Shc Phosphorylation In Myeloid Cells Is Regulated by Granulocyte ...

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hematopoietic cells through binding to specific, high af- finity, cell surface ... dermal growth factor; PAGE, polyacrylamide gel electrophoresis. protein-tyrosyl ...
THEJOURNAL OF BIOLOXAL. CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 7, Issue of February 18, pp. 5016-5021, 1994 Printed in U.S.A.

Shc PhosphorylationIn Myeloid Cells Is Regulated by Granulocyte Macrophage Colony-stimulating Factor, Interleukin-3, and Steel p210BClUABL$ Factor and Is Constitutively Increased by (Received for publication, June 21, 1993, and in revised form, October 5 , 1993)

Tetsuya MatsuguchiS, Ravi SalgiaS, Michael HallekS,Matthias EderS, Brian DrukerQ, Timothy J. ErnstS, and James D. Griffinh From the Wiuisions of %mor Immunologq a n d @Moleculara n d Cellular Biology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts -02116

Granulocytemacrophagecolony-stimulatingfactor, protein-tyrosylkinases,such as p21OBCWmL (24, 25), can interleukin-3, and steel factor induce proliferation of eliminate the need for IL-3 for growth of h u m a n or murine hematopoietic cells through bindingto specific, high af- myeloid cell lines.Also, tyrosine kinase inhibitors, which block finity, cell surface receptors. However, little is known receptor-associated kinase activity, inhibit the biological effects about post-receptor signal transduction pathways. Here of GM-CSF (26). we report that an SH2 domain containing protein preIdentification of the substrates that are tyrosyl phosphoviously implicated in the activation of p2lrM, Shc, is rylated by the various kinases activated by GM-CSF, IL-3, a n d transiently tyrosine phosphorylated in myeloid cells af- SF is likely tobe important in understanding the signal transter stimulation with granulocyte macrophage colonyduction pathways that lead to the control of cell proliferation. stimulating factor, interleukin-3, or steel factor. Also, In previous studies, we noted that each of these three factors Shc was found to be constitutively tyrosine phosphorylated in myeloid celllines made factor independent by could independently support proliferation of the human myexpression of p21PCwmL.AShc-associated140-kDa pro- eloid cell line M07 andthat there was overlap inthe proteins tein was identified, which was phosphorylated on tyro- that are tyrosyl phosphorylated in response to allthree of the sine residues transiently after cytokine stimulation andfactors (27, 28). In this study, we showthat each of these cytoconstitutively afterexpression of p210BCWABL.Thesekines induce rapid phosphorylationof Shc protein (29), a cytofindings suggest that Shc could play an important role plasmic protein that has been implicated in the activation of in a signal transduction pathway, which leads to the p21"" in response to epidermal growth factor (30). proliferation of myeloid cells. MATERIALSANDMETHODS Cells and Cell Culture-The human GM-CSF and IL-3-dependent Granulocytemacrophagecolony-stimulatingfactor (GM- cell line, M07, was obtained from Dr. Steve Clark, Genetics Institute, CSF),l interleukin-3 (IL-31, and steel factor (SF) have diverse Cambridge, MA, and was originally derived by Avanzi and colleagues (31) from the peripheral blood cells of an infantwith acute megakaryoeffects on the proliferation, differentiation, and activation of cytic leukemia. The cell line was cultured in Dulbecco's modified Eagle's blood cells and their precursors (1-7). These biological effects medium (Life Technologies, Inc.) supplemented with 20% fetal bovine are mediated through binding of the factors to specific, high serum, 10 ng/ml rh GM-CSF, and 10 ng/ml rh IL-3 (Genetics Institute). affinity cell surface receptors (5, 8-16). However, post-receptor The 32Dc13 cell line (32) was obtained from Joel Greenberger, Universignal transduction pathwaysare not well understood and are sity of Massachusetts Medical Center, Worcester, MA, and was cultured in RPMI 1640, 10% fetal bovine serum, and 10-15% medium condilikely to be composed of both mitogenic and differentiation tioned by the WEHI-3Bcell line as a source of murine IL-3. Other signals. Although the SF receptor, the product of c-kit, poshuman cell lines were obtained from the American Type Culture Colthe known lection. sessesintrinsicprotein-tyrosinekinaseactivity, components of the GM-CSF and IL-3 receptors do not have pGD210 (33), an expression plasmid of p210BCWABL, were obtained recognizable tyrosine kinase catalytic domains (9,11-13). How- from Richard van Etten and George Daley, Massachusetts Institute of GM-CSF or Technology. The sublines expressing p21OBCWmLweregenerated by ever, in all cells so far examined, which respond to IL-3, there are multiple cytoplasmic proteinsthat are tyrosine transfection of plasmid pGD210 into M07 cells and 32Dc13 cells by electroporation as previously described(34) using a Bio-Rad Gene Pulphosphorylated within seconds after factor stimulation (17sar (Richmond, CA) and selecting for G418-resistant sublines. All of 21). There is considerable evidence that the induction of this these lines were factor-independent and wereshownto express protein-tyrosyl kinase activityis critical for mediating the mi- p210BCWABL by immunoblotting as described below. togenic effects of each of the three factors. For example, intro- Generation of Antibodies to She Proteins-A cDNA encoding human duction of a known protein-tyrosyl kinase receptor, such as the Shc was a gift from Dr. P.G. Pelicci, University of Perugia, Italy. A epidermal growth factor receptor (22,23), or oncogene-encoded 285-base pair fragment (from nucleotides 1211 to1495 (29)), which contains the Shc SH2 domain, was isolated using polymerase chain reaction and cloned into the pGEX-3X vector. Bacterial cultures con* This work was supported by Public Health Services Grants CA taining the pGEX-3X-Shc SH2 plasmid were inducedwith 0.1 mM iso36167 and CA 34183. The costs of publication of this article were de- propyl-1-thio-P-D-galactopyranosideand lysedby sonication. The refrayed in part by the payment of page charges. This article must there- sulting glutathione 5'-transferase fusion protein was affinitypurified on fore be hereby marked "aduertisement" in accordance with 18 U.S.C. glutathione agarose beads as described (35, 36). A New Zealand White Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: Div.of Tumor Im- rabbit was immunized with a series of five triweekly subcutaneous munology, Dana-Farber Cancer Inst., 44 Binney St., Boston, MA02115. injections of purified fusion proteins to obtain high titer antisera. The antisera was affinity purified on a fusion protein column as described Tel.: 617-632-360; Fax: 617-632-4388. The abbreviations used are: GM-CSF, granulocyte macrophage (35, 36). The flow-throughwas used as precleared serum. Immunoblotting-Cellswerelysed in 1%Nonidet P-40, 150 mM colony-stimulatingfactor; IL-3, interleukin-3; SF, steel factor; EGF, epiNaC1, 50 mM Tris, pH 8.0, 0.5%deoxycholic acid, 0.1% SDScontaining dermal growth factor; PAGE, polyacrylamide gel electrophoresis.

