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• W2092583849 abstract "We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-γ2 in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-γ2was time- and dose-dependent. By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor. Erythropoietin-activated PLC-γ2 hydrolyzed purified [3H]GPI indicating that GPI hydrolysis and PLC-γ2 activation under erythropoietin stimulation were correlated. Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-γ2, whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects. Thus, our results suggest that erythropoietin regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-γ2activation. We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-γ2 in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-γ2was time- and dose-dependent. By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor. Erythropoietin-activated PLC-γ2 hydrolyzed purified [3H]GPI indicating that GPI hydrolysis and PLC-γ2 activation under erythropoietin stimulation were correlated. Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-γ2, whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects. Thus, our results suggest that erythropoietin regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-γ2activation. erythropoietin receptor α-minimum essential medium glycosylphosphatidylinositol Src homology domain 2 inositol phosphoglycan epidermal growth factor phospholipase C phosphatidylinositol-phospholipase C wild type high performance thin layer chromatography 2,5-anhydromannitol phosphatidylinositol monophosphate phosphatidylcholine Erythropoietin is essential for the survival, proliferation, and differentiation of the late erythroid progenitor cells (for review see Ref. 1Krantz S.B. Blood. 1991; 77: 419-434Crossref PubMed Google Scholar). Erythropoietin exerts its action on its target cells by binding to specific cell-surface receptors (for review see Ref. 2Koury M.J. Bondurant M.C. Eur. J. Biochem. 1992; 210: 649-663Crossref PubMed Scopus (255) Google Scholar). The erythropoietin receptor (Epo-R)1 belongs to the cytokine receptor superfamily, and its cytosolic domain lacks intrinsic kinase activity. Nevertheless, ligand binding leads to a rapid but transient tyrosine phosphorylation of cellular proteins, including the receptor itself (3Miura O. D'Andrea A. Kabat D. Ihle J.N. Mol. Cell. Biol. 1991; 11: 4895-4902Crossref PubMed Scopus (184) Google Scholar, 4Quelle F.W. Wojchowski D.M. J. Biol. Chem. 1991; 266: 609-614Abstract Full Text PDF PubMed Google Scholar, 5Dusanter-Fourt I. Casadevall N. Lacombe C. Muller O. Billat C. Fischer S. Mayeux P. J. Biol. Chem. 1992; 267: 10670-10675Abstract Full Text PDF PubMed Google Scholar). This phosphorylation is carried out via the activation of an associated tyrosine kinase, JAK2 (6Witthuhn B.A. Quelle F.W. Silvennoinen O. Yi T. Tang B. Miura O. Ihle J.N. Cell. 1993; 74: 227-236Abstract Full Text PDF PubMed Scopus (1008) Google Scholar, 7Miura O. Nakamura N. Quelle F.W. Witthuhn B.A. Ihle J.N. Aoki N. Blood. 1994; 84: 1501-1507Crossref PubMed Google Scholar). Epo-R tyrosine phosphorylation creates binding sites for Src homology domain 2 (SH2) containing proteins such as Grb2, Shc, Stat5, phosphatidylinositol 3-kinase, SHP1, and SHP2 that relay and amplify the signals (for review see Ref. 8Damen J.E. Krystal G. Exp. Hematol. 1996; 24: 1455-1459PubMed Google Scholar). The glycosylphosphatidylinositol (GPI) molecules with the general structure lipid-phosphate-inositol glycan have been isolated from plants, bacteria, yeast, parasitic protozoa, and mammalian cells (9Ferguson M.A. Williams A.F. Annu. Rev. Biochem. 1988; 57: 285-320Crossref PubMed Scopus (953) Google Scholar, 10Gaulton G.N. Pratt J.C. Semin. Immunol. 1994; 6: 97-104Crossref PubMed Scopus (34) Google Scholar, 11Thomas J.R. Dwek R.A. Rademacher T.W. Biochemistry. 1990; 29: 5413-5422Crossref PubMed Scopus (169) Google Scholar). In eukaryotic cells, GPI molecules may be attached to protein moieties thereby providing a membrane anchor. Free GPIs also exist under an uncomplexed form. Both forms of GPI have been shown to participate in signal transduction events (for review see Ref. 12Jones D.R. Varela-Nieto I. Int. J. Biochem. Cell Biol. 1998; 30: 313-326Crossref PubMed Scopus (95) Google Scholar). In different cellular systems GPI turnover is modulated by a variety of hormones, cytokines, and growth factors such as insulin (13Saltiel A.R. Cuatrecasas P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5793-5797Crossref PubMed Scopus (226) Google Scholar, 14Mato J.M. Kelly K.L. Abler A. Jarett L. J. Biol. Chem. 1987; 262: 2131-2137Abstract Full Text PDF PubMed Google Scholar), insulin growth factor-I (15Farese R.V. Nair G.P. Standaert M.L. Cooper D.R. Biochem. Biophys. Res. Commun. 