763) was found to be necessary for activation of Shc, p2lras, Raf-1, and mitogen-activated protein kinase as well as induction

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1 Proc. Natl. Acad. Sci. USA Vol. 92, pp , September 1995 Medical Sciences Identification of a viability domain in the granulocyte/ macrophage colony-stimulating factor receptor (8-chain involving tyrosine-750 ROGER C. INHORN*, NADIA CARLESSO*, MELISSA DURSTIN*, DAVID A. FRANKtt, AND JAMES D. GRIFFIN* Divisions of *Hematologic Malignancies and tmedical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; idepartment of Neurology, Division of Neuroscience, Children's Hospital, Boston, MA 02115; and Department of Neurobiology, Harvard Medical School, Boston, MA Communicated by Helen M. Ranney, Alliance Pharmaceutical, San Diego, CA, May 30, 1995 (received for review January 17, 1995) ABSTRACT The granulocyte/macrophage colonystimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. In this study we have investigated domains of the GMR 13-chain (GMR18) involved in maintaining cellular viability. Using a series of nested GMRI8 deletion mutants, we demonstrate that there are at least two domains of GMRj8 that contribute to viability signals. Deletion of amino acid residues causes a viability defect that can be rescued with fetal calf serum (FCS). Deletion of residues , in contrast, causes a further decrement in viability that can be only partially compensated by the addition of FCS. GMR,B truncated proximal to amino acid 517 will not support long-term growth under any conditions. Site-directed mutagenesis of tyrosine-750 (Y750), which is contained within the distal viability domain, to phenylalanine eliminates all demonstrable tyrosine phosphorylation of GMR.8. Cell lines transfected with mutant GMRI3 (Y750 -* F) have a viability disadvantage when compared to cell lines containing wild-type GMR that is partially rescued by the addition of FCS. We studied signal transduction in mutant cell lines in an effort to identify pathways that might participate in the viability signal. Although tyrosine phosphorylation of JAK2, SHPTP2, and Vav is intact in Y750 -> F mutant cell lines, Shc tyrosine phosphorylation is reduced. This suggests a potential role for Y750 and potentially Shc in a GM-CSF-induced signaling pathway that helps maintain cellular viability. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact The granulocyte/macrophage colony-stimulating factor (GM- CSF) receptor (GMR) is a heterodimeric receptor consisting of a unique a-chain (GMRa) and a common 3-chain (GMRf) shared by interleukin 3 (IL-3) and IL-5 receptors (1). GMRa alone binds GM-CSF with low affinity and cannot transmit mitogenic or viability signals. GMRJ3 together with GMRa reconstitutes high-affinity binding of GM-CSF as well as normal signal transduction upon binding of the ligand (2, 3). Signal transduction pathways activated by the GMR are not well understood. Although neither chain of the GMR possesses tyrosine kinase activity, there is abundant evidence that tyrosine phosphorylation plays an important role in signal transduction initiated by this cytokine (reviewed in ref. 1). Binding of GM-CSF to its receptor activates JAK2, a member of the Janus tyrosine kinase family (4). JAK2 thus initiates signaling pathways involving tyrosine phosphorylation via direct interaction with the GMR. Specific domains within GMRa and GMR3 may contain the information necessary to initiate distinct events, including proliferation, viability, and differentiation. We have recently demonstrated that a chimeric receptor containing only the extracellular and transmembrane sequences of GMRa and the cytoplasmic domain of GMR,B binds GM-CSF with low affinity but induces proliferation and maintains viability of Ba/F3 cells, suggesting that the cytoplasmic domain of GMRI3 contains sequences that are sufficient for these functions (5). Previous studies of GMRI3 deletion mutants have demonstrated that there are at least two distinct functional domains within the cytoplasmic portion of GMRI3 (4, 6, 7). A membrane proximal domain (amino acids ) was shown to