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• W1999722257 abstract "The vav proto-oncogene product (Vav), which is specifically expressed in hematopoietic cells, contains multiple structural motifs commonly used by intracellular signaling molecules. Although a variety of stimuli including erythropoietin (Epo) have been shown to tyrosine phosphorylate Vav, little is known about the Vav signal transduction pathway. Here, we have investigated the role of Vav in the Epo signaling pathway by characterizing its interaction with other proteins, using the human Epo-responsive cell line, F-36P. Immunoprecipitation and immunoblot analyses have demonstrated that Vav was associated with the Epo receptor (EpoR) in an Epo-independent manner and was tyrosine-phosphorylated after Epo stimulation. Furthermore, two phosphotyrosine proteins (pp70 and pp100) co-immunoprecipitated with the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) (p85) were identified as EpoR and Vav, respectively. The interaction between Vav and p85 was shown to be mediated through the SH2 domains of p85 by an in vitro binding assay and confirmed by the presence of in vitro PI3-kinase activity associated with Vav. Treatment of the cells with antisense-vav and -p85 abrogated Epo-induced cell proliferation and PI3-kinase activity. Finally, we found that JAK2 was associated with Vav in vivo and that Vav could be tyrosine-phosphorylated by activated JAK2 in vitro. These results suggest the possible role of JAK2 for tyrosine phosphorylation of Vav and involvement of Vav and PI3-kinase in Epo-induced proliferative signals. The vav proto-oncogene product (Vav), which is specifically expressed in hematopoietic cells, contains multiple structural motifs commonly used by intracellular signaling molecules. Although a variety of stimuli including erythropoietin (Epo) have been shown to tyrosine phosphorylate Vav, little is known about the Vav signal transduction pathway. Here, we have investigated the role of Vav in the Epo signaling pathway by characterizing its interaction with other proteins, using the human Epo-responsive cell line, F-36P. Immunoprecipitation and immunoblot analyses have demonstrated that Vav was associated with the Epo receptor (EpoR) in an Epo-independent manner and was tyrosine-phosphorylated after Epo stimulation. Furthermore, two phosphotyrosine proteins (pp70 and pp100) co-immunoprecipitated with the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) (p85) were identified as EpoR and Vav, respectively. The interaction between Vav and p85 was shown to be mediated through the SH2 domains of p85 by an in vitro binding assay and confirmed by the presence of in vitro PI3-kinase activity associated with Vav. Treatment of the cells with antisense-vav and -p85 abrogated Epo-induced cell proliferation and PI3-kinase activity. Finally, we found that JAK2 was associated with Vav in vivo and that Vav could be tyrosine-phosphorylated by activated JAK2 in vitro. These results suggest the possible role of JAK2 for tyrosine phosphorylation of Vav and involvement of Vav and PI3-kinase in Epo-induced proliferative signals. Erythropoietin (Epo), 1The abbreviations used are: EpoerythropoietinEpoRerythropoietin receptorPI3-kinasephosphatidylinositol3-kinase JAKJanus protein tyrosine kinaseSHSrc homologyVavthe vav proto-oncogene productTyr(P)phosphotyrosineODNoligodeoxynucleotideGSTglutathione S-transferasePTKprotein-tyrosine kinaseMAPKmitogen-activated protein kinaseGEFguanine nucleotide exchange factorGM-CSFgranulocyte macrophage-colony stimulating factorPAGEpolyacrylamide gel electrophoresisPIphosphatidylinositolILinterleukinASantisenseScomplementary senseSTATsignal transducers and activators of transcription a 34-kDa glycoprotein hormone, uniquely regulates the proliferation and differentiation of cells committed to the erythroid lineage (1Krantz S.B. Blood. 