©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Major SHPTP2-binding Protein That Is Tyrosine-phosphorylated in Response to Insulin (*)

(Received for publication, March 8, 1995; and in revised form, April 27, 1995)

Keishi Yamauchi (1) Vered Ribon (3) (2) Alan R. Saltiel (3) (2) Jeffrey E. Pessin (1)(§)

From the  (1)Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, the (2)Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48104, and the (3)Department of Signal Transduction, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, Michigan 48105

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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()(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) .

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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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) .


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).

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).


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.



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.


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 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) .


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.



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).


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 -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.


DISCUSSION

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 -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.


FOOTNOTES

*
This work was supported by Research Grants DK33823 and DK25295 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: IRS1, insulin receptor substrate-1; SH2, Src homology 2; PDGF, platelet-derived growth factor; CHO, Chinese hamster ovary; IR, insulin receptor.


ACKNOWLEDGEMENTS

We thank Dr. Gus Lienhard for providing the IRS1 antiserum.

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.


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