COMMUNICATION:
Interleukin-3 Induces the Association of the Inositol 5-Phosphatase SHIP with SHP2*

(Received for publication, December 18, 1996, and in revised form, February 11, 1997)

Ling Liu , Jacqueline E. Damen , Mark D. Ware and Gerald Krystal Dagger

From the The Terry Fox Laboratory, British Columbia Cancer Agency, University of British Columbia, Vancouver, British Columbia V5Z 1L3, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We recently purified and cloned a 145-kDa protein that becomes tyrosine phosphorylated and associated with Shc in response to multiple cytokines. Based on its predicated amino acid sequence and its enzymatic activity, we have called this protein SHIP, for rc omology 2-containing nositol hosphatase. To gain further insight into the intracellular pathways that this putative signal transduction intermediate might regulate we have investigated whether SHIP binds to intracellular proteins other than Shc. The results presented herein demonstrate that following interleukin-3 stimulation, SHIP binds to the tyrosine phosphatase, SHP2 (also called Syp, PTP1D, SHPTP2, and PTP2C) and that Shc is not present in these SHIP-SHP2 complexes. Time course studies reveal that SHIP's association with SHP2 is transient and is maximal at 10 min of stimulation with interleukin-3. We further show that the association of SHIP with SHP2 occurs through the direct interaction of the SH2 domain of SHIP with a pYXN(I/V) sequence within SHP2.


INTRODUCTION

Recently we (1) and Lioubin et al. (2) cloned the cDNA for a 145-kDa protein that becomes tyrosine phosphorylated and associated with Shc in response to multiple cytokines in hemopoietic cell lines. Based on its predicted amino acid sequence, which contains an amino-terminal Src homology (SH)1 2 domain, two phosphotyrosine binding (PTB) consensus sequences (i.e. NPXY sequences) (3), several proline-rich SH3 binding regions and two motifs highly conserved among inositol polyphosphate 5-phosphatases, we agreed to call this protein SHIP for SH2-containing inositol phosphatase (1, 2). Unlike most inositol polyphosphate 5-phosphatases that hydrolyze phosphatidylinositol 4,5-P2-bisphosphate and/or inositol 1,4,5-trisphosphate (4), SHIP selectively hydrolyzes the 5'-phosphate from inositol 1,3,4,5-tetraphosphate and phosphatidylinositol 3,4,5-trisphosphate (1), two inositol polyphosphates recently implicated in growth factor-mediated signaling (5-8). SHIP is also unique in that it is the only inositol polyphosphate 5-phosphatase cloned to date that possesses an SH2 domain, and we recently demonstrated that this SH2 domain is critical for SHIP's tyrosine phosphorylation and its association with Shc (9). To gain further insight into the possible function(s) of SHIP, we have sought to identify additional binding partners of SHIP and in this report present data indicating that SHIP associates with the tyrosine phosphatase SHP2 in response to IL-3 stimulation.


EXPERIMENTAL PROCEDURES

Reagents

The production and purification of COS cell-derived murine interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor were as described previously (10). The glutathione S-transferase (GST) fusion protein consisting of the 27-kDa amino-terminal of GST linked to the SH2 domain of murine SHIP (amino acids 7-133) (1) was expressed in Escherichia coli in pGEX-2T plasmids (Pharmacia Biotech Inc.) and purified from the sonicated bacteria using glutathione-agarose (Pharmacia) as described previously (1). Rabbit antiserum to SHIP was generated by immunizing animals with the GST-SHIP SH2 fusion protein described above. The 12-mer KRKGHEpYTNIKY, corresponding to the sequence flanking Tyr542 within SHP2 (11-13), was synthesized by the Sequencing Center at the University of Victoria (Victoria, BC, Canada) and the 11-mer STDpYSSGGSQG, corresponding to an intracellular region of the erythropoietin receptor (14), was generously provided by Dr. Taolin Yi (Cleveland Clinic Foundation, Cleveland, OH). The anti-SHP2 antibody used for immunoprecipitations was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and for Western blotting from Transduction Laboratories (Lexington, KY). The anti-phosphotyrosine (anti-PY) monoclonal antibody (mAb), was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Both affinity purified rabbit polyclonal antibodies to Shc (for immunoprecipitations) and mAb to Shc (for Western blotting) were obtained from Transduction Laboratories. Horseradish peroxidase-conjugated second antibodies were purchased from Jackson Immunoresearch (West Grove, PA). Protein grade Nonidet P-40 was from Calbiochem. The enhanced chemiluminescence Western blotting reagents were obtained from Pierce.