1991; 77: 419-434Crossref PubMed Google Scholar). The interaction of Epo with the erythropoietin receptor (EpoR) activates the EpoR itself and several cellular proteins, which lead to a cascade of biochemical events (2D'Andrea A.D. Lodish H.F. Wong G.G. Cell. 1989; 57: 277-285Abstract Full Text PDF PubMed Scopus (553) Google Scholar, 3Jones S.S. D'Andrea A.D. Haines L.L. Wong G.G. Blood. 1990; 76: 31-35Crossref PubMed Google Scholar, 4Youssoufian H. Longmore G. Neumann D. Yoshimura A. Lodish H.F. Blood. 1993; 81: 2223-2236Crossref PubMed Google Scholar). Although the EpoR itself does not contain a tyrosine kinase catalytic domain in its cytoplasmic domain, Epo rapidly induces the tyrosine phosphorylation of several cellular proteins such as Janus protein tyrosine kinase (JAK) 2 (5Witthuhn B.A. Quelle F.W. Silvennoinen O. Yi T. Tang B. Miura O. Ihre J.N. Cell. 1993; 74: 227-236Abstract Full Text PDF PubMed Scopus (1008) Google Scholar, 6Miura O. Nakamura N. Quelle F.W. Witthuhn B.A. Ihre J.N. Aoki N. Blood. 1994; 84: 1501-1507Crossref PubMed Google Scholar) and Shc (7Damen J.E. Liu L. Cutler R.L. Krystal G. Blood. 1993; 82: 2296-2303Crossref PubMed Google Scholar), as well as its own receptor (8Miura O. D'Andrea A. Kabat D. Ihre J.N. Mol. Cell. Biol. 1991; 11: 4895-4902Crossref PubMed Scopus (184) Google Scholar, 9Yoshimura A. Lodish H.F. Mol. Cell. Biol. 1992; 12: 706-715Crossref PubMed Scopus (92) Google Scholar, 10Dusanter-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). Furthermore, a number of other biochemical events have been associated with activation of the EpoR, including an increase in the activities of phosphatidylinositol (PI) 3-kinase (11Damen J.E. Mui A.L.-F. Puil L. Pawson T. Krystal G. Blood. 1993; 81: 3204-3210Crossref PubMed Google Scholar, 12Miura O. Nakamura N. Ihre J.N. Aoki N. J. Biol. Chem. 1994; 269: 614-620Abstract Full Text PDF PubMed Google Scholar), phospholipase C-γ1 (13Ren H.-Y. Komatsu N. Shimizu R. Okada K. Miura Y. J. Biol. Chem. 1994; 269: 19633-19638Abstract Full Text PDF PubMed Google Scholar), p21ras (14Torti M. Marti K.B. Altschuler D. Yamamoto K. Lapetina E.G. J. Biol. Chem. 1992; 267: 8293-8298Abstract Full Text PDF PubMed Google Scholar), Raf-1 (15Carroll M.P. Spivak J.L. McMahon M. Weich N. Rapp U.R. May W.S. J. Biol. Chem. 1991; 266: 14964-14969Abstract Full Text PDF PubMed Google Scholar), and mitogen-activated protein kinase (MAPK) (16Miura Y. Miura O. Ihre J.N. Aoki N. J. Biol. Chem. 1994; 269: 29962-29969Abstract Full Text PDF PubMed Google Scholar). Although the tyrosine phosphorylation of these intracellular proteins correlates with Epo-induced mitogenesis (8Miura O. D'Andrea A. Kabat D. Ihre J.N. Mol. Cell. Biol. 1991; 11: 4895-4902Crossref PubMed Scopus (184) Google Scholar), little is known about the sequence of these molecules and their importance to Epo-induced growth and differentiation in the target cells. erythropoietin erythropoietin receptor phosphatidylinositol Janus protein tyrosine kinase Src homology the vav proto-oncogene product phosphotyrosine oligodeoxynucleotide glutathione S-transferase protein-tyrosine kinase mitogen-activated protein kinase guanine nucleotide exchange factor granulocyte macrophage-colony stimulating factor polyacrylamide gel electrophoresis phosphatidylinositol interleukin antisense complementary sense