Functional and Biochemical Consequences of Abrogating the Activation of Multiple Diverse Early Signaling Pathways in Kit
2003; Elsevier BV; Volume: 278; Issue: 13 Linguagem: Inglês
10.1074/jbc.m207068200
ISSN1083-351X
AutoresBai Lin Tan, Hong Li, Veerendra Munugalavadla, Reuben Kapur,
Tópico(s)Platelet Disorders and Treatments
ResumoKit receptor tyrosine kinase and erythropoietin receptor (Epo-R) cooperate in regulating blood cell development. Mice that lack the expression of Kit or Epo-R die in utero of severe anemia. Stimulation of Kit by its ligand, stem cell factor activates several distinct early signaling pathways, including phospholipase Cγ, phosphatidylinositol 3-kinase, Src kinase, Grb2, and Grb7. The role of these pathways in Kit-induced growth, proliferation, or cooperation with Epo-R is not known. We demonstrate that inactivation of any one of these early signaling pathways in Kit significantly impairs growth and proliferation. However, inactivation of the Src pathway demonstrated the most profound defect. Combined stimulation with Epo also resulted in impaired cooperation between Src-defective Kit mutant and Epo-R and, to a lesser extent, with Kit mutants defective in the activation of phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between the Src-defective Kit mutant and Epo-R was associated with reduced transphosphorylation of Epo-R and expression of c-Myc. Remarkably, restoration of only the Src pathway in a Kit receptor defective in the activation of all early signaling pathways demonstrated a 50% correction in proliferation in response to Kit stimulation and completely restored the cooperation with Epo-R. These data demonstrate an essential role for Src pathway in regulating growth, proliferation, and cooperation with Epo-R downstream from Kit. Kit receptor tyrosine kinase and erythropoietin receptor (Epo-R) cooperate in regulating blood cell development. Mice that lack the expression of Kit or Epo-R die in utero of severe anemia. Stimulation of Kit by its ligand, stem cell factor activates several distinct early signaling pathways, including phospholipase Cγ, phosphatidylinositol 3-kinase, Src kinase, Grb2, and Grb7. The role of these pathways in Kit-induced growth, proliferation, or cooperation with Epo-R is not known. We demonstrate that inactivation of any one of these early signaling pathways in Kit significantly impairs growth and proliferation. However, inactivation of the Src pathway demonstrated the most profound defect. Combined stimulation with Epo also resulted in impaired cooperation between Src-defective Kit mutant and Epo-R and, to a lesser extent, with Kit mutants defective in the activation of phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between the Src-defective Kit mutant and Epo-R was associated with reduced transphosphorylation of Epo-R and expression of c-Myc. Remarkably, restoration of only the Src pathway in a Kit receptor defective in the activation of all early signaling pathways demonstrated a 50% correction in proliferation in response to Kit stimulation and completely restored the cooperation with Epo-R. These data demonstrate an essential role for Src pathway in regulating growth, proliferation, and cooperation with Epo-R downstream from Kit. receptor tyrosine kinase phospholipase Cγ phosphatidylinositol 3-kinase platelet-derived growth factor stem cell factor macrophage colony-stimulating factor colony-forming unit-erythroid erythropoietin Epo receptor amino acids wild type chimeric kit receptor enhanced green fluorescent protein phycoerythrin immunoprecipitation pyrazolopyrimidine Receptor tyrosine kinases (RTKs)1 trigger multitude of cellular events, including proliferation, survival, differentiation, and migration. In response to ligand-induced stimulation, RTKs undergo dimerization and autophosphorylation on several distinct cytoplasmic tyrosine residues (1van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar, 2Pawson T. Nature. 