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She Phosphorylation in Myeloid Cells 1 m phenylmethylsulfonylfluoride,20 pg/ml aprotinin,and 1 mM sodium orthovanadate (all reagents from Sigma) a t 1 x loR celldml. Immunoblotsusingantiphosphotyrosine monoclonal antibody 4G10 were performed a s described (19). Shc immunoblots were done with affinity purified anti-Shc antibody dissolved in 1 x TBST (10 n w TrisHCI (pH 8.0), 150 xnw NaCI, 0 . 0 5 8 ' h e e n 20) buffer at 67.5 ng/ml. Anti-vabl antibody 24-21 (37) was obtained from Naomi Rosenberg, Tufts Medical School, Boston, MA. Epidermal growth factor receptor was detected usingan EGFreceptor-specific monoclonal antibody ( c l l ) from ICN (Imine, CA). Immunoprecipitation and in Vitro Kinase Assay-The cell lysates were incubated with either affinity purified anti-Shc antibody or preimmune serum for 2 h a t 4 "C, and then protein A-Sepharose beads (Pharmacia LKB Biotechnology Inc.) were added for a n additional 1h a t 4 "C. The beads were washed3 times in the same buffer, suspended in sample buffer a s described (341, and heateda t 9 5"C for 5 min, and the eluted proteins were applied to SDS-polyacrylamide gels and detected by immunoblotting. For in vitro kinase assay, the immune complexes were collected on Protein A beads, washed 3 times with lysis buffer, suspended in kinase buffer (50 m Tris, pH 7.4, 10 m MgCl2, 1 m dithiothreitol, 10 p.v ATP), containing ["PIATP (24 nmol/pl, 7,000 Ci/ mmol, DuPont NEN) for 20 min a t 30 "C. Immunoprecipitates were then washed with1 x phosphate-buffered saline twice and suspended in sample buffer, heated at 95"C for 5 min, resolved by SDS-PAGE, and autoradiographed. Labeling of Cells withPzPIOrthophosphate-l x lo7 cells were washed with phosphate-free RPMI 1640 containing0.5% bovine serum albumin and cultured in the phosphate-free medium. After 3 h of incubation in the phosphate-free medium, the cells were incubatedfor an additional 2 h with the addition of 0.5 mCi of carrier-free 1"2Plorthophosphate (AmershamCorp.). Labeled cells were cultured withor without growth factors a t 37 "C for 10 min and lysed. Phosphoamino Acid Analysis-1 x lo7 cellswere "P-labeled as above, cultured with or without 20 ng/ml GM-CSF a t 37 "C for 10 min, and lysed. The lysates were immunoprecipitated with affinity-purified anti-Shcantibody,resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and autoradiographed. The excised bands from the membrane were directly submitted to 6 N HCI hydrolysis at 110 "C for90min.Thehydrolysatesweredriedandwashedwith double-distilled water. Samples were then subjected to two dimensional electrophoresis on TLC plates with pH 1.9 running buffer (25 volumes of 90% formic acid:78 volumes of acetic acid:897 volumes of H20) and then with pH 3.5 running buffer(5 pyridine:50 acetic acid:945 doubledistilled water). Unlabeled phosphoamino acid markers were visualized by spraying with 0.5% ninhydrin in acetone. "P-labeled amino acids were visualized by autoradiography.

