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• W2053987316 abstract "Erythroid cells terminally differentiate in response to erythropoietin binding its cognate receptor. Previously we have shown that the tyrosine kinase Lyn associates with the erythropoietin receptor and is essential for hemoglobin synthesis in three erythroleukemic cell lines. To understand Lyn signaling events in erythroid cells, the yeast two-hybrid system was used to analyze interactions with other proteins. Here we show that the hemopoietic-specific protein HS1 interacted directly with the SH3 domain of Lyn, via its proline-rich region. A truncated HS1, bearing the Lyn-binding domain, was introduced into J2E erythroleukemic cells to determine the impact upon responsiveness to erythropoietin. Truncated HS1 had a striking effect on the phenotype of the J2E line—the cells were smaller, more basophilic than the parental proerythoblastoid cells and had fewer surface erythropoietin receptors. Moreover, basal and erythropoietin-induced proliferation and differentiation were markedly suppressed. The inability of cells containing the truncated HS1 to differentiate may be a consequence of markedly reduced levels of Lyn and GATA-1. In addition, erythropoietin stimulation of these cells resulted in rapid, endosome-mediated degradation of endogenous HS1. The truncated HS1 also suppressed the development of erythroid colonies from fetal liver cells. These data show that disrupting HS1 has profoundly influenced the ability of erythroid cells to terminally differentiate. Erythroid cells terminally differentiate in response to erythropoietin binding its cognate receptor. Previously we have shown that the tyrosine kinase Lyn associates with the erythropoietin receptor and is essential for hemoglobin synthesis in three erythroleukemic cell lines. To understand Lyn signaling events in erythroid cells, the yeast two-hybrid system was used to analyze interactions with other proteins. Here we show that the hemopoietic-specific protein HS1 interacted directly with the SH3 domain of Lyn, via its proline-rich region. A truncated HS1, bearing the Lyn-binding domain, was introduced into J2E erythroleukemic cells to determine the impact upon responsiveness to erythropoietin. Truncated HS1 had a striking effect on the phenotype of the J2E line—the cells were smaller, more basophilic than the parental proerythoblastoid cells and had fewer surface erythropoietin receptors. Moreover, basal and erythropoietin-induced proliferation and differentiation were markedly suppressed. The inability of cells containing the truncated HS1 to differentiate may be a consequence of markedly reduced levels of Lyn and GATA-1. In addition, erythropoietin stimulation of these cells resulted in rapid, endosome-mediated degradation of endogenous HS1. The truncated HS1 also suppressed the development of erythroid colonies from fetal liver cells. These data show that disrupting HS1 has profoundly influenced the ability of erythroid cells to terminally differentiate. erythropoietin epo-receptor hematopoietic lineage cell-specific protein truncated HS1 Src homology glutathioneS-transferase phosphate-buffered saline β-galactosidase fetal calf serum polymerase chain reaction fluorescein isothiocyanate murine erythroleukemia mitogen-activated protein kinase Erythropoiesis, the process of red blood cell development, is primarily controlled by erythropoietin (epo).1 Several model systems have been used to study erythropoiesis in vitro. Although primary erythroid cells provide the ideal cell type for analysis, heterogeneity in preparations and insufficient numbers can preclude biochemical analysis of epo signaling. A number of erythroid cell lines have been derived which provide useful models for analyzing epo-induced signaling cascades, including the SKT6 and J2E lines (1.Todokoro K. Kanazawa S. Amanuma H. Ikawa Y. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4126-4130Crossref PubMed Scopus (74) Google Scholar, 2.Klinken S.P. Nicola N.A. Johnson G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8506-8510Crossref PubMed Scopus (83) Google Scholar). The J2E cell line was used in this study because it proliferates, remains viable, produces hemoglobin and undergoes morphological maturation in response to epo (2.Klinken S.P. Nicola N.A. Johnson G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8506-8510Crossref PubMed Scopus (83) Google Scholar, 3.Busfield S.J. Klinken S.P. Blood. 1992; 80: 412-419Crossref PubMed Google Scholar). Following epo stimulation of J2E cells, phosphorylation changes to the epo receptor, janus kinase-2 (JAK2), signal transducer and activator of transcription-5 (STAT5),ras-GTPase activating protein, phosphatidylinositol 3-kinase, phospholipase Cγ, and MAP-kinase are identical to the kinetics reported in other cell systems (4.Tilbrook P.A. Bittorf T. Busfield S.J. Chappell D. Klinken S.P. J. Biol. Chem. 1996; 271: 3453-3459Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar).Previously, we reported that epo-initiated signaling was disrupted in a J2E subclone (J2E-NR), which remained viable in the presence of epo but did not differentiate or undergo enhanced proliferation following hormonal stimulation (4.Tilbrook P.A. Bittorf T. Busfield S.J. Chappell D. Klinken S.P. J. Biol. Chem. 1996; 271: 3453-3459Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The tyrosine kinase Lyn was shown to be severely reduced in the J2E-NR cells, and reintroduction of Lyn restored the ability of the cells to terminally differentiate (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar). Lyn pre-associated with the epo receptor in parental J2E cells, and inhibition of its activity suppressed differentiation (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar). Chinet al. (6.Chin H. Arai A. Wakao H. Kamiyama R. Miyasaka N. Miura O. Blood. 