears to serve an essential role in the growth-promoting
actions of insulin in a fashion analogous to the Drosophila homologue of SHPTP2, corkscrew, which is required for
normal anterior/posterior
development(33, 34, 35, 36) . Even though these data suggest that SHPTP2 is a required positive
effector of tyrosine kinase signaling, its physiological targets have
not been identified. Based upon in vitro studies, it was
suggested that the insulin-stimulated association of SHPTP2 with IRS1
functioned to dephosphorylate the tyrosine-phosphorylated IRS1 protein (47) . However, expression of either wild-type SHPTP2 or a
catalytically inactive SHPTP2 mutant did not significantly affect the
insulin-stimulated tyrosine phosphorylation state of
IRS1(37, 38, 39) . Consistent with these
reports, we have observed that only a very small fraction (<4%) of
the total pool of tyrosine-phosphorylated IRS1 was associated with
SHPTP2 following insulin stimulation of 3T3L1 adipocytes. Although we
have been unable to quantitatively immunoprecipitate IRS1 from 3T3L1
cell extracts, semiquantitative analysis also indicated that only a
very small fraction (<2%) of the total SHPTP2 pool was
coimmunoprecipitated with tyrosine-phosphorylated IRS1. Taken together,
these data suggest that IRS1 is not the predominant target of SHPTP2
binding and/or function in 3T3L1 adipocytes. To identify the
insulin-stimulated substrate(s) and/or protein(s) associated with
SHPTP2, we screened cell extracts for tyrosine-phosphorylated proteins
that were coimmunoprecipitated with an SHPTP2 antibody. In this manner,
we have identified a predominant tyrosine-phosphorylated band in the
115-kDa region of SDS gels that was coimmunoprecipitated with SHPTP2
from insulin-stimulated CHO/IR and 3T3L1 adipocytes. The tyrosine
phosphorylation and/or association of this broad pp115 band with SHPTP2
was not observed following PDGF stimulation, although a doublet
migrating in this molecular mass range has been reported following
epidermal growth factor stimulation(17) . Consistent with this
observation, we have also detected a similar SHPTP2-associated pp115
protein in both epidermal growth factor- and nerve growth
factor-treated PC12 cells (data not shown). In addition to its
function as the predominant tyrosine-phosphorylated SHPTP2-binding
protein, pp115 also appears to be an in vivo substrate for the
SHPTP2 phosphatase. This speculation is consistent with the large
increase in the amount of tyrosine-phosphorylated pp115 that was
coimmunoprecipitated with SHPTP2 following expression of the
catalytically inactive SHPTP2 mutant. However, it should be noted that
in these studies, we were not able to distinguish between the amount of
pp115 bound to SHPTP2 versus the extent of tyrosine
phosphorylation. Interestingly, the pp115 protein was highly
diffuse, perhaps representing heterogeneity at the level of
phosphorylation or, alternatively, the presence of multiple, closely
related molecular mass phosphoproteins. In some gels, this region
appeared to be composed of several closely spaced but discrete protein
bands. However, this separation was quite variable, and we have not yet
found a gel system to reproducibly separate these bands. Since a
variety of proteins in the 115-125-kDa range have been shown to
be tyrosine-phosphorylated, we attempted to identify these components
by immunoprecipitation with known antibodies. However, we were unable
to coimmunoprecipitate any insulin-stimulated tyrosine-phosphorylated
proteins with antibodies directed against Jak1, Jak2, Jak3, Tyk2,
STAT2, Ras GTPase-activation protein, c-Dbl, c-Cbl, or the p120 Src
substrate. In addition, Grb2 and Crk have been observed to
coimmunoprecipitate several tyrosine-phosphorylated proteins in this
molecular mass range following T cell
activation(21, 45) . However, coimmunoprecipitation
with Grb2, Crk, and Nck antibodies was also unsuccessful in identifying
any insulin-stimulated tyrosine-phosphorylated proteins in this
molecular mass range. Recently, a tyrosine-phosphorylated
SHPTP2-associated 120-kDa protein was detected in NIH 3T3 cells
expressing high levels of the human insulin receptor(38) .
