SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Chernock, R. D. Articles by Ganju, R. K. Search for Related Content PubMed PubMed Citation Articles by Chernock, R. D. Articles by Ganju, R. K. Related Collections Signal Transduction Chemokines, Cytokines, and Interleukins Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 February 2001, Vol. 97, No. 3, pp. 608-615 CHEMOKINES SHP2 and cbl participate in -chemokine receptor CXCR4-mediated signaling pathways Rebecca D. Chernock, Rama P. Cherla, and Ramesh K. Ganju From the Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Stromal cell-derived factor (SDF)-1 and its receptor, CXCR4, play an important role in cell migration, embryonic development, and human immunodeficiency virus infection. However, the cellular signaling pathways that mediate these processes are not fully elucidated. We and others have shown that the binding of SDF-1 to CXCR4 activates phosphatidylinositol-3 kinase (PI-3 kinase), p44/42 mitogen-associated protein kinase, and the transcription factor nuclear factor-B, and it also enhances the tyrosine phosphorylation and association of proteins involved in the formation of focal adhesions. In this study, we examined the role of phosphatases in CXCR4-mediated signaling pathways. We observed significant inhibition of SDF-1-induced migration by phosphatase inhibitors in CXCR4-transfected pre-B lymphoma L1.2 cells, Jurkat T cells, and peripheral blood lymphocytes. Further studies revealed that SDF-1 stimulation induced robust tyrosine phosphorylation in the SH2-containing phosphatase SHP2. SHP2 associated with the CXCR4 receptor and the signaling molecules SHIP, cbl, and fyn. Overexpression of wild-type SHP2 increased SDF-1-induced chemotaxis. Enhanced activation of fyn and lyn kinases and the tyrosine phosphorylation of cbl were also observed. In addition, SDF-1 stimulation enhanced the association of cbl with PI-3 kinase, Crk-L, and 14-3-3 proteins. Our results suggest that CXCR4-mediated signaling is regulated by SHP2 and cbl, which collectively participate in the formation of a multimeric signaling complex. (Blood. 2001;97:608-615) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Chemokines and their receptors play important roles in immune and inflammatory responses through the regulation of cell migration and growth. In addition, chemokines participate in the pathogenesis of several diseases.1-5 Chemokine receptors act as coreceptors for the human immunodeficiency virus (HIV), and the expression of both chemokines and chemokine receptor homologues by Kaposi's sarcoma herpes virus (KSHV/HHV-8) has been implicated in the development of Kaposi's sarcoma.6,7 The -chemokine, stromal cell-derived factor (SDF), is a widely expressed ligand of the CXCR4 receptor.8-11 It is a potent chemotactic factor for mature leukocytes such as monocytes and T lymphocytes as well as for CD34+ progenitors.12-14 CXCR4 is a 7-transmembrane G protein-coupled receptor that is expressed by a variety of cells, including peripheral blood lymphocytes (PBLs), monocytes, thymocytes, pre-B cells, dendritic cells, and endothelial cells.15-17 CXCR4 serves as a coreceptor for T-cell tropic HIV-1 strains and plays an important role in HIV pathogenesis.1,17,18 In addition, CXCR4 and SDF-1 are critical for embryonic development. Targeted disruption of either protein leads to severe defects in mice embryos that are lethal.19-22 Although CXCR4 and/or SDF-1 play important roles in both physiologic and pathologic processes, the cellular signaling pathways that mediate these effects are not fully elucidated. Previously, we and others have shown that the association of SDF-1 with CXCR4 activates multiple signaling pathways.23-25 The tyrosine phosphorylation and association of proteins such as RAFTK, Crk, and paxillin, which are involved in the formation of focal adhesions, were enhanced upon SDF-1 stimulation.23,24 Phosphatidylinositol-3 kinase (PI-3 kinase), which is an essential component of signaling pathways that induce chemotaxis, was also activated.23,26 In addition, selective activation of the downstream signaling molecule, p44/42 mitogen-activated protein kinase (MAPK), but not p38 MAPK or Jun N-terminal kinase, was observed as well as the activation of the transcription factor, nuclear factor (NF)-B.23 In the present study, we have characterized the role of the tyrosine phosphatase SHP2 and adaptor molecule cbl in CXCR4-mediated signaling pathways. Protein tyrosine phosphatases (PTPs) play an important role in the regulation of signals generated by various stimuli.27-30 However, the role of individual PTPs in signaling is not uniform. SHP1, a PTP expressed predominantly in hematopoietic cells, and SHIP (SH2-containing inositol phosphatase) act as negative regulators of signaling.29-31 In contrast, the ubiquitously expressed PTP, SHP2, appears to play a positive role in growth factor-induced signaling pathways.32-35 In addition to their tyrosine phosphatase activity, SHP1 and SHP2 can function as adaptor molecules. Through their SH2 domains, SHP1 and SHP2 can bind to several proteins and then transduce signals.36-39 Both SHP1 and SHP2 play important roles in immune regulation and development.40-42 However, their roles in chemokine-mediated signaling are not fully understood. SHP1 was recently shown to mediate SDF-1-induced chemotaxis. Alterations in chemotactic response to SDF-1 were observed in hematopoietic cells derived from mice lacking SHP1 (moth-eaten or viable).43 Cbl is a 120-kd protein present in lymphocytes that can function as an adaptor molecule in tyrosine phosphorylation-dependent signaling. It is rapidly phosphorylated by Src-like kinases upon stimulation of cell surface receptors and binds to several other signaling molecules, including ZAP-70, Syk, PI-3 kinase, Crk-L, and Vav.44-48 Cbl also contains a ubiquitin-associated domain. It binds to and stimulates the ubiquitin-mediated degradation of active platelet-derived growth factor, epidermal growth factor, and colony-stimulating factor-1 receptors.49-51 In this way, cbl can act as a negative regulator of receptor and nonreceptor tyrosine kinases. In this study, we demonstrate that SDF-1 treatment of CXCR4-positive cells resulted in the tyrosine phosphorylation of SHP2 and cbl and the activation of fyn kinase. Furthermore, we show that SHP2 is constitutively associated with the CXCR4 receptor and is recruited into the protein tyrosine kinase machinery by forming a complex with SHIP, cbl, and fyn. Cbl was also shown to form an activation-induced multimeric complex with PI-3 kinase, 14-3-3, and Crk-L. Moreover, pretreatment of cells with the phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate reduced the SDF-1-induced migration of T and B cells. These results suggest that SHP2 and cbl are key mediators of SDF-1-induced functional responses.