1998; 91: 3734-3745Crossref PubMed Google Scholar) confirmed the binding of Lyn to the epo receptor and demonstrated that it may play a role in regulating the JAK/STAT pathway. Lyn is a member of the Src family of membrane-associated tyrosine kinases, which is present mainly in lympho/hemopoietic cells and is involved in signal transduction from numerous receptors (7.Yamanashi Y. Fukushige S.-I. Semba K. Sukegawa J. Miyajima N. Matsubara K.-I. Yamamoto T. Toyoshima K. Mol. Cell. Biol. 1987; 7: 237-243Crossref PubMed Scopus (164) Google Scholar, 8.Yamanashi Y. Kinoshita Y. Ichimori Y. Yamamoto T. Toyoshima K. Science. 1991; 251: 192-194Crossref PubMed Scopus (335) Google Scholar, 9.Cichowski K. McCormick F. Brugger J.S. J. Biol. Chem. 1992; 267: 5025-5028Abstract Full Text PDF PubMed Google Scholar, 10.Torigoe T. Saragovi H.U. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2674-2678Crossref PubMed Scopus (86) Google Scholar, 11.Torigoe T. O'Conner R. Santoli D. Reed J.C. Blood. 1992; 80: 617-624Crossref PubMed Google Scholar, 12.Corey S. Eguinoa A. Puyana-Theall K. Bolen J.B. Cantley L. Mollinedo F. Jackson T.R. Hawkins P.T. Stephens L.R. EMBO J. 1993; 12: 2681-2690Crossref PubMed Scopus (170) Google Scholar, 13.Corey S.J. Burkhardt A.L. Bolen J.B. Geahlen R.L. Tkatch L.S. Tweardy D.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4683-4687Crossref PubMed Scopus (205) Google Scholar, 14.Yamanashi Y. Fukui Y. Wongsasant B. Kinoshita Y. Ichimori Y. Toyoshima K. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1118-1122Crossref PubMed Scopus (184) Google Scholar, 15.Pleiman C.M. Clark M.R. Gauen L.K.T. Wintz S. Coggeshall K.M. Johnson G.L. Shaw A.S. Cambier J.C. Mol. Cell. Biol. 1993; 13: 5877-5887Crossref PubMed Google Scholar).Lyn is most closely related to the tyrosine kinase Lck which plays an essential role in T cell activation and development (16.Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). The SH2 domain of Lck binds to tyrosine-phosphorylated CD45 and ZAP-70, whereas its SH3 domain associates with phosphatidylinositol 3-kinase, p120, and HS1 (17.Autero M. Saharinen J. Pessa-Morikawa T. Soula-Rothhut M. Oetken C. Gassmann M. Bergman M. Alitalo K. Burn P. Gahmberg C.G. Mol. Cell. Biol. 1994; 14: 1308-1321Crossref PubMed Scopus (117) Google Scholar, 18.Duplay P. Thome M. Herve F. Acuto O. J. Exp. Med. 1994; 179: 1163-1172Crossref PubMed Scopus (153) Google Scholar, 19.Prasad K.V. Kapeller R. Janssen O. Repke H. Duke-Cohan J.S. Cantley L.C. Rudd C.E. Mol. Cell. Biol. 1993; 13: 7708-7717Crossref PubMed Scopus (136) Google Scholar, 20.Reedquist K.A. Fukazawa T. Druker B. Panchamoorthy G. Shoelson S.E. Band H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4135-4139Crossref PubMed Scopus (74) Google Scholar, 21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar). HS1, or LckBP1, is a 75-kDa intracellular protein expressed mainly in hemopoietic and lymphoid cells (22.Kitamura D. Kaneko H. Miyagoe Y. Ariyasu T. Watanabe T. Nucleic Acids Res. 1989; 17: 9367-9379PubMed Google Scholar) and is a major substrate for several Src family kinases (21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar, 23.Yamanashi Y. Okada M. Semba T. Yamori T. Umemori H. Tsunasawa S. Toyoshima K. Kitamura D. Watanabe T. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3631-3635Crossref PubMed Scopus (142) Google Scholar, 34.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). It contains a proline-rich region, an SH3 domain, an acidic α-helix, and a basic segment resembling the DNA-binding motif of the helix-turn-helix family, suggesting it could play a role in both signal transduction and transcriptional regulation (21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar). HS1 is phosphorylated following activation of B cell and T cell receptors (21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar, 23.Yamanashi Y. Okada M. Semba T. Yamori T. Umemori H. Tsunasawa S. Toyoshima K. Kitamura D. Watanabe T. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3631-3635Crossref PubMed Scopus (142) Google Scholar, 24.Yamanashi Y. Fukuda T. Nishizumi H. Inazu T. Higashi K. Kitamura D. Ishida T. Yamamura H. Watanabe T. Yamamoto T. J. Exp. Med. 1997; 185: 1387-1392Crossref PubMed Scopus (90) Google Scholar, 25.Takemoto Y. Sato M. Furuta M. Hashimoto Y. Int. Immunol. 1996; 8: 1699-1705Crossref PubMed Scopus (34) Google Scholar) but not after stimulation of IL-3, GM-CSF, or SCF receptors (26.Fukamachi H. Yamada N. Miura T. Kato T. Ishikawa M. Gulbins E. Altman A. Kawakami Y. Kawakami T. J. Immunol. 