©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Epidermal Growth Factor-induced Association of the SHPTP2 Protein Tyrosine Phosphatase with a 115-kDa Phosphotyrosine Protein (*)

Keishi Yamauchi , Jeffrey E. Pessin (§)

From the (1)Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Epidermal growth factor (EGF) stimulation of HepG2 and NIH 3T3 cells expressing high levels of the human EGF receptor (3T3/ER) resulted in the tyrosine phosphorylation of a 115-kDa protein that was co-immunoprecipitated with the Src homology 2 (SH2) domain containing protein tyrosine phosphatase, SHPTP2. In contrast, activation of the EGF receptor resulted in a relatively low level (<1%) of the total SHPTP2 pool associated with the tyrosine-autophosphorylated EGF receptor itself. Similarly, quantitative immunoprecipitations also demonstrated that only trace amounts of the total EGF receptor pool were associated with SHPTP2. Further, activation of the EGF receptor did not result in any significant tyrosine phosphorylation of SHPTP2 and/or the association of the 115-kDa protein with Grb2. In comparison, activation of Jurkat cells with a T cell receptor agonist monoclonal antibody resulted in the co-immunoprecipitation of a 120-kDa tyrosine-phosphorylated protein with Grb2 and a 105-kDa protein with SHPTP2. Thus, these data have identified the 115- and 105-kDa proteins as the predominant SHPTP2-associated phosphotyrosine proteins in EGF- and T cell receptor-activated cells, respectively.


INTRODUCTION

Protein phosphatases are typically thought to function as the inactivation arms of protein kinase-mediated signaling pathways. However, there are several recent examples whereby protein tyrosine-specific phosphatases play an essential positive signaling role. For example, in T cell receptor signaling, the CD45 protein tyrosine-specific phosphatase dephosphorylates the inhibitory carboxyl-terminal tyrosine phosphorylation site on Fyn and/or Lck allowing for activation of tyrosine protein kinase activity(1, 2, 3) . Similarly, recent studies have demonstrated that SHPTP2 protein tyrosine-specific phosphatase plays an important positive role in tyrosine kinase downstream signaling. Microinjection of SHPTP2-specific antibodies was observed to block insulin-stimulated DNA synthesis and expression of dominant interfering SHPTP2 mutants inhibited activation of mitogen-activated protein kinase, c-fos transcription, DNA synthesis, and fibroblast growth factor-stimulated Xenopus oocyte mesoderm induction(4, 5, 6, 7, 8) .

These data have provided substantial evidence demonstrating a positive signaling role for SHPTP2 in mediating tyrosine kinase growth factor receptor action. To identify potential physiological targets for SHPTP2, previous studies have observed that the SH2 domains of SHPTP2 result in the targeting to the tyrosine-phosphorylated epidermal growth factor (EGF)()receptor, platelet-derived growth factor receptor, and insulin receptor substrate-1 (IRS1)(9, 10, 11, 12, 13) . In addition, it has been reported that tyrosine-phosphorylated IRS1 functions as a substrate for SHPTP2 in vitro(14) . However, the tyrosine dephosphorylation of IRS1 is difficult to reconcile with a positive effector role for SHPTP2 tyrosine phosphatase activity, and a quantitative assessment of these associations has not yet been determined. In this paper, we have determined that only a small fraction of SHPTP2 associates with the EGF receptor and that a 115-kDa tyrosine-phosphorylated protein (pp115) is the major SHPTP2 binding protein in EGF-stimulated cells.


EXPERIMENTAL PROCEDURES

Cell Culture

HepG2 cells were obtained from the American Type Tissue Culture collection and were maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. NIH 3T3 cells overexpressing human EGF receptor (3T3/ER) were obtained from Dr. John Koland (University of Iowa). 3T3/ER cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. The human Jurkat T cell line and TCR monoclonal antibody (OKT3) were provided by Dr. Gary Koretzky (University of Iowa). The Jurkat T cells were maintained in RPMI 1640 containing 10% fetal bovine serum.

Immunoprecipitations and Western Blot Analysis

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 (500 µg) were diluted 5-fold using the lysis buffer without Triton X-100 and incubated with 4 µg of a carboxyl-terminal SHPTP2 polyclonal antibody (Santa Cruz) or an EGF receptor monoclonal antibody, LA1 (Upstate Biotechnology), 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. The whole cell extracts and the immunoprecipitates were then subjected to Western blot analysis using an amino-terminal SHPTP2 antibody (Transduction Laboratories), an EGF receptor antibody (Transduction Laboratories), or a phosphotyrosine antibody (PY20-HRP, Santa Cruz) and visualized with the enhanced chemical luminescence (ECL) detection system (Amersham Corp.).


