FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2009284265> ?p ?o ?g. }

• W2009284265 endingPage "17963" @default.
• W2009284265 startingPage "17953" @default.
• W2009284265 abstract "Src-like adaptor protein 2 (SLAP-2) is a hematopoietic adaptor protein previously implicated as a negative regulator of T-cell antigen receptor (TCR)-mediated signaling. SLAP-2 contains an SH3 and an SH2 domain, followed by a unique carboxyl-terminal tail, which is important for c-Cbl binding. Here we describe a novel role for SLAP-2 in regulation of the colony-stimulating factor 1 receptor (CSF-1R), a receptor tyrosine kinase important for growth and differentiation of myeloid cells. SLAP-2 co-immunoprecipitates with c-Cbl and CSF-1R in primary bone marrow-derived macrophages. Using murine myeloid cells expressing CSF-1R (FD-Fms cells), we show that SLAP-2 is tyrosine-phosphorylated upon stimulation with CSF-1 and associates constitutively with both c-Cbl and CSF-1R. In addition, we show that expression of a dominant negative form of SLAP-2 impairs c-Cbl association with the CSF-1R and receptor ubiquitination. Impaired c-Cbl recruitment also correlated with changes in the kinetics of CSF-1R down-regulation and trafficking. CSF-1-mediated differentiation of FD-Fms cells and activation of downstream signaling events was also enhanced in cells stably expressing dominant negative SLAP-2. Together, these results demonstrate that SLAP-2 plays a role in c-Cbl-dependent down-regulation of CSF-1R signaling. Src-like adaptor protein 2 (SLAP-2) is a hematopoietic adaptor protein previously implicated as a negative regulator of T-cell antigen receptor (TCR)-mediated signaling. SLAP-2 contains an SH3 and an SH2 domain, followed by a unique carboxyl-terminal tail, which is important for c-Cbl binding. Here we describe a novel role for SLAP-2 in regulation of the colony-stimulating factor 1 receptor (CSF-1R), a receptor tyrosine kinase important for growth and differentiation of myeloid cells. SLAP-2 co-immunoprecipitates with c-Cbl and CSF-1R in primary bone marrow-derived macrophages. Using murine myeloid cells expressing CSF-1R (FD-Fms cells), we show that SLAP-2 is tyrosine-phosphorylated upon stimulation with CSF-1 and associates constitutively with both c-Cbl and CSF-1R. In addition, we show that expression of a dominant negative form of SLAP-2 impairs c-Cbl association with the CSF-1R and receptor ubiquitination. Impaired c-Cbl recruitment also correlated with changes in the kinetics of CSF-1R down-regulation and trafficking. CSF-1-mediated differentiation of FD-Fms cells and activation of downstream signaling events was also enhanced in cells stably expressing dominant negative SLAP-2. Together, these results demonstrate that SLAP-2 plays a role in c-Cbl-dependent down-regulation of CSF-1R signaling. The colony-stimulating factor 1 receptor (CSF-1R) 4The abbreviations used are: CSF-1R, colony-stimulating factor-1 receptor; RTK, receptor tyrosine kinase; CSF-1, colony-stimulating factor-1; E3, ubiquitin-protein isopeptide ligase; TCR, T-cell antigen receptor; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PE, phycoerythrin; WT, wild type; IL-3, interleukin-3; PKC, protein kinase C; STAT3, signal transducers and activators of transcription 3; SLAP-1, -2, Src-like adaptor proteins 1 and 2. is a member of the type III receptor tyrosine kinase (RTK) family, which includes c-Kit, platelet-derived growth factor receptor α and β, and Flt3 (1Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3172) Google Scholar). All of the functions of colony-stimulating factor-1 (CSF-1) are mediated by CSF-1R, which is the primary regulator of the common myeloid lineage consisting of osteoclasts and mononuclear phagocytes (monocytes and macrophages) in vivo (2Dai X.M. Ryan G.R. Hapel A.J. Dominguez M.G. Russell R.G. Kapp S. Sylvestre V. Stanley E.R. Blood. 