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Reagents and materials Antibodies to SHP2, cbl, fyn, lyn, 14-3-3, and Crk-L were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antiphosphotyrosine antibody (4G10) was a generous gift from Dr Brian Druker (Oregon Health Sciences University, Portland, OR). Antibody to PI-3 kinase was from Upstate Biotechnology (Lake Placid, NY). The GST-SH2 domain of SHP2 was obtained from Santa Cruz Biotechnology. Electrophoresis reagents and the nitrocellulose membrane were obtained from Bio-Rad Laboratories (Hercules, CA). The phosphatase inhibitors PAO and sodium orthovanadate and the protease inhibitors leupeptin and  1-antitrypsin and all other reagents were acquired from Sigma Chemical Co (St Louis, MO). Construction of stable CXCR4 transfectants CXCR4 complementary DNA, tagged at the amino-terminus with a Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), was subcloned into the pcDNAIII expression vector and then stably transfected into cells from the murine pre-B lymphoma cell line, L1.2, as described.52-54 Geneticin (G418)-containing medium was used to select for transfectants. Cell-surface expression of CXCR4 on the L1.2 transfectants was confirmed by flow cytometry. Isolation of PBLs PBLs were generated from peripheral blood mononuclear cells as described.55,56 Briefly, peripheral blood mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation. After 2 rounds of adherence to plastic in RPMI 1640 with 10% fetal calf serum, 2 mM glutamine, 50 µg/mL penicillin, and 50 µg/mL streptomycin, the cells were grown in medium containing 5 µg/mL phytohemagglutinin for 3 days. The PBLs were then washed and grown in RPMI 1640 containing 15% fetal calf serum and 5% interleukin-2 (Advanced Biotechnologies, Columbia, MD). After 3 weeks, the activated T cells were found to be 50% to 60% positive for CXCR4 by flow cytometry and were then used for further studies. Cell culture The CXCR4 L1.2 cells were grown in RPMI 1640 with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 µg/mL penicillin, 50 µg/mL streptomycin, 55 µM 2-mercaptoethanol, and 0.8 mg/mL G418 (Gibco BRL, Grand Island, NY). Jurkat T cells were grown in RPMI 1640 with 10% fetal calf serum, 2 mM glutamine, 50 µg/mL penicillin, and 50 µg/mL streptomycin. Flow cytometry Cells were washed twice with phosphate-buffered saline (PBS), resuspended in 500 µL of wash buffer (PBS containing 5% fetal calf serum) and 12G5 anti-CXCR4 antibody, and then incubated at 4°C for 30 minutes. The cells were next washed 3 times with wash buffer, resuspended in 500 µL of wash buffer and fluorescein isothiocyanate-coupled secondary antibody, and then incubated at 4°C for 30 minutes. Thereafter, the cells were washed 3 times, resuspended in 500 µL PBS, and analyzed by flow cytometry. Stimulation of cells Cells were washed twice with RPMI 1640 and serum-starved for 2 hours at 37°C in 1 × Hank's buffered salt solution at a concentration of 10 × 106 cells/mL. The cells were then treated with 100 ng/mL SDF-1 at 37°C. After different time periods, cells were lysed in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 150 mM NaCl; 1 mM phenylmethylsulfonyl fluoride; 10 µg/mL each of aprotinin, leupeptin, and pepstatin; and 10 mM each of sodium vanadate, sodium fluoride, and sodium pyrophosphate). Total cell lysates were clarified by centrifugation at 10 000g for 10 minutes. Protein concentrations were determined by protein assay (Bio-Rad Laboratories). Immunoprecipitation and immunoblotting Equal amounts of protein from each sample were clarified by incubation with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 hour at 4°C. After the removal of protein A-Sepharose by brief centrifugation, the samples were incubated with a primary antibody for 2 hours or overnight at 4°C. The antibody-antigen complexes were immunoprecipitated by incubation with 50 µL protein A-Sepharose for 3 hours or overnight at 4°C. Nonspecific bound proteins were removed by washing the protein A-Sepharose beads 3 times with modified RIPA buffer and one time with PBS. Bound immunocomplexes were solubilized in 30 µL 2 × Laemmli buffer, separated on 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat milk protein for 1 hour and probed with primary antibody for 2 hours at room temperature or overnight at 4°C. Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescent system (Amersham Pharmacia Biotech). The immunoprecipitation and Western blotting data shown in "Results" are representative of findings from 3 independent experiments. Glutathione-S-transferase-fusion protein binding studies SH-PTP2 glutathione-S-transferase (GST)-fusion proteins were purchased from Santa Cruz Biotechnology. Fifty micrograms of GST-fusion protein were added to 1 mg cell lysate and incubated for 1 hour at 4°C. GST protein (Santa Cruz Biotechnology) was used as a control. The complexes were then preabsorbed with 50 µL Glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) and incubated for 3 hours or overnight at 4°C. The beads were centrifuged and washed 3 times with modified RIPA buffer and once with 1 × PBS. Bound proteins were eluted by boiling in Laemmli sample buffer and separated on 8% SDS-PAGE. Migration assays Cells were resuspended at 6.6 × 106/mL in RPMI 1640 medium containing 2.5% fetal calf serum. Twenty-four-well plates containing 5 µm porosity inserts (CoStar Corp, Kennebunk, ME) were used for the assays. A total of 25 ng/mL SDF-1 in 600 µL of medium was added to the bottom wells, and 150 µL of cells, untreated or treated with the phosphatase inhibitor PAO or sodium orthovanadate, was placed in the inserts. Controls were treated with appropriate solvents and SDF-1 under similar conditions. After 3 hours, cells that migrated to the bottom wells were collected and counted on a hemacytometer. Fyn, lyn, and p44/42 MAPK assays Cell lysates were immunoprecipitated with fyn, lyn, or Erk1 and Erk2 antibodies. For fyn and lyn kinase assays, the immune complexes were washed twice with RIPA buffer and twice with kinase buffer (20 mM HEPES, 50 mM NaCl, 10 µM Na3VO4, 5 mM MgCl2, and 5 mM MnCl2). The complexes were then incubated in kinase buffer containing 0.18 MBq (5 µCi) -32P-adenosine triphosphate (ATP) for 20 minutes at 30°C. The reaction was terminated by adding 4 × Laemmli sample buffer and boiling the samples for 5 minutes. Proteins were then separated on 8% SDS-PAGE and viewed by