signal transducers and activators of transcription The vav proto-oncogene product is specifically expressed in hematopoietic cells and encodes a 95-kDa protein (Vav) that contains multiple structural motifs commonly found in intracellular signaling molecules, including Src homology (SH) 2, SH3, and pleckstrin homology domains as well as a helix-loop-helix domain, a leucine zipper-like domain, and a Rho guanine nucleotide exchange factor (GEF) homology domain (17Katzav S. Martin-Zanca D. Barbacid M. EMBO J. 1989; 8: 2283-2290Crossref PubMed Scopus (426) Google Scholar, 18Adams J.M. Houston H. Allen J. Lints T. Harvey R. Oncogene. 1992; 7: 611-618PubMed Google Scholar, 19Coppola J. Bryant S. Koda T. Conway D. Barbacid M. Cell Growth & Differ. 1991; 2: 95-105PubMed Google Scholar, 20Bustelo X.R. Ledbetter J.A. Barbacid M. Nature. 1992; 356: 68-71Crossref PubMed Scopus (245) Google Scholar, 21Margolis B. Hu P. Katzav S. Li W. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (307) Google Scholar). Vav has been shown to be tyrosine-phosphorylated by cross-linking of the T-cell receptor (21Margolis B. Hu P. Katzav S. Li W. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (307) Google Scholar, 22Gulbins E. Coggeshall K.M. Baier G. Katzav S. Burn P. Altman A. Science. 1993; 260: 822-825Crossref PubMed Scopus (230) Google Scholar), immunoglobulin (Ig) M antigen receptor (23Bustelo X.R. Barbacid M. Science. 1992; 256: 1196-1199Crossref PubMed Scopus (170) Google Scholar), and CD19 (24Weng W.K. Jarvis L. LeBien T.W. J. Biol. Chem. 1994; 269: 32514-32521Abstract Full Text PDF PubMed Google Scholar) and in response to a variety of stimuli including interleukin-2 (IL-2) (25Evans G.A. Howard O.M. Erwin R. Farrar W.L. Biochem. J. 1993; 294: 339-342Crossref PubMed Scopus (52) Google Scholar), IL-3, granulocyte macrophage-colony stimulating factor (GM-CSF) (26Matsuguchi T. Inhorn R.C. Carlesso N. Xu G. Drucker B. Griffin J.D. EMBO J. 1995; 14: 257-265Crossref PubMed Scopus (134) Google Scholar), stem cell factor (27Alai M. Mui A.L. Cutler R.L. Bustelo X.R. Barbacid M. Krystal G. J. Biol. Chem. 1992; 267: 18021-18025Abstract Full Text PDF PubMed Google Scholar), platelet-derived growth factor (20Bustelo X.R. Ledbetter J.A. Barbacid M. Nature. 1992; 356: 68-71Crossref PubMed Scopus (245) Google Scholar), and epidermal growth factor (20Bustelo X.R. Ledbetter J.A. Barbacid M. Nature. 1992; 356: 68-71Crossref PubMed Scopus (245) Google Scholar, 21Margolis B. Hu P. Katzav S. Li W. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (307) Google Scholar). Recently, Epo has been demonstrated to induce tyrosine phosphorylation of Vav, and Vav may be involved in growth signaling from the EpoR (28Miura O. Miura Y. Nakamura N. Quelle F.W. Witthuhn B.A. Ihre J.N. Aoki N. Blood. 1994; 84: 4135-4141Crossref PubMed Google Scholar). However, the potential downstream signaling proteins interacting with Vav in Epo signaling pathway remain unclear. In the present study, we investigated the role of Vav in the Epo signaling pathway by characterizing its interaction with proteins that are known to be involved in Epo signal transduction. We observed a stable association of Vav with the EpoR and the physical interaction between Vav and p85 in response to Epo. We also examined the kinase responsible for the tyrosine phosphorylation of Vav and detected an Epo-induced association of JAK2 with Vav and tyrosine phosphorylation of Vav by JAK2. F-36P (kindly provided by Dr. S. Chiba, University of Tokyo, Japan), a human IL-3 and GM-CSF-dependent cell line, was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and 