2002; 99: 111-120Crossref PubMed Scopus (856) Google Scholar, 3Motoyoshi K. Int. J. Hematol. 1998; 67: 109-122Crossref PubMed Google Scholar, 4Bourette R.P. Rohrschneider L.R. Growth Factors. 2000; 17: 155-166Crossref PubMed Scopus (111) Google Scholar, 5van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1253) Google Scholar). Ligand binding induces CSF-1R dimerization and autophosphorylation on tyrosine residues that regulate both kinase activity and mediate binding to downstream SH2 and PTB domain-containing proteins. Recruitment of such proteins leads to activation of signaling cascades involved in determining the biological outcomes of CSF-1R activation as well as receptor down-regulation (6Wilhelmsen K. Burkhalter S. van der Geer P. Oncogene. 2002; 21: 1079-1089Crossref PubMed Scopus (67) Google Scholar, 7Hamilton J.A. J. Leukocyte Biol. 1997; 62: 145-155Crossref PubMed Scopus (168) Google Scholar, 8Weiss A. Schlessinger J. Cell. 1998; 94: 277-280Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). A number of adaptor proteins, including Gads/Mona, FMIP, DOK-2, and c-Cbl, have been implicated as negative regulators of CSF-1R signaling. c-Cbl possesses a variant SH2 domain (TKB domain) that mediates binding to activated tyrosine kinases and a RING finger domain demonstrated to confer E3 ubiquitin ligase activity, which promotes the ubiquitination of activated tyrosine kinases (9Meng W. Sawasdikosol S. Burakoff S.J. Eck M.J. Nature. 1999; 398: 84-90Crossref PubMed Scopus (248) Google Scholar, 10Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (917) Google Scholar). In c-Cbl-deficient macrophages, both CSF-1R ubiquitination and endocytosis of the ubiquitinated receptor are impaired (11Lee P.S. Wang Y. Dominguez M.G. Yeung Y.G. Murphy M.A. Bowtell D.D. Stanley E.R. EMBO J. 1999; 18: 3616-3628Crossref PubMed Scopus (254) Google Scholar). Subsequent to CSF-1R ubiquitination, activated CSF-1R is degraded intralysosomally (12Guilbert L.J. Stanley E.R. J. Biol. Chem. 1986; 261: 4024-4032Abstract Full Text PDF PubMed Google Scholar). The degradation of ligand-stimulated CSF-1R normally occurs rapidly, and the half-life of the CSF-1R decreases from 3 h to 5 min upon ligand binding (13Fixe P. Praloran V. Eur. Cytokine Netw. 1997; 8: 125-136PubMed Google Scholar, 14Carlberg K. Tapley P. Haystead C. Rohrschneider L. EMBO J. 1991; 10: 877-883Crossref PubMed Scopus (54) Google Scholar). Mutation of the tyrosine residue within the c-Cbl binding site (Tyr-969 in human CSF-1R) has been frequently observed in myelodysplasia and acute myeloblastic leukemia (15Ridge S.A. Worwood M. Oscier D. Jacobs A. Padua R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1377-1380Crossref PubMed Scopus (220) Google Scholar). Similarly, the transforming viral homologue of CSF-1R, v-Fms, lacks the c-Cbl binding site. Importantly, re-addition of the c-Cbl binding site to v-Fms decreases its transforming activity (16Mancini A. Koch A. Wilms R. Tamura T. J. Biol. Chem. 2002; 277: 14635-14640Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Src-like adaptor protein family proteins, SLAP and SLAP-2, have an amino-terminal myristoylation signal required for association with cell membranes, closely juxtaposed SH3 and SH2 domains, and a unique carboxyl-terminal region that mediates association with c-Cbl. Both SLAP and SLAP-2 have been demonstrated to function as negative regulators of T-cell antigen receptor (TCR) signaling via a mechanism requiring the c-Cbl binding region (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar, 