autoradiography. For p44/42 MAPK assays, the immune complexes were washed twice with RIPA buffer and twice with kinase buffer (50 mM HEPES, pH 7.4; 10 mM MgCl2; and 20 µM ATP). The complexes were then incubated in kinase buffer containing 7 µg of myelin basic protein (Upstate Biotechnology) and 0.18 MBq (5 µCi) of -32P-ATP for 30 minutes. Proteins were separated on 15% SDS-PAGE and viewed by autoradiography. Transient transfection Wild-type SHP2 (SHP2 WT) construct and control vector (kindly provided by Dr Benjamin G. Neel, Beth Israel Deaconess Medical Center) were transiently transfected into the JMC.T5 Jurkat cell line (kindly provided by Dr Hamid Band, Harvard Medical School) by the electroporation method as described.55 Briefly, 10 µg of plasmid carrying the control vector or SHP2 WT34,35 was added to the cell suspension (20 × 106 cells/mL) in a Gene Pulser cuvette and then incubated on ice for 10 minutes. Electric pulse was given by using the Gene Pulser II (Bio-Rad) set at 250 V, 950 µF. The cells were transferred to RPMI 1640 medium containing 10% fetal bovine serum and then grown for 72 hours. The expression of SHP2 was examined by immunoprecipitation as described above.     Results Top Abstract Introduction Materials and methods Results Discussion References Phosphatase inhibitors reduce SDF-1- induced migration of CXCR4 L1.2, Jurkat T cells, and PBLs SDF-1 has been shown to act as a potent chemoattractant for various cell types.8,9,12 We and others have recently shown that SDF-1-induced chemotaxis is regulated by PI-3 kinase.23,26 Because several signaling components are required for mediating chemotaxis, we analyzed the effect of the phosphatase inhibitors PAO and sodium orthovanadate on SDF-1-induced migration of CXCR4 transfectants, Jurkat T cells, and PBLs. PAO and sodium orthovanadate inhibit the catalytic activity of PTPs, which modulate growth factor-mediated chemotaxis.57 As shown in Figure 1A,B, PAO led to a dose-dependent decrease in SDF-1-induced chemotaxis in CXCR4 L1.2 and Jurkat cells as compared with cells treated with solvent controls. Similarly, sodium orthovanadate treatment reduced the migration of CXCR4 L1.2, Jurkat T cells, and PBLs in a dose-dependent manner (Figure 2A-C). At the highest concentration (1.0 µM), PAO reduced the migration of the CXCR4 transfectants to less than 5% (P  .001) and the Jurkat cells to less than 10% (P  .002) (Figure 1A,B). At 100 µM of sodium orthovanadate, the migration of the CXCR4 transfectants was reduced to 58% (P  .034), the Jurkat T cells to 42% (P  .002), and the PBLs to 39% (P  .009) (Figure 2A-C). Under similar conditions, the inhibitors had no effect on the viability of the cells (data not shown). In addition, the phosphatase inhibitors had no effect on SDF-1-induced p44/42 MAPK activation at 1 µM (PAO) and 100 µM (sodium orthovanadate), indicating that the observed inhibition of migration was not due to toxicity (data not shown). View larger version (13K): [in this window] [in a new window]   Figure 1. Effect of PAO on SDF-1-induced chemotaxis of CXCR4 L1.2 and Jurkat cells. CXCR4 L1.2 (A) or Jurkat (B) cells were pretreated with 0, 0.1, 0.3, or 1.0 µM concentration of PAO for 45 minutes. Migration toward 25 ng/mL SDF-1 was measured after 3 hours. Chemotaxis of cells in the absence of inhibitor is considered 100% migration. *P < .05; **P < .005. View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of sodium orthovanadate on SDF-1-induced chemotaxis of CXCR4 L1.2 cells, Jurkat cells, and PBLs. CXCR4 L1.2 (A), Jurkat (B), or PBL (C) cells were untreated (0) or pretreated with different concentrations of sodium orthovanadate for 45 minutes. Migration toward 25 ng/mL SDF-1 was measured after 3 hours. Chemotaxis of cells in the absence of inhibitor is considered 100% migration. *P < .05; **P < .005. SHP2 is tyrosine-phosphorylated upon SDF-1 treatment of CXCR4 L1.2 and Jurkat T cells Recently, the involvement of the tyrosine phosphatase, SHP1, in SDF-1-induced chemotaxis has been shown in cells derived from SHP1-deficient moth-eaten mice.43 SHP2 has also been shown to regulate migration induced by various growth factors and cytokines.57,58 To characterize the role of SHP2 in CXCR4-mediated signaling pathways, CXCR4 transfectants and Jurkat T cells were treated with SDF-1, and cell lysates were then analyzed for SHP2 tyrosine phosphorylation. Figure 3A-B (top panels) shows that stimulation with SDF-1 resulted in a marked increase in the tyrosine phosphorylation of SHP2. This phosphorylation of SHP2 was rapid, and maximum phosphorylation was obtained after 5 to 10 minutes. Equal amounts of SHP2 protein were present in each lane (Figure 3A-B, bottom panels). View larger version (30K): [in this window] [in a new window]   Figure 3. Tyrosine phosphorylation of SHP2 upon SDF-1 stimulation. CXCR4 L1.2 (A) or Jurkat (B) cells were either unstimulated (0) or stimulated with 100 ng/mL SDF-1 for varying time periods. Cells were then lysed and immunoprecipitated with anti-SHP2 antibody. The immune complexes were run on SDS-PAGE and immunoblotted with antiphosphotyrosine antibody (top panels), followed by anti-SHP2 antibody (bottom panels). C represents the antibody control, and TCL is 50 µL of the total cell lysate. IP indicates immunoprecipitation; WB, Western blot. SHP2 associates with the CXCR4 receptor SHP1 was recently shown to associate with CXCR4 upon SDF-1 stimulation.25 In the present study, we determined the association of SHP2 with CXCR4 upon SDF-1 stimulation of Jurkat cells. As shown in Figure 4, SHP2 constitutively associated with CXCR4, and this association was enhanced upon SDF-1 stimulation. View larger version (15K): [in this window] [in a new window]   Figure 4. CXCR4 associates with SHP2 upon SDF-1 stimulation. Lysates obtained from Jurkat cells unstimulated (0) or stimulated for varying time periods with 100 ng/mL SDF-1 were immunoprecipitated with CXCR4. The immune complexes were subjected to SDS-PAGE and immunoblotted with anti-SHP2 antibody. C represents the antibody control, and TCL is 50 µL of the total cell lysate. SHP2 associates with SHIP, fyn, and cbl upon SDF-1 stimulation of Jurkat T cells SHP2 has been shown to associate with several signaling components upon growth factor or cytokine stimulation.36-39 Here, we examined its association with other signaling molecules upon SDF-1 stimulation. As shown in Figure 5A-F, SHP2 associated with the phosphatase SHIP, the adaptor molecule cbl, and fyn kinase upon SDF-1 stimulation of Jurkat T cells. Association with SHIP was rapid, reached a maximum level at 5 to 10 minutes, and was reduced thereafter (Figure 5A). This