domains, with the activated erythropoietin receptor ...

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1993 81: 3204-3210

Phosphatidylinositol 3-kinase associates, via its Src homology 2 domains, with the activated erythropoietin receptor JE Damen, AL Mui, L Puil, T Pawson and G Krystal

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RAPID COMMUNICATION

Phosphatidylinositol 3-Kinase Associates, Via Its Src Homology 2 Domains, With the Activated Erythropoietin Receptor By Jacqueline E. Damen, Alice L.-F. Mui, Lorri Puil, Tony Pawson, and Gerald Krystal The erythropoietin receptor (EpR) belongs to a family of hematopoietin receptors whose members lack tyrosine kinase activity. Nonetheless, within minutes of binding Ep, a number of cellular proteins become transiently phosphorylated on tyrosine residues. One of these proteins, as w e and others have shown previously, is the EpR itself. To identify the remaining protein substrates, we have examined the antiphosphotyrosine immunoprecipitates of lysates from Ba/F3 cells expressing high levels of cell surface EpRs. W e now present data showing that, in responseto Ep, the 85-Kd regulatory subunit of phosphatidylinositol3-kinase (PI 3-kinase) becomes immunoprecipi-

table with antiphosphotyrosineantibodies. This appears to be due, in large part, to the specific association of PI 3-kinase with the tyrosine-phosphorylatedEpR, either directly or through a 93- or 70-Kd tyrosine-phosphorylatedintermediate. The activity of this EpR associated PI 3-kinase, assessed in anti-EpR immunoprecipitates, is maximal within 2 minutes of incubation with Ep and returns almost to baseline levels by 10 minutes. In vitro studies suggest that the interaction between PI 3-kinase and the activated EpR is mediated by the N- and C-terminal SH2 domains of p85 and tyrosine-phosphorylatedmotifs on the EpR. 0 1993 by The American Society of Hematology.

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tor (EGF) receptor, the colony stimulating factor-I (CSF-1) receptor, and the Steel factor (SF) receptor, it appears that several cytoplasmic proteins become not only tyrosine phosphorylated but also physically associated with these receptors after ligand binding. These cytoplasmic proteins include phospholipase C-7 1 (PLC-., l ) , I 4 , l 5 Ras GTPase-activating protein (GAP),'6,17 the Src family of tyrosine and phosphatidylinositol (PI) 3 - k i n a ~ e . ~ @ * ~ Cloning of these putative signaling proteins has shown that they all contain at least one Src homology 2 (SH2) domain, consisting of approximately 100 amino acids, that appears to direct their physical association with specific tyrosine phosphorylated regions within the activated recept01-s.~~ In the case of PI-3 kinase, a heterodimeric enzyme complex that phosphorylates PI, PI 4-phosphate (PI-CP), and PI 4,5bisphosphate (PI-4,5-P2) at the D-3 position of the inositol ring,26it is now generally believed that its 85-Kd subunit, which contains two SH2 domain^,'^ functions as an adaptor molecule that targets its associated catalytic 1 IO-Kd component,' to activated growth factor receptors.*' Interestingly, although the precise role of PI-3 kinase in regulating cell proliferation is unknown, it appears to be essential for PDGF-induced mit~genesis~'~~' and for transformation by the middle T-c-Src c o m p l e ~ . ~ ' . ~ ~ Based on these findings with receptor tyrosine kinases, and with the murine interleukin-2 (IL-2) receptor, which is another hematopoietin receptor superfamily member that has recently been shown to bind PI 3-kinase after ligand bindingz4we investigated whether PI 3-kinase might associate with the tyrosine-phosphorylated EpR. In this report, we show that Ep stimulates the association of PI 3-kinase with the activated EpR. This association appears to involve the SH2 domains of the p85 subunit of PI 3-kinase and either the activated EpR itself or a 93- or 70-Kd tyrosinephosphorylated intermediate.