Immunoprecipitations and Western Blotting

Murine B6SUtA1 cells maintained in RPMI 1640 with 10% fetal calf serum and 5 ng/ml granulocyte-macrophage colony-stimulating factor were growth factor-deprived for 4-6 h at 37 °C in RPMI 1640 containing 0.1% bovine serum albumin and then stimulated at 37 °C for the indicated times with murine IL-3 (400 ng/ml). The cells were then washed once with phosphate-buffered saline, solubilized at 2 × 107 cells/ml with 0.5% Nonidet P-40 in 4 °C phosphorylation solubilization buffer (PSB), and subjected to immunoprecipitation and Western blotting as described previously (15).

In Vitro Binding Assays

Cell lysates from B6SUtA1 cells, treated with or without IL-3 for 10 min at 37 °C, were either incubated immediately for 1 h at 4 °C with glutathione-agarose beads bearing a GST-fusion protein containing the SH2 domain of SHIP or immunoprecipitated first with anti-SHP2 antibodies and the immunoprecipitate supplemented with SDS to a final concentration of 1%, boiled for 5 min, and diluted 20-fold with PSB containing 0.5% Nonidet P-40 before incubation with the beads. The beads were then washed three times with the same buffer and boiled in SDS-sample buffer, and the eluted proteins were subjected to Western blot analysis with the anti-PY mAb, 4G10.

Phosphopeptide Inhibition Assay

B6SUtA1 cells were incubated with IL-3 for 10 min at 37 °C, and the cells were rapidly lysed with 0.5% Nonidet P-40 in PSB in the presence or the absence of 50 µM of either a tyrosine phosphorylated 12-mer corresponding to the sequence flanking Tyr542 within SHP2; this same 12-mer dephosphorylated with calf intestinal alkaline phosphatase (Promega Corp, Madison, WI), chromatographed through Sephadex G25 (Pharmacia) to remove the alkaline phosphatase, and shown to be dephosphorylated by immunoblot analysis with anti-PY mAbs or a control tyrosine phosphorylated 11-mer corresponding to an intracellular region of the erythropoietin receptor. The cell lysates were incubated with anti-SHIP antibodies, and the immune complexes were collected on protein A-Sepharose beads. The bound proteins were then eluted off the beads by heating at 100 °C for 3 min in SDS sample buffer and subjected to Western analysis, first with anti-SHP2 and then with anti-SHIP antibodies.


RESULTS AND DISCUSSION

IL-3 Stimulates the Co-precipitation of SHIP with SHP2

In previous studies we established that IL-3 stimulates the rapid association of SHIP with Shc (1, 15). As a preliminary test to see if SHIP might be interacting with other intracellular proteins, lysates from B6SUtA1 cells treated with and without IL-3 for 10 min at 37 °C were subjected to immunoprecipitation with anti-SHIP antibodies, and Western analysis was then carried out with anti-PY mAbs. Aside from Shc, only one heavily phosphorylated protein with an apparent molecular mass of approximately 70 kDa was present following IL-3 stimulation (Fig. 1A). Reprobing this blot with antibodies to various candidate proteins revealed that this 70-kDa protein was the tyrosine phosphatase, SHP2 (Fig. 1A, middle panel). A second reprobing of this blot with anti-SHIP antibodies (Fig. 1A, lower panel) demonstrated equal loading of the control and IL-3-stimulated lanes. To confirm this finding the reciprocal experiment was carried out, i.e., lysates from B6SUtA1 cells treated with and without IL-3 for 10 min at 37 °C were subjected to immunoprecipitation with anti-SHP2 antibodies, and then Western analysis was performed with anti-SHIP antibodies. As can be seen in the upper panel of Fig. 1B, SHIP co-precipitated with SHP2 after IL-3 stimulation. A reprobing with anti-SHP2 antibodies confirmed equal loading (Fig. 1B, lower panel).


Fig. 1. IL-3 stimulates the co-precipitation of SHIP with SHP2. A, lysates from B6SUtA1 cells treated with and without IL-3 for 10 min at 37 °C were subjected to immunoprecipitation with anti-SHIP antibodies and Western analysis with anti-PY mAbs (upper panel). The band immediately below Shc corresponds to the Ig heavy chain. The blot was reprobed with anti-SHP2 (middle panel) and anti-SHIP (lower panel) antibodies. B, similar cell lysates were immunoprecipitated with anti-SHP2 antibodies and Western analysis carried out with anti-SHIP antibodies (upper panel). The blot was reprobed with anti-SHP2 antibodies (lower panel). IB, immunoblot.
[View Larger Version of this Image (28K GIF file)]