association was further confirmed by the reverse immunoprecipitation of blotting SHP2 immunoprecipitates with SHIP (Figure 5B). Association of SHP2 with cbl was rapid and sustained for up to 20 minutes of stimulation with SDF-1 (Figure 5C). The cbl association was also reconfirmed by blotting SHP2 immunoprecipitates with cbl (Figure 5D). Furthermore, cbl was shown to associate with the amino-terminal SH2 domain of SHP2 when using GST-fusion protein for the immunoprecipitation (Figure 5E). Fyn constitutively associated with SHP2, was slightly enhanced after 2 to 5 minutes of stimulation with SDF-1, and was reduced thereafter (Figure 5F). View larger version (24K): [in this window] [in a new window]   Figure 5. SHP2 associates with SHIP, fyn, and cbl upon SDF-1 stimulation. Lysates obtained from Jurkat cells unstimulated (0) or stimulated with 100 ng/mL SDF-1 for various time periods were immunoprecipitated with either anti-SHIP (A), anti-SHP2 (B), anti-cbl (C), anti-SHP2 (D), the GST SH2-domain of SHP2 (E), or anti-Fyn (F). The immune complexes were then run on SDS-PAGE and immunoblotted with anti-SHP2 antibody (A,C,F) or anti-SHIP (B) or anti-cbl antibody (D,E). C represents the antibody control, and TCL is 50 µL of total cell lysate. SHP2 overexpression enhances SDF-1-induced migration To address the functional role of SHP2 in SDF-1-induced chemotaxis, we transiently transfected Jurkat cells with control vector or SHP2 WT. As shown in Figure 6A, a higher amount of SHP2 was expressed in SHP2 WT-transfected cells (lane 3) than in untransfected or control vector-transfected cells (lanes 1 and 2, respectively). Chemotaxis studies carried out with these transfectants revealed that overexpression of SHP2 WT protein enhanced SDF-1-induced chemotaxis by 50% to 60% as compared with cells overexpressing the control vector (Figure 6B). Similar results were obtained in 3 different transfection experiments. View larger version (18K): [in this window] [in a new window]   Figure 6. Overexpression of SHP2 in transfected JMC.T5 Jurkat cells enhances SDF-1-induced migration. (A) JMC.T5 Jurkat cells were untransfected or transfected with either control vector or SHP2 WT and then lysed with lysis buffer. Lysates (500 µg protein) from untransfected (lane 1), control vector-transfected (lane 2), or SHP2 WT-transfected (lane 3) cells were immunoprecipitated with SHP2 antibody. The immune complexes were then resolved on SDS-PAGE and blotted with SHP2 antibody. (B) The cells transfected with the control vector or SHP2 WT were subjected to migration assay with SDF-1 (50 ng/mL). The cells migrating to the bottom chamber were counted. *P < .005. Fyn and lyn kinases are activated upon SDF-1 stimulation of Jurkat T cells Because fyn associated with SHP2, we examined fyn and lyn kinase activation in SDF-1-stimulated cells. We found that both fyn and lyn kinases were activated after 2 minutes of SDF-1 treatment, although the activation of fyn was greater than that of lyn (Figure 7A-B). The activation of both fyn and lyn kinases was reduced after 5 to 10 minutes of stimulation. View larger version (26K): [in this window] [in a new window]   Figure 7. Fyn and Lyn are activated upon SDF-1 stimulation. Lysates obtained from Jurkat cells unstimulated (0) or stimulated with 100 ng/mL SDF-1 for various time periods were immunoprecipitated with anti-fyn (A) or anti-lyn (B) antibody. The immune complexes were subjected to in vitro kinase reactions. C represents the control antibody. Cbl is phosphorylated upon SDF-1 stimulation of Jurkat T cells and associates with PI-3 kinase, 14-3-3, and Crk-L Cbl acts as an adaptor protein in tyrosine-dependent signaling and has been shown to associate with several signaling molecules in response to a variety of stimuli.44-48 Because cbl associated with SHP2, we explored its phosphorylation and association with other signaling molecules in response to SDF-1 treatment. Stimulation with SDF-1 increased the tyrosine phosphorylation of cbl in Jurkat cells (Figure 8A, upper panel). Maximum phosphorylation was obtained after 2 to 5 minutes of stimulation and declined thereafter. Equal amounts of cbl protein were present in each lane (Figure 8A, bottom panel). Furthermore, cbl associated with PI-3 kinase and the adaptor proteins Crk-L and 14-3-3 (Figure 8B-F). Cbl associated constitutively with PI-3 kinase, and the association was only slightly increased after 5 minutes of treatment with SDF-1 (Figure 8B). Similar results were obtained when PI-3 kinase immunoprecipitates were immunoblotted with cbl (Figure 8C). The association of cbl with Crk-L was also enhanced upon SDF-1 stimulation (Figure 8D). This association was reconfirmed by immunoblotting Crk-L immunoprecipitates with cbl (Figure 8E). Cbl also associated with 14-3-3 upon SDF-1 stimulation (Figure 8F). View larger version (26K): [in this window] [in a new window]   Figure 8. Cbl is tyrosine-phosphorylated and associates with PI-3 kinase, Crk-L, and 14-3-3 upon SDF-1 stimulation. Lysates obtained from Jurkat cells were unstimulated (0) or stimulated with 100 ng/mL SDF-1 for various time periods and then lysed. The cell lysates were immunoprecipitated with anti-cbl antibody run on SDS-PAGE and immunoblotted with antiphosphotyrosine antibody (A, top panel), followed by anti-cbl antibody (A, bottom panel). Equivalent amounts of cell lysate (500 µg) were immunoprecipitated with anti-cbl (B,D,F), anti-PI-3 kinase (C), or anti-Crk-L (E) and then run on SDS-PAGE and immunoblotted with anti-PI-3 kinase (B), anti-cbl (C,E), anti-Crk-L (D), or anti-14-3-3 (F). C represents the antibody control, and TCL is 50 µL of total cell lysate.     