1995; 373: 573-580Google Scholar, 3Pawson T. Nash P. Genes Dev. 2000; 14: 1027-1047Google Scholar, 4Schlessinger J. Cell. 2000; 103: 211-225Google Scholar). These phosphorylated tyrosine residues become binding sites for a variety of Src homology 2 domain-containing enzymes and adaptor proteins such as phospholipase Cγ (PLC-γ), phosphatidylinositol 3-kinase p85 subunit (PI 3-kinase), Ras GTPase-activating protein, SHP2 phosphatase, Src kinases, Grb2, Grb7, and Shc (5Pawson T. Saxton T.M. Cell. 1999; 97: 675-678Google Scholar). In this manner, the phosphorylated tyrosine residues initiate signal transduction via several distinct early signaling pathways. A major unresolved question in the field of RTK signaling is whether these diverse signaling pathways result in redundant or nonredundant biological functions. Recent studies utilizing the platelet-derived growth factor (PDGF) RTK have begun to address some of these issues in nonhematopoietic cells (6Fambrough D. McClure K. Kazlauskas A. Lander E.S. Cell. 1999; 97: 727-741Google Scholar, 7Heuchel R. Berg A. Tallquist M. Ahlen K. Reed R.K. Rubin K. Claesson-Welsh L. Heldin C.H. Soriano P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11410-11415Google Scholar, 8Klinghoffer R.A. Mueting-Nelsen P.F. Faerman A. Shani M. Soriano P. Mol. Cell. 2001; 7: 343-354Google Scholar). However, relatively little is known about the biological consequence(s) of activation of diverse signaling pathways by RTKs in hematopoietic cells. In hematopoietic cells, the RTK Kit plays an essential role in regulating proliferation, survival, differentiation, and migration of stem and progenitor cells (9Broudy V.C. Blood. 1997; 90: 1345-1364Google Scholar, 10Linnekin D. Int. J. Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar, 11Boissan M. Feger F. Guillosson J.J. Arock M. J. Leukocyte Biol. 2000; 67: 135-148Google Scholar). The proto-oncogene Kit encodes the receptor for stem cell factor (SCF) and belongs to the type III receptor tyrosine kinase subfamily (9Broudy V.C. Blood. 1997; 90: 1345-1364Google Scholar, 10Linnekin D. Int. J. Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar, 11Boissan M. Feger F. Guillosson J.J. Arock M. J. Leukocyte Biol. 2000; 67: 135-148Google Scholar). This family of cytokine receptor includes the macrophage colony-stimulating factor (M-CSF) receptor, the PDGF receptor, and the Flk-2/Flk-3 receptor (1van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar). The structure of these receptors includes an extracellular domain with five Ig-like motifs, a single short membrane-spanning domain, and a cytoplasmic domain with tyrosine kinase activity (9Broudy V.C. Blood. 1997; 90: 1345-1364Google Scholar, 10Linnekin D. Int. J. Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar, 11Boissan M. Feger F. Guillosson J.J. Arock M. J. Leukocyte Biol. 2000; 67: 135-148Google Scholar). The kinase domain is separated by a kinase insert sequence that divides the kinase domain into an ATP binding region and phosphotransferase region (9Broudy V.C. Blood. 1997; 90: 1345-1364Google Scholar, 10Linnekin D. Int. J. Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar, 11Boissan M. Feger F. Guillosson J.J. Arock M. J. Leukocyte Biol. 2000; 67: 135-148Google Scholar). The product of the Kit gene is a transmembrane receptor composed of 976 amino acids (aa) with 519 extracellular aa, a transmembrane domain of 23 aa, and an intracellular tail of 433 aa (9Broudy V.C. Blood. 1997; 90: 1345-1364Google Scholar, 10Linnekin D. Int. J. Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar, 11Boissan M. Feger F. Guillosson J.J. Arock M. J. Leukocyte Biol. 2000; 67: 135-148Google Scholar). In addition to being expressed on hematopoietic cells, Kit is also expressed on cells of nonhematopoietic origin, including melanocytes, primordial germ cells, and interstitial cells of Cajal (9Broudy V.C. Blood. 1997; 90: 1345-1364Google Scholar). In hematopoietic cells, Kit can synergize with other growth factor receptors to promote survival, proliferation, and differentiation of multiple hematopoietic lineages (9Broudy V.C. Blood. 1997; 90: 1345-1364Google Scholar, 10Linnekin D. Int. J. Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar, 11Boissan M. Feger F. Guillosson J.J. Arock M. J. Leukocyte Biol. 2000; 67: 135-148Google Scholar). Intriguingly, the most profound phenotype due to the lack of Kit expression in mice is manifested in erythroid cells. Mutant mice that lack the expression of Kit (dominant white spotting, or W, mutants) demonstrate severe deficiencies in erythroid cell development (9Broudy V.C. Blood. 1997; 90: 1345-1364Google Scholar, 12Nocka K. Majumder S. Chabot B. Ray P. Cervone M. Bernstein A. Besmer P. Genes Dev. 1989; 3: 816-826Google Scholar). Kit-deficient mice exhibit a severe reduction of colony-forming unit-erythroid (CFU-E) progenitors in the fetal liver and die of anemia around day 16 of gestation (9Broudy V.C. Blood. 1997; 90: 1345-1364Google Scholar, 12Nocka K. Majumder S. Chabot B. Ray P. Cervone M. Bernstein A. Besmer P. Genes Dev. 1989; 3: 816-826Google Scholar). Epo-R-deficient mice also demonstrate a similar decrease in CFU-E progenitors and die of anemia between days 13 and 15 of gestation (13Wu H. Liu X. Jaenisch R. Lodish H.F. Cell. 1995; 83: 59-67Google Scholar), suggesting that erythroid progenitors cannot survive, proliferate, or differentiate unless both Kit and the Epo-R signal transduction pathways are functional. Recent studies have suggested that Epo and Epo-R interactions may contribute to this process by preventing erythroid progenitors from undergoing apoptosis by activating Stat5 and subsequently inducing the expression of an antiapoptotic protein, Bcl-xL (14Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Google Scholar). Consistent with these studies, mice deficient in the expression of Stat5 or Bcl-xL manifest a decrease in the number of erythroid progenitors due to enhanced apoptosis (14Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Google Scholar, 15Gregory T. Yu C. Ma A. Orkin S.H. Blobel G.A. Weiss M.J. Blood. 1999; 94: 87-96Google Scholar, 16Motoyama N. Kimura T. Takahashi T. Watanabe T. Nakano T. J. Exp. Med. 1999; 189: 1691-1698Google Scholar, 17Motoyama N. Wang F. Roth K.A. Sawa H. Nakayama K. Negishi I. Senju S. Zhang Q. Fujii S. Loh D.Y. Science. 1995; 267: 1506-1510Google Scholar). However, the role of Kit in erythroid cell development alone or in combination with Epo-R is poorly understood. To this end, we have recently demonstrated an essential role for Kit in proliferation of erythroid progenitors (18Kapur R. Zhang L. J. Biol. Chem. 2001; 276: 1099-1106Google Scholar). Further, we and others have also demonstrated that Kit synergizes with Epo-R in enhancing proliferation and survival of erythroid progenitors (18Kapur R. Zhang L. J. Biol. Chem. 2001; 276: 1099-1106Google Scholar, 19Wu H. Klingmuller U. Besmer P. Lodish H.F. Nature. 1995; 377: 242-246Google Scholar, 20Joneja B. Chen H.C. Seshasayee D. Wrentmore A.L. Wojchowski D.M. Blood. 1997; 90: 3533-3545Google Scholar, 21Wu H. Klingmuller U. Acurio A. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1806-1810Google Scholar, 22Jacobs-Helber S.M. Penta K. Sun Z. Lawson A. Sawyer S.T. J. Biol. Chem. 1997; 272: 6850-6853Google Scholar, 23Sui X. Krantz S.B. You M. Zhao Z. Blood. 