1988; 156: 1346-1352Crossref PubMed Scopus (30) Google Scholar, 16Kojima I. Kitaoka M. Ogata E. J. Biol. Chem. 1990; 265: 16846-16850Abstract Full Text PDF PubMed Google Scholar), nerve growth factor (17Chan B.L. Chao M.V. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1756-1760Crossref PubMed Scopus (102) Google Scholar, 18Represa J. Avila M.A. Miner C. Giraldez F. Romero G. Clemente R. Mato J.M. Varela-Nieto I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8016-8019Crossref PubMed Scopus (70) Google Scholar), interleukin-2 (19Merida I. Pratt J.C. Gaulton G.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9421-9425Crossref PubMed Scopus (71) Google Scholar), thyroid-stimulating hormone (20Martiny L. Antonicelli F. Thuilliez B. Lambert B. Jacquemin C. Haye B. Cell. Signal. 1990; 2: 21-27Crossref PubMed Scopus (33) Google Scholar), or erythropoietin in rat erythroid progenitor cells (21Devemy E. Billat C. Sartelet H. Martiny L. Haye B. Cell. Signal. 1994; 6: 523-529Crossref PubMed Scopus (9) Google Scholar). Then the hydrolysis of glycosylphosphatidylinositol could be another pathway in signal transduction. GPI hydrolysis generates diacylglycerol and the polar group of the lipid, an inositol phosphoglycan (IPG) which can act as a second messenger (for review see Ref. 22Varela-Nieto I. Leon Y. Caro H.N. Comp. Biochem. Physiol. 1996; 115: 223-241Crossref Scopus (89) Google Scholar). The dependence on increased receptor phosphorylation and GPI hydrolysis after growth factor stimulation remains to be conclusively defined. However, tyrosine kinase inhibitors were shown to be able to partially block epidermal growth factor (EGF)-stimulated GPI hydrolysis (23Clemente R. Jones D.R. Ochoa P. Romero G. Mato J.M. Varela-Nieto I. Cell. Signal. 1995; 7: 411-421Crossref PubMed Scopus (20) Google Scholar), and hydrolysis of GPI in response to insulin is reduced in cells bearing kinase-deficient insulin receptors (24Villalba M. Alvarez J.F. Russell D.S. Mato J.M. Rosen O.M. Growth Factors. 1990; 2: 91-97Crossref PubMed Scopus (26) Google Scholar, 25Suzuki S. Taneda Y. Hirai S. Yamamoto-Honda R. Toyota T. Diabetes. 1992; 41: 1373-1379Crossref PubMed Scopus (17) Google Scholar). The nature of the phospholipase C (PLC) responsible for hormone-induced GPI hydrolysis is still unknown. Mammalian GPI-PLC was partially purified from rat liver membranes (26Fox J.A. Soliz N.M. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2663-2667Crossref PubMed Scopus (107) Google Scholar) but has not yet been cloned. PI-PLC from Bacillus cereus was shown to facilitate the release of some PI-glycan-ethanolamine-anchored proteins as PI-PLC isolated from trypanosomes (27Hereld D. Krakow J.L. Bangs J.D. Hart G.W. Englund P.T. J. Biol. Chem. 1986; 261: 13813-13819Abstract Full Text PDF PubMed Google Scholar) and rat liver membranes (26Fox J.A. Soliz N.M. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2663-2667Crossref PubMed Scopus (107) Google Scholar, 28Kuppe A. Evans L.M. McMillen D.A. Griffith O.H. J. Bacteriol. 1989; 171: 6077-6083Crossref PubMed Google Scholar). Among eukaryote PI-PLC isoforms, only the γ1 and γ2 types contain SH2 and SH3 domains and become potential candidates for binding to phosphotyrosine residues during Epo receptor activation (29Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 30Pawson T. Gish G.D. Cell. 1992; 71: 359-362Abstract Full Text PDF PubMed Scopus (796) Google Scholar, 31Schlessinger J. Curr. Opin. Genet. & Dev. 1994; 4: 25-30Crossref PubMed Scopus (400) Google Scholar). Epo activation of a PLC was suggested in erythroid progenitor cells (32Mason-Garcia M. Clejan S. Tou J.-S. Beckman B.S. Am. J. Physiol. 1992; 262: C1197-C1203Crossref PubMed Google Scholar), and PLC-γ1 activation was evidenced in UT7 cells (33Ren H.-Y. Komatsu N. Shimizu R. Okada K. Miura Y. J. Biol. Chem. 1994; 269: 19633-19638Abstract Full Text PDF PubMed Google Scholar), but Epo activation of PLC-γ2has never been described, whereas PLC-γ2 activation by macrophage-colony-stimulating factor has been described in FDC-P1 cells (34Bourette R.P. Myles G.M. Choi J.