be essential for proliferation, activation of JAK2 and pim-1, and induction of c-myc. A second domain (amino acids ) was found to be necessary for activation of Shc, p2lras, Raf-1, and mitogen-activated protein kinase as well as induction of c-fos and c-jun. Truncation of GMR,3 at amino acid 626 did not affect the ability of the receptor to support short-term proliferation in fetal calf serum (FCS)-containing medium. Other studies, however, have indicated that activation of p2lras and Raf-1, in particular, is linked to proliferation or other biological functions in many cell types, including myeloid cells (8, 9), which raises the possibility that FCS might rescue subtle defects of GMRf3 truncated at amino acid 626. In this study we used nested deletion mutants of GMRf3 to define two distinct domains that participate in maintaining cellular viability. MATERIALS AND METHODS Cells and Cell Culture. Ba/F3, an IL-3-dependent murine pro-b-cell line, was transfected with a plasmid containing human GMRa (6) and a hygromycin-resistance selection marker (Ba/F3-GMRa; generous gift of Atsushi Miyajima, DNAX). Ba/F3-GMRa cells were cultured in RPMI 1640 medium, 10% FCS, 15% medium conditioned by the WEHI-3B cell line as a source of murine IL-3, and 0.5 mg of hygromycin B per ml (Boehringer Mannheim). Ba/F3-GMRa cells were then transfected by electroporation using a Bio-Rad Gene Pulser with nested GMRf3 deletion mutants subcloned into pme18sneo under control of the SRa promoter as described (6). G418-resistant clones were selected and analyzed for expression of GMR,B using a monoclonal antibody generated against the extracellular domain of GMR13. Two or more independent isolates of each deletion mutant were characterized. For viability assays, washed cells were resuspended in a 1:1 mixture of RPMI-1640 medium and AIM-V (GIBCO) with or without GM-CSF (Genetics Institute) and FCS. Viability was determined by trypan blue exclusion. To study signaling events, cells were stimulated with 10 ng of GM-CSF per ml for 10 min as indicated. Abbreviations: GM-CSF, granulocyte/macrophage colony-stimulating factor; GMR, GM-CSF receptor; GMRa, GM-CSF receptor a-chain; GMR,3, GM-CSF receptor,-chain; FCS, fetal calf serum; WT, wild-type; IL, interleukin. ITo whom reprint requests should be addressed at: Division of Hematologic Malignancies, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA

2 8666 Medical Sciences: Inhorn et al. A ae J.s Hours C Wild Type A763 A626 A544 A517 A45 -FCS D Wild Type A763 _ A 626 A 544 A 517 A 455 jfcs Relative Viability (%) FIG. 1. Viability of Ba/F3 cell lines transfected with GMRa and nested GMR,B deletion mutants. (A and B) Cell lines were grown in medium containing 1 ng of GM-CSF per ml in either the absence (A) or the presence (B) of 5% FCS. Data presented here are from one experiment representative of three separate determinations. o, GMR,B wild-type (WT); A, GMRf3 A763; *, GMR, A626; m, GMR,3 A544; A, GMRJ3 A517; 0, GMRj3 A455. (C and D) Viability at 72 hr was determined for each cell line and then normalized to viability of the Ba/F3-GMRaI3wT cell line. Data represent the mean ± SE from three separate experiments. Antibodies. Anti-JAK2 antibody was from Upstate Biotechnology (Lake Placid, NY), anti-shc antibody was from Transduction Laboratories (Lexington, KY), and anti-shptp2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Vav monoclonal antibodies were generated as described (10). Phosphotyrosine was detected with monoclonal antibody 4G10 (11). Polyclonal anti-gmri3 antibody was raised against the extracellular domain of GMRI expressed as a glutathione S-transferase (GST) fusion protein and was used for immunoprecipitations (3). Monoclonal anti-gmri3 antibody was a generous gift from Angel Lopez (Adelaide, Australia) and was used for immunoting. Immunoprecipitation and immunoting were performed as described (12). Site-Directed Mutagenesis. The Xho I-Xba I fragment of KH97 was subcloned into psk (Promega). The codon encoding tyrosine-750 (TAT) was mutated to phenylalanine (TlT) using synthetic oligonucleotides spanning the codon as primers for the PCR (13); then