18Pandey A. Ibarrola N. Kratchmarova I. Fernandez M.M. Constantinescu S.N. Ohara O. Sawasdikosol S. Lodish H.F. Mann M. J. Biol. Chem. 2002; 277: 19131-19138Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 19Holland S.J. Liao X.C. Mendenhall M.K. Zhou X. Pardo J. Chu P. Spencer C. Fu A. Sheng N. Yu P. Pali E. Nagin A. Shen M. Yu S. Chan E. Wu X. Li C. Woisetschlager M. Aversa G. Kolbinger F. Bennett M.K. Molineaux S. Luo Y. Payan D.G. Mancebo H.S. Wu J. J. Exp. Med. 2001; 194: 1263-1276Crossref PubMed Scopus (49) Google Scholar). In addition to c-Cbl, SLAP-2 associates with the Zap-70 and Syk cytoplasmic tyrosine kinases and promotes their degradation when co-expressed. Recent work has shown that SLAP functions as a negative regulator of the TCR by promoting c-Cbl-dependent ubiquitination of TCR chain ζ and down-regulation of the CD3 complex (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar, 20Sosinowski T. Killeen N. Weiss A. Immunity. 2001; 15: 457-466Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 21Myers M.D. Dragone L.L. Weiss A. J. Cell Biol. 2005; 170: 285-294Crossref PubMed Scopus (63) Google Scholar). In addition, both SLAP and SLAP-2 have been implicated as negative regulators of B-cell receptor signaling (19Holland S.J. Liao X.C. Mendenhall M.K. Zhou X. Pardo J. Chu P. Spencer C. Fu A. Sheng N. Yu P. Pali E. Nagin A. Shen M. Yu S. Chan E. Wu X. Li C. Woisetschlager M. Aversa G. Kolbinger F. Bennett M.K. Molineaux S. Luo Y. Payan D.G. Mancebo H.S. Wu J. J. Exp. Med. 2001; 194: 1263-1276Crossref PubMed Scopus (49) Google Scholar, 22Dragone L.L. Myers M.D. White C. Sosinowski T. Weiss A. J. Immunol. 2006; 176: 335-345Crossref PubMed Scopus (34) Google Scholar), and avian SLAP has been demonstrated to interfere with erythropoietin signaling in erythroblasts (23Lebigot I. Gardellin P. Lefebvre L. Beug H. Ghysdael J. Quang C.T. Blood. 2003; 102: 4555-4562Crossref PubMed Scopus (19) Google Scholar). Although SLAP and SLAP-2 have been implicated in the regulation of antigen receptor signaling, SLAP was originally discovered in the context of RTK signaling (24Pandey A. Duan H. Dixit V.M. J. Biol. Chem. 1995; 270: 19201-19204Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and ectopic expression of SLAP in fibroblasts shown to inhibit platelet-derived growth factor receptor-induced proliferation, suggesting that SLAP may also act as a negative regulator of RTK signaling (25Roche S. Alonso G. Kazlauskas A. Dixit V.M. Courtneidge S.A. Pandey A. Curr. Biol. 1998; 8: 975-978Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26Manes G. Bello P. Roche S. Mol. Cell. Biol. 2000; 20: 3396-3406Crossref PubMed Scopus (37) Google Scholar). Both SLAP and SLAP-2 are expressed in lymphoid tissues and cell lines, whereas SLAP-2 is also abundantly expressed in myeloid cell lines such as KG1a, OCI, AML-2, -3, and -5 (27Loreto M.P. McGlade C.J. Oncogene. 2003; 22: 266-273Crossref PubMed Scopus (10) Google Scholar). A recent study by Manes et al. (28Manes G.A. Masendycz P. Nguyen T. Achuthan A. Dinh H. Hamilton J.A. Scholz G.M. FEBS J. 2006; 273: 1791-1804Crossref PubMed Scopus (10) Google Scholar) demonstrated that SLAP-2 is expressed in murine bone marrow-derived macrophages and suggested a potential role for SLAP-2 in CSF-1 signaling. Here we provide evidence that SLAP-2 plays a role in c-Cbl recruitment to activated CSF-1 receptors and consequent down-regulation of CSF-1R signaling by promoting internalization and degradation of activated receptors. Bacterial and Mammalian Expression Constructs—Wild-type SLAP-2 and SLAP-2 mutants have been previously described (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). SLAP-2 