©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of SH2-containing Phosphotyrosine Phosphatase Syp in Erythropoietin Receptor Signal Transduction Pathways (*)

(Received for publication, October 25, 1994; and in revised form, January 5, 1995)

Tetsuzo Tauchi (1) (4)(§) Gen-Sheng Feng (4) (2) Randy Shen (4) (2) Maureen Hoatlin (5) Grover C. Bagby Jr. (5) David Kabat (6) Li Lu (1) (4) Hal E. Broxmeyer (1) (3)(¶)

From the  (1)Departments of Medicine (Hematology/Oncology), (2)Biochemistry and Molecular Biology, (3)Microbiology and Immunology, and the (4)Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and the (5)Division of Hematology and Medical Oncology and (6)Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The phosphorylation of proteins at tyrosine residues is critical in the growth signaling induced by Epo. This mechanism is regulated by the activities of both protein-tyrosine kinases and protein tyrosine phosphatases. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase, widely expressed in all tissues in mammals became phosphorylated on tyrosine after stimulation with Epo in M07ER cells engineered to express high levels of human EpoR. Syp was complexed with Grb2 in Epo-stimulated M07ER cells. Direct binding between Syp and Grb2 was also observed in vitro. Furthermore, Syp appeared to bind directly to tyrosine-phosphorylated EpoR in M07ER cells. Both NH(2)-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-trnsferase fusion proteins, were able to bind to the tyrosine-phosphorylated EpoR in vitro. These results suggest that Syp may be an important signaling component downstream of the EpoR and may regulate the proliferation and differentiation of hematopoietic cells.


INTRODUCTION

Erythropoietin (Epo) (^1)is the hematopoietic growth factor that regulates the proliferation and differentiation of cells committed to the erythroid lineage(1, 2) . Introduction of the Epo receptor (EpoR), a member of the cytokine receptor family, into interleukin-3-dependent cell lines confers on the cells the ability to proliferate in response to Epo and has provided a valuable model for studies on the signal transduction of the EpoR (3, 4, 5) . Stimulation of growth factor-dependent cell lines with Epo has been shown to induce a variety of immediate cellular responses, including the rapid tyrosine phosphorylation of the EpoR and a number of cellular substrates(6, 7, 8, 9) ; induction of transcription of several immediate-early genes(10, 11) ; induction of the association of phosphatidylinositol 3`-kinase with the EpoR(12, 13, 14, 15) ; activation of components of the Ras signaling pathway, including Shc phosphorylation and its association with Grb2; increases in GTP-bound Ras, and activation of Raf-1 kinase(16, 17, 18) . Recent studies have shown that a 130-kDa protein tyrosine kinase, Jak2, is physically associated with the membrane-proximal region of the EpoR and is activated upon Epo stimulation(19, 20) .

Although a general scheme is emerging for EpoR signaling events, the role of specific protein tyrosine phosphatases in modulating this pathway is not yet clear. Recently, two mammalian phosphotyrosine phosphatases, PTP1C and Syp, that contain two SH2 domains have been isolated(21, 22, 23, 24, 25, 26, 27, 28) . PTP1C (also called SHPTP1, HCP, and SHP) is primarily expressed by hematopoietic cells, while Syp (also called SHPTP2, PTP1D, and PTP2C) exhibited a wider tissue distribution. Syp is most similar in sequence to the Drosophila corkscrew gene product, which is apparently required for signaling down stream of the Torso receptor tyrosine kinase(27) . Syp becomes tyrosine-(27, 28, 29, 30, 31, 32, 33, 34) and threonine- (29) phosphorylated in response to several kinds of growth factor stimulation and binds to the ligand-activated platelet-derived growth factor receptor and epidermal growth factor receptor and c-kit receptor(27, 28, 29, 30, 31, 34) . In addition to binding to those transmembrane proteins, Syp also binds to the tyrosine-phosphorylated form of insulin receptor substrate-1 and p210bcr-abl fusion protein(35, 36) . Likewise, PTP1C binds to the activated c-kit receptor and to the interleukin-3 receptor in hematopoietic cells and becomes tyrosine phosphorylated upon stimulation with these growth factors(37, 38) . These observations suggested that PTP1C and Syp, like other SH2-containing proteins, may be common downstream targets of receptor tyrosine kinases. In the present study we investigated a potential association between EpoR and Syp. It was found that Syp bound directly to tyrosine-phosphorylated EpoR and Grb2 upon Epo stimulation. The association between EpoR and Syp was mediated in vitro by both SH2 domains of Syp. Grb2 also appeared to bind directly to tyrosine-phosphorylated EpoR. We infer from these results that Syp, like PTP1C, is involved in the control of proliferation and differentiation of hematopoietic cells.