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FIG.1. Identification of Shc proteins in various cell lines. Cell lysates were prepared from 4 x lo6 cells of: lane 1, NIH3T3; lane 2, M07; lane3 , M07 p210; lane 4, HL60; lane 5, Raji; lane 6, Jurkat; lane 7, HPB-ALL; lane 8, K562; lane 9, 32Dc13; lane 10, MEL. Cells were separated by 7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with affinity-purified anti-Shc antibodies.

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FIG.2. In vivo phosphorylation ofShc in M07 cells after cytokine treatment and p210 BCRJABL transfection.A and B , 1x lo7 cells were labeled with 0.5 mCi/ml I:"Plorthophosphate for 2 h, lysed, and immunoprecipitated with affinity-purified anti-Shc antibody (lanes 1 4 )or preimmune serum (lanes 6 and 7). The precipitates in each lane RESULTS are from: lane 1, unstimulated M07 cells; lane 2, M07 cells stimulated 20 ng/ml of GM-CSF; lane 3.20ng/ml of IL3; lane 4,20 for 10 min with Shc Protein Expression in Hematopoietic Cell Lines-Cell ng/ml of SF; lane 5, M07 p210 cells; lane 6, unstimulated M07 cells; lysates were prepared from the humanmyeloid cell lines, M07, lane 7, M07p210 cells. Immunoprecipitateswere resolved by SDSM07 transfected with p210RCWARL cDNA (M07 p210), HMO PAGE and autoradiographed for 12 h (panel A)or 72 h (panel B ) . C , and K562 cells, from the humanlymphoid cell lines, Jurkat and serum-starved M07 cells (1 x lo7 cells each) were in vivo labeled a s Raji cells, from the murinemyeloid cell lines, 32Dc13, and from above with ["ZPlorthophosphate and stimulated with 20 ng/ml of GMCSF for the indicated times, lysed, immunoprecipitated with affinitythe murine erythroleukemia cell line, MEL. Shc was detected purified anti-Shc antibodies, resolved by SDS-PAGE, and autoradioby immunoblotting with rabbit anti-human Shc antibody. As graphed.

shown in Fig. 1, two proteins,and p46Shc,were recognized in every hematopoietic cell line examined. In some cell in Fig. 2C, after stimulation with GM-CSF, Shc phosphorylalines (M07, M07p210, Raji, HPB-ALL, K562, and MEL cells) a tion was induced within 2 min anddecreased after 10min, but third proteinof 66 kDa was also observed. A similar protein was there was stillsignificant Shc phosphorylation a t 60 minafter detected in NIH3T3 cells as previously reported (29). In the the cytokine stimulation. Cytokines Stimulate Drosine Phosphorylation of Shc Proother cell lines, HL60, 32Dc13, and Jurkat,~ 6 6 was ~ ~barely ' detectable. teins-Because Shc is known to be phosphorylated in part on GM-CSI;: ZL-3, and Steel Factor Induce Phosphorylation of tyrosine residues by growth factors in fibroblasts (29), we exShc in a Human Myeloid Cell Line-Phosphorylation of Shc amined the tyrosine phosphorylation of Shc in myeloid cells was examined in factor-dependent M07 cells before and after treated with cytokines. Cell lysates from M07 cells unstimustimulation by GM-CSF, IL3, and SF for 10 min. M07 cells lated or stimulated withGM-CSF, IL3, or SF were immunoprewere labeled in vivo with 32P-orthophosphate, and the phos- cipitated with anti-Shc antibody followed by immunoblotting phorylation status of Shc was examinedby immunoprecipitat- with the anti-phosphotyrosine antibody, 4G10. The tyrosine ing with anti-Shc antibody and autoradiography.As shown in phosphorylation of p 5 P h Cwas increasedfollowing stimulation Fig. 2 B , phosphorylation of Shc was significantly increased by GM-CSF, IL3, and SF (Fig. 3A ). There was also an increase 6 after 6 cytokine ~ ~stimula~ following stimulation by each of these three cytokines. ~ 5 2 " ~ "in tyrosine phosphorylation of ~ was most prominently phosphorylated, whilea small amountof tion. We could not detect the tyrosine phosphorylation of ~ 4 6 ' ~ " because of comigration with immunoglobulin heavy chains. As phosphorylation was detected on ~ 4 6 " ~and ' ~ 6 6 ~As~ shown " .