1998; 92: 1142-1149Google Scholar, 24Pircher T.J. Geiger J.N. Zhang D. Miller C.P. Gaines P. Wojchowski D.M. J. Biol. Chem. 2001; 276: 8995-9002Google Scholar, 25Miller C.P. Heilman D.W. Wojchowski D.M. Blood. 2002; 99: 898-904Google Scholar). However, the role of early activating signal transduction pathways downstream from Kit in erythroid cell growth, proliferation, and cooperation with Epo-R is not known. Activated Kit binds signaling molecules at specific tyrosine residues: PLC-γ at tyrosine 728 (26Gommerman J.L. Sittaro D. Klebasz N.Z. Williams D.A. Berger S.A. Blood. 2000; 96: 3734-3742Google Scholar), PI 3-kinase at tyrosine 719 (28Serve H. Yee N.S. Stella G. Sepp-Lorenzino L. Tan J.C. Besmer P. EMBO J. 1995; 14: 473-483Google Scholar), Src class kinases at positions 567/569 (27Ueda S. Mizuki M. Ikeda H. Tsujimura T. Matsumura I. Nakano K. Daino H. Honda Zi.Z. Sonoyama J. Shibayama H. Sugahara H. Machii T. Kanakura Y. Blood. 2002; 99: 3342-3349Google Scholar, 29Timokhina I. Kissel H. Stella G. Besmer P. EMBO J. 1998; 17: 6250-6262Google Scholar, 30Linnekin D. DeBerry C.S. Mou S. J. Biol. Chem. 1997; 272: 27450-27455Google Scholar, 31Lennartsson J. Blume-Jensen P. Hermanson M. Ponten E. Carlberg M. Ronnstrand L. Oncogene. 1999; 18: 5546-5553Google Scholar), Grb2 at tyrosine 702 (32Thommes K. Lennartsson J. Carlberg M. Ronnstrand L. Biochem. J. 1999; 341: 211-216Google Scholar), and Grb7 at tyrosine 934 (32Thommes K. Lennartsson J. Carlberg M. Ronnstrand L. Biochem. J. 1999; 341: 211-216Google Scholar). Other classes of signaling proteins have also been reported to bind activated Kit with unknown sequence specificity (33Duronio V. Welham M.J. Abraham S. Dryden P. Schrader J.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1587-1591Google Scholar, 34Cutler R.L. Liu L. Damen J.E. Krystal G. J. Biol. Chem. 1993; 268: 21463-21465Google Scholar, 35Sattler M. Salgia R. Shrikhande G. Verma S. Pisick E. Prasad K.V. Griffin J.D. J. Biol. Chem. 1997; 272: 10248-10253Google Scholar, 36van Dijk T.B. van Den Akker E. Amelsvoort M.P. Mano H. Lowenberg B. von Lindern M. Blood. 2000; 96: 3406-3413Google Scholar, 37Jahn T. Seipel P. Urschel S. Peschel C. Duyster J. Mol. Cell. Biol. 2002; 22: 979-991Google Scholar). Studies in multiple cell types have shown that Kit carrying tyrosine to phenylalanine mutations at the critical residues fail to bind the associated signaling molecules and consequently fail to activate these signaling pathways (10Linnekin D. Int. J. Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar, 11Boissan M. Feger F. Guillosson J.J. Arock M. J. Leukocyte Biol. 2000; 67: 135-148Google Scholar). In this study, we took advantage of the ability of Tyr → Phe mutations in Kit to block the activation of downstream signaling molecules to comprehensively investigate the effect of lack of activation of five early signaling pathways in Kit-induced growth, proliferation, and cooperation with Epo-R in a relevant cell type. Utilizing an erythroid progenitor cell line (G1E-ER2) that closely mimics primary proerythroblasts (15Gregory T. Yu C. Ma A. Orkin S.H. Blobel G.A. Weiss M.J. Blood. 1999; 94: 87-96Google Scholar, 18Kapur R. Zhang L. J. Biol. Chem. 2001; 276: 1099-1106Google Scholar, 38Kapur R. Cooper R. Xiao X. Weiss M.J. Donovan P. Williams D.A. Blood. 1999; 94: 1915-1925Google Scholar, 39Kapur R. Cooper R. Zhang L. Williams D.A. Blood. 