RESULTS AND DISCUSSION

We and others have recently observed that SHPTP2 functions as a positive mediator in the regulation of tyrosine kinase receptor downstream signaling(4, 5, 6, 7, 8) . To identify potential targets for this phosphatase, we assessed the presence of phosphotyrosine-containing proteins that associated with SHPTP2 (Fig. 1). Stimulation of NIH 3T3/ER cells with 100 ng/ml EGF for 5 min resulted in a marked increase in the tyrosine phosphorylation of the EGF receptor (ER) following immunoprecipitation with an ER-specific antibody (Fig. 1A, lanes1 and 2). In contrast, immunoprecipitation of SHPTP2 primarily resulted in the co-immunoprecipitation of a 115-kDa protein and another band migrating at approximately 55 kDa (Fig. 1A, lanes3 and 4). Surprisingly, at this exposure level there was no detectable co-immunoprecipitation of the tyrosine-phosphorylated ER. To assess the presence of the ER in the SHPTP2 immunoprecipitates, panelA was overexposed (Fig. 1B). At this exposure level, we were able to detect a small amount of an EGF-stimulated phosphotyrosine band that co-migrated with the ER in the SHPTP2 immunoprecipitates (Fig. 1B, lanes3 and 4). In addition to the predominant EGF-stimulated 115-kDa protein, several other lower molecular weight bands were detected. These additional SHPTP2-associated phosphotyrosine-containing bands probably represent degradation products of the 115-kDa protein.


Figure 1: Identification of pp115 as the predominant tyrosine-phosphorylated protein associated with SHPTP2 following EGF stimulation of 3T3/ER cells. NIH 3T3/ER cells were incubated in the absence (lanes1 and 3) or presence (lanes2 and 4) of 100 ng/ml EGF for 5 min at 37 °C. Whole cell detergent lysates were prepared and immunoprecipitated with an ER-specific monoclonal antibody (ER, lanes1 and 2) or with a SHPTP2-specific polyclonal antibody (lanes3 and 4) as described under ``Experimental Procedures.'' The immunoprecipitates were then subjected to phosphotyrosine immunoblotting using the PY20-HRP antibody and visualized by the ECL method for 5 s (A) or for 2 min (B). IB, immunoblot; IP, immunoprecipitate.



Since previous studies have demonstrated that SHPTP2 can associate with EGF-stimulated tyrosine-phosphorylated ER(10) , we next determined the relative extent of SHPTP2 binding compared with the total cellular pool of ER and SHPTP2 (Fig. 2). Control and EGF-stimulated NIH 3T3/ER cells were initially immunoprecipitated with the SHPTP2 antibody and subjected to Western blotting with the ER antibody (Fig. 2A, lanes1 and 2). Under these conditions, we were unable to detect any significant level of ER protein by ER Western blotting. In contrast, when the supernatant from the initial SHPTP2 immunoprecipitate was subjected to a second round of immunoprecipitation using the ER antibody, a strong positive ER Western blotting signal was readily observed (Fig. 2A, lanes3 and 4). It should be noted that the apparent increase in the molecular weight of the ER following EGF stimulation (Fig. 2A, lane4) compared with unstimulated cells (Fig. 2A, lane3) reflects a decrease in electrophoretic mobility due to the phosphorylation of the ER.


Figure 2: Determination of the relative amount of ER associated with SHPTP2. 3T3/ER cells were either left untreated (lanes 1, 3, 5, and 7) or incubated with 100 ng/ml EGF (lanes 2, 4, 6, and 8) for 5 min at 37 °C. Whole cell detergent lysates were prepared and subjected to immunoprecipitation with the SHPTP2 polyclonal antibody (A) or with the ER monoclonal antibody (B) as described under ``Experimental Procedures.'' The resulting supernatants from the initial SHPTP2 immunoprecipitation in panelA were subjected to a second round of ER immunoprecipitation (lanes 3, 4, 7, and 8). The resulting supernatants from the initial ER immunoprecipitation in panelB were subjected to a second round of SHPTP2 immunoprecipitation (lanes 3, 4, 7, and 8). The immunoprecipitates were then Western blotted with an ER-specific antibody (lanes 1-4) or with a SHPTP2 specific antibody (lanes 5-8). IB, immunoblot; IP, immunoprecipitate.