RYTHROPOIETIN (Ep), a 34-Kd glycoprotein hormone, is the principal in vivo stimulator of mammalian erythropoiesis' and exerts its action by binding to a specific cell surface receptor present on relatively mature erythroid progenitor cells.2 Cloning of this Ep receptor (EpR), from both mouse3 and human4 cells, has shown that it belongs to a growing family of hematopoietin receptors whose members are characterized by having conserved cysteines and Trp-Ser-X-Trp-Ser motifs in their extracellular domains and no tyrosine kinase consensus sequence in their intracellular region^.^.^ However, although the EpR lacks tyrosine kinase activity, it, along with a number of intracellular proteins, becomes transiently phosphorylated on tyrosine residues within minutes of binding E P . ~ . The ' ~ tyrosine phosphorylation ofthese intracellular proteins appears to be essential for Ep-induced mitogenesis' and much effort has therefore been directed at identifying and characterizing these putative signal transduction intermediates. From studies with cell surface receptors bearing endogenous tyrosine kinase domains, such as the platelet-derived growth factor (PDGF) receptor, the epidermal growth fac-

From the Terry Fox Laboratory, B.C. Cancer Research Centre, Vancouver, Canada; and the Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada. Submitted February 4, 1993; accepted March 24, 1993. Supported by the National Cancer Institute of Canada (NCI-C) and the Medical Research Council of Canada (MRCC) with core support from the B.C. Cancer Foundation and the Cancer Control Agency ofBC. J.E.D. holds a Leukemia Research Fellowship, A I F.M. holds a MRCC Studentship, T.P. is a Terry Fox Cancer Research Scientist of the NCI-C, and G.K. is a Senior Scientist of the NCI-C. Address reprint requests to Gerald Krystal, PhD, Terry Fo.x Laboratory, B.C. Cancer Research Centre, 601 UT10th Ave, Vancouver, BC, V5Z IL3 Canada. The publication costs of this article were deJiayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1993 by The American Society of Hematology. 0006-49 71/93/82 12-0041$3.00/0 3204

MATERIALS AND METHODS

Reagents. The antiphosphotyrosine (anti-P-Tyr) monoclonal antibody, 4G IO, generated against phosphotyramine and shown to be highly specific for phosphotyrosine residue^,'^ and the anti-PI 3-kinase, specific for the pS5 subunit ofPI 3-kinase, were purchased from Upstate Biotechnology Inc (Lake Placid, NY). Recombinant Blood, Vol 81, No 12 (June 15). 1993: pp 3204-3210

From bloodjournal.hematologylibrary.org by guest on July 13, 2011. For personal use only. PI 3-KINASE BINDS TO THE ACTIVATED EP RECEPTOR

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Fig 1. Identification of proteins tyrosine phosphorylated in response to Ep. Equivalent numbers of Ba/F3 C5 cells were incubated for 10 minutes a t 37°C with ( + ) or without ( - ) Ep and the cells were then lysed and immunoprecipitated with the anti-P-Tyr MoAb, 4G10. (A) Phosphotyrosine-containingproteins were identified by immunoblotting with 4610. The numbers in bold print refer to the major tyrosinephosphorylated species. (B) The immunoblot shown in (A) was reprobed with an anti-C-terminal EpR antibody. (C) The same immunoblot was reprobed with an anti-PI 3-kinase antibody. Similar results were obtained in four different experiments.

human Ep was purified from culture supernatants of baby hamster cells expressing an Ep cDNA and was biotinylated as described previously.” Streptavidin-agarose beads and protein grade Nonidet P-40 (NP-40) were purchased from Calbiochem (San Diego, CA). Anti-N-terminal and anti-C-terminal EpR antibodies were generated in rabbits by immunizing with a peptide (covalently linked to KLH) corresponding to the predicted N- and C-terminal 15 amino acids ofthe murine EpR cDNA, respectively.” The antibodies were purified from serum by affinity binding to the N- and C-terminal peptides. The glutathione S-transferase (GST) fusion proteins, consisting of the 27-Kd amino-terminal of GST linked to the PI 3-kinase (N) SH2 domain (residues 3 I2 to 444 of bovine brain PI 3-kinase p85). the PI 3-kinase (C) SH2 domain (residues 612 to 722 of the 85-Kd subunit), the GAP (N) SH2 domain (residues I78 to 277 of human GAP), and the PLC-yI (N) SH2 domain (residues 547 to 659 of bovine PLC-yl) were expressed in Escherichia coli in pGEX-2T plasmids (Pharmacia LKB Biotechnology, Inc, Baie d’Urf6, Quebec, Canada) as described previ~usly.~’ After induction with I mmol/L isopropylthiogalactopyranoside, the bacteria were lysed by sonication and the fusion proteins recovered from clarified lysates by using glutathione-agarose (Pharmacia) as described.36 The amount of each GST fusion protein was determined by Coomassie blue staining and cross-linked at l mg/mL to CNBr-activated Sepharose 4B beads (Pharmacia). ”P-y ATP (3,000 Ci/ mmol) was obtained from Dupont (Mississauga, Ontario, Canada). The enhanced chemiluminescence (ECL) Western blotting reagents were purchased from Amersham Corp (Arlington Heights, IL). PI, PI-4-P, PI-4, 5-P2, L-a-phosphatidyl-L-serine (PS), and all other reagents were purchased from Sigma (St Louis, MO) unless otherwise indicated. Cells. The murine IL-3 (mlL-3)-dependent pro-B-cell line,