-L. Rohrschneider L.R. EMBO J. 1997; 16: 5880-5893Crossref PubMed Scopus (91) Google Scholar). In this report, we show that erythropoietin induces a rapid and transient hydrolysis of GPI and tyrosine phosphorylation of PLC-γ2. Both effects require the presence of the same tyrosine residues on the intracellular domain of Epo receptor. We show that Epo-activated PLC-γ2 is able to hydrolyze purified [3H]GPI. Our results suggest that tyrosine Y1(Tyr343), Y2 (Tyr401), Y7, and/or Y8(Tyr464 and/or Tyr479) are implicated in Epo-induced GPI hydrolysis and tyrosine phosphorylation of PLC-γ2, whereas tyrosines Y3 and/or Y4 (Tyr429 and/or Tyr431) seem to be involved in Epo-induced inhibition of GPI hydrolysis. Thus, our results strongly suggest that Epo regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-γ2activation. Purified recombinant human erythropoietin (specific activity of 120,000 units/mg) was from Roche Molecular Biochemicals. [1-3H]Ethan-1-ol-2-amine hydrochloride (26 Ci/mmol) and d-[6-3H]glucosamine hydrochloride (33 Ci/mmol) were from Amersham Pharmacia Biotech. Anti-PLC-γ2 antibodies (catalog number (Q-20) sc-407) were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphotyrosine antibodies (4G10) (catalog number 17-123) and anti-JAK2 antibodies (catalog number 06-255) were from Upstate Biotechnology, Inc. ECL substrate solution was from Amersham Pharmacia Biotech. α-MEM, Iscove Dulbecco's medium, and fetal calf serum were from Life Technologies, Inc. All other reagents were purchased from Sigma. The intracellular domain of the murine Epo receptor contains 8 tyrosines as follows: Tyr343(Y1), Tyr401 (Y2), Tyr429 (Y3), Tyr431(Y4), Tyr443 (Y5), Tyr460 (Y6), Tyr464(Y7), and Tyr479 (Y8) of the mature protein. Epo-R mutants were constructed as described previously (35Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar). A schematic representation of Epo-R wild type (WT) and mutants is done in Fig. 1. The Y1–2F3–4Y5–8 Epo-R mutant was kindly provided by Dr. U. Klingmüller (Zentrum fur Molekulare Biologie, Universitat Heidelberg, Germany). FDC-P1 cells were maintained in α-minimum essential medium (α-MEM) containing 10% fetal calf serum and 3% WEHI conditioned media as a source of interleukin-3. Stable transfections of murine Epo-R cDNA into FDC-P1 cells were done as described previously (35Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar). After transfection, Epo-sensitive cells were maintained in α-MEM containing 10% fetal calf serum and 2 units/ml Epo. Cells were grown with 3% WEHI as source of growth factor and metabolically labeled with [3H]ethanolamine (1.5 μCi/ml) or [3H]glucosamine (5 μCi/ml) for 20 h prior to assay. Four hours before the end of this period, cells were starved of growth factor in α-MEM containing 1% (v/v) of fetal calf serum. Cells were then washed twice with α medium, 25 mm HEPES, and aliquots of 10–20 × 106 cells were preincubated at 37 °C for 15 min before stimulation with 5 units/ml Epo for 0–30 min. Incubations were stopped with HClO4 (5% v/v). After centrifugation at 1200 × g, pellets and supernatants were stored at −20 °C before GPI and IPG quantitations. Lipids were extracted from the resultant pellets and analyzed by sequential acid/base silica gel thin layer chromatography (TLC, Silica Gel 60, Merck) as described previously (14Mato J.M. Kelly K.L. Abler A. Jarett L. J. Biol. Chem. 1987; 262: 2131-2137Abstract Full Text PDF PubMed Google Scholar) or by HPTLC (Silica Gel 60 aluminum-backed HPTLC plates, Merck) developed in chloroform/methanol/NH4OH/water (45:45:3.5:10, v/v). HPTLC plates were sprayed with EN3HANCE (NEN Life Science Products) and fluorographed on Kodak XAR-5 film. To quantitate incorporation of radiolabel into GPI, HPTLC plates were either scanned before fluorography on a Berthold LB 2821 automatic TLC linear analyzer, or 1-cm bands of TLC were scraped and their radioactivity content estimated by scintillation counting. IPG levels from perchlorate supernatants or from the aqueous phase obtained after GPI hydrolysis were determined by chromatography on a Dowex AG 1-X8 (200–400 mesh) column as described previously (13Saltiel A.R. Cuatrecasas P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5793-5797Crossref PubMed Scopus (226) Google Scholar, 21Devemy E. Billat C. Sartelet H. Martiny L. Haye B. Cell. Signal. 1994; 6: 523-529Crossref PubMed Scopus (9) Google Scholar). A sample of [3H]GPI from WT Epo-R FDC-P1 cells recovered after the basic thin layer chromatography was incubated in 25 mm sodium acetate, 0.16 msodium nitrite, pH 3.5, for 5 h at 37 °C (9Ferguson M.A. Williams A.F. Annu. Rev. Biochem. 