(Received for publication, October 25, 1994; and in revised form, January 5, 1995)
From the
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-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.
Erythropoietin (Epo) ()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.
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.
Figure 1:
Epo induces the tyrosine
phosphorylation of Syp- and Grb2-associated proteins in MO7ER cells. A, MO7ER cells (10 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) .
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.
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.
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.