However, the pp115 protein identified in this study has distinctly
different properties. For example, the previously described 120-kDa
protein could not be detected by immunoprecipitation with an SHPTP2
antibody. In addition, the tyrosine-phosphorylated 120-kDa protein was
only observed in cells expressing a catalytically inactive SHPTP2
mutant by precipitation with an amino-terminal SHPTP2 SH2 domain fusion
protein. In contrast, the pp115 protein identified in the present study
was readily detected by coimmunoprecipitation with an SHPTP2 antibody
in extracts from both control and wild-type SHPTP2-expressing cells.
Furthermore, we have not observed any precipitation of pp115 from
either CHO/IR or 3T3L1 adipocyte cell extracts with the SHPTP2 SH2
fusion protein (data not shown). In summary, we have identified the
predominant insulin-stimulated tyrosine-phosphorylated
SHPTP2-associated protein(s) as a diffuse pp115 band. The tyrosine
phosphorylation and association with SHPTP2 displayed a similar insulin
dose responsiveness compared with IRS1 and the insulin receptor
Note Added in Proof-After submission of this manuscript, a
report by Zhao et al. (Zhao, Z., Tan, Z., Wright, J. H.,
Diltz, C. D., Shen, S.-H., Krebs, E. G., and Fischer, E. H.(1995) J. Biol. Chem.270, 11765-11769) indicated that
an epidermal growth factor-stimulated tyrosine-phosphorylated 43-RDa
protein was coimmunoprecipitated with an inactive PTP2C mutant.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(3, 4, 5) and proteins of
46 and 52 kDa termed Shc (Src homology
2/
-collagen-related)(6, 7, 8) . These
molecules contain specific tyrosine phosphorylation sites that provide
recognition signals for the binding of Src homology 2 (SH2)
domain-containing proteins(9) . In the case of IRS1, tyrosine
phosphorylation results in the formation of a multisubunit signaling
complex composed of the small adapter proteins Grb2 and Nck,
phosphatidylinositol 3-kinase, and the recently identified
tyrosine-specific protein phosphatase,
SHPTP2(10, 11) .
Cell Culture
Chinese hamster ovary cells
expressing 3
10
human insulin receptors/cell
(CHO/IR) were isolated and maintained in minimal Eagle's medium
containing nucleotides plus 10% fetal bovine serum as described
previously(41) . 3T3L1 preadipocytes were obtained from the
American Type Culture Collection and were differentiated into the
adipocyte phenotype as described by Rubin et al.(42) .
Briefly, 3T3L1 preadipocytes were maintained in Dulbecco's
modified Eagle's medium containing 10% calf serum. The cells were
differentiated into adipocytes by replacement with fresh medium
containing 10% fetal bovine serum, 0.25 µM dexamethasone,
500 µM isobutylmethylxanthine, and 1 µg/ml insulin for
3 days. The cells were then medium-changed into Dulbecco's
modified Eagles's medium containing 10% fetal bovine serum for
8-14 days.
Expression Plasmids
The Myc epitope (MEQKLISEEDL)
was introduced into the amino terminus of SHPTP2 by cloning an
oligonucleotide linking upstream of the initiation methionine codon.
This domain was introduced both in the wild-type SHPTP2 cDNA
(Myc-WT-SHPTP2) and in the catalytically inactive SHPTP2 mutant in
which cysteine 459 was replaced with serine (Myc-C/S-SHPTP2) by
site-directed mutagenesis. These cDNAs were then subcloned into the
high efficiency mammalian expression vector CLDN(43) .Transfection of CHO/IR Cells by Electroporation
To
obtain a high degree of transfection efficiency necessary for
immunoprecipitation and Western blotting of whole cell extracts, CHO/IR
cells were electroporated with a total of 40 µg of plasmid DNA at
340 V and 960 microfarads as described previously(44) . Under
these conditions, 80-95% of the viable cell population was
functionally transfected as assessed by in situ
-galactosidase staining. Thirty-six h following transfection,
the cells were serum-starved for 6 h and either untreated or incubated
for 5 min in the presence of 100 nM insulin or 100 ng/ml PDGF
prior to the preparation of whole cell lysates.