2001; 97: 1975-1981Google Scholar) and primary fetal liver cells, we demonstrate that inactivation of any one of these five early signaling pathways in Kit significantly impairs growth and proliferation in response to Kit activation. The most profound defect was observed in Kit mutants impaired in the activation of the Src kinase pathway. Interestingly, when stimulated in combination with erythropoietin (Epo), the mutant of Kit deficient in the activation of Src kinase demonstrated a profound reduction in proliferation, which was associated with impaired transphosphorylation of Epo-R and expression of c-Myc. Remarkably, restoration of the Src kinase pathway in Kit, alone, in the absence of remaining four early signaling pathways restored the cooperation and transphosphorylation of Epo-R in response to Kit activation. These results demonstrate an essential role for the Src signaling pathway in regulating growth, proliferation, and cooperation with Epo-R downstream from Kit. G1E-ER2 cells have been described previously (15Gregory T. Yu C. Ma A. Orkin S.H. Blobel G.A. Weiss M.J. Blood. 1999; 94: 87-96Google Scholar). G1E-ER2 cells were grown in Iscove's modified Dulbecco's medium (Invitrogen) with 15% heat-inactivated fetal bovine serum (Fisher), recombinant Epo (2 units/ml) (Amgen, Thousand Oaks, CA), and recombinant rat SCF (50 ng/ml) (Amgen). The CHR gene was constructed from DNA encoding aa 1–513 of the human M-CSF receptor and aa 528–977 of the murine Kit receptor joined at an EcoRI site. Plasmid containing the human full-length M-CSF receptor cDNA (a kind gift of Dr. Sherr, St. Judes, Memphis, TN) was utilized. Forward (NotI-containing) and reverse (EcoRI-containing) primers corresponding to the start site and transmembrane region of the M-CSF receptor were utilized to perform PCR on the extracellular domain of the M-CSF receptor. Forward (EcoRI-containing) and reverse (XhoI-containing) primers corresponding to the transmembrane and the stop site were used to perform PCR on the transmembrane and the cytoplasmic domain of the murine Kit receptor. The PCR product was digested and ligated into the NotI and XhoI sites of MIEG3 bicistronic retroviral expression vector (40Yang F.C. Kapur R. King A.J. Tao W. Kim C. Borneo J. Breese R. Marshall M. Dinauer M.C. Williams D.A. Immunity. 2000; 12: 557-568Google Scholar). The sequence of the CHR was verified. To generate mutant CHRs, theNotI-XhoI WT CHR DNA fragment (2.9 kb) spanning the sites to be mutated was subcloned into Bluescript. The QuikChange site-directed mutagenesis kit (Stratagene) and primers containing the appropriate mutations were used to mutate tyrosine residues 567, 569, 702, 719, 728, and 745 to Phe. The NotI-XhoI fragment containing mutations at tyrosine residues 567, 569, 702, 719, 728, and 745 in murine Kit receptor was verified by sequencing released from Bluescript and religated into the NotI-XhoI site of MIEG3 retroviral vector. In some experiments, mutant CHR lacking all six tyrosine residues was used as a template to restore phenylalanine mutations at positions 567 and 569 back to tyrosine. Utilizing this bicistronic retroviral vector, we inserted the WT and the mutant CHR cDNAs upstream of the internal ribosome entry site (IRES) and the enhanced green fluorescence protein (EGFP) gene (see Fig. 1). To produce WT and mutant CHR viral supernatants for infection of G1E-ER2 cells, Phoenix ecotropic cells were transiently transfected with WT or the mutant CHR retroviral constructs using LipofectAMINE Plus reagent (Invitrogen). Supernatants were collected 48 h after transfection, filtered through 0.45-μm membranes, and used. Cells were infected with 2 ml of virus supernatant in the presence of 8 μg/ml polybrene. Virus-infected cells were harvested 48 h later, sorted by a fluorescence-activated cell sorter, and expanded in culture. G1E-ER2 cells expressing similar levels of EGFP and M-CSF receptors were utilized to perform all of the experiments described in these studies. Phycoerythrin (PE)-conjugated secondary monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used to detect