1988; 57: 285-320Crossref PubMed Scopus (953) Google Scholar) or with 1 unit/ml PI-PLC from B. cereus in 25 mm HEPES, pH 7.4, for 2 h at 37 °C (36Gaulton G.N. Kelly K.L. Pawlowski J. Mato J.M. Jarett L. Cell. 1988; 53: 963-970Abstract Full Text PDF PubMed Scopus (55) Google Scholar). Controls were done by incubating [3H]GPI with buffer alone. Reaction was terminated by the addition of chloroform/methanol (1:2), and the amount of radioactivity released in the aqueous phase after lipid extraction was determined. Results were expressed as the percentage of recovered radioactivity. Hydrolysis of purified [3H]GPI from WT FDC-P1 cells by mammalian PLC-γ2 was determined by incubation of Epo-activated PLC-γ2 from WT FDC-P1 cells bound on 2.5 mg of protein G beads or with PLC-γ2 from FDC-P1 ZERO obtained from a PLC-γ2 immunoprecipitation with 15 × 106 cells, in 250 μl of HEPES 25 mm, pH 7.4, for 2 h at 37 °C. Control was done by incubation of [3H]GPI with 2.5 mg of protein G beads in 250 μl of HEPES 25 mm, pH 7.4, for 2 h at 37 °C. Lipids were extracted with the same protocol as above. Then, [3H]GPI contained in the organic phase was reanalyzed by HPTLC and quantitated on Berthold analyzer before fluorography. IPG contained in the aqueous phase was quantitated as described previously (13Saltiel A.R. Cuatrecasas P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5793-5797Crossref PubMed Scopus (226) Google Scholar, 21Devemy E. Billat C. Sartelet H. Martiny L. Haye B. Cell. Signal. 1994; 6: 523-529Crossref PubMed Scopus (9) Google Scholar). A sample of presumed GPI labeled with [3H]glucosamine from WT Epo-R FDC-P1 cells recovered after the basic thin layer chromatography was hydrolyzed with nitrous acid. Then the deaminated glycan was reduced with sodium borotritide and subjected to solvolysis in 0.5m methanolic HCl for 12 h at 80 °C as described previously (37Ralton J.E. McConville M.J. J. Biol. Chem. 1998; 273: 4245-4257Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Samples were analyzed on Silica Gel 60 HPTLC plates developed for 10 cm in 1-propanol/acetone/water (9:6:5, v/v) (38Schneider P. Ralton J.E. McConville M.J. Fergusson M.A.J. Anal. Biochem. 1993; 210: 106-112Crossref PubMed Scopus (27) Google Scholar) and were scanned on a Berthold analyzer. 2,5-[3H]Anhydromannitol control was prepared by treating [3H]glucosamine as above. Starved FDC-P1 cells expressing the WT and mutant Epo-R were incubated with or without Epo (5 units/ml) for 1–10 min at 37 °C. Cells were washed once with phosphate-buffered saline and solubilized at 2 × 107cells/ml with 1% Brij 98 in solubilized buffer (10 mmTris-HCl, 5 mm EDTA, 150 mm NaCl, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mmNa2VO4, pH 7.4). Immunoprecipitations were done as described previously (5Dusanter-Fourt I. Casadevall N. Lacombe C. Muller O. Billat C. Fischer S. Mayeux P. J. Biol. Chem. 1992; 267: 10670-10675Abstract Full Text PDF PubMed Google Scholar) with either anti-PLC-γ2 or anti-JAK2 antibodies. Following SDS-polyacrylamide gel electrophoresis using 7.5% polyacrylamide gels, proteins were electrophoretically transferred to protean nitrocellulose membrane. Blots were blocked, incubated with anti-PY antibodies and then with horseradish peroxidase-conjugated second antibody before adding ECL substrate solution and exposing to Kodak X-Omat film. When necessary, blots were stripped by incubating the membrane for 30 min at 55 °C in 62.5 mm Tris-HCl, pH 6.7, 100 mmβ-mercaptoethanol, 2% SDS. Then the blot was washed twice with 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Tween 20 and reprobed as described previously. GPI identification was done with cells metabolically labeled for 20 h with [3H]ethanolamine, a radiolabeled precursor of GPI. Polar lipids were then extracted and resolved by sequential acid/base thin layer chromatography (TLC) or by HPTLC. During the first acid TLC, GPI did not migrate and was clearly separated from phosphatidylethanolamine which migrated at 9 cm (not shown). The material around the origin (2 cm) was recovered and analyzed on a second TLC or on HPTLC which was run in an alkaline solvant system. As shown in Fig. 2,A–C, a single peak of [3H]glycolipidic fraction was recovered between phosphatidylcholine (PC) and phosphatidylinositol monophosphate (PIP). The same fraction was also labeled in extracts from WT Epo-R FDC-P1 cells incubated with [3H]inositol or [3H]glucosamine instead of [3H]ethanolamine (not shown). Characterization of GPI was confirmed by testing the sensitivity of the radiolabeled product to several GPI-hydrolyzing conditions. The recovered [3H]ethanolamine glycolipid fraction was first treated with nitrous acid that hydrolyzes the glycosidic bond between inositol and glucosamine. After deamination by nitrous acid, about 65% of the labeled material