Ba/F3, kindly supplied by Dr A. Miyajima (DNAX Research lnstitute, Palo Alto, CA), was routinely cultured in RPMl containing 10% heat-inactivated fetal calf serum (FCS) and 3 nmol/L COScell-derived mIL-3. These cells were retrovirally infected with a JZen TKneo vector containing the murine EpR, as previously described,” and the present studies were performed with one of the high EpR-expressing clones, C5.” These cells were routinely maintained in RPMl containing 10%FCS and 0.5 U/mL Ep. Preparation of cell 1.vsate.s and immunoprecipitation. Ba/F3 clone C5 cells were incubated overnight in RPMI, I% FCS at 37OC, resuspended at 2 X IO’ cells/mL in RPMl containing 0.1% bovine serum albumin (BSA). and incubated with or without 50 U/mL Ep for IO minutes at 37°C. The cells were then washed and solubilized at 2 X lO’cells/mL with 0.5% NP-40 in phosphorylation solubilization buffer (PSB). ie. 50 mmol/L HEPES, pH 7.4, 100 mmol/L NaF, IO mmol/L NaPPi, 2 mmol/L Na,VO,, 2 mmol/L EDTA, and 2 mmol/L (NH& MO containing the protease inhibitors aprotinin (100 KIU/mL). leupeptin (40 pg/mL), and phenylmethylsulfonyl fluoride (PMSF: 1 mmol/L). After I hour of incubation at 4”C, insoluble material was removed by centrifugation for IO minutes at 16,000~andthe supernatants were gently rocked either for 4 hours with anti-P-Tyr-bound Sepharose beads33or for I hour with anti-PI 3-kinase antiserum or anti-C-terminal EpR antibody, followed by protein A-Sepharose beads (20 pL; Pharmacia) for an additional 2 hours at 4°C. The beads were then either prepared for PI 3-kinase assays (see below) or washed extensively with PSB containing 0. I % NP-40 and the bound proteins eluted with I 0 0 mmol/ L phenylphosphate (for anti-P-Tyr immunoprecipitates) or boiled in sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Biotinylated-Eplstreptavidin-agarose pirriJcation of the EpR.

From bloodjournal.hematologylibrary.org by guest on July 13, 2011. For personal use only. DAMEN ET AL

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Fig 2. Evidencefor the physical association of p85 with the activated EpR. (A) C5 cells, incubated for 1 0 minutes at 37°C with ( ) or without ( - ) b-Ep were lysed, exposed to streptavidin-agarose, and subjected to Western analysis, first using 4 6 1 0 (lanes 1 and 2) and then reprobing with an anti-PI 3-kinase antibody (lanes 3 and 4). (B) C5 cells, incubated for 1 0 minutes at 37°C with ( ) or without ( ) Ep, were lysed, immunoprecipitated with anti-PI 3-kinase, and subjected to Western analysis, first using 4 6 1 0 (lanes 1 and 2) and then reprobing with an anti-PI 3-kinase antibody to determine equivalence in loading (lanes 3 and 4). The position of the lgG heavy chain is indicated in (B), lanes 1 and 2.