Western Blot Analysis of Whole Cell Extracts
Whole
cell extracts were prepared by detergent solubilization in lysis buffer
(20 mM Hepes, pH 7.4, 1% Triton X-100, 2 mM EDTA, 100
mM sodium fluoride, 10 mM sodium pyrophosphate, 2
mM sodium orthovanadate, 1 mM phenylmethylsulfonyl
fluoride, 10 µM leupeptin, 10 µg/ml aprotinin, and 1.5
mM pepstatin) for 1 h at 4 °C. The resultant cell extracts
were subjected to Western blotting using an amino-terminal SHPTP2
antibody (Transduction Laboratories), a Myc epitope antibody (9E10;
Santa Cruz), or a phosphotyrosine antibody conjugated to horseradish
peroxidase (PY20-HRP; Santa Cruz) and visualization with the ECL
detection system (Amersham Corp.).Immunoprecipitations
Immunoprecipitation of the
whole cell lysates was performed by a 5-fold dilution of the
detergent-solubilized cell extracts (lysis buffer without Triton X-100)
and incubation with 4 µg of a carboxyl-terminal SHPTP2 polyclonal
antibody (Santa Cruz), monoclonal antibody 9E10, a Grb2 polyclonal
antibody (Santa Cruz), a Crk monoclonal antibody (Transduction
Laboratories), a Nck monoclonal antibody (Upstate Biotechnology, Inc.),
or an IRS1 rabbit polyclonal antiserum (a kind gift from Dr. Gus
Lienhard, Dartmouth Medical School) for 2 h at 4 °C. The primary
polyclonal and monoclonal antibodies were incubated with protein
A-agarose or protein G plus-agarose, respectively, for 1 h at 4 °C.
The resulting immunoprecipitates were then subjected to
SDS-polyacrylamide gel electrophoresis and Western blotted as described
above.
Insulin-stimulated Association of a 115-kDa
Phosphoprotein with SHPTP2
Insulin stimulation has previously
been observed to result in the specific association of SHPTP2 with
tyrosine-phosphorylated IRS1 (5, 9, 18) . To
assess the potential association of SHPTP2 with other effector
proteins, we initially examined the ability of an amino-terminal SHPTP2
antibody to coimmunoprecipitate phosphotyrosine-containing proteins
following growth factor stimulation (Fig. 1). Treatment of
CHO/IR cells with 100 nM insulin for 10 min resulted in the
coimmunoprecipitation of SHPTP2 with a tyrosine-phosphorylated 185-kDa
band identified as IRS1 (Fig. 1A, lanes1 and 2). However, the major insulin-dependent
tyrosine-phosphorylated species that coimmunoprecipitated with SHPTP2
migrated as a broad band at 115 kDa (Fig. 1A, lane2). In several experiments, the broad
tyrosine-phosphorylated 115-kDa protein band appeared to be composed of
multiple distinct species that were not always resolved (for example,
see Fig. 3). In contrast, stimulation of CHO/IR cells with 100
ng/ml PDGF resulted in the coimmunoprecipitation of the
tyrosine-phosphorylated PDGF receptors (
and
) as well as a
tyrosine-phosphorylated band of 68 kDa with SHPTP2 (Fig. 1A, lane3). However, the
amount of SHPTP2-coimmunoprecipitated tyrosine-phosphorylated 115-kDa
protein did not differ significantly between control and PDGF-treated
cells (Fig. 1A, lanes1 and 3). Western blots with an SHPTP2 antibody demonstrated that
approximately equal amounts of SHPTP2 protein were immunoprecipitated
under these conditions. Moreover, the 68-kDa tyrosine-phosphorylated
band observed in response to PDGF (Fig. 1A, lane3) comigrated with the SHPTP2 protein (Fig. 1A, lanes 4-6). These data are
consistent with previous reports demonstrating the direct tyrosine
phosphorylation and association of SHPTP2 with the PDGF receptor, but
not with the insulin receptor (18, 19, 24) .