10 ng/ml IL-3. Anti-JAK2 and EpoR polyclonal antibodies, anti-rat p85 PI3-kinase antiserum, and anti-human Vav monoclonal antibody were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-EpoR antiserum was kindly provided by Dr. O. Miura (Tokyo Medical and Dental University, Japan). Anti-phosphotyrosine (anti-Tyr(P)) monoclonal antibody (PY20) was from ICN Biomedicals, Inc. (Costa Mesa, CA). Anti-p85 monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY). The enhanced chemiluminescence detection kit was obtained from Amersham Corp. Recombinant human IL-3 and Epo were kindly provided from Kirin Brewery (Tokyo, Japan). All other reagents were purchased from commercial sources. Cells (1 × 107) were starved of growth factors for 24 h and then stimulated with or without Epo (50 units/ml) at 37 °C for the indicated periods. The cells were washed and lysed in lysis buffer (1% Nonidet P-40, 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA, 0.1% aprotinin, 1 mm phenylmethylsulfonyl fluoride, and 1 mmNa3VO4). After 15 min of incubation at 4 °C, the insoluble materials were removed by centrifugation for 15 min at 15,000 rpm at 4 °C. The supernatants were incubated with the indicated antibodies or antiserum for 1 h to overnight at 4 °C and immunoprecipitated with Protein A-Sepharose. The immunoprecipitates were washed four times with the lysis buffer and eluted by boiling for 5 min in Laemmli's SDS sample buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, the proteins were electrophoretically transferred onto a nitrocellulose membrane (Hybond-C super; Amersham Corp., UK) using a semi-dry transfer cell (Bio-Rad). The filter was blocked by incubation in TBS buffer (20 mm Tris-HCl, pH 7.6, 150 mm NaCl) containing 2% bovine serum albumin for 1 h at room temperature or overnight at 4 °C. The blots then were incubated for 1 h with an appropriate concentration of the primary antibody in TBS, washed three times for 5 min each with TBS-T (TBS buffer containing 0.1% Tween 20), and probed with a 1:1,000 dilution of biotinylated anti-mouse or anti-rabbit Ig followed by incubation with a 1:5,000 dilution of horseradish peroxidase-conjugated streptavidin (Caltag, South San Francisco, CA). After washing, the blots were visualized by using an enhanced chemiluminescence Western blotting detection system (Amersham Corp.). Short-term proliferative responses to Epo were examined by a [3H]thymidine incorporation assay. The cells (1 × 104) were incubated at 37 °C for 24 h in the presence or absence of 20 units/ml Epo before the addition of 0.25 μCi of [3H]thymidine. The cells were pulsed for 4 h and then harvested onto glass fiber filters. The filter strips were dried, and the amount of radioactivity present on each filter was determined. Agarose-conjugated glutathioneS-transferase (GST) -p85 SH2-N (amino acids 333–428) and SH2-C (amino acids 624–718) were purchased from Upstate Biotechnology Inc. For the in vitro binding assays, the GST fusion proteins were incubated in cell lysates at 4 °C for 1 h. The samples were washed four times with lysis buffer, eluted by boiling in Laemmli's SDS sample buffer, separated by SDS-PAGE, and immunoblotted with anti-Vav antibody. Cells (1 × 107) were stimulated with or without Epo, lysed, and immunoprecipitated with anti-p85, anti-Tyr(P), or anti-Vav antibody as described above. The immunoprecipitates were washed three times with lysis buffer, three times with 50 mm LiCl in 100 mm Tris-HCl, pH 7.4, and twice with TNE buffer (10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm EDTA) containing 100 μm Na3VO4. The