was recovered in the aqueous phase, and 10.5% of the radioactivity was recovered in the aqueous phase in control incubations. Moreover, we have shown 2,5-[3H]anhydromannitol production after having deaminated/reduced and methanolyzed the recovered [3H]glucosamine glycolipid fraction (Fig.3, A–C). We further investigated the cleavage of the purified recovered3H-labeled lipidic peak from WT Epo receptor-transfected FDC-P1 cells by PI-specific phospholipase C (PI-PLC) from B. cereus. After treatment, about 21% of the labeled material was recovered in the aqueous phase versus control 5%. These values are in good agreement with previously published results showing the sensitivity of GPI from various tissues to B. cereusPI-PLC (24Villalba M. Alvarez J.F. Russell D.S. Mato J.M. Rosen O.M. Growth Factors. 1990; 2: 91-97Crossref PubMed Scopus (26) Google Scholar, 39Eardley D.D. Koshland M.E. Science. 1991; 251: 78-81Crossref PubMed Scopus (103) Google Scholar). Altogether, our results show that the lipidic products extracted from FDC-P1 WT Epo receptor-transfected cells and migrating between PIP and PC corresponded to GPI. GPI hydrolysis was assessed by measuring both GPI or inositol phosphate glycan (IPG) levels (its hydrolysis product) from cells metabolically labeled with [3H]ethanolamine. Epo (5 units/ml) induced a rapid and transient GPI hydrolysis with a parallel IPG release within 1 min in FDC-P1 cells transfected with WT Epo-R. Then the IPG level rapidly decreased and returned to control values after 30 min of Epo exposure. No GPI hydrolysis was detected after Epo stimulation of non-transfected FDC-P1 cells (Fig.4). To assay the possible involvement of PLC-γ2 in Epo-induced GPI hydrolysis, we first investigated its tyrosine phosphorylation in WT Epo-R FDC-P1 cells upon Epo stimulation. As shown in Fig.5 A, Epo induced the tyrosine phosphorylation of a 142-kDa protein immunoprecipitated with anti-PLC-γ2 antibodies. This effect was maximal after 1–2 min of Epo stimulation and then decreased after 5 min of stimulation. Reprobing the blot with anti-PLC-γ2antibodies confirmed that the 142-kDa protein was indeed PLC-γ2. Epo-induced PLC-γ2 tyrosine phosphorylation was dose-dependent as shown in Fig.5 B and the Epo effect was maximal at 0.1 units/ml. In order to know whether tyrosine residues of the Epo-R play a significant role in Epo-induced PLC-γ2 tyrosine phosphorylation, we used interleukin-3-dependent FDC-P1 cells transfected with the wild-type (WT) Epo-R and with Epo-R devoid of tyrosine residue (ZERO) (Fig. 1). Then, cells were stimulated for 1 min with Epo 5 units/ml. Cellular extracts were immunoprecipitated with anti-PLC-γ2 antibodies and analyzed by Western blotting. As shown in Fig. 6, in contrast to FDC-P1 cells expressing WT Epo-R, Epo did not induce the tyrosine phosphorylation of PLC-γ2 in FDC-P1 cells ZERO, suggesting that the tyrosine residues of the intracellular domain of the Epo-R were required for Epo-induced tyrosine phosphorylation of PLC-γ2. Reprobing the blot with anti-PLC-γ2antibodies showed that the same amounts of PLC-γ2 had been immunoprecipitated from each sample. Moreover, JAK2 immunoprecipitations carried on the same cellular extracts followed by an anti-phosphotyrosine blot showed that these cells were stimulated by Epo. PLC-γ2 immunoprecipitations from FDC-P1 cells expressing various Epo-R mutants were achieved after Epo stimulation. As shown in Fig. 7, PLC-γ2 tyrosine phosphorylation was found to occur in mutants Y1, F1Y2, F1Y5–8, and in Y1–2F3–4Y5–8 upon Epo stimulation. It was more important in Y1–2F3–4Y5–8 FDC-P1 cells than in WT FDC-P1 cells. In F1Y3–4 and Y1–6 FDC-P1 cells, an inhibition of PLC-γ2tyrosine phosphorylation was seen after Epo stimulation. It was probably due to the presence of Tyr428–431 which inhibited PLC-γ2 phosphorylation. This hypothesis was confirmed by the enhanced PLC-γ2 activation observed in Epo-stimulated Y1–2F3–4Y5–8 FDC-P1 cells. Altogether, our results suggest that Tyr343, Tyr401, and Tyr464 and/or Tyr479are involved in PLC-γ2 activation, whereas Tyr429 and/or Tyr431 reduced this PLC-γ2 activation. Treatment of recovered [3H]GPI with Epo-activated PLC-γ2bound on protein G beads from immunoprecipitates of WT or ZERO-FDC-P1 cells resulted in an hydrolysis of about 50.2 and 9.8%, respectively (Fig. 8 A). Analysis of the resultant products showed a greater IPG content released in the aqueous phase in samples treated with Epo-activated PLC-γ2 from WT FDC-P1 cells than from ZERO FDC-P1 cells (Fig. 8 A). To the contrary, analysis of [3H]GPI remaining in the organic phase