the antibody to the extracellular domain of the CHR (M-CSF receptor; 2-4A5; Santa Cruz Biotechnology). G1E-ER2 cells (1 × 106) expressing WT or mutant CHRs were incubated at 4 °C for 30 min with 1 μg of the primary antibody. Cells were washed three times with phosphate-buffered saline containing 0.1% bovine serum albumin (Sigma) and incubated with a secondary antibody for 30 min at 4 °C, washed as above, and analyzed by a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson, San Jose, CA). The effect of M-CSF and Epo on proliferation of G1E-ER2 cells was assayed by thymidine incorporation. 96-Well tissue culture plates were utilized for these studies. G1E-ER2 cells expressing WT or mutant CHRs were plated at 5 × 104 cells/well for 48 h, either in the absence or presence of M-CSF (50 ng/ml) and/or Epo (2 units/ml). Subsequently, 1.0 μCi of [3H]thymidine was added to each well for 6–8 h at 37 °C. Cells were then harvested using an automated cell harvester (96-well harvester; Brandel, Gaithersburg, MD), and thymidine incorporation was determined in a scintillation counter. The effect of M-CSF and/or Epo on cell growth and viability was performed by plating 1 × 105 cells in a six-well tissue culture plate in replicates of three for 24 and 48 h, after which cells were subjected to trypan blue and counted under the microscope. Viable cells were scored at various time points. Fetal liver cells were harvested from day 12.5 WT embryos. Single cell suspensions were prepared and incubated with or without retrovirus expressing the WT CHR or CHR mutants containing tyrosine to phenylalanine mutations at positions 567 and 569 as described above. After infection, cells were plated in triplicate in α-methylcellulose (Stemcell Technologies, Vancouver, Canada) with Epo (2 units/ml) or Epo plus M-CSF (2 units/ml Epo and 100 ng/ml M-CSF). Benzidine-positive colonies were counted 2–3 days after plating. Immunoprecipitations (IPs) were performed as previously described (41Kapur R. Majumdar M. Xiao X. McAndrews-Hill M. Schindler K. Williams D.A. Blood. 1998; 91: 879-889Google Scholar). Briefly, cells expressing either the WT or the mutant CHR deficient in the activation of Src kinases (CHR 567/569) or Src add-back (CHR 567/569B) CHR mutants were stimulated for the indicated times. Thereafter, cells were lysed in lysis buffer (10 mm K2HPO4, 1 mm EDTA, 50 mm EGTA, 10 mmMgCl2, 1 mm Na2VO4, 50 mm β-glycerol phosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstatin A (pH 7.2)). Lysates were clarified by centrifugation at 10,000 × g, 4 °C for 30 min. IP was performed by incubating equivalent amounts of cell lysates with either anti-Epo-R or an anti-Kit or anti-M-CSF receptor antibody overnight at 4 °C (all from Santa Cruz Biotechnology). Protein A- or protein G-Sepharose beads (Amersham Biosciences) were used to collect the antigen-antibody complexes. IPs were separated by SDS-PAGE), and proteins were electrophoretically transferred onto nitrocellulose membranes (Bio-Rad). After blocking residual binding sites on the transfer membrane by incubating the membrane with 5% milk overnight, Western blot analysis using an anti-phosphotyrosine antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and Supersignal West Dura extended duration detection system (Pierce) was utilized according to the manufacturer's instructions. For Western blot analysis, 1–2 × 106 WT CHR and CHR 567/569 were plated in duplicate six-well tissue culture plates for 12 and 24 h at 37 °C in the presence or absence of M-CSF (100 ng/ml) and/or Epo (2 units/ml). Thereafter, cells were harvested and lysed in lysis buffer as described above. An equal amount of protein was fractionated on a 10% SDS-PAGE gel and electrophoretically transferred to nitrocellulose membrane. Expression of c-Myc was determined by using an anti-c-Myc antibody (Santa Cruz Biotechnology). Expression of Bcl-xL was determined by using an anti-Bcl-xL antibody (Invitrogen). Activation of Stat5 was determined by utilizing a phosphospecific Stat5 antibody (Cell Signaling, Beverly, MA). To analyze the role of diverse early signaling pathways activated via Kit in response to SCF stimulation in erythroid cells, we utilized an erythroid progenitor cell line G1E-ER2 (15Gregory T. Yu C. Ma A. Orkin S.H. Blobel G.A. Weiss M.J. Blood. 