1994; 152: 642-652PubMed Google Scholar). Significantly, Lyn has been shown to associate with HS1 in B and T cells (23.Yamanashi Y. Okada M. Semba T. Yamori T. Umemori H. Tsunasawa S. Toyoshima K. Kitamura D. Watanabe T. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3631-3635Crossref PubMed Scopus (142) Google Scholar, 24.Yamanashi Y. Fukuda T. Nishizumi H. Inazu T. Higashi K. Kitamura D. Ishida T. Yamamura H. Watanabe T. Yamamoto T. J. Exp. Med. 1997; 185: 1387-1392Crossref PubMed Scopus (90) Google Scholar). Like Lyn knockout mice, studies on HS1-deficient mice have revealed a central role for HS1 in B cell responsiveness (27.Hibbs M.L. Tarlinton D.M. Armes J. Grail D. Hodgson G. Maglitto R. Stacker S.A. Dunn A.R. Cell. 1995; 83: 301-311Abstract Full Text PDF PubMed Scopus (615) Google Scholar, 28.Taniuchi I. Kitamura D. Maekawa Y. Fukuda T. Kishi H. Watanabe T. EMBO J. 1995; 14: 3664-3678Crossref PubMed Scopus (108) Google Scholar). While this manuscript was in preparation, HS1 was shown to bind the novel hematopoietic progenitor kinase (HPK1) in erythroid cells (29.Nagata Y. Kiefer F. Watanabe T. Todokoro K. Blood. 1999; 93: 3347-3354Crossref PubMed Google Scholar).In this study we attempted to identify downstream effectors of Lyn in erythroid cells using a yeast two-hybrid screen of wild type and a kinase inactive mutant (Y397F) of Lyn. Of the seven Lyn-interacting proteins identified, we report here on the interaction between Lyn and HS1, and the crucial role of HS1 for epo-induced differentiation of erythroid cells.DISCUSSIONIn this manuscript we have shown that the known Src kinase substrate HS1 associates with Lyn in erythroid cells. We have also demonstrated that a truncated HS1 (tHS1) markedly interferes with the phenotype of erythroid cells and impairs their ability to proliferate and differentiate. By disrupting the Lyn/HS1 interaction, we have generated a cascade of events which had a profound effect on erythroid maturation. These data indicate that HS1 plays a pivotal role in regulating intracellular signaling within erythroid cells and support the recent prediction by Nagata et al. (29.Nagata Y. Kiefer F. Watanabe T. Todokoro K. Blood. 1999; 93: 3347-3354Crossref PubMed Google Scholar) that “ … HS1 is likely to be involved in erythroid proliferation and differentiation. . . . . ”Introduction of a truncated form of HS1 into erythroid cells produced significant morphological, biochemical, and functional perturbations. The truncated mutant spanned the carboxyl-terminal, Lyn-binding region of HS1 that is sequentially phosphorylated by kinases (49.Ruzzene M. Brunati A.M. Marin O. Donella-Deana A. Pinna L.A. Biochemistry. 1996; 35: 5327-5332Crossref PubMed Scopus (43) Google Scholar), but it did not include the amino-terminal Hax1-binding region (50.Suzuki Y. Demoliere C. Kitamura D. Takeshita H. Deuschle U. Watanabe T. J. Immunol. 1997; 158: 2736-2744PubMed Google Scholar). Strikingly, cells expressing tHS1 were smaller, more basophilic, and replicated much more slowly. In addition, epo-induced differentiation was almost totally blocked as the cells failed to mature morphologically and did not synthesize hemoglobin. These data demonstrate that tHS1 acted in a dominant negative fashion and emphasize the importance of a fully functional HS1 to erythroid maturation.The tHS1 mutant had several biochemical effects on the erythroid cells, which may account for its dominant negative activity. First, it greatly reduced the level of Lyn protein within the cells, and as a consequence a marked decrease in tyrosine phosphorylation of intracellular proteins was detected. This result is compatible with our previous observation that the J2E-NR subclone expressed low levels of Lyn, and tyrosine phosphorylation of proteins was substantially reduced (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar). As Lyn associates with the epo receptor (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar, 6.Chin H. Arai A. Wakao H. Kamiyama R. Miyasaka N. Miura O. Blood. 1998; 91: 3734-3745Crossref PubMed Google Scholar), transmission of signals from the receptor via this kinase would be significantly diminished in the transfected cells. Second, the GATA-1 content fell dramatically in these cells. GATA-1 is a key transcription factor involved with erythroid development (51.Tsai S. Martin D.I.K. Zon L.I. D'Andrea A.D. Wong G.G. Orkin S.H. Nature. 1989; 339: 446-451Crossref PubMed Scopus (663) Google Scholar, 52.Pevny L. Lin C.-S., V., D.A. Simon M.C. Orkin S.H. Constantini F. Development. 1995; 121: 163-172Crossref PubMed Google Scholar), and in its absence erythroid precursors arrest at the proerythroblast stage (53.Weiss M.J. Keller G. Orkin S.H. Genes Dev. 1994; 8: 1184-1197Crossref PubMed Scopus (480) Google Scholar). Thus, the loss of GATA-1 protein may play a significant role in the inability of cells bearing tHS1 to mature morphologically or to produce hemoglobin.In addition to reducing the levels of Lyn and GATA-1, tHS1 had a significant effect on endogenous HS1. While the epo receptor was phosphorylated normally in cells containing the mutant HS1 (Fig. 6), endogenous HS1 was degraded rapidly in endosomes/lysosomes after exposure to epo (Fig. 7), indicating that the truncated mutant promoted this proteolysis. It is likely that degradation of endogenous HS1 prevented transmission of signals by this molecule. Thus, tHS1 uncovered an unexpected mechanism for dominant negative action. This observation warrants further investigation with the recent association of signaling molecules with degradation viz. the SOCS family of negative regulators of cytokine action target Janus kinases for degradation by associating with elongins (54.Zhang J.G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausmann G. Kile B.J. Kent S.B. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2071-2076Crossref PubMed Scopus (517) Google Scholar, 55.Kamura T. Sato S. Haque D. Liu L. Kaelin Jr., W.G. Conaway R.C. Conaway J.W. Genes Dev. 1998; 12: 3872-3881Crossref PubMed Scopus (498) Google Scholar), and c-cbl regulates receptor ubiquitination and endocytosis (56.Lee P.S.W. Wang Y. Dominguez M.G. Yeung Y.-G. Murphy M.A. Bowtell D.D.L. Stanely E.R. EMBO J. 1999; 18: 3616-3628Crossref PubMed Scopus (251) Google Scholar, 57.Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar).In this study HS1 was identified as a Lyn-binding protein through a yeast two-hybrid screen, which was confirmed by direct associationin vitro, together with co-immunoprecipitation in vivo. In addition to intracellular co-localization within erythroid cells, the proline-rich region of HS1 was shown to bind the SH3 domain of Lyn, similar to the Lck/HS1 interaction reported previously (21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar). Lyn has been shown to associate with HS1 in B and T cells (45.Takemoto Y. Furuta M. Sato M. Findell P.R. Ramble W. Hashimoto Y. J. Immunol. 1998; 161: 625-630PubMed Google Scholar), but this is the first report that we are aware of where Lyn interacts with HS1 in erythroid cells. Although HS1 and Lyn were constitutively associated in erythroid cells, the interaction increased appreciably after epo activation (Fig. 2 C), which is consistent with previous observations in T cells where the SH3 domain of Lck, or Lyn, binds to HS1 in the absence of stimulation, then the SH2 domain of these kinases associates with HS1 upon receptor activation, increasing the interaction (25.Takemoto Y. Sato M. Furuta M. Hashimoto Y. Int. Immunol. 1996; 8: 1699-1705Crossref PubMed Scopus (34) Google Scholar).HS1 is linked with several kinases. Here we demonstrated HS1 co-immunoprecipitated with Lyn, and two other Src kinases (Lck and Fyn) in J2E cells (Fig. 2 B); tHS1 could potentially interfere with signaling from these molecules. HS1 also associates with the novel hemopoietic kinase HPK1 in the erythroid SKT6 cell line (29.Nagata Y. Kiefer F. Watanabe T. Todokoro K. Blood. 1999; 93: 3347-3354Crossref PubMed Google Scholar). In addition, Takemoto et al. (45.Takemoto Y. Furuta M. Sato M. Findell P.R. Ramble W. Hashimoto Y. J. Immunol. 1998; 161: 625-630PubMed Google Scholar) suggested that the HS1 association with the SH3 domain of Grb2 may regulate the Grb2 and Src signaling pathways. Together these results suggest that HS1 may mediate signals emanating from several kinases and play a crucial role in transmitting intracellular signals within erythroid cells. Erythropoiesis, the process of red blood cell development, is primarily controlled by erythropoietin (epo).1 Several model systems have been used to study erythropoiesis in vitro. Although primary erythroid cells provide the ideal cell type for analysis, heterogeneity in preparations and insufficient numbers can preclude biochemical analysis of epo signaling. A number of erythroid cell lines have been derived which provide useful models for analyzing epo-induced signaling cascades, including the SKT6 and J2E lines (1.Todokoro K. Kanazawa S. Amanuma H. Ikawa Y. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4126-4130Crossref PubMed Scopus (74) Google Scholar, 2.Klinken S.P. Nicola N.A. Johnson G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8506-8510Crossref PubMed Scopus (83) Google Scholar). The J2E cell line was used in this study because it proliferates, remains viable, produces hemoglobin and undergoes morphological maturation in response to epo (2.Klinken S.P. Nicola N.A. Johnson G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8506-8510Crossref PubMed Scopus (83) Google Scholar, 3.Busfield S.J. Klinken S.P. Blood. 1992; 80: 412-419Crossref PubMed Google Scholar). Following epo stimulation of J2E cells, phosphorylation changes to the epo receptor, janus kinase-2 (JAK2), signal transducer and activator of transcription-5 (STAT5),ras-GTPase activating protein, phosphatidylinositol 3-kinase, phospholipase Cγ, and MAP-kinase are identical to the kinetics reported in other cell systems (4.Tilbrook P.A. Bittorf T. Busfield S.J. Chappell D. Klinken S.P. J. Biol. Chem. 1996; 271: 3453-3459Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar). Previously, we reported that epo-initiated signaling was disrupted in a J2E subclone (J2E-NR), which remained viable in the presence of epo but did not differentiate or undergo enhanced proliferation following hormonal stimulation (4.Tilbrook P.A. Bittorf T. Busfield S.J. Chappell D. Klinken S.P. J. Biol. Chem. 1996; 271: 3453-3459Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The tyrosine kinase Lyn was shown to be severely reduced in the J2E-NR cells, and reintroduction of Lyn restored the ability of the cells to terminally differentiate (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar). Lyn pre-associated with the epo receptor in parental J2E cells, and inhibition of its activity suppressed differentiation (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar). Chinet al. (6.Chin H. Arai A. Wakao H. Kamiyama R. Miyasaka N. Miura O. Blood. 