is greater in sample hydrolyzed with Epo-activated PLC-γ2 from ZERO FDC-P1 than from WT FDC-P1 cells (Fig.8, B and C). This demonstrated that PLC-γ2 tyrosine phosphorylation is required for GPI hydrolysis. Moreover, as described previously, Epo induced a rapid IPG release within 1 min in FDC-P1 cells transfected with WT Epo-R, and no change of IPG levels was observed in FDC-P1 cells ZERO after Epo stimulation (Fig. 9). IPG levels were also determined upon Epo stimulation (5 units/ml, 1 min) in metabolically labeled FDC-P1 cells expressing various Epo-R mutants. These experiments show an excellent correlation between Epo-induced PLC-γ2 tyrosine phosphorylation and Epo-induced GPI hydrolysis. Indeed, after Epo stimulation, IPG levels were increased in Y1 and in F1Y2 mutants which only retains a single tyrosine at positions 343 and 401, respectively. These cells differ from FDC-P1 ZERO by only one tyrosine. Nevertheless, IPG level in Y1 or Y2 mutants was about half the level obtained in WT-Epo-R FDC-P1 transfected cells. IPG level was greatly increased after Epo stimulation in F1Y5–8 mutant, but IPG increase was also lower than in WT-Epo-R FDC-P1 transfected cells. In Y1–2F3–4Y5–8, IPG level obtained after Epo stimulation was greater than in WT-Epo-R FDC-P1-transfected cells. In F1Y3–4 and Y1–6mutants, Epo induced an inhibition of IPG basal level (Fig. 9).Figure 9Epo-induced GPI hydrolysis in FDC-P1 cells transfected with various Epo-R mutants. FDC-P1 cells expressing either the wild-type Epo receptor or Epo-R mutants were labeled with [3H]ethanolamine (1.5 μCi/ml) for 20 h, deprived of growth factor for 4 h, and then stimulated for 1 min with 5 units/ml Epo (+) or with vehicle alone (−). IPG release was determined as described under “Experimental Procedures” in WT cells and Epo-R mutants. Data are the means of three independent experiments.View Large Image Figure ViewerDownload (PPT) Our results show that FDC-P1 cells contain GPI molecules that are hydrolyzed to produce IPG after Epo stimulation. After TLC or HPTLC analysis, GPI migrated as a single peak between PC and PIP as described previously in Epo-sensitive cells (21Devemy E. Billat C. Sartelet H. Martiny L. Haye B. Cell. Signal. 1994; 6: 523-529Crossref PubMed Scopus (9) Google Scholar) and in other cells (14Mato J.M. Kelly K.L. Abler A. Jarett L. J. Biol. Chem. 1987; 262: 2131-2137Abstract Full Text PDF PubMed Google Scholar, 37Ralton J.E. McConville M.J. J. Biol. Chem. 1998; 273: 4245-4257Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 36Gaulton G.N. Kelly K.L. Pawlowski J. Mato J.M. Jarett L. Cell. 1988; 53: 963-970Abstract Full Text PDF PubMed Scopus (55) Google Scholar). The material of this peak exhibited all the characteristics of GPI. Indeed, it could be labeled with [3H]inositol and [3H]glucosamine in addition to [3H]ethanolamine. Moreover, the GPI structure of this lipid was confirmed by its partial sensitivity to PI-PLC from B. cereus and nitrous deamination of the free amino group on glucosamine. Presence of glucosamine in 3H-recovered glycolipidic fraction was also confirmed by 2,5-anhydromannitol obtained after deamination/reduction and methanolysis of this molecule. Altogether these results indicate that the labeled glycolipid fraction corresponds to GPI. It has been previously shown that GPI displays various degrees of sensitivity to bacterial PI-PLCs cleavage (14Mato J.M. Kelly K.L. Abler A. Jarett L. J. Biol. Chem. 1987; 262: 2131-2137Abstract Full Text PDF PubMed Google Scholar, 19Merida I. Pratt J.C. Gaulton G.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9421-9425Crossref PubMed Scopus (71) Google Scholar,23Clemente R. Jones D.R. Ochoa P. Romero G. Mato J.M. Varela-Nieto I. Cell. Signal. 1995; 7: 411-421Crossref PubMed Scopus (20) Google Scholar, 24Villalba M. Alvarez J.F. Russell D.S. Mato J.M. Rosen O.M. Growth Factors. 1990; 2: 91-97Crossref PubMed Scopus (26) Google Scholar, 36Gaulton G.N. Kelly K.L. Pawlowski J. Mato J.M. Jarett L. Cell. 1988; 53: 963-970Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 39Eardley D.D. Koshland M.E. Science. 1991; 251: 78-81Crossref PubMed Scopus (103) Google Scholar). Epo stimulation of Epo receptor-transfected FDC-P1 cells metabolically labeled with [3H]glucosamine induced the same level of GPI hydrolysis with release of water-soluble IPG forms. In contrast to GPI molecules involved in insulin signal transduction in H35 hepatoma cells which does not contain ethanolamine (14Mato J.M. Kelly K.L. Abler A. Jarett L. J. Biol. Chem. 1987; 262: 2131-2137Abstract Full Text PDF PubMed Google Scholar, 40Mato J.M. Kelly K.L. Abler A. Jarett L. Corkey B.E. Cashel J.A. Zopf D. Biochem. Biophys. Res. Commun. 