immunoprecipitates were resuspended in 30 μl of TNE buffer, followed by the addition of 10 μl of PI (2 mg/ml in 10 mm Tris-HCl, pH 7.4, 1 mm EGTA) and 10 μl of 100 mmMgCl2. The reactions were initiated by adding 20 μCi of [γ-32P]ATP and 2 μl of 100 mm ATP in 20 mm MgCl2 and then were incubated for 10 min at 25 °C. The reactions were terminated with 100 μl of 1n HCl and 200 μl of CHCl3-MeOH (1:1), vortexed briefly, and separated into phases by centrifugation (2,000 rpm) for 10 min. The CHCl3 phases were spotted onto oxalate-treated thin layer chromatography (TLC) plates and developed using a solvent system of CHCl3:MeOH:H2O:ammonium hydroxide (90:70:14.6:5.4). The PI3-phosphate was visualized by autoradiography and quantified by Bio-Imaging analyzer BAS2000 (Fuji Film, Tokyo). Antisense (AS) and complementary sense (S) ODNs that targeted the translation initiation regions of the regulatory subunit of phosphatidylinositol 3-kinase (p85) and the proto-oncogenevav were synthesized in fully phosphorothioated forms. The sequence of each 20-mer was as follows: AS-p85 (5′-TGGTACCCCTCAGCACTCAT-3′), S-p85 (5′-ATGAGTGCTGAGGGGTACCA-3′), AS-vav (5′-CATTGGCGCCACAGCTCCAT-3′), S-vav(5′-ATGGAGCTGTGGCGCCAATG-3′). The F-36P cells (1 × 104cells) were exposed to ODNs at the indicated concentration in the defined medium for 6 h prior to the addition of Epo (50 units/ml). After 18 h of incubation, the cells were labeled for 4 h with 0.25 μCi of [3H]thymidine prior to harvest. For the analysis of the expression of p85 and Vav, the ODN-treated cells were lysed, and total cell lysates were separated by SDS-PAGE, followed by anti-p85 or anti-Vav immunoblotting as described above. The JAK2 and Vav proteins were immunoprecipitated from serum-starved F-36P cells as described above and resuspended in the kinase buffer (40 mm HEPES, pH 7.4, 10 mmMgCl2, 3 mm MnCl2). Sepharose beads-conjugated Vav protein immunoprecipitated from Epo-unstimulated cells were mixed with the immunoprecipitated JAK2 kinases from the cells stimulated with or without Epo. The kinase reactions were initiated by the addition of 200 mm ATP, incubated for 15 min at 25 °C, and terminated by the addition of SDS sample buffer. The reaction mixtures were boiled for 5 min, separated by SDS-PAGE, and transferred to a nitrocellulose membrane, followed by immunoblotting with anti-Tyr(P) as described above. All of the experiments described under “Results” were carried out at least twice and yielded similar results. To examine the growth signaling pathway from the EpoR, the human nonlymphoid leukemia cell line F-36P was utilized. F-36P has an absolute dependence on IL-3 or GM-CSF and can be induced to proliferate in response to Epo (data not shown) (29Chiba S. Takaku F. Tange T. Shibuya K. Misawa C. Sasaki K. Miyagawa K. Yazaki Y. Hirai H. Blood. 1991; 78: 2261-2268Crossref PubMed Google Scholar). Although a previous study has shown that Epo induces tyrosine phosphorylation of Vav and suggested that Vav may play an important role in growth signaling from the EpoR (28Miura O. Miura Y. Nakamura N. Quelle F.W. Witthuhn B.A. Ihre J.N. Aoki N. Blood. 1994; 84: 4135-4141Crossref PubMed Google Scholar), little is known about the molecules involved in the Vav signaling pathway. To identify any phosphorylated proteins that might interact with Vav, F-36P cells were stimulated with Epo, and anti-Vav immunoprecipitates were examined for tyrosine-phosphorylated proteins by anti-phosphotyrosine (anti-Tyr(P)) and anti-Vav immunoblotting (Fig. 1). Consistent with the previous study (28Miura O. Miura Y. Nakamura N. Quelle F.W. Witthuhn B.A. Ihre J.N. Aoki N. Blood. 