Discussion Top Abstract Introduction Materials and methods Results Discussion References The -chemokine, SDF-1, and its cognate receptor, CXCR4, play a critical role in embryonic development, neuronal patterning, and HIV pathogenesis.1,17,19-22 The association of SDF-1 with CXCR4 also plays an important role in the immune system through the regulation of myeloid progenitor proliferation and the trafficking of leukocytes.12-14 However, the signaling pathways behind these normal and disease processes are only beginning to be understood. SDF-1-induced activation of CXCR4 is coupled to the JAK/STAT, PI-3 kinase, p44/p42 MAPK, and NF-B pathways.23-26 Recently, SDF-1 was shown to induce phosphorylation of the CXCR4 receptor at serine and tyrosine residues and association of the CXCR4 receptor with the tyrosine phosphatase, SHP1.25,59 Furthermore, the altered chemotactic response to SDF-1 observed in hematopoietic cells derived from SHP1-deficient mice indicates that SHP1 mediates SDF-1-induced chemotaxis.43 In this study, we have further assessed the role of PTPs in SDF-1-initiated signaling events. PTPs can regulate various cellular functions by initiating and terminating signals. We observed that the SDF-1-induced chemotaxis of CXCR4+ cells was significantly reduced by the tyrosine phosphatase inhibitors PAO and sodium orthovanadate. However, PAO and sodium orthovanadate did not affect SDF-1-induced p44/42 MAPK activation. The strong inhibition of migration by tyrosine phosphatase inhibitors suggests that tyrosine phosphatases are important mediators of SDF-1-induced chemotaxis. PAO and sodium orthovanadate have been reported to suppress platelet-derived growth factor-induced migration and cell spreading of lung carcinoma cells by inhibiting SHP2 activity.51,60 We found that SDF-1 stimulation of T cells enhanced the tyrosine phosphorylation of SHP2, which has been shown to be tyrosine-phosphorylated in response to stimulation by growth factors and cytokines.32-35 Furthermore, we observed that overexpression of SHP2 WT enhanced SDF-1-induced chemotaxis. In other studies, SHP2 has been shown to regulate chemotaxis induced by integrins. Its role in cell migration has also been confirmed in SHP2/ knock-out mice; fibroblasts derived from these mice have slower spreading and motility as compared with cells from WT animals.35 SHP2 has been shown to regulate cell adhesion, spreading, and migration by modulating tyrosine phosphorylation of focal adhesion proteins.35,58,61 We and others have recently shown that SDF-1 induces the tyrosine phosphorylation of components of focal adhesion complexes.23,62 SHP2 was shown to associate with the CXCR4 receptor and the signaling molecules SHIP and PI-3 kinase. Tyrosine-phosphorylated SHP2 can act as an adaptor molecule and has been shown to bind to several signaling molecules, including growth factor receptors and the adhesion molecule PECAM-1.32-35,38 Recently, another PTP, SHP1, was shown to bind to the CXCR4 receptor.25 SHIP, which is a 145-kd inositol-5-phosphatase that selectively hydrolyzes the 5' phosphate from both inositol 1,3,4,5 tetraphosphate (IP4) and phosphatidylinositol 3,4,5, triphosphate (PIP3), has previously been shown to bind to SHP2 in various cell types.31,63,64 SHIP seems to play an important role in chemokine-mediated signaling: Hematopoietic cells derived from SHIP/ knock-out mice showed enhanced chemotactic responses to SDF-1 as compared with the cells from WT mice.65 SHP2 also binds to PI-3 kinase, which recently was shown to regulate SDF-1-induced chemotaxis.23,26 Furthermore, SHIP may down-regulate PI-3 kinase-initiated signaling events through inhibition of protein kinase B activation by decreasing the levels of PIP3.66 Tyrosine phosphorylation of the adaptor protein cbl was enhanced upon SDF-1 stimulation. Therefore, cbl may play a role in SDF-1-induced chemotaxis in T cells. Cbl is a prominent component of signaling events downstream of a variety of cell surface receptors, including T cells, B cells, epidermal growth factor, integrins, and cytokines.44-48 It is involved in ubiquitination and the endocytic degradation of various receptors.49-51 We observed that SDF-1 enhanced the association of cbl with SHP2, PI-3 kinase, Crk-L, and 14-3-3. The reduction of cbl tyrosine phosphorylation and its association with membrane-localized PI-3 kinase activity have been shown in Src-family kinase mutants, which were defective in their ability to spread on fibronectin-coated surfaces. Furthermore, cbl antisense nucleotides and PI-3 kinase inhibitors also blocked the spreading of WT cells.47 Association of cbl with Crk-L was recently shown to be involved in Ba/F3 cell chemotactic events.67 Cbl also associates with 14-3-3 proteins upon SDF-1 stimulation. In addition, T-cell activation mediated by anti-CD3 has been shown to lead to the enhanced association of cbl with 14-3-3 proteins.68,69 These proteins bind to a wide variety of regulatory proteins and thereby participate in signaling events leading to cell differentiation and proliferation.70-72 The Src-related tyrosine kinases fyn and lyn were also activated upon SDF-1 stimulation, and fyn associated with SHP2. Fyn and lyn have been shown to associate with cbl and SHP2 and thereby regulate their functions.73-75 Both of these kinases play important roles in various growth factor and T- and B-cell receptor signaling by initiating changes in the phosphorylation of signaling intermediates that modulate further downstream events.76 Taken together, our studies suggest that the tyrosine phosphatase SHP2 and the adaptor cbl are key components of CXCR4-mediated signaling events induced by SDF-1. SHP2 associates with CXCR4 and forms a multiprotein signaling complex with cbl, SHIP, and fyn. Cbl also forms an activation-induced complex with PI-3 kinase, Crk-L, and 14-3-3 proteins. The formation of these large multimeric signaling complexes may result in the activation of multiple downstream effectors, which mediate the various functional effects of the CXCR4 receptor.     Acknowledgments We are thankful to Dr Jerome E. Groopman for his advice and support for our research project. We also thank Stephanie Brubaker for technical help, Janet Delahanty for editing, Daniel Kelley for preparation of the figures, and Simone Jadusingh for facilitating receipt of the reagents for the experiments.     Footnotes Submitted May 24, 2000; accepted October 4, 2000. Supported by National Institutes of Health grant CA76950 (R.K.G.). The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Ramesh K. Ganju, Division of Experimental Medicine, Harvard Institutes of Medicine-BIDMC, 4 Blackfan Circle, Room 343, Boston, MA 02115; e-mail: rganju{at}caregroup.harvard.edu.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Premack BA, Schall TJ. Chemokine receptors: gateways to inflammation and infection. Nat Med. 1996;2:1174-1178[CrossRef][Medline] [Order article via Infotrieve]. 2. Rollins BJ. Chemokines. Blood. 1997;90:909-928[Free Full Text]. 3. Bokoch GM. Chemoattractant signaling and leukocyte activation. Blood. 1995;86:1649-1660[Free Full Text]. 4. Kunkel SL. Through the looking glass: the diverse in vivo activities of chemokines. J Clin Invest. 1999;104:1333-1334[Medline] [Order article via Infotrieve]. 5. Luster AD. Chemokineschemotactic cytokines that mediate inflammation. N Engl J Med. 1998;338:436-445[Free Full Text]. 6. Boshoff C, Weiss RA. Kaposi's sarcoma-associated herpesvirus. Adv Cancer Res. 1998;75:57-86[Medline] [Order article via Infotrieve]. 7. Bais C, Santomasso B, Coso O, et al. G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator. Nature. 