an Eae I-BstEII fragment flanking this codon was recloned into psk-gmr,3. The identity of the mutation as well as the fidelity of the PCR were confirmed by dideoxynucleotide sequencing. The Xho I-Xba I fragment of the mutated psk-gmri3 was subcloned into pme18sneo, which was used for transfections into Ba/F3-GMRa as described above. RESULTS Nested Deletion Mutants of GMRp Identify Multiple Viability Domains. Previous studies measured the proliferation of Ba/F3 cells transfected with GMRa and nested deletion mutants of GMRf3 (6). Cells grown in FCS were able to proliferate in response to GM-CSF with GMR,B truncations as proximal as amino acid 517. Since factors in FCS can activate p2lras and other signaling pathways, we reconstructed several of these constructs in Ba/F3 cells and studied their growth in the presence and absence of FCS. Ba/F3 cells expressing GMRa were transfected with either full-length GMRB (Ba/ F3-GMRaI3wT) or GMRJ3 truncated at amino acid 455 (Ba/ F3-GMRac,q455), 517 (Ba/F3-GMRac3A5l7), 544 (Ba/F3- GMRaI3A544), 626 (Ba/F3-GMRaI3A626), or 763 (Ba/F3- GMRaI,3763). Cell lines expressed equivalent amounts of GMRf3 as assessed by immunoting (data not shown). Although Ba/F3-GMRa3,3763 and Ba/F3-GMRal3wT cell lines remain >90% viable during 72 hr of culture in serum-free medium containing 1 ng of GM-CSF per ml, Ba/F3- GMRa3g,626 was only 50% viable at 72 hr, and cell lines containing more proximal GMR,B truncations died (Fig. LA). These data suggest that a viability domain is located between amino acids 627 and 763. In the presence of FCS, Ba/F3- GMRa,3A626 maintains nearly 100% viability with 1 ng of GM-CSF per ml, Ba/F3-GMRa3A455 does not maintain long- Proc. Natl. Acad. Sci. USA 92 (1995) term viability, and Ba/F3-GMRaI3A5l7 and Ba/F3- GMRaf3As544 display intermediate phenotypes (Fig. 1B). Compared to Ba/F3-GMRaI3wT cells, Ba/F3-GMRaI3A626 cells have impaired viability that is rescued by the addition of FCS (Fig. 1 C and D). Ba/F3-GMRca3A5l7 and Ba/F3- GMRa3A544 display a further decrement in viability that is not compensated by FCS, and Ba/F3-GMRaI3A455 is not viable, even in the presence of FCS and GM-CSF concentrations as high as 100 ng/ml. Thus, these GMRI3 deletion mutants identify up to three domains potentially involved in viability signaling: a region between amino acids 627 and 763 that can be complemented by FCS, a domain between amino acids 517 and 626 that cannot be rescued by FCS, and the proximal region between amino acids 455 and 516 that contains the box 1/box 2 homology and JAK2 binding domains. Mutation of Y750 Reduces Tyrosine Phosphorylation of GMRi3. In an effort to identify signaling pathways associated with viability in myeloid cells, we considered the possibility that tyrosine phosphorylation of GMR,3 was important in initiating a viability signal. Previous work demonstrated that deletion of amino acids , which comprises the distal viability domain identified in our studies, greatly reduced GM-CSF-induced tyrosine phosphorylation of GMR,B (6). This domain contains two tyrosine residues, Y695 and Y750. Y750, unlike Y695, is flanked by acidic residues (FEGYVEL) and thus represents the most likely tyrosine phosphorylation site (14). We therefore mutated this residue to phenylalanine and transfected mutant and WT GMR,B cdna into Ba/F3 cell lines stably transfected with human GMRa. Five WT and four mutant (Ba/F3-GMRa43F750) polyclonal cell lines were established. Data presented here compare individual clones that were representative of the other isolates. The Y750 mutation markedly reduced the level of GMRf3 tyrosine phosphorylation relative to WT GMRf3 (Fig. 2A). The A P GMRI3IP WT GM-CSF rt GMRf Ptyr - A d* ] IgH GMR,B UmiME B GM-CSF I.0 U FIG. 2. Tyrosine phosphorylation of GMRj3 in Ba/F3-GMRaPwT and Ba/F3-GMRac3F7so cell lines. (A) Lysates were immunoprecipitated with rabbit polyclonal anti-gmr,3 antiserum. Western s were performed with 4G10 anti-phosphotyrosine (upper) or monoclonal anti-gmri3 (lower) antiserum. (B) Lysates were subjected to SDS/PAGE, transferred to nitrocellulose, and then ted with 4G10 anti-phosphotyrosine antiserum.