SHIP Associates Transiently with SHP2

We then explored the kinetics of assembly of SHIP-SHP2 complexes by carrying out time course studies with B6SUtA1 cells. Specifically, following IL-3 stimulation at 37 °C for the indicated times, the cells were lysed and the lysates subjected to immunoprecipitation with anti-SHIP antibodies, and the precipitates were examined by Western analysis with anti-PY antibodies. As can be seen in the top panel of Fig. 2, the 145/135-kDa SHIP doublet was tyrosine phosphorylated in B6SUtA1 cells to some extent even in the absence of growth factor stimulation, as we shown previously (1) and in Fig. 1A. Following IL-3 stimulation, however, its level of tyrosine phosphorylation increased substantially, peaking between 5 and 12.5 min and then declining. The maximal association of SHP2 with SHIP occurred at 10 min (second panel, Fig. 2), and this coincided with the maximal tyrosine phosphorylation of SHIP-associated SHP2 (top panel, Fig. 2). Interestingly, a reprobing with anti-Shc antibodies suggested that Shc association with SHIP might be reaching maximal levels slightly sooner (i.e. at 5 min). Reprobing of this blot with anti-SHIP antibodies confirmed equal loading (Fig. 2, lowest panel). The maximal association of SHIP with SHP2 may thus occur slightly later than with Shc.


Fig. 2. The kinetics of IL-3-induced association of SHIP with SHP2. B6SUtA1 cells, treated with and without IL-3 for the indicated times at 37 °C, were lysed, immunoprecipitated with anti-SHIP antibodies, and subjected to Western analysis with anti-PY antibodies (top panel). This blot was reprobed with anti-SHP2 (second panel), anti-Shc (third panel), and anti-SHIP (lowest panel) antibodies. The tyrosine phosphorylated band at about 90 kDa was also seen with preimmune serum. IB, immunoblot.
[View Larger Version of this Image (58K GIF file)]


SHP2 Does Not Associate with Shc Following IL-3 Stimulation

Because IL-3 stimulates the association of SHIP with both Shc and SHP2, it is conceivable that all three proteins form a complex. However, the slight difference in the kinetics of association of SHP2 and Shc with SHIP (seen consistently in three separate experiments) suggested this might not be the case. To test this further, lysates from IL-3-stimulated B6SUtA1 cells were immunoprecipitated with either anti-Shc (lane 1) or anti-SHP2 (lane 2) antibodies and the immunoprecipitates subjected to Western analysis with anti-PY mAbs (Fig. 3A, top panel). As expected, both antibodies co-precipitated SHIP, but there was no evidence of a 70-kDa band in the anti-Shc immunoprecipitate. Reprobing this blot with anti-Shc (Fig. 3A, middle panel) and anti-SHP2 (Fig. 3A, lower panel) antibodies confirmed that SHP2 did not co-precipitate with Shc. However, it is conceivable that the anti-Shc and anti-SHP2 antibodies we used immunoprecipitated only a subset of the total cellular Shc and SHP2 (e.g. perhaps the epitopes these antibodies recognized on Shc and SHP2 were not accessible in SHIP-Shc-SHP2 complexes). To test this depletion studies were undertaken in which total cell lysates were incubated with either anti-Shc or anti-SHP2 antibodies under the same conditions used in Fig. 3A and the supernatants subjected to Western analysis with anti-SHP2 (Fig. 3B, top panel) and anti-Shc (Fig. 3B, lower panel) antibodies. As can be seen by comparing lanes 1, 2, and 3 in Fig. 3B, total cell lysates (lane 1) were effectively depleted of Shc (lane 2) and SHP2 (lane 3) with these antibodies. We also tested the sensitivity of the anti-Shc and anti-SHP2 antibodies used for Western blotting by immunoblotting total cell lysates from 2 × 106 (lane 1), 2 × 105 (lane 4) and 2 × 104 (lane 7) cells and found that Shc and SHP2 could easily be detected when total cell lysate from as few as 2 × 104 cells were loaded onto the gel. Because the immunoprecipitates shown in Fig. 3A utilized 2 × 107 cells and because all the Shc and SHP2 were effectively immunoprecipitated from these cells, complexes containing both SHP2 and Shc, if present, would constitute less than <FR><NU>1</NU><DE>1000</DE></FR>th of the Shc or SHP2 present in these cells. Thus it appears that SHIP forms separate complexes with Shc and SHP2.