Insulin Concentration Dependence of pp115 Tyrosine
Phosphorylation
To determine the insulin sensitivity of pp115
tyrosine phosphorylation and association with SHPTP2, we treated both
CHO/IR and 3T3L1 adipocytes with various concentrations of insulin for
10 min. The cells were detergent-solubilized and immunoprecipitated
with an SHPTP2 antibody, followed by phosphotyrosine immunoblotting (Fig. 2). In these experiments, a small amount of pp115 was
coimmunoprecipitated with SHPTP2 in untreated CHO/IR cells (Fig. 2A, lane1). Incubation of the
cells with insulin resulted in a dose-dependent increase in the extent
of tyrosine-phosphorylated pp115 in the SHPTP2 immunoprecipitates (Fig. 2A, lanes 2-5). Insulin treatment
of 3T3L1 adipocytes also resulted in a similar dose response of
coimmunoprecipitated pp115 (Fig. 2B, lanes
1-4). In multiple experiments, the ED for both
cell types was
1 nM. As observed in Fig. 1, the
amount of tyrosine-phosphorylated IRS1 coimmunoprecipitated by the
SHPTP2 antibody in both the CHO/IR and 3T3L1 adipocyte cell extracts
was relatively low compared with that of pp115. In addition, the
insulin concentration dependence of pp115 tyrosine phosphorylation and
association with SHPTP2 was similar to that observed for insulin
receptor
-subunit, IRS1, and Shc tyrosine phosphorylation (data
not shown).
Time Course of pp115 Tyrosine Phosphorylation
To
examine the rapidity of these events, we next determined the insulin
time dependence of pp115 tyrosine phosphorylation (Fig. 3). In
CHO/IR cells, approximately half-maximal tyrosine phosphorylation of
pp115 was observed in the SHPTP2 immunoprecipitates after only a
0.5-min stimulation with insulin (Fig. 3A, lanes1 and 2). Maximal tyrosine phosphorylation of
pp115 occurred by 2 min and remained at this level over the next 60 min (Fig. 3A, lanes 3-7). Similarly, maximal
phosphorylation of IRS1 (185 kDa) and the insulin receptor
-subunit (95 kDa) was observed between 0.5 and 2 min and declined
slightly over the following 60-min time period (data not shown).
Insulin stimulation of 3T3L1 adipocytes for 0.5 min resulted in a
partial increase in the tyrosine phosphorylation and association of
pp115 with SHPTP2 (Fig. 3B, lanes1 and 2). Maximal tyrosine phosphorylation of the
SHPTP2-associated pp115 protein occurred by 2 min and remained
unchanged following 15 min of insulin stimulation (Fig. 3B, lanes 3-5).