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FIG.3. Shc tyrosinephosphorylation in MO7 cells after cytokine treatment andp210 BCWABL transfection. A, lysates from 1 X lo7 serum-starved M07 cells unstimulated (lane 1)or stimulated for 10 min with 20 ng/ml of GM-CSF(lane Z),20 ng/ml of I L 3 (lane 3), and 20 ndml of SF (lane 4 ) , and M07 p210 cells (lanes 5 and 6 )were immunoprecipitated with affinity-purified anti-Shc antibody (lanes 1-5) or preimmune serum (lane 6).Immunoprecipitates were subjected to SDS-PAGE, transferred to nitrocellulose and blotted with anti-phosphoty2.osine antibody, 4G10. Lysates from the same number of M07 (lane 7) and M07 p210 cells (lane 8)were immunoblotted with affinity-purifiedanti-Shc antibody. B , serum-starved M07 cells (1 x lo7 cells each lane) were stimulated with the indicated concentration of either GM-CSF, IL-3, or SF for 10 min, immunoprecipitated with affinity-purified anti-Shc antibody, and immunoblotted with 4G10. C, serum-starved M07 cells (1x lo7 cells each) were stimulated for the indicated times with 20 nglml GM-CSF, lysed, immunoprecipitatedwith affinity-purifiedanti-Shc antibodies, and blotted with ~ ' M07 cells and M07 p210 cells. Positions of phosphoserine (S), phospho4G10. D , Phosphoamino acid analysis of ~ 5 2 in~ GM-CSF-stimulated threonine (T), and phosphotyrosine (Y) markers are indicated.

shown in Fig. 3B, a maximum responseof p52Shctyrosine phosphorylation was obtained with either 20 ng/ml of GM-CSF, 20 ng/ml of IL3, or 20 ng/ml of SF, and higherconcentrations of the cytokines did not produce any further increaseof Shc tyrosine phosphorylation.After stimulationwith GM-CSF, tyrosine phosphorylation of ~ 5 2 was ~ ~induced ' within 2 min and declined after 10 min, and there was no detectable tyrosine phosphorylation after 60 min (Fig. 3C).Phosphoamino acidanalysis revealed that GM-CSF phosphorylated ~ 5 2primarily ~ ~ " on serine and to a lower level on tyrosine residues (Fig. 30). Thus, these three cytokines induce rapid, transient tyrosine phosphorylation and rapid, more prolonged serine phosphorylation of ~52'~". In contrast, fetal calf serum did not induce tyrosine phosphorylation of Shc (datanot shown). Constitutive7)rosinePhosphorylation of Shc Proteins in Cells Expressing p210BcRiAsL-As shown in Fig. 2A, after 32P labeling, Shc phosphorylation was much higher in M07 p210 cells than after cytokine stimulation of M07 cells. Further, Shcwas constitutivelyheavilytyrosinephosphorylated in p210BCWABL-expressing cells (Fig. 3A ).A doublet a t 52 kDa was observed in thepresence of p210BCWABL. This doublet is likely to representtwo forms of p52Shc, because it wasalso observed following anti-p-tyr immunoprecipitation and anti-Shcblotting (data not shown).Shc was also heavily tyrosine phosphorylated in 32Dc13 cells expressing p21OBCWmL(data notshown). Phosphoamino acid analysis revealed marked phosphorylation of tyrosine residues and lesser phosphorylation of serine residues (Fig. 30). In cells expressing p210BCWABL, the extent of Shc phosphorylation was equivalent whether or not cells were exposed to IL-3 (data not shown). Cytokines andp210BCRlABL Induce 7'yrosine Phosphorylation of a 140-kDa Shc-associated Protein-When the lysates from

M07 p210 cells were immunoprecipitated with anti-Shc and immunoblotted with anti-phosphotyrosine antibody(Fig. 3A), a coprecipitating proteinwith a molecular mass of approximately 140 kDa was detected. A 140-kDa protein was also observed to coprecipitate with Shc in M07p210cells after in vivo labeling with 32P(Fig. 2 A ) . The 140-kDa protein also coprecipitated with Shc in M07 cells transiently after cytokine stimulation, although the protein was more difficult to visualize than in p210BCWABL-transfected cells (Fig. 3A). In some experiments, but not others, a 60-kDa protein was also detected in antiphosphotyrosineblots of anti-Shc immunoprecipitates (Fig. 3A ). Immunoprecipitation with preimmune serumdid not precipitate a similar protein (Fig 3 A , lane 6).It is not known whether cytokines and p210BCWABL stimulate the association of this 140-kDa protein with Shc or the phosphorylation of a preassociated 140-kDa protein. Because of the known association of Shc with the EGF receptor (29), we asked if Shc is associated with the EGFreceptor in myeloid cells. Using crossimmunoprecipitationhmmunoblottingwith anti-Shc and antiEGF receptor antibodies, no association could be detected in M07 cells (data not shown). Furthermore, we could not detect expression of EGF receptor in thiscell line by immunoblotting, and treatmentof the cell line with EGF in the absence of serum did not altertyrosine phosphorylation of any cellular proteins, including Shc (data not shown). These data suggest that this unknown 140-kDa phosphoprotein is distinct from the EGF receptor in myeloid cells. In Vitro Kinase Assays with Shc Proteins in Hematopoietic Cells-Because Shc hasbeen shown to be physically associated with the EGF receptor in fibroblasts after EGF stimulation (29), we asked if Shc is associated with any kinase inmyeloid cells by performing in vitrokinase assayswith anti-Shc immu-