MATERIALS AND METHODS

Cytokines and Antibodies

Purified recombinant human Epo was purchased from Amgen Inc. (Thousand Oaks, CA). Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was a gift from Dr. Douglas E. Williams (Immunex Corp., Seattle, WA). Polyclonal anti-Syp Ab (raised against GST fusion protein containing Syp residues 2-216) was described previously(27) . Affinity purified anti-Grb2 Ab (raised against a peptide corresponding to residues 195-217 mapping at the carboxyl terminus of human Grb2) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phosphotyrosine mAb (PY20), anti-Grb2 mAb (GR81), and anti-Syp mAb were obtained from Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine mAb (4G10) and affinity purified anti-human EpoR Ab (raised against a GST fusion protein corresponding to a portion of the extracellular domain of the human EpoR) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Plasmids

The retroviral vector encoding hEpoR, pSFF-hEpoR, was constructed from a three-way ligation of the following fragments: a BamHI/XhoI pSFF vector fragment, BgIII/BssHII, and a BssMDRVHII/XhoI fragment from the hEpoR-encoding plasmid p18 (a gift from G. Wong, Genetics Institute, Cambridge, MA).

Cell Lines

Retroviral packaging cell lines -2 (39) and PA12 (40) cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum. The GM-CSF-dependent hematopoietic cell line MO7e (kindly provided by Dr. Jim Ihle, St. Jude Children's Hospital) was maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum and 100 units/ml GM-CSF. -2 and PA12 cells were transfected with retroviral vectors encoding hEpoR to produced helper-free virions by ping-pong amplification as described previously(41, 42) . MO7e cells were infected with EpoR-encoding virions and selected for growth in Epo. One of the high EpoR-expressing clones, designated M07ER was routinely maintained in RPMI containing 20% fetal bovine serum and 0.5 unit/ml Epo or 100 units/ml GM-CSF. Before Epo stimulation, exponentially growing M07ER cells were washed and incubated for 18 h at 37 °C in serum-free RPMI 1640 medium containing 0.5% bovine serum albumin (Sigma). After ``growth factor-starvation,'' cells were washed once with serum-free medium and then exposed to Epo (40 unuts/ml) for 5 min.

Immunoblotting and Immunoprecipitation

Immunoblotting was performed as described previously(36, 43) .

In Vitro Binding Assays

GST-Syp SH2 NC (amino acids 2-216), GST-Syp SH2-N (amino acids 2-109), and GST-Syp SH2-C (amino acids 108-216) were freshly prepared for the in vitro binding experiments. GST-Grb2 (amino acids 1-217) and GST rasGAP (amino acids 171-448) were purchased from Santa Cruz Biotechnology. For in vitro binding assays, glutathione-Sepharose beads (Pharmacia LKB Biotechnol.) with bound fusion proteins (approximately 5 µg of fusion protein/binding reaction) were incubated in cell lysates at 4 °C for 90 min. The beads were washed four times with TNGN (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 100 mM sodium fluoride, and 2 mM sodium orthovanadate) washing buffer (36) before analysis.

To test for direct binding, immunoprecipitation proteins were boiled with 100 µl of SDS-lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 0.2% Triton X-100 (Bio-Rad), 1% SDS, 1 mM phenylmethylsulfonyl fluoride, 0.15 unit/ml aprotinine, 10 µg/ml leupeptin, 100 mM sodium fluoride, and 2 mM sodium orthovanadate). Then eluted proteins were diluted with Triton X-100 lysis buffer (44) to 1 ml, so that the final concentration of SDS was 0.1%. The samples were incubated with GST-fusion proteins immobilized on sepharose beads and then analyzed by SDS-polyacrylamide gel electrophoresis.

To dissociate and denature the preexisting protein complexes, MO7ER cells stimulated in the presence or absence of Epo were lysed in a small volume of SDS-lysis buffer. After centrifugation, the supernatants were heated to 95 °C for 5 min, and then diluted with Triton X-100 lysis buffer(44) . The samples were incubated with GST-Grb2 fusion proteins or with anti-Syp Ab and then analyzed by SDS-polyacrylamide gel electrophoresis.