3 A k i F750 *- Wr [GM] = ngimi -FCS Aj Medical Sciences: Inhorn et al B 100 lol Hours C 109 0' 7i0 X 6(a 0-4 8( ' 0 - Wi Proc. Natl. Acad. Sci. USA 92 (1995) D S C- F750 [GMJ= 0.1 ng/ml 10 &-* WT +FCS Viability of Ba/F3-GMRaP3wT and Ba/F3-GMRa13PF75o cell lines. (A and B) Cell lines were grown in 1 ng of GM-CSF per ml in either FIG. 3. the absence (A) or the presence (B) of 5% FCS. Data points represent the mean ± SE of triplicate determinations. (C and D) Cell lines were grown in 0.1 ng of GM-CSF per ml with (C) or without (D) 5% FCS. reduced GMR,3 tyrosine phosphorylation does not, however, result from a general decrease in tyrosine phosphorylation (Fig. 2B). The decreased tyrosine phosphorylation seen in the range of 130 kda in the mutant cell line likely results, at least in part, from the decreased phosphorylation of GMRf3. Mutant Cell Lines Have a Viability Defect in the Absence of Serum. Viability of the mutant cell lines was assessed to determine whether loss of GMRf3 tyrosine phosphorylation was associated with a physiological phenotype. There is a reproducible decrease in viability of Ba/F3-GMRai3F750 grown in 1 ng of GM-CSF per ml in the absence of serum when compared to Ba/F3-GMRaI3wT (Fig. 3 A and B). Normal viability is restored in the presence of FCS. Culture of Ba! F3-GMRa3F75o cells in 0.1 ng of GM-CSF per ml accentuates the viability defect, and viability is only partially restored by the addition of FCS (Fig. 3 C and D). Activation of JAK2 Is Intact in Mutant Cell Lines. Signaling pathways known to be activated by GM-CSF were studied to determine if a specific biochemical abnormality could be associated with the Y750 mutant receptor. The JAK2 tyrosine kinase is activated by GM-CSF in cells containing WT GMR (4), and JAK2 can directly phosphorylate GMRf3 in insect cells coinfected with baculoviruses expressing these proteins (R.C.I. and J.D.G., unpublished data). JAK2 is tyrosine phosphorylated in response to GM-CSF to a similar degree in WT and mutant cell lines (Fig. 4). Tyrosine Phosphorylation of Vav and SHPTP2 Is Intact in Mutant Cell Lines. Three SH2-containing signaling proteins known to be activated by GM-CSF-namely, Vav, SHPTP2, and Shc-were next studied. Vav is a hematopoietically restricted protein that contains one SH2 domain and two SH3 domains (15). Vav is tyrosine phosphorylated in response to GM-CSF, possibly by JAK2 (10). Fig. 5A shows a similar degree of tyrosine phosphorylation in WT and mutant cell lines after GM-CSF stimulation. JAK2 IP m- '1 GM-CSF Ptyr - JAK2 o m a - JAK2 F750 [GL= 1 ngfml *- Wr FCS Tyrosine phosphorylation of JAK2 in Ba/F3-GMRaP3wT FIG. 4. and Ba/F3-GMRa3F75so cell lines. (A) Lysates were immunoprecipitated with anti-jak2 antiserum. Western s were performed with 4G10 anti-phosphotyrosine (upper) or anti-jak2 (lower) antiserum. 0- ei 0 F750 M1= 0.1 nghni SHPTP2 (also known as Syp, PTP1D, or PTP2C) is a protein containing two SH2 domains as well as an intrinsic tyrosine phosphatase domain (16, 17). SHIPTP2 functions as an adaptor protein in platelet-derived growth factor receptor signaling, linking the receptor to the Grb2/Sos pathway (18, 19). In this system, SHPTP2 binds to the receptor via its SH2 domain, and Grb2 binds to SHPTP2 after it is inducibly tyrosine phosphorylated. Fig. SB demonstrates that GM-CSF stimulates tyrosine phosphorylation of the 75-kDa SHPTP2 in WT and mutant cell lines. Coprecipitation of prominently phosphorylated 95-kDa and 130-kDa proteins was also observed in both cell lines. Shc Phosphorylation Is Reduced in Mutant Cell Lines. Shc is an SH2-containing molecule that