Shc Phosphorylation in Myeloid Cells

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FIG. 4. In vitro kinase assay of Shc immunoprecipitates from M07 cells. Serum-starved M 0 7 cells unstimulated(lane 1 ) and stimulated for 10 min with 20 ng/ml of GM-CSF (lane 2), 20 ng/ml of IL3 (lane 3 ) , 2 0 ng/ml of SF (lane 4 ) , and M 0 7 p210 cells (lanes 5 and 6) (1 x lo7 cells each) were lysed and immunoprecipitated with affinity-purified anti-Shc antibody (lanes 1 5 ) or with preimmune serum (lane 6). Immunoprecipitateswereincubatedwith ["2PlATP inkinase buffer, washed with 1 x phosphate-buffered saline, separated by SDS-PAGE, and autoradiographed.

noprecipitates of MO7 and M07p210cells. As seen inFig. 4, a weak kinase activity was coimmunoprecipitated with Shc in M07 cells, which resulted in the phosphorylation of p52Shcand immunoglobulin heavychain. This weak kinase activity is thought to be specific to Shc, because it was not detected in immunoprecipitate with preimmune serum (lane 6). The significance of this weak kinase activity is obscure, because it was not increased with cytokine stimulation. In contrast,increased kinase activity was coimmunoprecipitated with Shc in p210BCWABL overexpressing cells, resulting in the significant phosphorylation of p52Shc,immunoglobulin heavy chain, anda 140-kDa protein. A small amountof phosphorylation of a 210kDa protein was also detected. Possible Association of Shc with p21@'cRtABL in M07p210 Cells-Because the coimmunoprecipitation of a 210-kDa protein with Shc wasdetected in M07p210 cells in several different experiments(Fig. 2B, Fig. 3A, Fig. 41, but not in M07cells, we asked if Shc coprecipitates with p210BCWABL. Cell lysate from M07p210 cells was immunoprecipitated with anti-Shc antibody and blottedwith an anti-ab1 monoclonal antibody (Fig. 5 ) . A single protein of 210 kDa was visualized (lane 1), suggesting coprecipitation of Shc and p210BCWABL. This band was not seen with control antibodies (lane2) and could not be detected in M07 cells that did not express p210BCWABL (data not shown). Each of these cell lines has abundant cabl, we and neverdetectedassociation of Shc and cabl. However, the amount of p210BCWABL that could coprecipitate with Shc is likely to be small, because the 210-kDa protein was barely detectable in anti-phosphotyrosine immunoprecipitates (Fig. 3A), and only a small amountof phosphorylation of a 210-kDa protein was detected in the anti-Shc vitro in kinase assay(Fig. 4). These results suggest that a minor fraction of ~ 2 1 0 ~ " associates with Shc in p210Bcr'Ab'expressing cells. DISCUSSION

In this study we show that Shc proteins are widely expressed in hematopoietic cell lines and rapidly and transiently phosphorylated, in part on tyrosine residues, in response to three mitogenic growth factors for myeloid cells, IL-3, GM-CSF, and steel factor. In contrast, fetal calf serum did not affect Shc phosphorylation in myeloid cells. Further, we demonstrate that introduction of p210BCWABL into the same cell line results in constitutive marked tyrosine phosphorylation of Shc proteins, suggesting that Shc directly is or indirectly a substrate for the p210BCWABL tyrosine kinase. Shc is a novel transforming protein first identified by Pelicci and colleagues (29) by screening a P3HR1 Burkitt lymphoma

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FIG.5. Association of p210Bcr/Ab1 with Shc in p210 transfected cells. Lysates from 1 x 10' serum-starved M 0 7 p210 cells were immunoprecipitated with affinity-purified anti-Shc antibodies (lane 1 ) or precleared serum (lane 2). separated by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-vabl antibody.