RESULTS AND DISCUSSION

Epo Induces the Tyrosine Phosphorylation of Syp- and Grb2-associated Proteins in M07ER Cells

Stimulation with Epo induced or enhanced the appearance of a series of tyrosine-phosphorylated proteins in M07ER cells (Fig. 1A, left). The tyrosine-phosphorylated protein with molecular mass of 72 kDa was the EpoR (Fig. 1A, right).


Figure 1: Epo induces the tyrosine phosphorylation of Syp- and Grb2-associated proteins in MO7ER cells. A, MO7ER cells (10^7 cells/ml) were treated with Epo (40 units/ml) for 5 min at 37 °C. Cell lysates were analyzed by 7.5% SDS-polyacrylamide gel, and immunoblotted (BLOT) with a mixture of anti-phosphotyrosine mAbs (4G10 + PY20) (left). Cell lysates were immunoprecipitated (IP) with anti-EpoR Ab. Immunoprecipitates were separated by 7.5% SDS-polyacrylamide gel and immunoblotted with anti-phosphotyrosine mAbs (4G10 + PY20) (right). B, cell lysates were immunoprecipitated with polyclonal anti-Syp or affinity purified anti-Grb2 and immunoblotted with anti-Syp mAbs (left) or anti-Grb2 mAbs (right). C, cell lysates were immunoprecipitated with polyclonal anti-Syp, affinity purified anti-Grb2 or non-immune rabbit immunoglobin antibodies as a control (C, as noted for immunoprecipitation), and immunoblotted with anti-phosphotyrosine mAbs (4G10 + PY20). The background band at 50 kDa is the immunoglobin heavy chain from the immunoprecipitation. Molecular mass markers are in kilodaltons. Similar results were obtained in two independent experiments.



In order to analyze the interaction of Syp and Grb2 with other proteins in M07ER cells, it was first shown, via immunoprecipitation, that stimulation of M07ER cells with Epo had no effect on protein level of Syp and Grb2 (Fig. 1B). To investigate the tyrosine phosphorylation status of Syp and Grb2 and their associated proteins in response to Epo, M07ER cells were lysed and incubated with anti-Syp or anti-Grb2 antibodies and the precipitated proteins were analyzed by immunoblotting with anti-phosphotyrosine mAbs (Fig. 1C). Epo-induced tyrosine phosphorylation of a 70-kDa protein which migrated similarily to the size of EpoR and Syp in anti-Syp immunoprecipitates (Fig. 1C). This 70-kDa tyrosine-phosphorylated protein was also detected in anti-Syp immunoprecipitation from the SDS- and heat-denatured cell lysates (data not shown). Therefore, Epo induced tyrosine phosphorylation of Syp. Epo did not induce the tyrosine phosphorylation of Grb2 (24 kDa) (Fig. 1C). However, anti-Grb2 immunoprecipitates obtained from Epo-stimulated M07ER cells contained a number of phosphotyrosine-containing proteins with apparent sizes of 130, 72, 52, and 46 kDa (Fig. 1C). The 52- and 46-kDa proteins, which were present in anti-Grb2 immunoprecipitates obtained from Epo-stimulated M07ER cells, most likely represent tyrosine-phosphorylated species of Shc oncogene product(18) .

Syp and Grb2 Bind Directly to the Tyrosine-phosphorylated EpoR in Vitro

The interactions of phosphotyrosine-containing proteins with Syp or Grb2 in Epo-stimulated M07ER cells were further examined in vitro using the SH2 domains of Syp and Grb2 expressed as GST fusion proteins immobilized to gluathionine-Sepharose. The samples were washed, and then the bound proteins were separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine mAbs (Fig. 2A). The 72-kDa phosphotyrosine-containing protein was complexed with GST-Syp SH2 fusion proteins only in the lysate from Epo stimulated M07ER cells (Fig. 2A). The 72-kDa phosphotyrosine-containing protein and several other phosphotyrosine-containing proteins (approximate molecular weights = 145, 130, and 52) complexed with GST-Grb2 in the lysate from Epo-treated cells (Fig. 2A).