G2A contains a point mutation changing amino acid 2 to alanine, SH2* has a point mutation changing the arginine residue at amino acid position 120 to lysine, and in the ΔC truncation mutant a stop codon was introduced such that it lacks the last 70 carboxyl-terminal amino acids. SLAP-2 GST fusion proteins were prepared as previously described (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). SLAP and SLAP-2 (WT, G2A, and SH2*) constructs were subcloned into the HSC retroviral vector (provided by Dr. James Ellis) at SalI and BamHI sites for use in preparation of ecotropic virus. FD-Fms Cell Culture, Cell Stimulation, and Lysis—Murine myeloid FDC-P1 cells expressing wild-type Fms (FD-Fms cell line) were kindly provided by Dr. Larry R. Rohrschneider (Fred Hutchinson Cancer Research Center). FD-Fms cells were cultured at 37 °C, 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS) and 5% WEHI cell supernatant (as a source of IL-3). WEHI supernatant was prepared by culturing cells in RPMI 1640 medium and 10% FBS for 7–10 days. Before FD-Fms cells were stimulated with CSF-1, they were starved of IL-3 by washing twice with phosphate-buffered saline (PBS) and culturing in DMEM with 10% FBS for 5–6 h. Cells were then suspended at 10–20 × 106 cells/ml in serum-free DMEM and 100 ng/ml rmCSF-1 (R&D Systems) at 37 °C for various times before pelletting and lysis in cold Nonidet P-40 lysis buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mm EGTA (pH 8.0) freshly supplemented with complete protease inhibitors (Roche Applied Science), 1 mm sodium orthovanadate, and 10 mm NaF. Retroviral Infection of FD-Fms Cells—The ecotropic Phoenix packaging cell line (generously provided by Dr. Ian Clarke) was cultured in DMEM with 10% FBS and transiently transfected using Lipofectamine (Invitrogen) with the HSC IRES-eGFP retroviral vectors containing wild-type (WT), and mutant forms SLAP-2. Viral particle containing supernatants were collected, and carryover cells/debris was removed by passing through a 45-μm filter (Millipore) and used directly for spin infection of FD-Fms cells. Briefly, 1.5 × 106 FD-Fms cells were suspended in 3 ml of retroviral supernatant supplemented with 8 μg/ml Polybrene (Hexadimethrine Bromide, from Sigma). Cells were centrifuged in 50-ml Falcon tubes at 1800 rpm (650 × g) for 90 min at room temperature. Cells were resuspended, and a second spin was performed. The cells were suspended in fresh viral supernatant plus Polybrene and incubated overnight at 32 °C and 5% CO2. Media was then changed back to DMEM with 10% FBS and 5% WEHI supernatant, and cells were expanded for 4 days. GFP-positive cells were collected using fluorescence-assisted cell sorting, expanded, and resorted as necessary to select comparable levels of WT, G2A, and SH2* SLAP-2 protein levels. Differentiation of FD-Fms Cells—FD-Fms cell lines (HSC, G2A, WT, and SH2*) were washed twice, suspended in DMEM with 10% FBS and seeded at 5000 cells/ml in DMEM with 10% FBS and 2500 units of hCSF-1/ml (provided by Dr. E. Richard Stanley) in 10-ml cultures. Cells were analyzed by flow cytometry using FlowJo software (Tree Star) on day 3 of culture to measure side-scatter (SSC) and CD11b (Mac-1). Cells were incubated with Fc block (anti CD16/32 antibody) prior to incubation with PE-anti-CD11b or isotype control (Rat IgG2b) antibodies. Dead cells were excluded from the analysis by staining with propidium iodide. Preparation and Stimulation of Primary Bone Marrow-derived Macrophages—Primary bone marrow-derived macrophage were prepared from the femur bone marrow of 6-week-old mice. To remove differentiated cells, bone marrow was suspended in RPMI supplemented with 10% fetal bovine serum (Invitrogen), 5 units of penicillin C/ml, and 5 mg of streptomycin sulfate/ml for 2 h. Adherent cells were discarded, while nonadherent cells were counted, and plated at 1.25 × 105 cells/ml in DMEM with 10% FBS and 2,500 units of recombinant hCSF-1/ml for 7–10 days in 10-cm culture plates. Adherent bone marrow-derived macrophages were starved of CSF-1 for 24 h and stimulated by washing once with serum-free DMEM at 37 °C, and then stimulated for various times in serum-free DMEM supplemented with 100 ng/ml rmCSF-1. Stimulated cells were subsequently lysed in Nonidet P-40 buffer, and soluble protein lysates were used for immunoprecipitations as described. Immunoprecipitation and Western Blotting—Cells were lysed in Nonidet P-40 lysis buffer with complete protease inhibitors and 1 mm sodium orthovanadate. Lysates were cleared by centrifugation at 14,000 rpm (20,800 × g) at 4 °C for 10 min and precleared by incubation with 50 μl of 20% (v/v) protein G-Sepharose beads or with protein A-Sepharose beads (Sigma) at 4 °C for 30 min. Precleared lysates were incubated with antibodies (described below) and either protein G- or protein A-Sepharose beads as described above and then incubated at 4 °C with gentle rotation for 90 min. Immune complexes were washed four times in 1 ml of cold Nonidet P-40 wash buffer (Nonidet P-40 lysis buffer with 0.1% Nonidet P-40, without EGTA, and 20 mm HEPES (pH 7.5)), and bound proteins were eluted by boiling for 5 min in 2× SDS sample buffer. Eluted proteins were resolved by SDS-PAGE on 10% gels. Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (PerkinElmer Life Sciences) and incubated in blocking solution for a minimum of 30 min prior to the addition of antibodies. Bound antibodies were detected by using enhanced chemiluminescence reagent (ECL, PerkinElmer Life Sciences). In Vitro Binding Assays and Far Western—GST fusion proteins were expressed in DH5α bacteria and purified on glutathione-Sepharose beads (Amersham Biosciences). In vitro binding assays were done with FD-Fms cell lysates (1 mg/ml) either unstimulated or stimulated with rmCSF-1. Lysates were incubated with 4 μg of GST fusion proteins for 90 min at 4 °C. Following several washes, bound proteins were eluted in 2× SDS sample buffer and resolved by SDS-PAGE. Membranes were stained with Coomassie Blue dye to ensure equal loading of fusion proteins. For the far Western, GST-SLAP-2 was eluted from Sepharose beads by incubating beads in 3 mg/ml glutathione in 50 mm Tris, pH 8.0, then dialyzing in PBS before quantifying. Anti-CSF-1R immunoprecipitates from the FD-Fms cell line were subjected to SDS-PAGE, and transferred to Nitrocellulose (Amersham Biosciences) membranes. The membranes were blocked in TBST (10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% (v/v) Tween 20) with 5% (w/v) skim milk powder for 1 h at room temperature, followed by incubation with 10 μg/ml GST-SLAP-2 in blocking solution supplemented with 10 mm dithiothreitol overnight at 4 °C. Following three 5-min washes with TBST, membranes were incubated with anti-GST antibody in blocking solution for 1 h, washed again, then incubated with protein-A linked horseradish peroxidase for 40 min. Finally, membranes were washed (4 × 5 min and 1 × 10 min) with TBST before ECL detection was used. Antibodies—Polyclonal SLAP-2 was produced as previously described (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). 