1998;391:86-89[CrossRef][Medline] [Order article via Infotrieve]. 8. Hamada T, Tashiro K, Tada H, et al. Isolation and characterization of a novel secretory protein, stromal cell-derived factor-2 (SDF-2) using the signal sequence trap method. Gene. 1996;176:211-214[CrossRef][Medline] [Order article via Infotrieve]. 9. Bleul CC, Farzan M, Choe H, et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature. 1996;382:829-833[CrossRef][Medline] [Order article via Infotrieve]. 10. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996;272:872-877[Abstract]. 11. Loetscher M, Geiser T, O'Reilly T, Zwahlen R, Baggiolini M, Moser B. Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes. J Biol Chem. 1994;269:232-237[Abstract/Free Full Text]. 12. Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med. 1997;185:111-120[Abstract/Free Full Text]. 13. Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci U S A. 1997;94:1925-1930[Abstract/Free Full Text]. 14. Forster R, Kremmer E, Schubel A, et al. Intracellular and surface expression of the HIV-1 coreceptor CXCR4/fusin on various leukocyte subsets: rapid internalization and recycling upon activation. J Immunol. 1998;160:1522-1531[Abstract/Free Full Text]. 15. Gupta SK, Lysko PG, Pillarisetti K, Ohlstein E, Stadel JM. Chemokine receptors in human endothelial cells: functional expression of CXCR4 and its transcriptional regulation by inflammatory cytokines. J Biol Chem. 1998;273:4282-4287[Abstract/Free Full Text]. 16. Hesselgesser J, Halks-Miller M, DelVecchio V, et al. CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons. Curr Biol. 1997;7:112-121[CrossRef][Medline] [Order article via Infotrieve]. 17. Littman DR. Chemokine receptors: keys to AIDS pathogenesis? Cell. 1998;93:677-680[CrossRef][Medline] [Order article via Infotrieve]. 18. Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol. 1999;17:657-700[CrossRef][Medline] [Order article via Infotrieve]. 19. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature. 1996;382:635-638[CrossRef][Medline] [Order article via Infotrieve]. 20. Ma Q, Jones D, Borghesani PR, et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci U S A. 1998;95:9448-9453[Abstract/Free Full Text]. 21. Tachibana K, Hirota S, Iizasa H, et al. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature. 1998;393:591-594[CrossRef][Medline] [Order article via Infotrieve]. 22. Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature. 1998;393:595-599[CrossRef][Medline] [Order article via Infotrieve]. 23. Ganju RK, Brubaker SA, Meyer J, et al. The alpha-chemokine, stromal cell-derived factor-1alpha, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J Biol Chem. 1998;273:23169-23175[Abstract/Free Full Text]. 24. Davis CB, Dikic I, Unutmaz D, et al. Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5. J Exp Med. 1997;186:1793-1798[Abstract/Free Full Text]. 25. Vila-Coro AJ, Rodriguez-Frade JM, Martin De Ana A, Moreno-Ortiz MC, Martinez-A C, Mellado M. The chemokine SDF-1alpha triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway. FASEB J. 1999;13:1699-1710[Abstract/Free Full Text]. 26. Sotsios Y, Whittaker GC, Westwick J, Ward SG. The CXC chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide 3-kinase in T lymphocytes. J Immunol. 1999;163:5954-5963[Abstract/Free Full Text]. 27. Neel BG. Role of phosphatases in lymphocyte activation. Curr Opin Immunol. 1997;9:405-420[CrossRef][Medline] [Order article via Infotrieve]. 28. Streuli M. Protein tyrosine phosphatases in signaling. Curr Opin Cell Biol. 1996;8:182-188[CrossRef][Medline] [Order article via Infotrieve]. 29. Haque SJ, Harbor P, Tabrizi M, Yi T, Williams BR. Protein-tyrosine phosphatase Shp-1 is a negative regulator of IL-4- and IL-13-dependent signal transduction. J Biol Chem. 1998;273:33893-33896[Abstract/Free Full Text]. 30. Dong Q, Siminovitch KA, Fialkow L, Fukushima T, Downey GP. Negative regulation of myeloid cell proliferation and function by the SH2 domain-containing tyrosine phosphatase-1. J Immunol. 1999;162:3220-3230[Abstract/Free Full Text]. 31. Huber M, Helgason CD, Damen JE, et al. The role of SHIP in growth factor induced signalling. Prog Biophys Mol Biol. 1999;71:423-434[CrossRef][Medline] [Order article via Infotrieve]. 32. Frearson JA, Alexander DR. The phosphotyrosine phosphatase SHP-2 participates in a multimeric signaling complex and regulates T cell receptor (TCR) coupling to the Ras/mitogen-activated protein kinase (MAPK) pathway in Jurkat T cells. J Exp Med. 1998;187:1417-1426[Abstract/Free Full Text]. 33. Nakamura K, Cambier JC. B cell antigen receptor (BCR)-mediated formation of a SHP-2-pp120 complex and its inhibition by Fc gamma RIIB1-BCR coligation. J Immunol. 1998;161:684-691[Abstract/Free Full Text]. 34. Gu H, Griffin JD, Neel BG. Characterization of two SHP-2-associated binding proteins and potential substrates in hematopoietic cells. J Biol Chem. 1997;272:16421-16430[Abstract/Free Full Text]. 35. Oh ES, Gu H, Saxton TM, et al. Regulation of early events in integrin signaling by protein-tyrosine-phosphatase SHP2. Mol Cell Biol. 1999;19:3205-3215[Abstract/Free Full Text]. 36. Bone H, Dechert U, Jirik F, Schrader JW, Welham MJ. SHP1 and SHP2 protein-tyrosine phosphatases associate with betac after interleukin-3-induced receptor tyrosine phosphorylation: identification of potential binding sites and substrates. J Biol Chem. 1997;272:14470-14476[Abstract/Free Full Text]. 37. Yu Z, Su L, Hoglinger O, Jaramillo ML, Banville D, Shen SH. SHP-1 associates with both platelet-derived growth factor receptor and the p85 subunit of phosphatidylinositol 3-kinase. J Biol Chem. 1998;273:3687-3694[Abstract/Free Full Text]. 38. Sagawa K, Kimura T, Swieter M, Siraganian RP. The protein-tyrosine phosphatase SHP-2 associates with tyrosine-phosphorylated adhesion molecule PECAM-1 (CD31). J Biol Chem. 1997;272:31086-31091[Abstract/Free Full Text]. 39. Ong SH, Lim YP, Low BC, Guy GR. SHP2 associates directly with tyrosine phosphorylated p90 (SNT) protein in FGF-stimulated cells. Biochem Biophys Res Commun. 1997;238:261-266[CrossRef][Medline] [Order article via Infotrieve]. 40. Shultz LD, Schweitzer PA, Rajan TV, et al. Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Cell. 1993;73:1445-1454[CrossRef][Medline] [Order article via Infotrieve]. 41. Shultz LD, Rajan TV, Greiner DL. Severe defects in immunity and hematopoiesis caused by SHP-1 protein-tyrosine-phosphatase deficiency. Trends Biotechnol. 1997;15:302-307[CrossRef][Medline] [Order article via Infotrieve]. 