cDNA library with a c-fes SH2 probe. The human Shc mRNA was found to encode two different proteins in an invitro transcription-translation system(29). Anti-Shc SH2 antibodies were shown to immunoprecipitate three proteins of approximate molecular mass 46, 52, and 66kDa. The 46- and 52-kDa proteins comigrated with the two Shc proteins produced by in vitro translation, but the relationship of the 66-kDa protein to the 46- and 52-kDa proteins hasnot yet been clarified. A single SH2 domain was found at amino acids 378-471, with the highest sequence homology to the SH2domains of Src andCrk. An N-terminal glycine/proline-rich domain from amino acids 233377 was homologous to collagen, and thegene was named Shc for "Src homologous and collagen" (29). There is growing evidence that Shc isinvolved in signaling from some growth factor receptors. Pelicci et al. have shown that Shc proteins associate with the EGF receptor and were phosphorylated on tyrosine in response to EGF in Rat-1 fibroblasts and in response to nerve growth factorin PC12 cells (29). In some cells, Shc may be involved in a mitogenic signal transduction pathway. Evidence for this comes from the observation that overexpression of Shc in murinefibroblasts is transforming (29). Furthermore, in Rat-2cells transformed by temperature sensitive v-src or v-fps mutants, p46, p52, and p66 were all rapidly tyrosine phosphorylated upon activation of the oncogenes, suggesting that Shcproteins could participate in mediating the transformation of fibroblasts by these tyrosine kinase oncogenes (38). Possible mechanisms whereby Shc might participateboth in have re~normal ' ~ ~ ' signaling pathways and in transformation cently been identified. Shc proteins were found to complex with a poorly phosphorylated 23-kDa protein in oncogene-transformed cells or EGF-stimulated fibroblasts (38). This 23-kDa protein has been identified as murine Grb2 (30,39). Grb2 is a SH2- and SH3-containing protein that has recently been reported to be associated with a Ras exchange factor, Sos, and linktyrosine kinases to Ras signaling(30,39-42).Overall, these resultssuggest that Shc proteins can link tyrosine kinase receptors to activation of ~21"" throughGrb2. Hematopoietic growth factors are known to activate p21"" (43, 4 4 1 , although the mechanism involved is unknown. Activation of a tyrosine kinase is required, buttyrosine phosphorylation of rasGAP does not appear to be needed, and we have failed to find any consistent increase in the tyrosine phosphorylation of rasGAP in two different factor-dependent cell lines,

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M07 and 32Dc13, in response to GM-CSF.2 In contrast, the readily demonstrable increase in phosphorylation of Shc proteins inresponse to IL-3, GM-CSF, and steelfactor suggestthat activation of nucleotide exchange through GrbWSem-5 pathway could lead to activation of p21m" in hematopoietic cells, and this pathway should be investigated further. In preliminary studies, we detected the coimmunoprecipitation of Grb2 and hSos with Shc in M07p210 cells.3 We have found that after stimulation of myeloid cells by either growth factors or p210BCwML,tyrosine phosphorylation of a 140-kDa Shc-associated protein was readily detected. In fibroblasts, Shc has been shown to associate with the EGF receptor after stimulation of cells with EGF (29). Because the molecular weightof the EGFreceptor is near this weight range, we asked if the EGFreceptor is associated with Shc in myeloid cells. Immunoprecipitation of cell lysates from M07p210 cells with anti-Shc antibody and blotting against anti-EGFreceptor antibody did not give any evidence for coprecipitation. Hematopoietic cells are not generally believed to have functional EGF receptors, and in unpublished studies with the M07cell line, we have shown that exposure of cells to a n optimum concentration of recombinant human EGF in the absence of serum did not lead to any detectable changes in the tyrosine phosphorylation of cellular proteins. Thus, it seems likely that the 140-kDa protein, which associates with the Shc proteins in hematopoietic cells after cytokine stimulation andintroduction of p210BCwML,is distinct from the EGF receptor. Another potential candidate is human Sos, a 165-kDa protein shown to associate with Grb2/Shc in fibroblasts transformed by v-src (39). We detected coimmunoprecipitation of hSos with S ~ C However, the coimmunoprecipitating hSos proteindidnot comigrate with the tyrosine phosphorylated 140-kDa protein. The antibody used in these studies,as in previous studies with Shc (291, was raised against an SH2 domain and cross-reaction of this antibody with another protein containing a n SH2 domain, which happens tobe tyrosine phosphorylatedin response to the same cytokines and oncogenes, should be considered. However, the antibody didnot react with any 140-kDa proteins by direct immunoblotting of cell lysates (Fig. 1). It isof interest that anti-Shc immunoprecipitates were found to contain a kinase capable of phosphorylating Shc and the 140-kDa protein in cells expressing p21OBCWMLbut not in cells lacking p21OBCWABL, even if treated withGM-CSF or IL-3. "he identity of thekinasepresentinanti-Shc precipitates in (+) cells is unknown. The most likely candidate is p21OBCWML p210BCwABL itself, because a minor fraction was found to coimmunoprecipitatewith Shc (Fig. 51, and Shc is phosphorylated mainly on tyrosine (Fig. 30). However, whether or not Shc is directly phosphorylated by p210bcr'ab' has not yet been examined. The mitogenic signal generated by p210BCwABL in IL-3 dependent cell lines is not well understood. Mutations, which inactivate theab1 tyrosine kinase, block transforming activity, but critical substrates for the kinase have notbeen identified. It has been suggested that p210BCwMLcould activate some pathways of signal transduction normally activated by IL-3 or other growth factors by phosphorylating proteins involved in signal transduction (34,45), but there previously has been little data for this notion. We demonstrated recently that rasGAP and two associated proteins, p190 and p62 were heavily phosphorylated on tyrosine in a series of independently derived subclones of 32D cells expressing p210BCwMLand in several