To demonstrate that the SHPTP2 antibody was effective, the initial SHPTP2 immunoprecipitation was Western blotted for the presence of SHPTP2 (Fig. 2A, lanes5 and 6). Under these conditions, the SHPTP2 antibody quantitatively immunoprecipitated 100% of the total SHPTP2 protein pool present in the whole cell detergent extracts (data not shown). In addition, the supernatant from the initial SHPTP2 immunoprecipitation was then subjected to immunoprecipitation with the ER antibody. As expected, the immunoprecipitation of ER from the initial SHPTP2-cleared supernatant was unable to immunoprecipitate any detectable SHPTP2 protein (Fig. 2A, lanes7 and 8).

In a complementary approach, control and EGF-stimulated NIH 3T3/ER cells were initially immunoprecipitated with an ER antibody and subsequently subjected to Western blotting with the ER antibody (Fig. 2B, lanes1 and 2). Under these conditions the ER antibody was able to immunoprecipitate greater than 95% of the total pool of ER protein present in the whole cell detergent extracts (data not shown). However, there was no detectable ER co-immunoprecipitated SHPTP2 protein (Fig. 2B, lanes5 and 6). Similarly, SHPTP2 immunoprecipitation of the ER-cleared supernatants did not result in the co-immunoprecipitation of the ER (Fig. 2B, lanes3 and 4) but did immunoprecipitate the SHPTP2 protein (Fig. 2B, lanes7 and 8). Thus, these data indicated that essentially an undetectable level of the total SHPTP2 protein pool was associated with the ER. Similarly, the total amount of the ER associated with SHPTP2 was also relatively low. The fact that a detectable, albeit small amount, of tyrosine-phosphorylated ER was co-immunoprecipitated with SHPTP2 (Fig. 1) probably reflects a greater sensitivity of the PY20-HRP antibody compared with the SHPTP2 and ER antibody used in the Western blots (Fig. 2). Nevertheless, all these data were consistent with the ER playing a minor role in the association of SHPTP2 whereas the predominant SHPTP2 binding phosphotyrosine protein in EGF-stimulated 3T3/ER cells was apparently the 115-kDa species.

To determine if the EGF-stimulated association of SHPTP2 with the tyrosine-phosphorylated 115-kDa protein was unique to NIH 3T3 cells genetically engineered to express high levels of the human ER, we directly compared the 3T3/ER cells with the human hepatoblastoma cell line, HepG2 (Fig. 3). Phosphotyrosine immunoblotting of whole cell detergent extracts demonstrated the EGF-stimulated tyrosine phosphorylation of the ER and proteins of approximately 55 and 42 kDa in the 3T3/ER cells (Fig. 3A, lanes1 and 2). The 55- and 42-kDa proteins most likely reflect the tyrosine phosphorylation of Shc and ERK2. In any case, EGF treatment of HepG2 cells resulted in a significantly weaker tyrosine phosphorylation of the ER, consistent with the lower levels of the ER in the HepG2 cells compared with the 3T3/ER cell line (Fig. 3A, lanes3 and 4). As previously observed, SHPTP2 immunoprecipitation of extracts from EGF-stimulated 3T3/ER cells resulted in the predominant co-immunoprecipitation of the 115-kDa phosphotyrosine-containing protein with a relatively low amount of the tyrosine phosphorylated ER (Fig. 3B, lanes1 and 2). Similarly, EGF stimulation of the HepG2 cells also demonstrated that the predominant SHPTP2 co-immunoprecipitated phosphotyrosine-containing protein was pp115 (Fig. 3B, lanes3 and 4). As expected, a small amount of tyrosine-phosphorylated ER was detected in the SHPTP2 immunoprecipitate of HepG2 cell extracts (Fig. 2B, lane4) when the gel was overexposed (data not shown). These data demonstrate that the EGF-stimulated tyrosine phosphorylation and association of pp115 with SHPTP2 do not result from high level of ER expression and also occur in a typical EGF-responsive cell line.


Figure 3: Comparison of EGF-stimulated tyrosine phosphorylation and association of SHPTP2 with pp115 in 3T3/ER and HepG2 cells. NIH 3T3/ER and HepG2 cells were incubated in the absence (lanes1 and 3) or presence (lanes2 and 4) of 100 ng/ml EGF for 5 min at 37 °C. A, whole cell detergent lysates were prepared and directly subjected to Western blotting using the phosphotyrosine antibody PY20-HRP. B, the detergent lysates prepared in A were immunoprecipitated with the SHPTP2 polyclonal antibody and subjected to Western blotting using the phosphotyrosine antibody PY20-HRP. IB, immunoblot; IP, immunoprecipitate.