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Ba/F3 clone C5 cells were incubated overnight in RPMI at 37°C. resuspended in Hanks' Balanced Salt Solution (HBSS) containing 2% FCS and 0.02% NaN, (HFN) buffer at 2 X IO' cells/mL, and then stimulated with 50 nmol/L biotinylated Ep (b-Ep) for 10 minutes at 37°C in the absence or presence of a 100-fold excess of unlabeled Ep (to control for nonspecific binding to streptavidin agarose). The cells were then solubilized using a final concentration of 8 mmol/L 3[(3-cholamidopropyl)-dimethyammonio]-l-propanesulfonate (CHAPS) in HBSS containing 2 mmol/L Na,VO, and 2 mmol/L (NH,),MO. After I hour at 4"C, insoluble material was removed by centrifugation for 10 minutesat 16,000gand thesupernatants were incubated for 2 hours at 4°C with 10 mg of streptavidin-agarose beads. The beads were exhaustively washed with HFN buffer containing 8 mmol/L CHAPS and bound proteins eluted by boiling for 3 minutes in SDS sample buffer containing 5% @-mercaptoethanol (@-ME)and analyzed by SDS-PAGE. Wesfern bloi ana/-vsis. After SDS-PAGE with 7.5% polyacrylamide gels, proteins were electrophoretically transferred onto Immobilon PVDF membranes (Millipore, MA) at 100 V for 90 minutes at 4°C using 10 mmol/L CAPS, pH 10.0, and 10%methanol. Residual binding sites on the membranes were blocked by incubation in Tris-buffered saline (TBS 10 mmol/L Tris-CI, pH 8.0, 150 mmol/L NaCI) containing 5% BSA plus 5% skim-milk powder for 1 hour at 23°C. Blots were then washed in TBST (TBS with 0.05% Tween-20) and incubated for I hour in TBST with I .O pglmL antibody. Blots were then washed three times for 5 minutes each with TBST and probed with a I :10,000 dilution of goat antimouse or goat antirabbit horseradish peroxidase conjugate (Jackson Labs, Bar Harbor, ME) for 45 minutes at 23°C. After washing, blots were incubated with enhanced chemiluminescence substrate solution and exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) to visualize immunoreactive bands. In some experiments, blots were stripped with 62.5 mmol/LTris-CI, pH 6.8,2% SDS, 100 mmol/L b-ME at 50°C for 30 minutes, reblocked, washed, and reprobed with either anti-PI 3-kinase (p85 subunit) or anti-N-terminal-EpR antibody, using goat antirabbit horseradish peroxidase conjugate (Jackson Labs) at 1: 10,000 as second antibody. I n viiro PI .?-kinase assay. Immunoprecipitated protein A beads were washed with the following buffers: ( I ) PBS; (2) 100 mmol/L Tris-CI, pH 7.4,50 mmol/L LiCI; (3) 10 mmol/L Tris-CI, pH 7.4, 0.1 mol/L NaCI. I mmol/L EDTA. The assay was performed directly with the beads in the presence of 25 mmol/L HEPES, pH 7.4, and 10 mmol/L MgCI,. A mixture of PI, PIA, 5-P,, and PS (l:l:2) at a total concentration of 0.2 mglmL was dispersed by sonication in 25 mmol/L HEPES, pH 7.4, 1 mmol/L EDTA and then added to the reaction mixture in a final volume of 50 pL. The reaction was initiated by adding 10 to 20 pCi of [y3'P]ATP (3,000 Ci/mmole; DuPont-New England Nuclear) and terminated by adding 20 pLof6 N HCI. The products were extracted into chloroform:methanol ( I :1) and separated by thin-layer chromatography (TLC) on oxalate-treated silica gel plates (Merck, Richmond, British Columbia, Canada) as previously d e ~ r i b e d . 2Unlabeled ~ lipid standards, PI-4-P and PI-4, 5-P,, were cochromatographed with the samples and visualized by exposure to iodine vapor. Binding io SH2 domains. NP-40 lysates from C5 cells, incubated in the presence and absence of Ep, were mixed at 4°C with Sepharose beads containing GST fusion proteins ( 1 mL of lysate with 20 pL of packed beads containing 20 pg of fusion protein) for 3 hours. The beads were then washed four times with PSB, 0. I% NP40 and bound proteins eluted by boiling with 100 pL of SDS sample buffer for 3 minutes. RESULTS AND DISCUSSION