10 μl (3 ug) of affinity-purified SLAP-2 antibody was used in immunoprecipitation experiments, and a concentration of 0.3 μg/ml was used for immunoblotting. The following antibodies and conditions were used: anti-phosphotyrosine 4G10 (UBI), 1:1000 for Western blotting; rabbit anti-STAT3 (UBI), 1:1000; anti-phospho-STAT3 (UBI), 1:1000; a 1:1 mix of mouse anti-c-Cbl (clone 17, BD Transduction Laboratories) and clone 7G10 (UBI), 1:1000; rabbit anti-Fms (CSF-1R) (UBI) was used at 2 μl for immunoprecipitation; 1:1 mix of rabbit anti-pY-CSF receptor (CSF-1R) phosphotyrosine 723 and phosphotyrosine 809 (Cell Signaling), 1:1000; rabbit anti-CSF-1R clone C-20 (Santa Cruz Biotechnology), 1:1000; antiubiquitin (Covance) 1:1000; anti-transferrin receptor (Zymed Laboratories Inc.) 1:1000; anti-GST (UBI), 1:1000; sheep anti-mouse antibody (1:6000 dilution); and protein A (1:3000 dilution) conjugated to horseradish peroxidase were used to detect bound primary mouse monoclonal antibodies and polyclonal antibodies, respectively. Blocking solutions varied according to the manufacturer's recommendations. PE-conjugated anti-CD11b (clone M1/70), rat IgG2b, and anti-CD32/16 antibodies used for flow cytometry were purchased from eBioscience. Subcellular Fractionation—FD-Fms cells (2.5 × 107) were washed with phosphate-buffered saline (PBS) and lysed in 1 ml of hypotonic lysis buffer (10 mm Tris-HCl (pH 8.0), 1 mm MgCl2) containing complete protease inhibitors and 1 mm sodium orthovanadate. Cells were passed through a Dounce homogenizer until all cells were lysed, as checked by trypan blue staining. Lysed cells were adjusted back to isotonic conditions by the addition of 5 m NaCl to a final concentration of 150 mm. Lysates were centrifuged at 3,000 rpm (900 × g) for 10 min at 4 °C. The pellet representing the nuclear fraction was discarded. The supernatant from the first spin was centrifuged in a Beckman tabletop ultracentrifuge by using the TLA-45 rotor at 43,000 rpm (100,000 × g) for 30 min at 4 °C. The pellet representing the membrane fraction was resuspended in extraction buffer (1% SDS, 1% Triton X-100, 1% sodium deoxycholate) in 10 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1 mm MgCl2. The supernatant was adjusted to 0.1% SDS-0.1% Triton X-100/0.1% sodium deoxycholate. Biotinylation of Surface Proteins—FD-Fms cells were stimulated for various times with 100 ng/ml rmCSF-1 (R&D Systems) at 37 °C. After stimulation, cells were cooled on ice for 5 min and washed twice with cold PBS. Cells were then incubated with biotin (EZ-Link NHS-SS-Biotin, Pierce) diluted at 0.2 mg/ml in biotinylation buffer (154 mm NaCl, 10 mm HEPES, 3 mm KCl, 1 mm MgCl2, 0.1 mm CaCl2, 10 mm glucose, pH 7.6) for 1 h at 4 °C. After labeling, cells were washed twice with cold PBS, blocked for 5 min in DMEM (10% FBS, 100 mm glycine) at 4°C, washed twice in cold PBS, and lysed in PLC lysis buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 10% (v/v) glycerol, 1% (v/v) Triton-X-100, 1 mm EGTA (pH 8.0) freshly supplemented with complete protease inhibitors (Roche Applied Science), 1 mm sodium orthovanadate, and 10 mm NaF). Biotin-labeled proteins were recovered by incubating ∼1 mg of lysate with 30 μl of streptavidin beads (ImmunoPure Immobilized Streptavidin, Pierce) overnight. Beads were washed 4× with PLC wash buffer and boiled in SDS sample buffer. Samples were analyzed by Western blotting against CSF-1R. SLAP-2 Associates with c-Cbl and CSF-1R in Primary Bone Marrow Macrophages and in FD-Fms Cells—To investigate the role of SLAP-2 in CSF-1R signaling, we performed immunoprecipitations from untreated or CSF-1-stimulated bone marrow-derived macrophage cell lysate using anti-SLAP-2 and anti-CSF-1R antibodies. Anti-phosphotyrosine Western blotting revealed that several tyrosine-phosphorylated proteins co-precipitated with SLAP-2 following CSF-1 stimulation compared with pre-immune control serum (Fig. 1A, upper panel). The tyrosine-phosphorylated protein of ∼120 kDa was identified as c-Cbl (Fig. 