Fig. 3. SHP2 does not associate with Shc following IL-3 stimulation. In A, lysates from B6SUtA1 cells, treated with and without IL-3 for 10 min at 37 °C, were subjected to immunoprecipitation with anti-Shc (lane 1) or anti-SHP2 (lane 2) antibodies, and Western analysis was carried out with anti-PY mAbs (upper panel). This blot was reprobed with anti-Shc (middle panel) and anti-SHP2 (lower panel) antibodies. In B, 1-ml lysates from 2 × 107 B6SUtA1 cells treated with IL-3 for 10 min at 37 °C were subjected or not (lanes 1, 4, and 7) to immunoprecipitation with anti-Shc (lanes 2, 5, and 8) or anti-SHP2 (lanes 3, 6, and 9) and 100 µl of the supernatants, added directly (lanes 1, 2, and 3) or after diluting by <FR><NU>1</NU><DE>10</DE></FR>th (lanes 2, 5, and 8) or <FR><NU>1</NU><DE>100</DE></FR>th (lanes 3, 6, and 9), analyzed by Western blotting with anti-SHP2 (upper panel) and anti-Shc (lower panel) antibodies. IB, immunoblot.
[View Larger Version of this Image (27K GIF file)]


The SH2 Domain of SHIP Interacts Directly with a pYXN Sequence within SHP2

We showed previously using surface plasmon resonance that the SH2 domain of SHIP is capable of interacting directly with the pY317XN sequence (i.e. pYVNV) of Shc (9). Because SHP2 has also been shown to have two YXN sequences (i.e. Y542TNI and Y580ENV (11, 13)) that can become tyrosine phosphorylated following growth factor stimulation (16, 17), we asked if SHIP and SHP2 might be interacting via their SH2 and pYXN motifs, respectively. To test this, a bead-bound GST-fusion protein containing the SH2 domain of SHIP was incubated with lysates from B6SUtA1 cells treated with and without IL-3 for 10 min at 37 °C, and the bound material subjected to Western analysis with anti-PY mAbs. As can be seen in Fig. 4A (left panel), tyrosine phosphorylated bands corresponding to the positions of Shc, as expected (1), and SHP2 bound to the SH2 domain of SHIP following IL-3 stimulation. A reprobing of this blot with anti-SHP2 antibodies confirmed that the 70-kDa band was indeed SHP2 (Fig. 4A, right panel). No binding was observed when identical experiments were carried out with beads bearing GST alone (data not shown). To determine if this interaction was direct and not mediated through another protein, anti-SHP2 immunoprecipitates from B6SUtA1 cells treated with and without IL-3 were boiled in SDS to disrupt any complexes and then diluted and incubated with SHIP-SH2 bound beads. Western analysis using anti-SHP2 antibodies revealed that SHP2 from IL-3-stimulated cells still bound to these beads (Fig. 4B). This was further confirmed by Far Western analysis using the SH2 domain of SHIP. This probe was found to bind selectively to the tyrosine phosphorylated form of SHP2 (data not shown).


Fig. 4. The SH2 domain of SHIP interacts directly with a pYXN sequence within SHP2. A, lysates from B6SUtA1 cells treated with and without IL-3 for 10 min at 37 °C were incubated with a bead-bound GST-fusion protein containing the SH2 domain of SHIP, and the bound material was subjected to Western analysis with anti-PY mAbs (left panel). A reprobing of this blot with anti-SHP2 antibodies is shown in the right panel. B, lysates from B6SUtA1 cells treated without (lane 1) and with (lane 2) IL-3 for 10 min at 37 °C were immunoprecipitated with anti-SHP2 antibodies, boiled in SDS to disrupt any complexes, diluted, and incubated with SHIP-SH2 bound beads, and the bound material was subjected to Western analysis with anti-SHP2 antibodies. C, lysates from B6SUtA1 cells, stimulated with IL-3 for 10 min at 37 °C, were immunoprecipitated with anti-SHIP antibodies in the presence and the absence (lane 4) of either a pYXN 12-mer phosphopeptide corresponding to the sequence flanking Tyr542 within SHP2 (lane 1), this same 12-mer dephosphorylated with alkaline phosphatase (lane 2), or a control 11-mer phosphopeptide corresponding to the sequence flanking Tyr443 within the erythropoietin receptor (lane 3), and Western analysis was carried out with anti-SHP2 antibodies. A reprobing with anti-SHIP antibodies demonstrated equal loading (lower panel). IB, immunoblot.
[View Larger Version of this Image (23K GIF file)]