42. Qu CK, Yu WM, Azzarelli B, Cooper S, Broxmeyer HE, Feng GS. Biased suppression of hematopoiesis and multiple developmental defects in chimeric mice containing Shp-2 mutant cells. Mol Cell Biol. 1998;18:6075-6082[Abstract/Free Full Text]. 43. Kim CH, Qu CK, Hangoc G, et al. Abnormal chemokine-induced responses of immature and mature hematopoietic cells from motheaten mice implicate the protein tyrosine phosphatase SHP-1 in chemokine responses. J Exp Med. 1999;190:681-690[Abstract/Free Full Text]. 44. Donovan JA, Wange RL, Langdon WY, Samelson LE. The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor. J Biol Chem. 1994;269:22921-22924[Abstract/Free Full Text]. 45. Andoniou CE, Lill NL, Thien CB, et al. The Cbl proto-oncogene product negatively regulates the Src-family tyrosine kinase Fyn by enhancing its degradation. Mol Cell Biol. 2000;20:851-867[Abstract/Free Full Text]. 46. Lee PS, Wang Y, Dominguez MG, et al. The Cbl protooncoprotein stimulates CSF-1 receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation. EMBO J. 1999;18:3616-3628[CrossRef][Medline] [Order article via Infotrieve]. 47. Meng F, Lowell CA. A beta 1 integrin signaling pathway involving Src-family kinases, Cbl and PI-3 kinase is required for macrophage spreading and migration. EMBO J. 1998;17:4391-4403[CrossRef][Medline] [Order article via Infotrieve]. 48. Feshchenko EA, Langdon WY, Tsygankov AY. Fyn, Yes, and Syk phosphorylation sites in c-Cbl map to the same tyrosine residues that become phosphorylated in activated T cells. J Biol Chem. 1998;273:8323-8331[Abstract/Free Full Text]. 49. Barinaga M. A new finger on the protein destruction button. Science. 1999;286:223-225[Free Full Text]. 50. Yokouchi M, Kondo T, Houghton A, et al. Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7. J Biol Chem. 1999;274:31707-31712[Abstract/Free Full Text]. 51. Levkowitz G, Waterman H, Ettenberg SA, et al. Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol Cell. 1999;4:1029-1040[CrossRef][Medline] [Order article via Infotrieve]. 52. Ponath PD, Qin S, Post TW, et al. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils [see comments]. J Exp Med. 1996;183:2437-2448[Abstract/Free Full Text]. 53. Wu L, Ruffing N, Shi X, et al. Discrete steps in binding and signaling of interleukin-8 with its receptor. J Biol Chem. 1996;271:31202-31209[Abstract/Free Full Text]. 54. Wu L, Gerard NP, Wyatt R, et al. CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Nature. 1996;384:179-183[CrossRef][Medline] [Order article via Infotrieve]. 55. Ganju RK, Dutt P, Wu L, et al. Beta-chemokine receptor CCR5 signals via the novel tyrosine kinase RAFTK. Blood. 1998;91:791-797[Abstract/Free Full Text]. 56. Alfano M, Schmidtmayerova H, Amella CA, Pushkarsky T, Bukrinsky M. The B-oligomer of pertussis toxin deactivates CC chemokine receptor 5 and blocks entry of M-tropic HIV-1 strains. J Exp Med. 1999;190:597-605[Abstract/Free Full Text]. 57. Qi JH, Ito N, Claesson-Welsh L. Tyrosine phosphatase SHP-2 is involved in regulation of platelet-derived growth factor-induced migration. J Biol Chem. 1999;274:14455-14463[Abstract/Free Full Text]. 58. Manes S, Mira E, Gomez-Mouton C, Zhao ZJ, Lacalle RA, Martinez AC. Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility. Mol Cell Biol. 1999;19:3125-3135[Abstract/Free Full Text]. 59. Haribabu B, Richardson RM, Fisher I, et al. Regulation of human chemokine receptors CXCR4. Role of phosphorylation in desensitization and internalization. J Biol Chem. 1997;272:28726-28731[Abstract/Free Full Text]. 60. Chintala SK, Kyritsis AP, Mohan PM, et al. Altered actin cytoskeleton and inhibition of matrix metalloproteinase expression by vanadate and phenylarsine oxide, inhibitors of phosphotyrosine phosphatases: modulation of migration and invasion of human malignant glioma cells. Mol Carcinog. 1999;26:274-285[CrossRef][Medline] [Order article via Infotrieve]. 61. Yu DH, Qu CK, Henegariu O, Lu X, Feng GS. Protein-tyrosine phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion. J Biol Chem. 1998;273:21125-21131[Abstract/Free Full Text]. 62. Wang JF, Park IW, Groopman JE. Stromal cell-derived factor-1 stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C. Blood. 2000;95:2505-2513[Abstract/Free Full Text]. 63. Liu L, Damen JE, Ware MD, Krystal G. Interleukin-3 induces the association of the inositol 5-phosphatase SHIP with SHP2. J Biol Chem. 1997;272:10998-11001[Abstract/Free Full Text]. 64. Lecoq-Lafon C, Verdier F, Fichelson S, et al. Erythropoietin induces the tyrosine phosphorylation of GAB1 and its association with SHC, SHP2, SHIP, and phosphatidylinositol 3-kinase. Blood. 1999;93:2578-2585[Abstract/Free Full Text]. 65. Kim CH, Hangoc G, Cooper S, et al. Altered responsiveness to chemokines due to targeted disruption of SHIP. J Clin Invest. 1999;104:1751-1759[Medline] [Order article via Infotrieve]. 66. Jacob A, Cooney D, Tridandapani S, Kelley T, Coggeshall KM. FcgammaRIIb modulation of surface immunoglobulin-induced Akt activation in murine B cells. J Biol Chem. 1999;274:13704-13710[Abstract/Free Full Text]. 67. Uemura N, Griffin JD. The adapter protein Crkl links Cbl to C3G after integrin ligation and enhances cell migration. J Biol Chem. 1999;274:37525-37532[Abstract/Free Full Text]. 68. Liu YC, Elly C, Yoshida H, Bonnefoy-Berard N, Altman A. Activation-modulated association of 14-3-3 proteins with Cbl in T cells. J Biol Chem. 1996;271:14591-14595[Abstract/Free Full Text]. 69. Liu YC, Liu Y, Elly C, Yoshida H, Lipkowitz S, Altman A. Serine phosphorylation of Cbl induced by phorbol ester enhances its association with 14-3-3 proteins in T cells via a novel serine-rich 14-3-3-binding motif. J Biol Chem. 1997;272:9979-9985[Abstract/Free Full Text]. 70. Garcia-Guzman M, Dolfi F, Russello M, Vuori K. Cell adhesion regulates the interaction between the docking protein p130(Cas) and the 14-3-3 proteins. J Biol Chem. 1999;274:5762-5768[Abstract/Free Full Text]. 71. Luk SC, Ngai SM, Tsui SK, Fung KP, Lee CY, Waye MM. In vivo and in vitro association of 14-3-3 epsilon isoform with calmodulin: implication for signal transduction and cell proliferation. J Cell Biochem. 1999;73:31-35[CrossRef][Medline] [Order article via Infotrieve]. 72. Tzivion G, Luo Z, Avruch J. A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature. 1998;394:88-92[CrossRef][Medline] [Order article via Infotrieve]. 73. Tang H, Zhao ZJ, Huang XY, Landon EJ, Inagami T. Fyn kinase-directed activation of SH2 domain-containing protein-tyrosine phosphatase SHP-2 by Gi protein-coupled receptors in Madin-Darby canine kidney cells. J Biol Chem. 1999;274:12401-12407[Abstract/Free Full Text]. 74. Anderson SM, Burton EA, Koch BL. Phosphorylation of Cbl following stimulation with interleukin-3 and its association with Grb2, Fyn, and phosphatidylinositol 3-kinase. J Biol Chem. 1997;272:739-745[Abstract/Free Full Text]. 