1999; 94: 87-96Google Scholar). These cells were utilized to specifically examine the role of Kit and Epo-R in the context of a cell type that is most affected due to mutations in Kit and Epo-R. We and others have previously shown that these cells mimic primary proerythroblasts and respond to both SCF and Epo (15Gregory T. Yu C. Ma A. Orkin S.H. Blobel G.A. Weiss M.J. Blood. 1999; 94: 87-96Google Scholar, 18Kapur R. Zhang L. J. Biol. Chem. 2001; 276: 1099-1106Google Scholar). Since G1E-ER2 cells express endogenous Kit receptors, we constructed eight chimeric Kit receptors (CHRs) to bypass endogenous Kit receptors (Fig. 1). The CHR approach to investigate the role of a specific signaling pathway in nonhematopoietic cells downstream from a RTK, such as the PDGF receptor, has been previously described (6Fambrough D. McClure K. Kazlauskas A. Lander E.S. Cell. 1999; 97: 727-741Google Scholar, 42DeMali K.A. Kazlauskas A. Mol. Cell. Biol. 1998; 18: 2014-2022Google Scholar). The M-CSF receptor and Kit belong to the same subfamily of RTKs but possess distinct ligand binding specificity (1van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar). G1E-ER2 cells do not express endogenous M-CSFR (Fig.2 B, left panel) and show no response to M-CSF stimulation (Fig.2 D, panel A). Based on these observations, we cloned a cDNA encoding a protein consisting of the extracellular domain of the human M-CSFR and the transmembrane and the cytoplasmic domain of murine Kit (Fig. 1). This CHR is activated upon binding M-CSF but signals in a fashion similar to endogenous Kit receptor (Fig. 2, C and D (panel B)). Eight mutant chimeric receptor cDNAs were constructed, encoding tyrosine to phenylalanine mutations in the cytoplasmic domain of Kit. These mutant receptors cannot bind and activate signaling molecules (10Linnekin D. Int. J. Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar, 11Boissan M. Feger F. Guillosson J.J. Arock M. J. Leukocyte Biol. 2000; 67: 135-148Google Scholar, 26Gommerman J.L. Sittaro D. Klebasz N.Z. Williams D.A. Berger S.A. Blood. 2000; 96: 3734-3742Google Scholar, 27Ueda S. Mizuki M. Ikeda H. Tsujimura T. Matsumura I. Nakano K. Daino H. Honda Zi.Z. Sonoyama J. Shibayama H. Sugahara H. Machii T. Kanakura Y. Blood. 2002; 99: 3342-3349Google Scholar, 28Serve H. Yee N.S. Stella G. Sepp-Lorenzino L. Tan J.C. Besmer P. EMBO J. 1995; 14: 473-483Google Scholar, 29Timokhina I. Kissel H. Stella G. Besmer P. EMBO J. 1998; 17: 6250-6262Google Scholar, 31Lennartsson J. Blume-Jensen P. Hermanson M. Ponten E. Carlberg M. Ronnstrand L. Oncogene. 1999; 18: 5546-5553Google Scholar, 43Kissel H. Timokhina I. Hardy M.P. Rothschild G. Tajima Y. Soares V. Angeles M. Whitlow S.R. Manova K. Besmer P. EMBO J. 2000; 19: 1312-1326Google Scholar). We mutated the binding sites for Src kinases at positions 567 and 569 of the Kit cytoplasmic domain, PI 3-kinase at position 719, PLC-γ at position 728, Grb2 at position 702, an additional mutation at position 745, a double mutant encoding tyrosine to phenylalanine mutations at positions 567 and 569, and a “naked” receptor that encodes tyrosine to phenylalanine mutations at all six positions (Fig. 1). For expression, biochemical and functional analysis of the WT CHR, we cloned this receptor into a bicistronic retroviral vector MIEG3 (Fig.2 A) that expresses the EGFP via an internal ribosome entry site and generated viral supernatants as described under “Experimental Procedures.” We have previously reported the use of this vector in generating high transduction of hematopoietic cells (40Yang F.C. Kapur R. King A.J. Tao W. Kim C. Borneo J. Breese R. Marshall M. Dinauer M.C. Williams D.A. Immunity. 