1987; 146: 764-770Crossref PubMed Scopus (109) Google Scholar), our results show that GPI molecules containing ethanolamine are involved in Epo signal transduction. Bacterial PI-PLCs can partially mimic the effects of the extracellular ligand on GPI through the generation of diacylglycerol and IPG (13Saltiel A.R. Cuatrecasas P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5793-5797Crossref PubMed Scopus (226) Google Scholar, 41Macaulay S.L. Larkins R.G. Cell. Signal. 1990; 2: 9-19Crossref PubMed Scopus (14) Google Scholar, 42Vila M.C. Milligan G. Standaert M.L. Farese R.V. Biochemistry. 1990; 29: 8735-8740Crossref PubMed Scopus (61) Google Scholar), suggesting that the formation of IPG from GPI is due to the activation of a phospholipase C. A GPI-specific PLC has been purified and cloned from T. brucei (43Bulow R. Overath P. J. Biol. Chem. 1986; 261: 11918-11923Abstract Full Text PDF PubMed Google Scholar) and peanuts (44Bütikofer P. Brodbeck U. J. Biol. Chem. 1993; 268: 17794-17802Abstract Full Text PDF PubMed Google Scholar). It is able to hydrolyze GPI-anchored proteins and phosphatidylinositol. However, this enzyme is not active on free GPI phospholipids (45Jones D.R. Avila M.A. Sanz C. Varela-Nieto I. Biochem. Biophys. Res. Commun. 1997; 233: 432-437Crossref PubMed Scopus (33) Google Scholar). A mammalian GPI-PLC has been partially purified from rat liver membranes (26Fox J.A. Soliz N.M. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2663-2667Crossref PubMed Scopus (107) Google Scholar) but has not been cloned. The presence of two different GPI-PLC activities has also been reported in brain membranes (46Fouchier F. Baltz T. Rougon G. Biochem. J. 1990; 269: 321-327Crossref PubMed Scopus (33) Google Scholar). Our results strongly suggest that Epo induced the hydrolysis of GPI through the activation of PLC-γ2. Indeed, PLC-γ2immunoprecipitated from Epo-stimulated FDC-P1 cells was able to hydrolyze GPI molecules purified from these cells. The time course of Epo-induced PLC-γ2 tyrosine phosphorylation displays a good parallelism with the kinetics of Epo-induced GPI hydrolysis. Moreover, we observed a full correlation between the ability for mutated Epo receptors to activate PLC-γ2 and to induce IPG release. PLC-γ2 is expressed mainly in hematopoietic cells (47Noh D.-Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-114Crossref PubMed Scopus (255) Google Scholar). In FDC-P1 cells, macrophage-colony-stimulating factor induces activation of PLC-γ2 and its tyrosine phosphorylation (34Bourette R.P. Myles G.M. Choi J.-L. Rohrschneider L.R. EMBO J. 1997; 16: 5880-5893Crossref PubMed Scopus (91) Google Scholar). Here, we observed that Epo stimulation of WT FDC-P1 cells induced a rapid tyrosine phosphorylation and dephosphorylation of PLC-γ2 since maximal tyrosine phosphorylation was detected between 1 and 2 min and decreased after this time. Although Epo-induced PLC-γ2 has not been previously reported, it has been shown that Epo activates PLC-γ1 in the UT-7 human cell line (33Ren H.-Y. Komatsu N. Shimizu R. Okada K. Miura Y. J. Biol. Chem. 1994; 269: 19633-19638Abstract Full Text PDF PubMed Google Scholar). The mechanism of PLC-γ1 activation by Epo was not reported. Our results indicate that the tyrosine residues of the Epo receptor are involved in the activation of PLC-γ2. Indeed, Epo receptors devoid of tyrosine residue on their intracellular domain did not activate PLC-γ2. In contrast, Epo induced the activation of PLC-γ2 in FDC-P1 cells expressing Epo receptors with either the first (Tyr343) or second tyrosine (Tyr401) residue albeit to a reduced level compared with cells expressing normal Epo receptors. Nevertheless, our results indicate that the main activation site for PLC-γ2 is located in the C-terminal part of the receptor, suggesting that the seventh or eighth tyrosine residue could be involved in this activation. Yet, we could not show that PLC-γ2 is associated with the activated Epo-R and coimmunoprecipitate with it as for PLC-γ1 in UT7/Epo cells (33Ren H.