serves as a linker protein between several receptors and the Grb2/Sos pathway (20-23). We have previously reported that Shc is tyrosine phosphorylated after stimulation with GM-CSF, IL-3, or Steel factor (12). GM-CSF also induces tyrosine phosphorylation of Shc in Ba/F3 cells expressing WT GMR, but the degree of phosphorylation is markedly reduced in Ba/F3-GMRa3F750 cells (Fig. 6A). Also, the 140-kDa tyrosine-phosphorylated protein that is known to coprecipitate with Shc (12) is detected in anti-shc immunoprecipitates from Ba/F3-GMRaP3wT, but not Ba/F3-GMRaIiF75o, cells. In Ba/F3-GMRa cell lines transfected with nested GMRf3 deletion mutants, Shc tyrosine phosphorylation is completely absent in Ba/F3-GMRa3A544 cell lines (Fig. 6B) as well as in cell lines containing more proximal GMR,B deletions (data not shown). Shc tyrosine phosphorylation is present in Ba/F3- GMRaf3A626 cell lines, although it is reduced compared to WT cells (Fig. 6B). In multiple experiments using several independently derived cell lines, the amount of Shc tyrosine phos- A VavlP GM-CSF Ptyr - * - Vav 49 -.g g Vav _s-a B SHPTP21P GM-CSF Ptyr - SHPTP i 4gFF _ -SHPTP2 l:igh FIG. 5. Tyrosine phosphorylation of Vav and SHPTP2 in Ba/F3- GMRa(3wT and Ba/F3-GMRa3F75so cell lines. (A) Lysates were immunoprecipitated with anti-vav antiserum. Western s were performed with 4G10 anti-phosphotyrosine (upper) or anti-vav (lower) antiserum. (B) Lysates were immunoprecipitated with anti- SHPTP2 antiserum. Western s were performed with 4G10 antiphosphotyrosine (upper) or anti-shptp2 (lower) antiserum.

4 8668 Medical Sciences: Inhorn et al. A ShcIP B A544 ShcIP GMCF-+ A626 GM-CSF - + h p Ptyr - Ptyr - p52 Shctp52Shc Shc Shc 3333 FIG. 6. Tyrosine phosphorylation of Shc in Ba/F3-GMRa,3wT and GMRB mutant cell lines. (A) Coinparison of GMR,3 Ba/F3- GMRa4wT and Ba/F3-GMRafPF75o cell lines. Lysates were immunoprecipitated with anti-shc antiserum. Western s were performed with 4G10 antiphosphotyrosine (upper) or anti-shc (lower) antiserum. Migration of the coprecipitating tyrosine-phosphorylated 140-kDa protein is also indicated. (B) Comparison of Ba/F3-GMRa3,544 and Ba/F3-GMRa83A626 cell lines. Shc immunoprecipitations and Western s were performed as described in A. phorylation observed in Ba/F3-GMRaI3A626 cell lines is intermediate between that seen in Ba/F3-GMRaPF375o and Ba/F3- GMRa3WT cell lines. DISCUSSION GM-CSF has a number of important biological effects on myeloid lineage cells, including induction of proliferation of immature cells and activation of mature neutrophils. We present data that support the emerging view that multiple signaling pathways are initiated by information contained within specific domains of GMRI3. Previous work (6) demonstrated that deletions of GMRP as proximal as amino acid 517 were able to support survival and proliferation when transfected cells were grown with GM-CSF and FCS. Deletion of amino acids , however, made cells incompetent to proliferate. This domain has subsequently been shown to be necessary to activate JAK2 (4), which is therefore likely to initiate a signaling pathway necessary for proliferation. In the present work we have reconstructed the nested GMR,B deletion mutants in Ba/F3 cells along with full-length GMRa and studied cellular viability in the presence and absence of serum. Although Ba/F3-GMRaI3wT and Ba/F3- GMRaf3A763 cell lines grow well when cultured with 1 ng of GM-CSF per ml in the absence of serum, Ba/F3-GMRafA&626 cell lines are only 50% viable after 72 hr in culture, suggesting there is a viability domain between amino acids 627 and 763 that can