2000; 12: 557-568Google Scholar). After infection of G1E-ER2 cells, EGFP-positive cells were sorted to homogeneity and utilized to perform functional and biochemical studies. Flow cytometry was utilized to examine the expression of M-CSFR in G1E-ER2 cells expressing the WT CHR. Fig. 2 B(left panel) demonstrates complete lack of M-CSFR expression in parental untransduced G1E-ER2 cells stained with a PE-conjugated antibody against M-CSFR (y axis). Fig.2 B (right panel) demonstrates 100% co-expression of both EGFP and M-CSFR in G1E-ER2 cells transduced with the WT CHR. To determine whether ligand-induced phosphorylation of the WT CHR is similar to endogenous Kit receptor, we starved the cells for 6 h of serum and growth factors, and stimulated them with either 100 ng/ml recombinant rat SCF or human M-CSF for 10 min, after which cells were lysed and subjected to immunoprecipitation using an anti-Kit antibody or an anti-M-CSFR antibody and subjected to Western blot analysis using an anti-phosphotyrosine antibody. As shown in Fig. 2 C, stimulation of G1E-ER2 cells expressing the WT CHR with SCF (lane 2) or M-CSF (lane 4) resulted in comparable tyrosine phosphorylation, suggesting that the WT CHR biochemically behaves in a fashion similar to endogenous Kit receptor. The phosphorylation of various CHR mutants examined in the present study was also similar to WT CHR except for CHR mutants impaired in the activation of Src and PI 3-kinase pathway. These two mutant CHRs demonstrated a slight decrease in phosphorylation compared with WT CHR (data not shown). Next, we analyzed the function of WT CHR by examining proliferation in response to M-CSF stimulation. Consistent with the lack of M-CSFR expression in G1E-ER2 cells shown earlier (Fig. 2 B,left panel), the addition of M-CSF to parental untransduced G1E-ER2 cells did not induce proliferation and did not cooperate with Epo-R to enhance proliferation (Fig. 2 D,panel A). In contrast, and as expected and previously shown (18Kapur R. Zhang L. J. Biol. Chem. 2001; 276: 1099-1106Google Scholar), stimulation of G1E-ER2 cells with SCF resulted in significant proliferation, which was further augmented in the presence of Epo (Fig. 2 D, panel A). Consistent with the expression and the biochemical observations noted above (Fig. 2, B and C), G1E-ER2 cells expressing the WT CHR demonstrated a similar increase in proliferation in response to M-CSF stimulation, as seen with SCF stimulation of endogenous Kit receptors (Fig. 2 D, panel B). This increase in proliferation was further augmented in the presence of Epo, to levels observed in response to endogenous Kit activation with SCF and Epo (Fig. 2 D, panel B). Collectively, these data demonstrate that the WT CHR appears to function in a fashion similar to the endogenous Kit receptor in G1E-ER2 cells. To determine the effect of abrogating the activation of diverse early signaling pathways in Kit-induced proliferation/growth and cooperation with Epo-R, we expressed all eight CHR mutants in G1E-ER2 cells and examined M-CSF induced growth and proliferation over a 2-day culture period by trypan blue exclusion. Fig. 3demonstrates similar co-expression of all eight CHR mutants (y axis) and EGFP (x axis) in G1E-ER2 cells as determined by flow c
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