* B. Druker, K. Okuda, M. Hallek, and J. Griffin, unpublished observations. T. Matsuguchi, R. Salgia, M. Hallek, M. Eder, B. Druker, T. Emst, and J. D. Griffin, unpublished data.

human Phl(+) cell lines (34). Further, p21OBCWABL, rasGAP, p62, and p190 were shown to co-immunoprecipitate from several of these lines, indicating that they likely existed as a complex in vivo (34). Because rasGAP is both a regulator of p2lrU8 function and also a potential downstreameffector molecule for ~21"" (46), the aberrant tyrosine phosphorylation of rasGAP could augment p21rQ"-mediatedsignal transduction events,although this has notbeen directly tested. BecauseShc proteinsare tyrosinephosphorylated inresponse to both growth factors and p210BCwABL, the results presented here suggest that Shc proteins could represent a site of overlap between the signal transduction pathways activated by hematopoietic growth factors and by p210BCwML.It is of interest that Shc, like pl2OrasGAP, may function to regulate p21ra8 function, and additional studies to better define the role of p2lnsinthetransformation of hematopoieticcells by p210BCwABL are warranted. REFERENCES Cannistra, S . A,, and Griffin, J. D. (1988) Semin. Hematol. 25, 173-188 Clark, S . C., and Kamen, R. (1987)Science 236,1229-1237 Metcalf, D., and Nicola, N. A. (1992) Blood 79,2861-2866 Metcalf. D. (1986)Blood 67.257-267 Bernstein, A., Forrester, L.,'Reith, A. D., Dubreuil, P., and Rottapel, R. (1991) Semin. Hematol. 28, 138-142 6 Broxmeyer, H. E.,and Williams, D. E. (1988) Crit. Rev. Oncol. Hematol. 8, 173-226 7 Gasson, J. C., Baldwin, G. C., Sakamoto, K. M., and DiPersio, J. F. (1990)Prog. Clin. Biol. Res. 352, 375384 8. Cannistra, S . A., Groshek, P., Garlick, R., Miller, J., and Griffin, J. D. (1990) Proc. Natl. Acad. Sei. U.S. A. 87, 93-97 9. Gearing, D. P., King, J. A., Gough, N. M., and Nicola, N. A. (1989) EMBO J. 8, 36673676 .10.~Gorman, D. M., Itoh, N., Kitamura, T.,Schreurs, J., Yonehara, S., Yahara, I., Arai, K., and Miyajima, A. (1990) Proc. Natl. Acad. Sci. U.S. A. 87,54595463 11. Hayashida, K., Kitamura, T., Gorman, D. M., Arai, K , Yokota, T., and Miyajima, A. (1990)Proc. Natl. Acad. Sci. U.S. A. 87,9655-9659 12. Itoh, N., Yonehara, S . , Schreurs, J., Gorman, D. M., Maruyama, K., Ishii, A,, Yahara, I., Arai, K , and Miyajima, A. (1990) Science 247,324-327 13. Kitamura, T.,Sato, N., Arai, K., and Miyajima, A. (1991) Cell 66, 1165-1174 14. Kuwaki, T., Kitamura, T., Tojo, A,, Matsuki, S., Tamai, Y., Miyazono, K., and Takaku, F. (1989)Biochem. Biophys. Res. Commun. 161, 16-22 15. Park, L.S., and Gillis, S . (1990) Prog. Clin. Biol. Res. 362, 189-196 16. Zsebo, K. M., Wypych, J., McNiece, I. K, Lu, H. S., Smith, K. A., Karkare, S . B., Sachdev, R. K., Yuschenkoff, V. N., Birkett,N.C.,Williams, L. R., Satyagal, V. N., Tung, W., Bosselman, R. A,, Mendiaz, E.A,, and Langley, K. E. (1990) Cell 69,19&201 17. Isfort, R. J., Stevens, D., May, W. S . , and Ihle,J. N. (1988) Proc. Natl. Acad. Sci. U.S. A. 85,7982-7986 18. Isfort, R. J.. and Ihle. J. N. (1990) Growth Factors 2, 213-220 19. Kanakura, Y., Druker, B., Cannistra, S . A,, Furukawa, Y., Torimoto, Y., and Griffin, J. D. (1990)Blood 76, 706-715 20. Morla, A. O., Schreurs, J., Miyajima, A., and Wang, J.Y. (1988)Mol. Cell. Biol. 8,2214-2218 21. Sorensen, P., Mui, A. L., and Krystal, G . (1989) J. Biol. Chem. 264, 1925319258 22. Pierce, J. H., Ruggiero, M., Fleming, T. P., Di, F. P.P., Greenberger, J. S., Varticovski, L., Schlessinger, J., Rovera, G., and Aaronson, S . A. (1988) Science 239,628-631 23. Collins, M. K., Downward, J., Miyajima, A,, Maruyama, K, Arai, K., and Mulligan, R. C. (1988) J. Cell. Physiol. 137, 293-298 24. Druker, B., Okuda, K , Matulonis, U., Salgia, R.,Roberts,T., and Griffin, J. D. (1992)Blood 79,2215-2220 25. McLaughlin, J., Chianese, E., and Witte, 0.N. (1987) Proc. Natl. Acad. Sci. U.S. A. 84,6558-6562 26. McColl, S . R., DiPersio, J. F., Caon, A. C., Ho, P., and Naccache, P. H. (1991) Blood 78, 1842-1852 27. Hallek, M., Druker, B., LePisto, E. M., Wood, K. W., Emst, T. J., and Griffin, J. D.(1992) J. Cell. Physiol. 153, 176-186 28. Kanakura, Y., Druker, B., Wood, K. W., Mamon, H. J., Okuda, K , Roberts, T. M., and Griffin, J. D. (1991)Blood 77,243-248 29. Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Grignani, F., Pawson, T.,and Pelicci, P. G . (1992) Cell 70, 93-104 30. Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R.,Li, W., Batzer, A., Thomas, S., Brugge, J., Pelicci, P. G., Schlessinger, J., and Pawson, T.(1992) Nature 360,689-692 31. Avanzi, G . C.,Lista, P., Giovinazzo, B., Miniero, R., Saglio, G., Benetton, G., Coda, R., Catorretti, G., and Pegoram, L. (1987)Br: J. Haematol. 69,359366 32. Greenberger, J. S., Sakakeeny, M. A,, Humphries, R. K., Eaves, C. J., and Eckner, R.J. (1983)Proc. Natl. Acad. Sci. U.S. A. 80,2931-2935 33. Daley, G . Q., Van, E. R. A., and Baltimore, D. (1990) Science 247,824-830 34. Druker, B., Okuda, K., Matulonis, U., Salgia, R.,Roberts,T., and Griffin, J. D. (1992) Blood 79,2215-2220 1. 2. 3. 4. 5