As a first step towards identifying the intracellular pro-

teins that are tyrosine phosphorylated in response to Ep,

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Fig 3. p85 is not associated with the inactive EpR. C5 cells were incubated for 2 hours at 4°C with ( ) or without ( -) b-Ep and then washed, lysed, and added to steptavidin-agarose (lane 1) or shifted up to 37°C for 5 minutes before washing, lysing, and mixing with streptavidin-agarose (lane 2). (A) An anti-C-terminal EpR immunoblot. (B) The blot shown in (A) was reprobed with anti-P-Tyr antibodies. (C) The blot shown in (A) was reprobed with anti-PI 3-kinase antibodies.

anti-P-Tyr immunoprecipitations were performed with lysates from Ba/F3 cells engineered to express high numbers of cell surface murine EpRs." These cells, designated Ba/F3 clone C5 and shown previously to proliferate in response to low levels of Ep,I3 were treated with or without Ep for 10 minutes at 37°C. As can be seen in Fig 1A, Western analysis of these anti-P-Tyr immunoprecipitates, using anti-P-Tyr antibodies, showed that Ep stimulates the appearance of 6 prominent tyrosine phosphorylated bands at 145, 130, 93, 72,70, and 56 Kd. Minor, Epspecific, tyrosine-phosphorylated bands were also seen with molecular masses of 1 10,85, and 50 Kd. Reprobing this blot with antibody to the EpR showed, as weI3 and others10*"have shown previously, that the major 72-Kd band is the tyrosine phosphorylated form of the EpR (Fig IB). (Preclearing of Epstimulated C5 cell lysates with anti-EpR antibodies showed that the EpR is the only component of this 72-Kd band [data not shown].) As a start towards identifying the remaining tyrosine-phosphorylated proteins, the blot was reprobed with antibody to the p85 subunit of PI 3-kinase (Fig IC). This showed that, in response to Ep, the p85 subunit of PI 3-kinase is substantially increased in anti-P-Tyr immunoprecipitates and that it comigrates with the minor 85-Kd band seen in Fig 1A. These experiments suggest that Ep stimulates the tyrosine phosphorylation of the p85 subunit of PI 3-kinase and/or its physical association with a tyrosine-phosphorylated protein in these cells. Because PI 3-kinase has been shown previously to become physically associated with several activated, tyrosine kinase-containing cell surface receptor^*^^^ and, more recently, with certain nontyrosine kinase-containing recep t ~ r s , ~we ~ .next ~ ' wanted to determine if p85 was physically associated with the activated EpR. However, for technical

reasons, our rabbit anti-EpR antibodiescould not be used in conjunction with the rabbit anti-p85 antiserum from UBI (for immunoprecipitation and immunoblots). We therefore incubated C5 cells for I O minutes at 37°C with or without b-Ep and cell lysates were exposed to streptavidin-agarose to enrich for EpRs (and, potentially, EpR-associated proteins). An anti-P-Tyr Western blot of the streptavidin-agarosebound material showed, as expected, that b-Ep bound tightly to a major 72-Kd phosphoprotein (Fig 2A, lanes 1 and 2) that, upon reblotting with anti-EpR antibody, was shown to be the EpR (data not shown). Minor tyrosinephosphorylated bands were also seen at 93,70, and 56 Kd in this EpR-enriched preparation (Fig 2A, lanes 1 and 2), corresponding, most likely, to the major tyrosine-phosphorylated species seen in Fig 1A. Importantly, reprobing ofthis blot with anti-PI 3-kinase antibody showed that b-Ep also enriched for the p85 subunit of PI 3-kinase (Fig 2A, lanes 3 and 4). The converse was also performed, ie, lysates from C5 cells, incubated for 10 minutes at 37°C in the presence or absence of Ep, were immunoprecipitated with antibodies to PI 3-kinase. An anti-P-Tyr immunoblot showed that, in the presence of Ep, three bands at 93,72, and 70 Kd were consistently precipitated with anti-PI 3-kinase (Fig 2B, lanes 1 and 2). If lysates from Epstimulated C5 cells were precleared with anti-EpR antibody before immunoprecipitation with anti-PI 3-kinase, the 72-Kd protein was not seen in anti-P-Tyr immunoblots, indicating that this was the tyrosine-phosphorylated EpR (data not shown). Reprobing of lanes 1 and 2 with anti-PI 3-kinase showed equal loading of PI 3-kinase in lanes 3 and 4 and indicated that very little, if any, of total cellular p85 is tyrosine phosphorylated in response to Ep. This is consistent with very recent studies in which IL-4 was shown to dramatically increase PI 3-kinase