To investigate whether this interaction was mediated by a pYXN sequence within SHP2, lysates from B6SUtA1 cells stimulated with IL-3 for 10 min at 37 °C were immunoprecipitated with anti-SHIP antibodies in the presence and the absence of a pYXN 12-mer phosphopeptide corresponding to the sequence flanking Tyr542 within SHP2, this same 12-mer dephosphorylated with alkaline phosphatase, or a control 11-mer phosphopeptide corresponding to the sequence flanking Tyr443 within the erythropoietin receptor. As can be seen in Fig. 4C, only the tyrosine phosphorylated YXN sequence of SHP2 was capable of disrupting the SHIP-SHP2 complex. A reprobing with anti-SHIP antibodies demonstrated equal loading (Fig. 4C, lower panel). Interestingly, although these results at first glance appear to be somewhat at odds with very recent degenerate phosphopeptide library studies that suggest that the optimal ligand for the SH2 domain of SHIP is pY(Y/D)X(L/I/V) (18), it is worthy of note that the amino acid in the plus three position of both the pY542XN sequence (i.e. an isoleucine) and the pY580XN sequence (i.e. a valine) within SHP2 (11, 13) are quite consistent with this sequence. Moreover, the amino acids carboxyl-terminal to the Tyr317 of Shc are VNV (19). Thus, although SHIP and Grb2 may be competing for the same motifs within SHP2 and Shc, they may be doing so by interacting with different amino acids within these motifs, i.e. pYXN in the case of Grb2 and pYXX(L/I/V) in the case of SHIP. We cannot say at this time whether SHIP binds via its SH2 domain to the pY542XN or/and the pY580XN motif of SHP2.

SHP2 is a ubiquitously expressed tyrosine phosphatase that, like its Drosophila homologue, corkscrew (20), appears to play a positive role in mediating most (21-25) but not all (26, 27) extracellular signals. However, very little is known to date concerning how it mediates these signals. Some insight into how it may function as a positive regulator, at least in part, comes from studies showing that it can bind via its SH2 domains to activated receptors (28, 29) and activate the Ras pathway (16). Specifically, it has been shown to bind via its SH2 domains to the activated platelet-derived growth factor receptor and subsequently, once tyrosine phosphorylated, attract Grb2 via the latter's SH2 domain (16, 29). However, the role of SHP2's tyrosine phosphorylation remains controversial and appears unnecessary for and perhaps even inhibitory to Ras activation in some growth factor systems (30). In addition to this function as an adaptor molecule, more recent studies suggest that SHP2's phosphatase activity is also essential for SHP2 to act as a positive regulator (21, 23, 24, 30-32).

In the case of IL-3 signaling, our finding that IL-3 stimulates the tyrosine phosphorylation of SHP2 is consistent with a previous report by Welham et al. in which they show that IL-3 stimulates both the tyrosine phosphorylation and activation of SHP2 and its binding via one of its pYXN motifs to Grb2 via the latter's SH2 domain (33). Interestingly, studies from both our laboratory (9) and others (2, 34, 35) suggest that SHIP, which may be restricted to hemopoietic progenitors and endothelial cells (36), plays a negative role in growth factor-mediated signaling. This is intriguing given that two of its binding partners, Shc and SHP2, are thought to transmit positive signals, at least in part, by interacting with Grb2. Because we showed previously that SHIP and Grb2 bind to the same site on Shc (15) and herein on SHP2, it is tempting to speculate that SHIP may act as a negative regulator, at least in part, by competing with Grb2 for these positive regulators. Because we have recently shown that the tyrosine phosphorylation of SHIP is critical for its ability to bind to Shc in vivo (9), it is also tempting to speculate that SHP2's association with and possible dephosphorylation of SHIP serves to inactivate the latter and permit proliferation. Studies are currently underway in our laboratory to test this possibility.


FOOTNOTES

*   This work was supported by the National Cancer Institute of Canada and the Medical Research Council of Canada with core support from the British Columbia Cancer Foundation and the British Cancer Cancer Agency.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada supported by funds from the Canadian Cancer Society. To whom correspondence should be addressed: Terry Fox Laboratory, BC Cancer Research Centre, 601 West 10th Ave., Vancouver, BC V5Z 1L3, Canada. Tel.: 604-877-6070; Fax: 604-877-0712.
1   The abbreviations used are: SH2, Src homology 2 domain; anti-PY, anti-phosphotyrosine; GST, glutathione S-transferase; IL-3, interleukin-3; mAb, monoclonal antibody; PSB, phosphorylation solubilization buffer.

ACKNOWLEDGEMENTS

We thank Vivian Lam for excellent technical assistance and Christine Kelly for typing the manuscript.


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