Recently it was reported that activation of the T cell antigen receptor (TCR) resulted in the tyrosine phosphorylation of proteins in the 116-kDa range that were associated with the small adapter proteins Crk and Grb2(15, 16) . To determine if the SHPTP2-associated 115-kDa protein was potentially related to these proteins, we compared the effect of EGF on HepG2 cells with that of TCR activation on Jurkat T cells (Fig. 4). As expected, EGF stimulation of HepG2 cells resulted in the co-immunoprecipitation of the 115-kDa tyrosine-phosphorylated protein with the SHPTP2 antibody (Fig. 4A, lanes1 and 2). Incubation of Jurkat cells with an activating TCR antibody (OKT3) resulted in the predominant tyrosine phosphorylation of a 105-kDa protein that was co-immunoprecipitated with the SHPTP2 antibody (Fig. 4A, lanes3 and 4). The other major band observed at approximately 25 kDa following TCR activation represents the light chain of the OKT3 monoclonal antibody. We next assessed whether the SHPTP2-associated proteins were also bound to Grb2 by co-immunoprecipitation with a Grb2 antibody (Fig. 4B). Immunoprecipitation of Grb2 from EGF-stimulated HepG2 cells demonstrated the presence of tyrosine-phosphorylated 52- and 46-kDa proteins, which probably represent the Shc proteins (Fig. 4B, lanes1 and 2). Due to the relatively low level of ER in these cells, the tyrosine-phosphorylated ER was only observed when the gel was overexposed (data not shown). Importantly, the SHPTP2-associated 115-kDa protein was not co-immunoprecipitated with Grb2. However, as previously reported(15, 16) , activation of the TCR resulted in the Grb2 co-immunoprecipitation of a 120-kDa tyrosine-phosphorylated band (Fig. 4B, lanes3 and 4). This band did not co-migrate with the SHPTP2-associated 105-kDa protein observed in these cells. Thus, the 115- and 105-kDa SHPTP2-associated proteins were unrelated to the previously described Grb2-associated tyrosine-phosphorylated protein. Furthermore, we have attempted to identify these components by immunoprecipitation with known antibodies against tyrosine-phosphorylated proteins in this molecular weight range. However, we have been unable to co-immunoprecipitate SHPTP2 with any EGF-stimulated tyrosine-phosphorylated proteins using antibodies directed against Jak1, Jak2, Jak3, Tyk2, STAT2, FAK, rasGAP, c-Dbl, c-Cbl, or the p120 Src substrate (data not shown).


Figure 4: Comparison of SHPTP2- and Grb2-associated phosphotyrosine proteins in HepG2 and Jurkat T cells. HepG2 cells were incubated in the absence (lane1) or presence (lane2) of 100 ng/ml EGF for 5 min at 37 °C. Jurkat T cells (JKT) were incubated in the absence (lane3) or presence of the TCR agonist antibody OKT3 (lane4). Whole cell detergent lysates were prepared and were either immunoprecipitated with a SHPTP2 antibody (A) or with a Grb2 antibody (B). The immunoprecipitates were then subjected to Western blotting using the phosphotyrosine antibody PY20-HRP. IB, immunoblot; IP, immunoprecipitate.



In summary, the data presented in this paper demonstrate that a protein of 115 kDa is a significant EGF-stimulated SHPTP2-associated tyrosine-phosphorylated protein in NIH 3T3/ER and HepG2 cells. Although SHPTP2 can bind to the autophosphorylated ER, the extent of this association is relatively minor and only accounts for a small fraction of the total cellular ER and SHPTP2 pool. Since we have not yet identified the nature of the 115-kDa protein and currently no antibodies are available, we have not been able to determine the relative extent of SHPTP2 association with pp115. However, based upon the tyrosine phosphorylation signal, we speculate that this protein is both a substrate for the ER and is the major SHPTP2 binding protein. In addition, an apparently related 105-kDa protein was detected in Jurkat T cells, and neither the 115- nor 105-kDa proteins were found to associate with Grb2. Clearly, the identification of the physiological function of pp115 is an important issue necessary to determine the molecular role of SHPTP2 in ER signaling.


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: EGF, epidermal growth factor; ER, epidermal growth factor receptor; IRS1, insulin receptor substrate-1; TCR, T cell antigen receptor.


ACKNOWLEDGEMENTS

We thank Dr. John Koland for providing the human EGF receptor expressing NIH 3T3 cells. We also thank Dr. Gary Koretzky for providing the Jurkat T cells and the OKT3 monoclonal antibody.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.