Shc Phosphorylation in Myeloid Cells 35. qichenbaum, F., Ando, K., DeCaprio, J. A,, and Griffin, J. D. (1993)J. Biol. Chem. 268,41134119 36. Kaelin, W. G . , Pallas, D. C., DeCaprio, J. A,, Kaye, F. J., and Livingston, D. M. (1991)Cell 64,521-532 37. Schiff, M. L., Burns, M.C., Kanopka, J. B., Clark, S., Witte, 0.N.,and Rosenberg, N.(1986)J. virol. 87, 1182-1190 38. McGlade, J., Cheng, A,, Pelicci, G., Pelicci, P. G . , and Pawson, T.(1992)P r o c . Natl. had.Sci. U.S. A. 89, 8869-8873 39. Egan, S. E., Giddings, B.W., Brooks, M.W., Buday, L., Sizeland, A. M., and Weinberg, R.A. (1993)Nature 363,4561 40. Gale, N. W., Kaplan, S., Lowenstein, E. J., Schlessinger, J., and Bar-Sagi, D. (1993)Nature 363,88-92

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41. Li, N., Baker, A., Daly, R., Yajnik, V., Skolnick, E., Chardin, P., Bar-Sagi, D., Margolis, B., and Schlessinger, J. (1993)Nature 363,8587 42. Rozakis-Admk, M., Fernlwy, R., Wade, J., Pawson, T.,and Bowtell. D. (1993) Nature 363,8345 43. Duronio, V.,Welham, M., Abraham, S., Dryden, P., and Schrader, J. W. (1992) Proc. Natl. Acad. Sei. U.S. A. 89, 1587-1591 44. Satoh, T.,Nakafuku, M., Miyajima, A,, and Kaziro, Y. (1991)Proc. Nut[. Acad. Sci. 88,3314-3318 45. Laneuville, P., Heisterkamp, N., and Groffen, J. (1991) Oncogene 6, 275-282 46. Martin, G . A,, Yatani, A., Clark, R.,Conroy, L., Polakis, P., Brown, A. M., and McCormick, F. (1992)Science 255,192-194