Figure 2: Both Syp and Grb2 bind to tyrosine-phosphorylated EpoR directly. A, cell lysates from MO7ER cells treated with or without Epo were incubated with GST-SypSH2NC or GST-Grb2 fusion proteins or GST alone for 90 min at 4 °C. Samples were analyzed by 7.5% SDS-polyacrylamide gel and immunoblotted with anti-phosphotyrosine mAbs (4G10 + PY20). B, cell lysates from MO7ER cells were incubated with GST-SypSH2NC or GST-Grb2 fusion proteins or GST alone, and then immunoblotted with anti-EpoR Ab. C, cell lysates from MO7ER cells treated with or without Epo were immunoprecipitated with anti-EpoR and then immunoblotted with antiphosphotyrosine mAbs (4G10 + PY20) (first and second lanes). Cell lysates from MO7ER cells treated with Epo were immunoprecipitated with anti-EpoR Abs. The immunoprecipitated proteins were boiled with 1% SDS at 95 °C for 5 min, and then eluted proteins were incubated with GST-SypSH2NC (third lane) or GST-Grb2 (fifth lane) or GST-rasGAP (seventh lane) fusion proteins or GST alone (fourth, sixth, and eighth lanes). The samples were washed and the bound proteins were immunoblotted with anti-phosphotyrosine mAbs (4G10 + PY20) (second to eighth lanes). Similar results were obtained in each of two separate experiments.



To test whether the 72-kDa phosphotyrosine-containing protein that binds to Syp and Grb2 corresponds to the tyrosine-phosphorylated EpoR, cell lysates from M07ER cells were incubated with GST-SypSH2 or GST-Grb2 fusion proteins, then analyzed by immunoblotting with anti-EpoR Abs (Fig. 2B). Indeed, the 72-kDa phosphotyrosine-containing protein is the EpoR that complexed with GST-Syp SH2, and GST-Grb2 in the cell lysates from Epo-stimulated M07ER cells in vitro (Fig. 2B). However, this experiment does not address whether Syp and Grb2 bind directly to the tyrosine-phosphorylated EpoR. To test for direct binding, we lysed M07ER cells and incubated with anti-EpoR Abs. These immunoprecipitated proteins were boiled in 1% SDS, and then eluted proteins were incubated with GST-Syp SH2, GST-Grb2, and GST-rasGAP fusion proteins or GST alone. The unbound proteins were removed and immunoblotted with anti-phosphotyrosine (Fig. 2C). GST-Syp SH2 and GST-Grb2 were still able to bind to tyrosine-phosphorylated EpoR under denatured conditions; however, GST-rasGAP and GST alone did not bind to the EpoR (Fig. 2C). These results demonstrate that Syp and Grb2 bind directly to tyrosine-phosphorylated EpoR.

Differential Binding to GST-Syp Fusion Proteins Containing the Amino or Carboxyl SH2 Domains to EpoR

To further define the domains of Syp that were responsible for binding EpoR specifically, additional GST fusion proteins were examined, containing either the amino-terminal Syp SH2 domain (SH2N) or the carboxyl Syp SH2 domain (SH2C). As shown in Fig. 3, the individual SH2-N or SH2-C domains were both able to bind to tyrosine-phosphorylated EpoR; however, the GST fusion protein containing the SH2-N domain of Syp exhibited higher binding affinity for the EpoR than the SH2-C domain (Fig. 3). It has been shown recently that the P.Tyr-Val/Ile/Thr-X-Val/Leu/Ile is selected by the Syp NH(2)-terminal SH2 domain from a degenerate peptide library (45) . It has also shown that the Syp SH2 domains recognize a YTAV sequence in the beta subunit platelet-derived growth factor receptor (30, 31) . In this regard, the sequence surrounding tyrosine 427 (pYTLI) within the carboxyl-terminal region of human EpoR is most likely a Syp-binding site.


Figure 3: Differential binding of GST-Syp fusion proteins containing the amino or carboxyl SH2 domains to EpoR. Immobilized GST fusion proteins containing the amino (SypSH2N) or carboxyl (SypSH2C) SH2 domains or a fusion protein with both SH2 domains (SypSH2NC) were incubated, at final concentration of 10, 50, or 100 nM, with cell lysates of Epo-stimulated MO7ER cells. The resulting complexes were resolved by SDS-polyacrylamide gel and immunoblotted with anti-phosphotyrosine mAbs (4G10 + PY20). Similar results were obtained in each of two separate experiments.