1994; 84: 4135-4141Crossref PubMed Google Scholar), Epo induced the tyrosine phosphorylation of Vav, and identical levels of Vav protein were present in each lane. In addition, a tyrosine-phosphorylated 70-kDa protein (pp70) also was observed after Epo stimulation. It has been shown that Epo induces tyrosine phosphorylation of the EpoR itself which causes a shift in its gel mobility from 66 to 72 kDa (8Miura O. D'Andrea A. Kabat D. Ihre J.N. Mol. Cell. Biol. 1991; 11: 4895-4902Crossref PubMed Scopus (184) Google Scholar, 9Yoshimura A. Lodish H.F. Mol. Cell. Biol. 1992; 12: 706-715Crossref PubMed Scopus (92) Google Scholar). To confirm that the co-immunoprecipitated pp70 represents the tyrosine-phosphorylated EpoR and to examine whether Vav binds to the EpoR constitutively or inducibly after Epo stimulation, anti-EpoR immunoprecipitates were subjected to anti-Vav immunoblotting. Fig.2 A shows that Vav was co-immunoprecipitated with anti-EpoR in an Epo-independent manner. Furthermore, Vav co-immunoprecipitated unphosphorylated EpoR with or without Epo stimulation and phosphorylated EpoR after Epo stimulation (Fig.2 B), whereas neither normal rabbit serum nor preimmune mouse serum co-immunoprecipitated Vav or EpoR, respectively. These results indicate that Vav constitutively associates with the EpoR and suggest that this binding may be independent of tyrosine phosphorylation of the EpoR.Figure 2Stable association of Vav with the EpoR.F-36P cells were stimulated with or without Epo at 37 °C for 10 min.A, the cells were lysed, immunoprecipitated (IP) with anti-EpoR or normal rabbit serum (NRS), and separated by SDS-PAGE followed by immunoblotting with anti-Vav. B, the lysates were immunoprecipitated with anti-Vav or preimmune mouse serum (pre) and subjected to anti-EpoR immunoblotting. The positions of the tyrosine-phosphorylated EpoR (EpoR-PY) or -unphosphorylated EpoR (EpoR) are indicated witharrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previous studies have shown that the regulatory subunit of phosphatidylinositol 3-kinase (p85) can bind to tyrosine-phosphorylated EpoR (11Damen J.E. Mui A.L.-F. Puil L. Pawson T. Krystal G. Blood. 1993; 81: 3204-3210Crossref PubMed Google Scholar, 12Miura O. Nakamura N. Ihre J.N. Aoki N. J. Biol. Chem. 1994; 269: 614-620Abstract Full Text PDF PubMed Google Scholar). Recently, Gobert et al. (30Gobert S. Porteu F. Pallu S. Muller O. Sabbah M. Dusanter-Fourt I. Courtois G. Lacombe C. Gisselbrecht S. Mayeux P. Blood. 1995; 86: 598-606Crossref PubMed Google Scholar) have reported that a truncated EpoR, which had no tyrosine residues and no longer bound PI3-kinase, can mediate Epo-induced activation of PI3-kinase and suggested an alternative pathway for PI3-kinase activation (30Gobert S. Porteu F. Pallu S. Muller O. Sabbah M. Dusanter-Fourt I. Courtois G. Lacombe C. Gisselbrecht S. Mayeux P. Blood. 1995; 86: 598-606Crossref PubMed Google Scholar). To test for the substrates associated with p85, anti-p85 immunoprecipitation and anti-phosphotyrosine immunoblotting were performed (Fig.3). Epo stimulation led to no tyrosine phosphorylation of p85 as previously reported (11Damen J.E. Mui A.L.-F. Puil L. Pawson T. Krystal G. Blood. 1993; 81: 3204-3210Crossref PubMed Google Scholar, 12Miura O. Nakamura N. Ihre J.N. Aoki N. J. Biol. Chem. 1994; 269: 614-620Abstract Full Text PDF PubMed Google Scholar). However, some tyrosine-phosphorylated proteins were associated with p85 after Epo stimulation, including two that migrated at approximately 100 (pp100) and 70 kDa (pp70). Consistent with previous reports (11Damen J.E. Mui A.L.