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levels at this temperature (Fig 3C). Thus, because PI 3-kinase was not associated with the EpR after incubation ofC5 cells with b-Ep at 4°C. it is most likely not associated with the receptor before Ep stimulation and EpR phosphorylation. To determine whether the 110-Kd catalytic component of PI 3-kinase is also associated with the activated EpR, PI 3-kinase assays were performed with anti-EpR immunoprecipitates. As can be seen from Fig 4A, only when Ep was

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Fig 4. Association of PI 3-kinase activity with the EpR and with tyrosine-phosphorylatedproteins. (A) C5 cells were incubated for 5 minutes at 37°C with (lanes 2 and 4) or without (lanes 1 and 3) Ep and lysates immunoprecipitated with anti-P-Tyr (lanes 1 and 2). anti-C-EpR (lanes 3 and 4). or anti-PI 3-kinase (lane 5) antibodies. Immunoprecipitated protein A beads were washed, PI 3-kinase assays performed, and TLC performed as described in Materials and Methods. (B) C5 cells were incubated with Ep for 0, 1,2,5, and 10 minutes at 37°C before lysing, immunoprecipitating with anti-CEpR antibodies, and measuring PI 3-kinase activity as in (A).

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97.4activity in anti-P-Tyr immunoprecipitates from FDCP-2 cell lysates, but did not stimulate the tyrosine phosphorylation of ~ 8 5 . ”Thus, after Ep stimulation, p85 is most likely brought down with antiphosphotyrosineantibodiesbecause of its association with the tyrosine-phosphorylated EpR (and/or a 93- or 70-Kd tyrosine-phosphorylated protein). To ascertain whether p85 is associated with the EpR before ligand binding, C5 cells were incubated with b-Ep for 2 hours at 4°C and then either processed directly for streptavidin-agarose affinity chromatography or shifted up first to 37°C for 5 minutes. We found, wing anti-EpR immunoblotting, that shifting up to 37°C resulted in a slight loss in total EpRs (Fig 3A). We also found, upon reblotting with anti-PTyr antibodies, that the EpR was significantly tyrosine phosphorylated only at 37°C (Fig 3B). Stripping and reprobing with anti-PI 3-kinase showed that, even though there was less total EpR at 37°C. the p85 subunit of PI 3-kinase was only substantially increased above background

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Fig 5. Binding of the activated EpR by bacteral GST-SH2 fusion proteins. Bacterially produced GST fusion proteins containing the p85 C-terminal SH2 (lane 1). the p85 N-terminal SH2 (lane 2), the GAP N-terminalSH2 (lane 3). and the PLC-71 N-terminal SH2 (lane 4) were immobilized on Sepharose 48 beads and mixed with lysates from C5 cells incubated with ( ) or without ( - ) Ep for 10 minutes at 37°C. Bound proteins were released by boiling for 1 minute in SDS sample buffer and resolved by SDS-PAGEand Western analysis using (A) anti-P-Tyr antibodies and (B) anti-EpR antibodies (lane 2 was subjected to anti-EpR immunoblotting).