1A, lower panel). Notably the association of SLAP-2 with c-Cbl was observed in unstimulated cells but was enhanced following treatment with CSF-1. A similar constitutive association between SLAP-2 and c-Cbl has also been observed in primary thymocytes (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). A tyrosine-phosphorylated protein of 180 kDa that co-migrated with the CSF-1R was also observed (Fig. 1A, upper panel), although anti-CSF-1R antibody was not sensitive enough to confirm its identity. A phospho-specific antibody (pY-CSF-1R) directed against phosphorylated tyrosine residues Tyr-807 and Tyr-721 on the activated CSF-1R was able to detect the CSF-1R that was co-precipitated with SLAP-2 (Fig. 1B). Due to a high level of background, and the low level expression, endogenous SLAP-2 could not be detected upon subsequent re-blotting of the SLAP-2 immunoprecipitates (data not shown). To further investigate the role of SLAP-2 in CSF-1R signaling, we utilized the FDC-P1 myeloid cell line, FD-Fms, which expresses wild-type murine CSF-1R (29Rohrschneider L.R. Metcalf D. Mol. Cell. Biol. 1989; 9: 5081-5092Crossref PubMed Scopus (62) Google Scholar, 30Bourette R.P. Myles G.M. Carlberg K. Chen A.R. Rohrschneider L.R. Cell Growth & Differ. 1995; 6: 631-645PubMed Google Scholar). Immunoprecipitation of endogenous SLAP-2 from lysates of unstimulated or CSF-1-stimulated cells probed with anti-SLAP-2 revealed bands corresponding to the p28 and p25 forms of SLAP-2 that have previously been described in hematopoietic cell lines and tissues (Fig. 1C, bottom panel) (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). Endogenous SLAP-2 was specifically co-immunoprecipitated with tyrosine-phosphorylated proteins of 120 and 180 kDa following CSF-1 stimulation (Fig. 1C, top panel). Re-blotting identified the 120-kDa band as c-Cbl and showed that C-Cbl associated with SLAP-2 prior to CSF-1R stimulation (Fig. 1C, middle panel). Again, anti-CSF-1R antibody was not sensitive enough to detect the co-immunoprecipitated CSF-1R; however, using the pY-CSF-1R antibody we were able to demonstrate that the CSF-1R is co-precipitated with SLAP-2 in FD-Fms cells (Fig. 1D). To determine which regions of SLAP-2 mediate its association with c-Cbl and CSF-1R, GST-pulldown assays were carried out using lysates from FD-Fms cells. Recombinant wild-type (WT) GST-SLAP-2 associated with tyrosine-phosphorylated proteins of similar size observed in SLAP-2 immunoprecipitates, and subsequent immunoblotting confirmed the identity of c-Cbl and CSF-1R, respectively (Fig. 2B). Similarly, a GST fusion of the related SLAP molecule also bound to c-Cbl and the CSF-1R. In this system, SLAP-2 association with the receptor appeared to be constitutive, whereas SLAP binding to CSF-1R was strongly induced by CSF-1 stimulation (Fig. 2B). In addition, c-Cbl binding to both GST-SLAP-2 and GST-SLAP was strongly induced by CSF-1 stimulation (Fig. 2B). A GST-SLAP-2-ΔC fusion protein, which lacks the entire carboxyl-terminal region (ΔC, Fig. 2A), was unable to associate with c-Cbl, consistent with previous studies that indicated the C-terminal region of SLAP-2 mediates its association with c-Cbl (Fig. 2B, second panel) (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar, 19Holland S.J. Liao X.C. Mendenhall M.K. Zhou X. Pardo J. Chu P. Spencer C. Fu A. Sheng N. Yu P. Pali E. Nagin A. Shen M. Yu S. Chan E. Wu X. Li C. Woisetschlager M. Aversa G. Kolbinger F. Bennett M.K. Molineaux S. Luo Y. Payan D.G. Mancebo H.S. Wu J. J. Exp. Med. 2001; 194: 1263-1276Crossref PubMed Scopus (49) Google Scholar, 27Loreto M.P. McGlade C.J. Oncogene. 