-F. Puil L. Pawson T. Krystal G. Blood. 1993; 81: 3204-3210Crossref PubMed Google Scholar, 12Miura O. Nakamura N. Ihre J.N. Aoki N. J. Biol. Chem. 1994; 269: 614-620Abstract Full Text PDF PubMed Google Scholar), pp70 was identified as the tyrosine-phosphorylated EpoR, and p85 associates with the EpoR after Epo stimulation (Fig. 4 A). We then examined whether the pp100 that binds to p85 corresponds to the tyrosine-phosphorylated Vav. Fig. 4 B (left panel) shows that Vav was detected in anti-p85 immunoprecipitates. Furthermore, as shown in Fig. 4 B (right panel), anti-Vav immunoprecipitates blotted with p85 revealed an association of Vav with p85. The binding of p85 to the tyrosine-phosphorylated Vav strongly suggests that this binding may be mediated through the SH2 domains of p85. To address this possibility, the cell lysates were incubated with agarose-conjugated GST fusion protein containing either the amino-terminal SH2 domain of p85 (SH2N) or the carboxyl-terminal SH2 domain (SH2C) and analyzed by immunoblotting with anti-Vav. Fig.5 shows that both the SH2 domains in the cell lysates from the Epo-stimulated cells can bind Vav in vitro, although the carboxyl-terminal SH2 domain binds to Vav with a higher affinity than does the amino-terminal SH2 domain. To better clarify the interaction between Vav and PI3-kinase, in vitro PI3-kinase assays were performed with anti-Tyr(P) and anti-Vav immunoprecipitates. We found that the Epo-induced in vitro PI3-kinase activity was associated with Vav (Fig. 6). Although the PI3-kinase activity of the anti-Vav immunoprecipitates was approximately 1/100 that of the anti-p85 immunoprecipitates after Epo stimulation, the activity of anti-Vav immunoprecipitates resulted in accounting for approximately 80% that of anti-Tyr(P) immunoprecipitates (data not shown). These results suggest that Epo stimulation leads to the association of PI3-kinase with tyrosine-phosphorylated Vav via the SH2 domain(s) of p85.Figure 4Epo-induced association of p85 with the EpoR and Vav. F-36P cells were stimulated with or without Epo at 37 °C for 10 min. A, the cells were lysed and immunoprecipitated (IP) with anti-p85 (left panel) and anti-EpoR antibody (right panel). The immunoprecipitates were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-EpoR (left panel) or anti-p85 antibody (right panel).B, the cells lysates were immunoprecipitated with anti-p85 (left panel) and anti-Vav (right panel) followed by immunoblotting with anti-Vav (left panel) and anti-p85 (right panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5In vitro association of Vav with the amino- and carboxyl-terminal SH2 domains of p85. Cell lysates from F-36P cells treated with or without Epo were incubated with agarose-conjugated GST-p85 SH2N, SH2C, or GST alone for 1 h at 4 °C. The samples were eluted by boiling in SDS sample buffer, separated by SDS-PAGE, and immunoblotted with anti-Vav antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Epo-induced association of the PI3-kinase activity with Vav. The cells were incubated for 10 min at 37 °C with or without Epo, and the lysates were immunoprecipitated withanti-p85, anti-PTyr, or anti-Vav. The immunoprecipitates (IP) were subjected to an in vitro PI3-kinase assay, and the reaction products were analyzed by thin layer chromatography. The positions of phosphatidylinositol phosphate (PIP) and the origin (Ori) are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previous studies have demonstrated that the membrane proximal region of the EpoR, which is crucial for growth signaling (31D'Andrea A.D. Yoshimura A. Youssoufian H. Zon L.I. Koo J.W. Lodish H.F. Mol. Cell. Biol. 1991; 11: 1980-1987Crossref PubMed Scopus (225) Google Scholar, 32Maruyama K. Miyata K. Yoshimura A. J. Biol. Chem. 1994; 269: 5976-5980Abstract Full Text PDF PubMed Google Scholar), also is required for the activation of JAK2 (5Witthuhn B.A. Quelle F.W. Silvennoinen O. Yi T. Tang B. Miura O. Ihre J.N. Cell. 1993; 74: 227-236Abstract Full Text PDF PubMed Scopus (1008) Google Scholar, 6Miura O. Nakamura N. Quelle F.W. Witthuhn B.A. Ihre J.N. Aoki N. Blood. 