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added to C5 cells did anti-EpR and anti-P-Tyr immunoprephosphorylated residues could still function in mitogenic signalling, it is conceivable that an Ep-induced conformacipitated preparations possess significant PI 3-kinase activity. This finding was consistent with those of our previous tional change rather than tyrosine phosphorylation of the receptor is critical for PI 3-kinase binding. To distinguish study showing that PI 3-kinase is not associated with the EpR until the latter was bound by its ligand (ie, as shown in among these possibilities, we are currently examining the Fig 3). A kinetic analysis of PI 3-kinase association with the effect of various EpR truncations on PI 3-kinase binding to EpR indicated that maximal PI 3-kinase activity was the EpR and to the 97- and 70-Kd proteins. reached by 2 minutes and returned almost to baseline by 10 Relevant to our data is the very recent finding by Linneminutes (Fig 4B). kin et a14' that a 97-Kd tyrosine-phosphorylated protein is Lastly, because the SH2 domains of the 85-Kd subunit of physically associated with the EpR in EpR-positive Ba/F3 PI 3-kinase have been shown recently to bind with high cells and that this protein may be a tyrosine kinase. Also of affinity to tyrosine-phosphorylated PDGF r e c e p t o r ~ , 2 ~ . ~interest ~ are the recent reports that a pp IO0 is tyrosine phosEGF receptor^,"*^^ CSF- 1 receptor^,^' SF receptor^,^' and phorylated in response to IL-3, granulocyte-macrophagecolinsulin-like growth factor receptors,23 we investigated ony-stimulating factor, or Ep in EpR-transfected FDC-P 1 and that a 97-Kd tyrosine-phosphorylated protein whether the p85 subunit of PI 3-kinase bound via its SH2 becomes associated with PI 3-kinase after IL-3 stimulation regions to the activated EpR. For these studies, GST fusion proteins, consisting of the amino terminus of GST, linked of FDCP-2 cells.37It remains to be determined whether the 93-Kd protein we observe in C5 cells is related to the 97to either the N- or C-terminal SH2 domains of p85, the N-terminal SH2 domain of GAP, or the N-terminal SH2 and/or 100-Kd proteins reported by these groups. domain of PLC-7 1 were expressed and cross-linkedto SephACKNOWLEDGMENT arose beads.35NP-40-solubilized lysates from C5 cells, incubated in the presence or absence of Ep for 10 minutes at We thank Vivian Lam for excellent technical assistance and Baila 37"C, were mixed with the various cross-linked beads and, Lazarus for typing the manuscript. after extensive washing, tightly associated proteins were released by boiling with SDS sample buffer and subjected to REFERENCES Western analysis with anti-P-Tyr antibodies (Fig 5A). The 1. Zanjani ED, Ascensao JL: Erythropoietin. Transfusion 29:46, results suggested that both the N- and C-terminal SH2 do1989 mains of p85 were capable of binding tightly to three tyro2. Wognum AW, Krystal G, Eaves CJ, Eaves AC, Lansdorp PM: Increased erythropoietin-receptor expression on CD34' bone sine-phosphorylated proteins present in lysates from Ep marrow cells from patients with chronic myeloid leukemia. Blood treated C5 cells; two minor phosphoproteinswith molecular 79:642, 1992 masses of 93 and 70 Kd and a major tyrosine-phosphory3. DAndrea AD, Lodish HF, Wong GG: Expression cloning of lated 72-Kd protein. Upon reprobing with anti-EpR antithe murine erythropoietin receptor. Cell 57:277, 1989 body, the 72-Kd protein was shown to be the tyrosine-phos4. Jones SS, DAndrea AD, Haines LL, WongGG:Humanerythphorylated EpR (Fig 5B). Because of the insensitivity of the ropoietin receptor: Cloning, expression, and biologic characterizaanti-EpR antibody, it could not be determined if the minor tion. 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Sawada T, Tsuda H, Mon XU, Kawakita M, Takatsuki K: EpR and PI 3-kinase may therefore associate via its SH2 Erythropoietin rapidly reduces phosphorylation of a membrane domains directly with the tyrosine-phosphorylatedEpR. Alprotein in a murine erythroleukemia cell line. Int J Hematol ternatively, because the EpR does not possess the YMXM 54:109, 1991 consensus sequence for PI 3-kinase binding that is present 8. Choi H-S, Bailey SC, Donahue KA, Vanasse GJ, Sytkowski in the kinase insert region of the PDGFR family,38in the AJ: Purification and characterization of the erythropoietin-sensiIRSl p r ~ t e i n ,and ~ ~in, ~the polyoma middle T antigen,38PI tive membranephosphoprotein, pp43. J Biol Chem 265:4143, 1990 9. Miura 0, D'Andrea A, Kabat D, Ihle JN: Induction of tyro3-kinase may bind indirectly to the EpR via either the tyrosine phosphorylation by the erythropoietin receptor correlates with sine-phosphorylated93- or 70-Kd protein because both are mitogenesis. 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