SHPTP2 Preferentially Associates with pp115 in
Insulin-treated 3T3L1 Adipocytes
Consistent with previous
studies demonstrating the specific association of
tyrosine-phosphorylated IRS1 with SHPTP2, we have observed the presence
of IRS1 in the SHPTP2 immunoprecipitates from CHO/IR and 3T3L1
adipocyte cell extracts (Figs. 1-3). However, the relative
phosphotyrosine signal intensity of IRS1 was low compared with that of
the pp115 protein. Since antibodies to the pp115 protein were not
available, we therefore determined the relative amount of IRS1 that was
associated with SHPTP2 compared with the total amount of
tyrosine-phosphorylated IRS1 (Fig. 4). As previously observed,
treatment of 3T3L1 adipocytes with insulin stimulated the association
of SHPTP2 with tyrosine-phosphorylated IRS1, pp115, and a band that
comigrated with the apparent mobility of Shc (Fig. 4A, lanes 1 and 2). However, immunoprecipitation of the
resulting supernatant from the first SHPTP2 immunoprecipitation with an
IRS1 antiserum demonstrated that the majority of
tyrosine-phosphorylated IRS1 was not associated with SHPTP2 (Fig. 4A, lanes3 and 4). In
addition, two tyrosine-phosphorylated proteins distinct from pp115 were
also detected. These could represent either other proteins associated
with IRS1, including the insulin receptor -subunit, or more likely
degradation productions of IRS1. In any case, the
tyrosine-phosphorylated IRS1 present in the second immunoprecipitation
was
96% of the total amount of IRS1 immunoprecipitated from the
cell extracts, whereas the percentage of IRS1 coimmunoprecipitated with
SHPTP2 was
4%. To determine the efficiency of the initial SHPTP2
immunoprecipitation, SHPTP2 immunoblotting was performed on the initial
SHPTP2 immunoprecipitates (Fig. 4A, lanes5 and 6), and the resulting supernatants were
subjected to a second round of SHPTP2 immunoprecipitation (Fig. 4A, lanes 7 and 8). These data
demonstrate that the initial immunoprecipitation with the SHPTP2
antibody quantitatively immunoprecipitates all of the SHPTP2 protein
present in the cell extract. The small amount of immunoblotted SHPTP2
protein detected in Fig. 4A (lane7)
represents spill-over from lane6 and was not due to
incomplete clearance from the initial SHPTP2 immunoprecipitation.
80% of the total tyrosine-phosphorylated IRS1 pool.
Nevertheless, the IRS1 immunoprecipitate from insulin-stimulated cell
extracts displayed barely detectable levels of immunoreactive SHPTP2
protein (Fig. 4B, lanes 5 and 6). In
contrast, when the supernatants from the initial IRS1
immunoprecipitation were incubated with an SHPTP2 antibody, essentially
all of the SHPTP2 protein was detected (Fig. 4B, lanes7 and 8). Even though the amount of
SHPTP2 protein detected in the initial IRS1 immunoprecipitate from
extracts of insulin-stimulated cells was relatively low (Fig. 4B, lane6), after correction
for the efficiency of IRS1 immunoprecipitation (Fig. 4B, lanes 2 and 4), we estimate
that <2% of the total SHPTP2 protein pool was associated with
tyrosine-phosphorylated IRS1.
pp115 Does Not Associate with Grb2, Nck, or Crk
We
next attempted to determine whether pp115 was related to other known
tyrosine-phosphorylated proteins of this approximate molecular mass
range. Following insulin stimulation, CHO/IR cell extracts were
immunoprecipitated with antibodies to c-Dbl, STAT2, Jak1, Jak2, Jak3,
Tyk2, Ras GTPase-activating protein, and the p120 Src substrate. These
antibodies failed to demonstrate the presence of any specific
tyrosine-phosphorylated proteins in phosphotyrosine immunoblots (data
not shown). In addition, a very weak pp120 tyrosine-phosphorylated band
was detected in c-Cbl immunoprecipitates; however, it was not
insulin-dependent (data not shown). We next determined whether the
small adapter proteins Crk, Nck, and Grb2 could specifically interact
with pp115 (Fig. 5). As controls, SHPTP2 immunoprecipitation of
extracts from untreated and insulin-stimulated CHO/IR cells
demonstrated the coimmunoprecipitation of tyrosine-phosphorylated
pp115, IRS1, and a small amount of Shc (Fig. 5A, lanes1 and 2). In contrast,
immunoprecipitation with a Crk (Fig. 5A, lanes3 and 4) or Nck (lanes5 and 6) antibody did not indicate the presence of pp115. Similarly,
immunoprecipitation with a Grb2 antibody (Fig. 5B, lanes3 and 4) clearly demonstrated the
insulin-dependent coimmunoprecipitation of tyrosine-phosphorylated IRS1
and Shc, but not pp115. Furthermore, the amount of
tyrosine-phosphorylated IRS1 coimmunoprecipitated by the Grb2 antibody
was substantially greater than that coimmunoprecipitated by the SHPTP2
antibody (Fig. 5B, lanes1 and 2). Consistent with Fig. 4, these data indicate that
the IRS1Grb2 complex does not contain significant amounts of the
SHPTP2 protein. The Grb2 and SHPTP2 immunoprecipitates also contained
similar amounts of a phosphotyrosine band that migrated with the
molecular mass of Shc. This finding is in agreement with previous
studies demonstrating that tyrosine-phosphorylated Shc is a major
Grb2-binding protein(43, 46) .