1994; 84: 1501-1507Crossref PubMed Google Scholar) and the tyrosine phosphorylation of Vav (28Miura O. Miura Y. Nakamura N. Quelle F.W. Witthuhn B.A. Ihre J.N. Aoki N. Blood. 1994; 84: 4135-4141Crossref PubMed Google Scholar). This suggests that Vav plays an important role in growth signaling. On the other hand, p85 has been shown to associate with the carboxyl-terminal region of the EpoR, a domain that may not play a role in growth signaling (12Miura O. Nakamura N. Ihre J.N. Aoki N. J. Biol. Chem. 1994; 269: 614-620Abstract Full Text PDF PubMed Google Scholar). To clarify the functional role of Vav and p85 in EpoR signaling pathway, we utilized antisense (AS) and complementary sense (S) oligodeoxynucleotides (ODNs) that targeted the translation initiation regions of the proto-oncogene vav and the regulatory subunit of phosphatidylinositol 3-kinase (p85) in fully phosphorothioated forms. Epo-stimulated F-36P cells were incubated with AS- or S- ODNs for 24 h and analyzed by immunoblotting. Treatment of AS-vav and AS-p85 resulted in significant decreases in the expression of Vav and p85 compared with the sense treatment (Fig.7, A and C). If Vav significantly contributes to the proliferation signaling from the EpoR, down-regulation of the expression of Vav should result in the inhibition of cell growth. To test this hypothesis, the effect of AS-vav on cell proliferation was examined by a [3H]thymidine incorporation assay. Fig. 7 A shows that AS-vav treatment inhibited the Epo-dependent proliferation of F-36P cells in a dose-dependent manner after co-incubation for 24 h, a time point when cell viability in all cultures was >90% (data not shown), whereas S-vav had no significant effect. This indicates that the observed inhibition of cell growth was due to antisense-mediated loss of Vav and not to growth suppression by ODN degradation products. Since we have observed the physical interaction between Vav and p85, we next examined the effect of AS-vav treatment on Epo-induced activation of PI3-kinase. F-36P cells were incubated in defined medium with Epo and 10 μm AS- or S-vav for 24 h. The cells were lysed, and in vitro PI3-kinase assays were carried out with anti-p85 immunoprecipitates. As shown in Fig. 7 B, treatment of cells with AS-vav resulted in a marked inhibition of PI3-kinase activity, suggesting the possible role of Vav on Epo-induced PI3-kinase activity. To confirm the involvement of Vav-p85 pathway in Epo-induced proliferation, we subsequently examined the effect of AS-p85 on cell proliferation. Although the cell viability in all cultures was >90% and no significant differences of the viability were observed with or without AS-ODN treatment (data not shown), AS-p85 treatment significantly suppressed the [3H]thymidine uptake (Fig.7 C), as observed in AS-vav treatment. It is important to identify the possible tyrosine kinases responsible for the tyrosine phosphorylation of Vav in the Epo signaling pathway. An important advance toward understanding cytokine actions was provided by the recently d" @default.
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