75. Deckert M, Elly C, Altman A, Liu YC. Coordinated regulation of the tyrosine phosphorylation of Cbl by Fyn and Syk tyrosine kinases. J Biol Chem. 1998;273:8867-8874[Abstract/Free Full Text]. 76. Kennedy JS, Raab M, Rudd CE. Signaling scaffolds in immune cells. Cell Calcium. 1999;26:227-235[CrossRef][Medline] [Order article via Infotrieve]. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: S. N. Zaman, M. E. Resek, and S. M. Robbins Dual acylation and lipid raft association of Src-family protein tyrosine kinases are required for SDF-1/CXCL12-mediated chemotaxis in the Jurkat human T cell lymphoma cell line J. Leukoc. Biol., October 1, 2008; 84(4): 1082 - 1091. [Abstract] [Full Text] [PDF] Y. Tanaka, N. Tanaka, Y. Saeki, K. Tanaka, M. Murakami, T. Hirano, N. Ishii, and K. Sugamura c-Cbl-Dependent Monoubiquitination and Lysosomal Degradation of gp130 Mol. Cell. Biol., August 1, 2008; 28(15): 4805 - 4818. [Abstract] [Full Text] [PDF] A. Prasad, Z. Qamri, J. Wu, and R. K. Ganju Slit-2/Robo-1 modulates the CXCL12/CXCR4-induced chemotaxis of T cells J. Leukoc. Biol., September 1, 2007; 82(3): 465 - 476. [Abstract] [Full Text] [PDF] B. Sargin, C. Choudhary, N. Crosetto, M. H. H. Schmidt, R. Grundler, M. Rensinghoff, C. Thiessen, L. Tickenbrock, J. Schwable, C. Brandts, et al. Flt3-dependent transformation by inactivating c-Cbl mutations in AML Blood, August 1, 2007; 110(3): 1004 - 1012. [Abstract] [Full Text] [PDF] C. Voena, C. Conte, C. Ambrogio, E. Boeri Erba, F. Boccalatte, S. Mohammed, O. N. Jensen, G. Palestro, G. Inghirami, and R. Chiarle The Tyrosine Phosphatase Shp2 Interacts with NPM-ALK and Regulates Anaplastic Lymphoma Cell Growth and Migration Cancer Res., May 1, 2007; 67(9): 4278 - 4286. [Abstract] [Full Text] [PDF] B. Tomkowicz, C. Lee, V. Ravyn, R. Cheung, A. Ptasznik, and R. G. Collman The Src kinase Lyn is required for CCR5 signaling in response to MIP-1beta and R5 HIV-1 gp120 in human macrophages Blood, August 15, 2006; 108(4): 1145 - 1150. [Abstract] [Full Text] [PDF] Z. Feng, G. R. Dubyak, M. M. Lederman, and A. Weinberg Cutting Edge: Human beta Defensin 3--A Novel Antagonist of the HIV-1 Coreceptor CXCR4 J. Immunol., July 15, 2006; 177(2): 782 - 786. [Abstract] [Full Text] [PDF] H. Schabath, S. Runz, S. Joumaa, and P. Altevogt CD24 affects CXCR4 function in pre-B lymphocytes and breast carcinoma cells J. Cell Sci., January 15, 2006; 119(2): 314 - 325. [Abstract] [Full Text] [PDF] M. Zhao, R. G. DiScipio, A. G. Wimmer, and I. U. Schraufstatter Regulation of CXCR4-Mediated Nuclear Translocation of Extracellular Signal-Related Kinases 1 and 2 Mol. Pharmacol., January 1, 2006; 69(1): 66 - 75. [Abstract] [Full Text] [PDF] M. Kucia, R. Reca, K. Miekus, J. Wanzeck, W. Wojakowski, A. Janowska-Wieczorek, J. Ratajczak, and M. Z. Ratajczak Trafficking of Normal Stem Cells and Metastasis of Cancer Stem Cells Involve Similar Mechanisms: Pivotal Role of the SDF-1-CXCR4 Axis Stem Cells, August 1, 2005; 23(7): 879 - 894. [Abstract] [Full Text] [PDF] Y. Le, M. Honczarenko, A. M. Glodek, D. K. Ho, and L. E. Silberstein CXC Chemokine Ligand 12-Induced Focal Adhesion Kinase Activation and Segregation into Membrane Domains Is Modulated by Regulator of G Protein Signaling 1 in Pro-B Cells J. Immunol., March 1, 2005; 174(5): 2582 - 2590. [Abstract] [Full Text] [PDF] S. M. Carlin, T. J. Resink, M. Tamm, and M. Roth Urokinase signal transduction and its role in cell migration FASEB J, February 1, 2005; 19(2): 195 - 202. [Abstract] [Full Text] [PDF] A. Prasad, A. Z. Fernandis, Y. Rao, and R. K. Ganju Slit Protein-mediated Inhibition of CXCR4-induced Chemotactic and Chemoinvasive Signaling Pathways in Breast Cancer Cells J. Biol. Chem., March 5, 2004; 279(10): 9115 - 9124. [Abstract] [Full Text] [PDF] N. Sharfe, A. Freywald, A. Toro, and C. M. Roifman Ephrin-A1 Induces c-Cbl Phosphorylation and EphA Receptor Down-Regulation in T Cells J. Immunol., June 15, 2003; 170(12): 6024 - 6032. [Abstract] [Full Text] [PDF] A. Z. Fernandis, R. P. Cherla, and R. K. Ganju Differential Regulation of CXCR4-mediated T-cell Chemotaxis and Mitogen-activated Protein Kinase Activation by the Membrane Tyrosine Phosphatase, CD45 J. Biol. Chem., March 7, 2003; 278(11): 9536 - 9543. [Abstract] [Full Text] [PDF] I. Schwering, A. Brauninger, U. Klein, B. Jungnickel, M. Tinguely, V. Diehl, M.-L. Hansmann, R. Dalla-Favera, K. Rajewsky, and R. Kuppers Loss of the B-lineage-specific gene expression program in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma Blood, February 15, 2003; 101(4): 1505 - 1512. [Abstract] [Full Text] [PDF] S. Kanda, Y. Mochizuki, and H. Kanetake Stromal Cell-derived Factor-1alpha Induces Tube-like Structure Formation of Endothelial Cells through Phosphoinositide 3-Kinase J. Biol. Chem., January 3, 2003; 278(1): 257 - 262. [Abstract] [Full Text] [PDF] A. Ptasznik, E. Urbanowska, S. Chinta, M. A. Costa, B. A. Katz, M. A. Stanislaus, G. Demir, D. Linnekin, Z. K. Pan, and A. M. Gewirtz Crosstalk Between BCR/ABL Oncoprotein and CXCR4 Signaling through a Src Family Kinase in Human Leukemia Cells J. Exp. Med., September 2, 2002; 196(5): 667 - 678. [Abstract] [Full Text] [PDF] S. J. McLeod, A. H. Y. Li, R. L. Lee, A. E. Burgess, and M. R. Gold The Rap GTPases Regulate B Cell Migration Toward the Chemokine Stromal Cell-Derived Factor-1 (CXCL12): Potential Role for Rap2 in Promoting B Cell Migration J. Immunol., August 1, 2002; 169(3): 1365 - 1371. [Abstract] [Full Text] [PDF] A. Z. Fernandis, R. P. Cherla, R. D. Chernock, and R. K. Ganju CXCR4/CCR5 Down-modulation and Chemotaxis Are Regulated by the Proteasome Pathway J. Biol. Chem., May 10, 2002; 277(20): 18111 - 18117. [Abstract] [Full Text] [PDF] M. Ticchioni, C. Charvet, N. Noraz, L. Lamy, M. Steinberg, A. Bernard, and M. Deckert Signaling through ZAP-70 is required for CXCL12-mediated T-cell transendothelial migration Blood, May 1, 2002; 99(9): 3111 - 3118. [Abstract] [Full Text] [PDF] N. C. Ottoson, J. T. Pribila, A. S. H. Chan, and Y. Shimizu Cutting Edge: T Cell Migration Regulated by CXCR4 Chemokine Receptor Signaling to ZAP-70 Tyrosine Kinase J. Immunol., August 15, 2001; 167(4): 1857 - 1861. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Chernock, R. D. Articles by Ganju, R. K. Search for Related Content PubMed PubMed Citation Articles by Chernock, R. D. Articles by Ganju, R. K. Related Collections Signal Transduction Chemokines, Cytokines, and Interleukins Social Bookmarking                   What's this?

	Copyright © 2001 by American Society of Hematology         Online ISSN: 1528-0020