be complemented by unknown components of serum (Fig. 1B). The existence of a distal viability domain is supported by recent work that shows that Ba/F3-GMRaA3544 cell lines died by apoptosis in the absence of serum despite a transient mitogenic response to GM-CSF (24). Interestingly, expression of an activated Ras protein in Ba/F3-GMRac5s44 cell lines complemented the viability defect. Since Ras activation maps to amino acids (7), the Ras pathway may account for part or all of the viability signal. Comparison of Ba/F3-GMRaI3A&l7 and Ba/F3-GMRaf3A544 to Ba/F3- GMRaI3A626 suggests that an additional contribution to viability maps between amino acids 518 and 626 (Fig. 1B). Using site-directed mutagenesis, we showed that mutation of Y750 to phenylalanine markedly reduces tyrosine phosphorylation of GMR3 (Fig. 24). This result suggests either that Y750 is, in fact, the major site of GMRf3 tyrosine phosphorylation or that Y750 is required for phosphorylation of other tyrosine residues in GMR/3, possibly by activating a tyrosine kinase that subsequently phosphorylates additional sites on GMRI3. Proc. Natl. Acad. Sci. USA 92 (1995) Ba/F3-GMRaf3F75o cell lines have impaired viability relative to cells transfected with WT GMR (Fig. 3). The survival curves generated at 1 ng of GM-CSF per ml (Fig. 3 A and B) are similar to those seen with Ba/F3-GMRa13A626 cell lines (Fig. 1 A and B), which suggests that Y750 may initiate the major viability signal originating within the domain. Interestingly, the viability defect is more pronounced at lower concentrations of GM-CSF (compare Fig. 3A with C), and the survival disadvantage is only partially compensated by serum. Ba/F3-GMRa3F750 cell lines demonstrate markedly reduced tyrosine phosphorylation of Shc in response to GM-CSF (Fig. 64), while tyrosine phosphorylation of JAK2, Vav, and SHPTP2 is intact (Figs. 4 and 5). Although Shc may in fact bind to phosphorylated Y750 via its SH2 domain, we have not been able to demonstrate direct interaction of Shc with GMR,3, by either in vivo coprecipitation or in vitro precipitation with Shc SH2 GST fusion proteins (R.C.I. and J.D.G., unpublished results). In contrast, Lanfrancone et al. (25) have demonstrated coprecipitation of Shc and GMR,3, although far-western s with a Shc SH2 GST fusion protein did not recognize GMRf3. Shc may therefore interact with GMRf3 via an intermediate linker protein. Alternatively, Y750 may bind to and activate an SH2-containing tyrosine kinase required for Shc phosphorylation. Previous studies demonstrated that Shc tyrosine phosphorylation was absent in Ba/F3-GMRaI3A626 cell lines (6). In this study, however, we show that GM-CSF induces Shc phosphorylation in Ba/F3-GMRaI3A626 cell lines (Fig. 6B), although phosphorylation is reduced compared to Ba/F3-GMRaPwT cell lines. Ba/F3-GMRa3A544 cell lines do not support Shc phosphorylation, which suggests that the region between amino acids 544 and 626 as well as tyrosine-750 may contribute to Shc activation. Ba/F3-GMRa3F750 cell lines appear to be more defective than Ba/F3-GMRa38A626 cell lines, suggesting that the amino acid 626 deletion may remove domains that are inhibitory as well as stimulatory for phosphorylation of Shc. Further studies are warranted, but our studies establish Y750 as a key component in the pathways that regulate Shc. In conclusion, these results establish the presence of at least two viability domains within GMRj and demonstrate that the contribution of the distal domain to viability can be complemented by unknown components within serum. 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