Association between pp115 and SHPTP2 Is Increased by
Expression of a Catalytically Inactive SHPTP2 Mutant
Since the
series of coimmunoprecipitations described above failed to identify the
nature of this insulin-stimulated tyrosine-phosphorylated protein, we
further characterized the binding interaction between SHPTP2 and the
pp115 protein by increased expression of SHPTP2 (Fig. 6).
Mammalian expression plasmids were constructed that contained an
amino-terminal Myc epitope tag with either a wild-type SHPTP2 cDNA
(Myc-WT-SHPTP2) or a catalytically inactive SHPTP2 point mutant in
which cysteine 459 was replaced by serine (Myc-C/S-SHPTP2).
Quantitative expression of these cDNAs by electroporation into CHO/IR
cells (44) resulted in an 3-fold increase in the total
amount of SHPTP2 protein levels compared with cells transfected with
the empty vector alone (data not shown). As expected,
immunoprecipitation with the Myc epitope monoclonal antibody (9E10) did
not result in the coimmunoprecipitation of the SHPTP2 protein from
mock-transfected cells (Fig. 6A, lanes1 and 2). In contrast, the SHPTP2 protein was readily
observed in the 9E10 immunoprecipitates from CHO/IR cells transfected
with either wild-type SHPTP2 (Myc-WT-SHPTP2) or the
phosphatase-inactive SHPTP2 mutant (Myc-C/S-SHPTP2) (Fig. 6A, lanes 3-6). Furthermore,
insulin treatment had no significant effect on the expression levels of
the SHPTP2 proteins (Fig. 6, compare lanes3 and 5 with lanes4 and 6,
respectively).
-subunit were also apparently
coimmunoprecipitated with the 9E10 monoclonal antibody. However, the
presence of these proteins in the immunoprecipitates from
mock-transfected cells indicates that they were nonspecifically
immunoprecipitated with the 9E10 antibody (Fig. 6C, lanes1 and 2). Nevertheless, these data
clearly demonstrate that the degree of pp115 tyrosine phosphorylation
and/or its extent of association with SHPTP2 largely depends upon the
protein tyrosine-specific phosphatase activity of SHPTP2.
-subunit itself. Furthermore, since pp115 was
tyrosine-phosphorylated with a slightly slower time course than IRS1,
we speculate that pp115 functions as a direct substrate of the insulin
receptor kinase, which subsequently associates with SHPTP2. In
addition, the increased association and tyrosine phosphorylation of
pp115 observed in cells expressing a catalytically inactive SHPTP2
mutant strongly suggest that pp115 functions as an in vivo substrate for the SHPTP2 phosphatase activity. The fact that
SHPTP2 appears to be an essential upstream effector for
insulin-dependent stimulation of mitogen-activated protein kinase
signaling also argues for a role of pp115 in this pathway.
Clarification of these issues and identification of the physiological
function of pp115 will require the molecular cloning and isolation of
pp115-specific antibodies.
We thank Dr. Gus Lienhard for providing the IRS1
antiserum.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.