-Y. Komatsu N. Shimizu R. Okada K. Miura Y. J. Biol. Chem. 1994; 269: 19633-19638Abstract Full Text PDF PubMed Google Scholar). This was probably due to the rapid dissociation of the SH2 domains of PLC-γ2from the phosphorylated tyrosine residues of the Epo receptor (48Rhee S.G. Trends Biochem. Sci. 1991; 16: 297-301Abstract Full Text PDF PubMed Scopus (193) Google Scholar). Previous results have indicated that an intact tyrosine kinase activity is required for GPI hydrolysis in response to growth factors in Chinese hamster ovary cells carrying normal human insulin receptors. These cells hydrolyze up to 70% of their GPI within 2 min after the addition of 0.1 nm insulin, whereas cells expressing a mutant cDNA (Lys-1018 to Ala) that encodes a receptor lacking kinase activity (49Chou C.K. Dull T.J. Russell D.S. Gherzi R. Lebwohl D. Ullrich A. Rosen O.M. J. Biol. Chem. 1987; 262: 1842-1847Abstract Full Text PDF PubMed Google Scholar) hydrolyzes only 20–30% in response to insulin (24Villalba M. Alvarez J.F. Russell D.S. Mato J.M. Rosen O.M. Growth Factors. 1990; 2: 91-97Crossref PubMed Scopus (26) Google Scholar). More recently, Clemente et al. (23Clemente R. Jones D.R. Ochoa P. Romero G. Mato J.M. Varela-Nieto I. Cell. Signal. 1995; 7: 411-421Crossref PubMed Scopus (20) Google Scholar) had indicated that tyrosine kinase inhibitors partially block GPI hydrolysis in parallel to the inhibition of both EGF receptor autophosphorylation and EGF-induced cell proliferation. Our results also indicate that the tyrosine residues Tyr429and/or Tyr431 are involved in deactivation of PLC-γ2 phosphorylation and in GPI hydrolysis inhibition. Indeed, Epo-induced PLC-γ2 tyrosine phosphorylation and GPI hydrolysis in mutants F1Y3–4 are lower than in controls without Epo but are greater in mutants Y1–2F3–4Y5–8 (deprived of Tyr429 and Tyr431) than in WT FDC-P1 cells. Then tyrosines Tyr429 and/or Tyr431 appear to be negative regulators of GPI hydrolysis. The Tyr429 was identified as the protein tyrosine phosphatase SHP1 in the cytoplasmic domain of the Epo-R (50Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (842) Google Scholar). Thus, our results suggest that PLC-γ2 could be a substrate for Epo-activated SHP1. The levels of cell surface expression of Epo-R have been already published for the cell lines used in this study (35Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar) except for the mutant Y1–2F3–4Y5–8 which expressed 880 ± 50 Epo receptors. Most Epo-R mutants used in our experiments have been previously shown to be able to mediate Epo-induced Stat5 activation (35Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar). Moreover, the ZERO Epo-R mutant which did not activate Stat5 (35Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar) mediates Epo-induced JAK2 activation (Fig. 6). These results show that all Epo-R mutants used in our experiments were activated by Epo. Although the levels of the cell surface expression are not identical, it should be noted that in WT FDC-P1 cells which expressed about 2100 Epo receptors or in Y1–2F3–4 Y5–8, we could detect a good level of phosphorylation of PLC-γ2 and GPI hydrolysis, whereas in mutant ZERO (without tyrosine residue) or in F1Y3–4 which expressed about 5700 and 7700 Epo receptors, respectively, we could not observe any PLC-γ2activation nor any GPI hydrolysis. From our results, we conclude that the levels of both Epo receptors are not correlated to GPI hydrolysis. A low level of PLC-γ2 tyrosine phosphorylation in immunoprecipitates of control cells was often observed. Nevertheless, no tyrosine phosphorylation of the Epo receptor was observed in these cells (not shown). This low level of PLC-γ2 could be attributed to unidentified molecules present in the fetal calf serum required to maintain cell survival during Epo starvation. The GPI/IPG pathway in Epo signal transduction has already been described in rat erythroid progenitor cells. Erythropoietin-stimulation of these cells increases GPI hydrolysis with a parallel increase in IPG levels, and purified erythroid rat IPG partially mimicked Epo-proliferative effects on erythroid colonies (21Devemy E. Billat C. Sartelet H. Martiny L. Haye B. Cell. Signal. 1994; 6: 523-529Crossref PubMed Scopus (9) Google Scholar). We have also shown that IPG induced Raf-1 and mitogen-activated protein kinase (p44 form) activation, and we also suggested that protein kinase C could be involved in this activation (51Devemy E. Billat C. Haye B. Cell. Signal. 1997; 9: 41-46Crossref PubMed Scopus (30) Google Scholar). Data presented here suggest a direct link between Epo receptor tyrosine phosphorylation leading to activation of PLC-γ2 and GPI hydrolysis and provide complementary information on the GPI/IPG pathway in Epo-proliferative effects. We thanks Marie-Line Sowa for excellent technical work." @default.
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• W2092583849 title "Erythropoietin Induces Glycosylphosphatidylinositol Hydrolysis" @default.
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