1998; 91: 3734-3745Crossref PubMed Google Scholar) confirmed the binding of Lyn to the epo receptor and demonstrated that it may play a role in regulating the JAK/STAT pathway. Lyn is a member of the Src family of membrane-associated tyrosine kinases, which is present mainly in lympho/hemopoietic cells and is involved in signal transduction from numerous receptors (7.Yamanashi Y. Fukushige S.-I. Semba K. Sukegawa J. Miyajima N. Matsubara K.-I. Yamamoto T. Toyoshima K. Mol. Cell. Biol. 1987; 7: 237-243Crossref PubMed Scopus (164) Google Scholar, 8.Yamanashi Y. Kinoshita Y. Ichimori Y. Yamamoto T. Toyoshima K. Science. 1991; 251: 192-194Crossref PubMed Scopus (335) Google Scholar, 9.Cichowski K. McCormick F. Brugger J.S. J. Biol. Chem. 1992; 267: 5025-5028Abstract Full Text PDF PubMed Google Scholar, 10.Torigoe T. Saragovi H.U. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2674-2678Crossref PubMed Scopus (86) Google Scholar, 11.Torigoe T. O'Conner R. Santoli D. Reed J.C. Blood. 1992; 80: 617-624Crossref PubMed Google Scholar, 12.Corey S. Eguinoa A. Puyana-Theall K. Bolen J.B. Cantley L. Mollinedo F. Jackson T.R. Hawkins P.T. Stephens L.R. EMBO J. 1993; 12: 2681-2690Crossref PubMed Scopus (170) Google Scholar, 13.Corey S.J. Burkhardt A.L. Bolen J.B. Geahlen R.L. Tkatch L.S. Tweardy D.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4683-4687Crossref PubMed Scopus (205) Google Scholar, 14.Yamanashi Y. Fukui Y. Wongsasant B. Kinoshita Y. Ichimori Y. Toyoshima K. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1118-1122Crossref PubMed Scopus (184) Google Scholar, 15.Pleiman C.M. Clark M.R. Gauen L.K.T. Wintz S. Coggeshall K.M. Johnson G.L. Shaw A.S. Cambier J.C. Mol. Cell. Biol. 1993; 13: 5877-5887Crossref PubMed Google Scholar). Lyn is most closely related to the tyrosine kinase Lck which plays an essential role in T cell activation and development (16.Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). The SH2 domain of Lck binds to tyrosine-phosphorylated CD45 and ZAP-70, whereas its SH3 domain associates with phosphatidylinositol 3-kinase, p120, and HS1 (17.Autero M. Saharinen J. Pessa-Morikawa T. Soula-Rothhut M. Oetken C. Gassmann M. Bergman M. Alitalo K. Burn P. Gahmberg C.G. Mol. Cell. Biol. 1994; 14: 1308-1321Crossref PubMed Scopus (117) Google Scholar, 18.Duplay P. Thome M. Herve F. Acuto O. J. Exp. Med. 1994; 179: 1163-1172Crossref PubMed Scopus (153) Google Scholar, 19.Prasad K.V. Kapeller R. Janssen O. Repke H. Duke-Cohan J.S. Cantley L.C. Rudd C.E. Mol. Cell. Biol. 1993; 13: 7708-7717Crossref PubMed Scopus (136) Google Scholar, 20.Reedquist K.A. Fukazawa T. Druker B. Panchamoorthy G. Shoelson S.E. Band H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4135-4139Crossref PubMed Scopus (74) Google Scholar, 21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar). HS1, or LckBP1, is a 75-kDa intracellular protein expressed mainly in hemopoietic and lymphoid cells (22.Kitamura D. Kaneko H. Miyagoe Y. Ariyasu T. Watanabe T. Nucleic Acids Res. 1989; 17: 9367-9379PubMed Google Scholar) and is a major substrate for several Src family kinases (21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar, 23.Yamanashi Y. Okada M. Semba T. Yamori T. Umemori H. Tsunasawa S. Toyoshima K. Kitamura D. Watanabe T. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3631-3635Crossref PubMed Scopus (142) Google Scholar, 34.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). It contains a proline-rich region, an SH3 domain, an acidic α-helix, and a basic segment resembling the DNA-binding motif of the helix-turn-helix family, suggesting it could play a role in both signal transduction and transcriptional regulation (21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar). HS1 is phosphorylated following activation of B cell and T cell receptors (21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar, 23.Yamanashi Y. Okada M. Semba T. Yamori T. Umemori H. Tsunasawa S. Toyoshima K. Kitamura D. Watanabe T. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3631-3635Crossref PubMed Scopus (142) Google Scholar, 24.Yamanashi Y. Fukuda T. Nishizumi H. Inazu T. Higashi K. Kitamura D. Ishida T. Yamamura H. Watanabe T. Yamamoto T. J. Exp. Med. 1997; 185: 1387-1392Crossref PubMed Scopus (90) Google Scholar, 25.Takemoto Y. Sato M. Furuta M. Hashimoto Y. Int. Immunol. 1996; 8: 1699-1705Crossref PubMed Scopus (34) Google Scholar) but not after stimulation of IL-3, GM-CSF, or SCF receptors (26.Fukamachi H. Yamada N. Miura T. Kato T. Ishikawa M. Gulbins E. Altman A. Kawakami Y. Kawakami T. J. Immunol. 1994; 152: 642-652PubMed Google Scholar). Significantly, Lyn has been shown to associate with HS1 in B and T cells (23.Yamanashi Y. Okada M. Semba T. Yamori T. Umemori H. Tsunasawa S. Toyoshima K. Kitamura D. Watanabe T. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3631-3635Crossref PubMed Scopus (142) Google Scholar, 24.Yamanashi Y. Fukuda T. Nishizumi H. Inazu T. Higashi K. Kitamura D. Ishida T. Yamamura H. Watanabe T. Yamamoto T. J. Exp. Med. 1997; 185: 1387-1392Crossref PubMed Scopus (90) Google Scholar). Like Lyn knockout mice, studies on HS1-deficient mice have revealed a central role for HS1 in B cell responsiveness (27.Hibbs M.L. Tarlinton D.M. Armes J. Grail D. Hodgson G. Maglitto R. Stacker S.A. Dunn A.R. Cell. 1995; 83: 301-311Abstract Full Text PDF PubMed Scopus (615) Google Scholar, 28.Taniuchi I. Kitamura D. Maekawa Y. Fukuda T. Kishi H. Watanabe T. EMBO J. 1995; 14: 3664-3678Crossref PubMed Scopus (108) Google Scholar). While this manuscript was in preparation, HS1 was shown to bind the novel hematopoietic progenitor kinase (HPK1) in erythroid cells (29.Nagata Y. Kiefer F. Watanabe T. Todokoro K. Blood. 1999; 93: 3347-3354Crossref PubMed Google Scholar). In this study we attempted to identify downstream effectors of Lyn in erythroid cells using a yeast two-hybrid screen of wild type and a kinase inactive mutant (Y397F) of Lyn. Of the seven Lyn-interacting proteins identified, we report here on the interaction between Lyn and HS1, and the crucial role of HS1 for epo-induced differentiation of erythroid cells. DISCUSSIONIn this manuscript we have shown that the known Src kinase substrate HS1 associates with Lyn in erythroid cells. We have also demonstrated that a truncated HS1 (tHS1) markedly interferes with the phenotype of erythroid cells and impairs their ability to proliferate and differentiate. By disrupting the Lyn/HS1 interaction, we have generated a cascade of events which had a profound effect on erythroid maturation. These data indicate that HS1 plays a pivotal role in regulating intracellular signaling within erythroid cells and support the recent prediction by Nagata et al. (29.Nagata Y. Kiefer F. Watanabe T. Todokoro K. Blood. 1999; 93: 3347-3354Crossref PubMed Google Scholar) that “ … HS1 is likely to be involved in erythroid proliferation and differentiation. . . . . ”Introduction of a truncated form of HS1 into erythroid cells produced significant morphological, biochemical, and functional perturbations. The truncated mutant spanned the carboxyl-terminal, Lyn-binding region of HS1 that is sequentially phosphorylated by kinases (49.Ruzzene M. Brunati A.M. Marin O. Donella-Deana A. Pinna L.A. Biochemistry. 1996; 35: 5327-5332Crossref PubMed Scopus (43) Google Scholar), but it did not include the amino-terminal Hax1-binding region (50.Suzuki Y. Demoliere C. Kitamura D. Takeshita H. Deuschle U. Watanabe T. J. Immunol. 1997; 158: 2736-2744PubMed Google Scholar). Strikingly, cells expressing tHS1 were smaller, more basophilic, and replicated much more slowly. In addition, epo-induced differentiation was almost totally blocked as the cells failed to mature morphologically and did not synthesize hemoglobin. These data demonstrate that tHS1 acted in a dominant negative fashion and emphasize the importance of a fully functional HS1 to erythroid maturation.The tHS1 mutant had several biochemical effects on the erythroid cells, which may account for its dominant negative activity. First, it greatly reduced the level of Lyn protein within the cells, and as a consequence a marked decrease in tyrosine phosphorylation of intracellular proteins was detected. This result is compatible with our previous observation that the J2E-NR subclone expressed low levels of Lyn, and tyrosine phosphorylation of proteins was substantially reduced (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar). As Lyn associates with the epo receptor (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar, 6.Chin H. Arai A. Wakao H. Kamiyama R. Miyasaka N. Miura O. Blood. 1998; 91: 3734-3745Crossref PubMed Google Scholar), transmission of signals from the receptor via this kinase would be significantly diminished in the transfected cells. Second, the GATA-1 content fell dramatically in these cells. GATA-1 is a key transcription factor involved with erythroid development (51.Tsai S. Martin D.I.K. Zon L.I. D'Andrea A.D. Wong G.G. Orkin S.H. Nature. 1989; 339: 446-451Crossref PubMed Scopus (663) Google Scholar, 52.Pevny L. Lin C.-S., V., D.A. Simon M.C. Orkin S.H. Constantini F. Development. 1995; 121: 163-172Crossref PubMed Google Scholar), and in its absence erythroid precursors arrest at the proerythroblast stage (53.Weiss M.J. Keller G. Orkin S.H. Genes Dev. 1994; 8: 1184-1197Crossref PubMed Scopus (480) Google Scholar). Thus, the loss of GATA-1 protein may play a significant role in the inability of cells bearing tHS1 to mature morphologically or to produce hemoglobin.In addition to reducing the levels of Lyn and GATA-1, tHS1 had a significant effect on endogenous HS1. While the epo receptor was phosphorylated normally in cells containing the mutant HS1 (Fig. 6), endogenous HS1 was degraded rapidly in endosomes/lysosomes after exposure to epo (Fig. 7), indicating that the truncated mutant promoted this proteolysis. It is likely that degradation of endogenous HS1 prevented transmission of signals by this molecule. Thus, tHS1 uncovered an unexpected mechanism for dominant negative action. This observation warrants further investigation with the recent association of signaling molecules with degradation viz. the SOCS family of negative regulators of cytokine action target Janus kinases for degradation by associating with elongins (54.Zhang J.G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausmann G. Kile B.J. Kent S.B. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2071-2076Crossref PubMed Scopus (517) Google Scholar, 55.Kamura T. Sato S. Haque D. Liu L. Kaelin Jr., W.G. Conaway R.C. Conaway J.W. Genes Dev. 1998; 12: 3872-3881Crossref PubMed Scopus (498) Google Scholar), and c-cbl regulates receptor ubiquitination and endocytosis (56.Lee P.S.W. Wang Y. Dominguez M.G. Yeung Y.-G. Murphy M.A. Bowtell D.D.L. Stanely E.R. EMBO J. 1999; 18: 3616-3628Crossref PubMed Scopus (251) Google Scholar, 57.Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar).In this study HS1 was identified as a Lyn-binding protein through a yeast two-hybrid screen, which was confirmed by direct associationin vitro, together with co-immunoprecipitation in vivo. In addition to intracellular co-localization within erythroid cells, the proline-rich region of HS1 was shown to bind the SH3 domain of Lyn, similar to the Lck/HS1 interaction reported previously (21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar). Lyn has been shown to associate with HS1 in B and T cells (45.Takemoto Y. Furuta M. Sato M. Findell P.R. Ramble W. Hashimoto Y. J. Immunol. 1998; 161: 625-630PubMed Google Scholar), but this is the first report that we are aware of where Lyn interacts with HS1 in erythroid cells. Although HS1 and Lyn were constitutively associated in erythroid cells, the interaction increased appreciably after epo activation (Fig. 2 C), which is consistent with previous observations in T cells where the SH3 domain of Lck, or Lyn, binds to HS1 in the absence of stimulation, then the SH2 domain of these kinases associates with HS1 upon receptor activation, increasing the interaction (25.Takemoto Y. Sato M. Furuta M. Hashimoto Y. Int. Immunol. 1996; 8: 1699-1705Crossref PubMed Scopus (34) Google Scholar).HS1 is linked with several kinases. Here we demonstrated HS1 co-immunoprecipitated with Lyn, and two other Src kinases (Lck and Fyn) in J2E cells (Fig. 2 B); tHS1 could potentially interfere with signaling from these molecules. HS1 also associates with the novel hemopoietic kinase HPK1 in the erythroid SKT6 cell line (29.Nagata Y. Kiefer F. Watanabe T. Todokoro K. Blood. 1999; 93: 3347-3354Crossref PubMed Google Scholar). In addition, Takemoto et al. (45.Takemoto Y. Furuta M. Sato M. Findell P.R. Ramble W. Hashimoto Y. J. Immunol. 1998; 161: 625-630PubMed Google Scholar) suggested that the HS1 association with the SH3 domain of Grb2 may regulate the Grb2 and Src signaling pathways. Together these results suggest that HS1 may mediate signals emanating from several kinases and play a crucial role in transmitting intracellular signals within erythroid cells. In this manuscript we have shown that the known Src kinase substrate HS1 associates with Lyn in erythroid cells. We have also demonstrated that a truncated HS1 (tHS1) markedly interferes with the phenotype of erythroid cells and impairs their ability to proliferate and differentiate. By disrupting the Lyn/HS1 interaction, we have generated a cascade of events which had a profound effect on erythroid maturation. These data indicate that HS1 plays a pivotal role in regulating intracellular signaling within erythroid cells and support the recent prediction by Nagata et al. (29.Nagata Y. Kiefer F. Watanabe T. Todokoro K. Blood. 1999; 93: 3347-3354Crossref PubMed Google Scholar) that “ … HS1 is likely to be involved in erythroid proliferation and differentiation. . . . . ” Introduction of a truncated form of HS1 into erythroid cells produced significant morphological, biochemical, and functional perturbations. The truncated mutant spanned the carboxyl-terminal, Lyn-binding region of HS1 that is sequentially phosphorylated by kinases (49.Ruzzene M. Brunati A.M. Marin O. Donella-Deana A. Pinna L.A. Biochemistry. 1996; 35: 5327-5332Crossref PubMed Scopus (43) Google Scholar), but it did not include the amino-terminal Hax1-binding region (50.Suzuki Y. Demoliere C. Kitamura D. Takeshita H. Deuschle U. Watanabe T. J. Immunol. 1997; 158: 2736-2744PubMed Google Scholar). Strikingly, cells expressing tHS1 were smaller, more basophilic, and replicated much more slowly. In addition, epo-induced differentiation was almost totally blocked as the cells failed to mature morphologically and did not synthesize hemoglobin. These data demonstrate that tHS1 acted in a dominant negative fashion and emphasize the importance of a fully functional HS1 to erythroid maturation. The tHS1 mutant had several biochemical effects on the erythroid cells, which may account for its dominant negative activity. First, it greatly reduced the level of Lyn protein within the cells, and as a consequence a marked decrease in tyrosine phosphorylation of intracellular proteins was detected. This result is compatible with our previous observation that the J2E-NR subclone expressed low levels of Lyn, and tyrosine phosphorylation of proteins was substantially reduced (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar). As Lyn associates with the epo receptor (5.Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar, 6.Chin H. Arai A. Wakao H. Kamiyama R. Miyasaka N. Miura O. Blood. 1998; 91: 3734-3745Crossref PubMed Google Scholar), transmission of signals from the receptor via this kinase would be significantly diminished in the transfected cells. Second, the GATA-1 content fell dramatically in these cells. GATA-1 is a key transcription factor involved with erythroid development (51.Tsai S. Martin D.I.K. Zon L.I. D'Andrea A.D. Wong G.G. Orkin S.H. Nature. 1989; 339: 446-451Crossref PubMed Scopus (663) Google Scholar, 52.Pevny L. Lin C.-S., V., D.A. Simon M.C. Orkin S.H. Constantini F. Development. 1995; 121: 163-172Crossref PubMed Google Scholar), and in its absence erythroid precursors arrest at the proerythroblast stage (53.Weiss M.J. Keller G. Orkin S.H. Genes Dev. 1994; 8: 1184-1197Crossref PubMed Scopus (480) Google Scholar). Thus, the loss of GATA-1 protein may play a significant role in the inability of cells bearing tHS1 to mature morphologically or to produce hemoglobin. In addition to reducing the levels of Lyn and GATA-1, tHS1 had a significant effect on endogenous HS1. While the epo receptor was phosphorylated normally in cells containing the mutant HS1 (Fig. 6), endogenous HS1 was degraded rapidly in endosomes/lysosomes after exposure to epo (Fig. 7), indicating that the truncated mutant promoted this proteolysis. It is likely that degradation of endogenous HS1 prevented transmission of signals by this molecule. Thus, tHS1 uncovered an unexpected mechanism for dominant negative action. This observation warrants further investigation with the recent association of signaling molecules with degradation viz. the SOCS family of negative regulators of cytokine action target Janus kinases for degradation by associating with elongins (54.Zhang J.G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausmann G. Kile B.J. Kent S.B. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2071-2076Crossref PubMed Scopus (517) Google Scholar, 55.Kamura T. Sato S. Haque D. Liu L. Kaelin Jr., W.G. Conaway R.C. Conaway J.W. Genes Dev. 1998; 12: 3872-3881Crossref PubMed Scopus (498) Google Scholar), and c-cbl regulates receptor ubiquitination and endocytosis (56.Lee P.S.W. Wang Y. Dominguez M.G. Yeung Y.-G. Murphy M.A. Bowtell D.D.L. Stanely E.R. EMBO J. 1999; 18: 3616-3628Crossref PubMed Scopus (251) Google Scholar, 57.Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar). In this study HS1 was identified as a Lyn-binding protein through a yeast two-hybrid screen, which was confirmed by direct associationin vitro, together with co-immunoprecipitation in vivo. In addition to intracellular co-localization within erythroid cells, the proline-rich region of HS1 was shown to bind the SH3 domain of Lyn, similar to the Lck/HS1 interaction reported previously (21.Takemoto Y. Furuta M. Li X.K. Strong-Sparks W.J. Y. H. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar). Lyn has been shown to associate with HS1 in B and T cells (45.Takemoto Y. Furuta M. Sato M. Findell P.R. Ramble W. Hashimoto Y. J. Immunol. 1998; 161: 625-630PubMed Google Scholar), but this is the first report that we are aware of where Lyn interacts with HS1 in erythroid cells. Although HS1 and Lyn were constitutively associated in erythroid cells, the interaction increased appreciably after epo activation (Fig. 2 C), which is consistent with previous observations in T cells where the SH3 domain of Lck, or Lyn, binds to HS1 in the absence of stimulation, then the SH2 domain of these kinases associates with HS1 upon receptor activation, increasing the interaction (25.Takemoto Y. Sato M. Furuta M. Hashimoto Y. Int. Immunol. 1996; 8: 1699-1705Crossref PubMed Scopus (34) Google Scholar). HS1 is linked with several kinases. Here we demonstrated HS1 co-immunoprecipitated with Lyn, and two other Src kinases (Lck and Fyn) in J2E cells (Fig. 2 B); tHS1 could potentially interfere with signaling from these molecules. HS1 also associates with the novel hemopoietic kinase HPK1 in the erythroid SKT6 cell line (29.Nagata Y. Kiefer F. Watanabe T. Todokoro K. Blood. 1999; 93: 3347-3354Crossref PubMed Google Scholar). In addition, Takemoto et al. (45.Takemoto Y. Furuta M. Sato M. Findell P.R. Ramble W. Hashimoto Y. J. Immunol. 1998; 161: 625-630PubMed Google Scholar) suggested that the HS1 association with the SH3 domain of Grb2 may regulate the Grb2 and Src signaling pathways. Together these results suggest that HS1 may mediate signals emanating from several kinases and play a crucial role in transmitting intracellular signals within erythroid cells. Assistance with confocal microscopy was kindly provided by P. Rigby and S. Codey, University of Western Australia. Recombinant human epo (Eprex) was a generous gift from Dr. J. Adams (Jansen-Cilag). We also thank Drs. S. Cory, J. Adams, and T. Metz for kindly providing the cell lines ME17, clone 11, and clone 24." @default.
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• W2053987316 title "HS1 Interacts with Lyn and Is Critical for Erythropoietin-induced Differentiation of Erythroid Cells" @default.
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