Grb2 Binds Directly to Syp in Epo-treated M07ER Cells

Various experiments have suggested that the sequence surrounding Syp-Y546 conforms to the consensus binding sequence p.Tyr-X-Asn-X for the SH2 domain of Grb2(27, 46, 47) . Indeed, phosphorylation of this site in SHPTP2, human homologue of Syp, creates a binding site for Grb2 after platelet-derived growth factor stimulation(48) . PTP1C also contains this conserved sequence (21, 24, 25) . We wished to determine whether Syp forms a physiological complex with Grb2 in Epo-stimulated M07ER cells. To test for complex formation, we used both coimmunoprecipitation and in vitro binding assays. Cell lysates were immunoprecipitated with anti-Syp or anti-Grb2, and these immune complexes were then analyzed by immunoblotting with anti-Syp (Fig. 4A). Syp could be detected in anti-Grb2 immunoprecipitates from Epo-treated M07ER cells, but not from control cell lysates (Fig. 4A). In a similar experiment, Grb2 was detected in anti-Syp immunoprecipitates from Epo-stimulated M07ER cells (Fig. 4B). To test whether Grb2 can bind directly to Syp after Epo stimulation, the protein complexes in the cell lysates were dissociated and denatured by 95 °C heat and SDS treatment prior to anti-Syp immunoprecipitation or GST-Grb2 binding assays. These protein complexes were analyzed by anti-Syp or anti-P-Tyr immunoblotting (Fig. 5). Grb2 was still able to bind to Syp, which migrated similarly to the 72-kDa Grb2-bound tyrosine-phosphorylated proteins even under denatured conditions (Fig. 5). These results indicate that Syp and Grb2 form complexes directly in Epo-stimulated M07ER cells. However, tyrosine-phosphorylated EpoR and Syp are migrated similarly, and this experiment does not address whether the 70-kDa Grb2-bound tyrosine phosphorylated protein is related to Syp.


Figure 4: Co-immunoprecipitation of Syp and Grb2 in Epo stimulated MO7ER cells. Cell lysates from Epo-stimulated or unstimulated MO7ER cells were immunoprecipitated with polyclonal anti-Syp or affinity purified anti-Grb2. The immunoprecipitates were subjected to immunoblot analysis with anti-Syp mAb (A) or anti-Grb2 mAb (B). Similar results were obtained in two independent experiments.




Figure 5: Grb2 binds to tyrosine-phosphorylated Syp. Cell lysates for Epo-stimulated or unstimulated MO7ER cells were denatured by heating at 95 °C for 5 min in the presence of 1% SDS and then incubated with either anti-Syp Abs or GST-Grb2 fusion proteins or GST proteins. These samples were analyzed by 7.5% SDS-polyacrylamide gel, immunoblotted with either anti-Syp mAb or anti-phosphotyrosine mAbs (4G10 + PY20). Similar results were obtained in two independent experiments.



In summary we have identified one of the tyrosinephosphorylated proteins induced by Epo as the phosphotyrosine phosphatase Syp. Syp appeared to bind directly to the tyrosine-phosphorylated EpoR and Grb2 in vitro upon Epo stimulation. Syp may act as a ``bridge'' or an ``adaptor'' between tyrosine-phosphorylated EpoR and Grb2. In this regard, it is possible Syp may play a role in propagation of the Epo signal to the Ras signaling pathway. We have previously shown that Syp is physically complexed with BCR-ABL fusion protein and constitutively phosphorylated on tyrosine in BCR-ABL transformed cells (36) . Syp also appears to be a downstream target of c-kit receptor (34) . Taken together, our results strongly suggest that Syp, a ubiquitously expressed phosphotyrosine phosphatase, might play an important role in the regulation of hematopoiesis. Determination of the distinct functions of Syp in this process is critical for understanding the dynamic interaction between protein tyrosine kinases and phosphotyrosine phosphatases in control of hematopoiesis.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants R37 CA36464, R01 HL46549, and R01 HL49202 from the National Institutes of Health and the National Cancer Institute (to H. E. B.). 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.

§
Visiting investigator from Tokyo Medical College, 1st Department of Internal Medicine, Tokyo, Japan.

To whom correspondence should be addressed: Walther Oncology Center, Indiana University School of Medicine, 975 W. Walnut St., Rm. 501, Indianapolis, IN 46202-5121. Tel.: 317-274-7510; Fax: 317-274-7592.

(^1)
The abbreviations used are: Epo, erythropoietin; GM-CSF, granulocyte-macrophage colony-stimulating factor; GST, glutathione Stransferase; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We thank Rebecca Miller and Shantay Glover for typing the manuscript.


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