2003; 22: 266-273Crossref PubMed Scopus (10) Google Scholar). This form of SLAP-2 also showed a significant reduction in CSF-1R binding suggesting that this region also stabilizes SLAP-2 binding to the receptor. A mutant form of SLAP-2 with an inactivating mutation in the SH2 domain (Fig. 2A, SH2*) failed to bind CSF-1R (Fig. 2B, third panel), suggesting that the SLAP-2 SH2 domain is required for receptor association in the presence or absence of growth factor stimulation. These observations contradict those of Manes et al., who observed that neither mutation of the SH2 domain nor deletion of the carboxyl terminus had an effect on CSF-1R binding when co-expressed in HEK293T cells (28Manes G.A. Masendycz P. Nguyen T. Achuthan A. Dinh H. Hamilton J.A. Scholz G.M. FEBS J. 2006; 273: 1791-1804Crossref PubMed Scopus (10) Google Scholar). To address whether the association between SLAP-2 and CSF-1R is direct, CSF-1R was immunoprecipitated from FD-Fms cell lysates and separated by SDS-PAGE, and then transferred to nitrocellulose membrane. The membrane was incubated with purified recombinant GST-SLAP-2 and immunoblotted with anti-GST antibody to detect bound GST-SLAP-2. As shown in Fig. 2C, GST-SLAP-2 bound to a band at the size expected for CSF-1R as well as another slower migrating protein. Subsequent reprobing of the same membrane with anti-CSF-1R confirmed that the bound GST-SLAP-2 overlapped with the CSF-1R signal and suggests that SLAP-2 associates with CSF-1R directly both before and after stimulation (Fig. 2C). SLAP-2 Is Tyrosine-phosphorylated upon CSF-1 Stimulation—When endogenous SLAP-2 was immunoprecipitated from lysates of CSF-1-stimulated FD-Fms cells we observed slower migrating species in addition to the two SLAP isoforms expressed in these cells. (Fig. 3A). Previously we and others (28Manes G.A. Masendycz P. Nguyen T. Achuthan A. Dinh H. Hamilton J.A. Scholz G.M. FEBS J. 2006; 273: 1791-1804Crossref PubMed Scopus (10) Google Scholar) reported that SLAP-2 is phosphorylated on serine residues when ectopically expressed, but tyrosine phosphorylation had not been reported. To test whether the slower migrating SLAP-2 species observed following CSF-1 stimulation is also tyrosine-phosphorylated, SLAP-2 was immunoprecipitated from a cell line engineered to overexpress WT SLAP-2 and immunoblotted with anti-phosphotyrosine (Fig. 3B). A tyrosine-phosphorylated band that co-migrated with the transient higher molecular weight form of SLAP-2 was observed, suggesting that SLAP-2 is transiently tyrosine-phosphorylated upon activation of the CSF-1R. When SLAP-2 immunoprecipitates were incubated with phosphatase, the anti-phosphotyrosine reactivity was lost and accompanied by a change in electrophoretic mobility (Fig. 3C). Taken together, these results suggest that SLAP-2 becomes tyrosine-phosphorylated upon CSF-1 stimulation. SLAP-2 Modulates c-Cbl Association and Down-regulation of the CSF-1R—To examine the role of SLAP-2 in CSF-1R signaling and down-regulation, we created FD-Fms stable cell lines expressing wild-type SLAP-2 (WT) or forms of SLAP-2 harboring mutations that disrupt myristoylation (G2A) and inactivate the SH2 domain (SH2*). Stably transduced cell lines (HSC, WT, G2A, and SH2*) were stimulated with CSF-1 at 4 °C (to slow the kinetics of receptor down-regulation), and membrane fractions were isolated and blotted for CSF" @default.
• W2009284265 created "2016-06-24" @default.
• W2009284265 creator A5005172437 @default.
• W2009284265 creator A5018232373 @default.
• W2009284265 creator A5018488437 @default.
• W2009284265 creator A5032495270 @default.
• W2009284265 creator A5037450756 @default.
• W2009284265 date "2007-06-01" @default.
• W2009284265 modified "2023-09-30" @default.
• W2009284265 title "The Src-like Adaptor Protein 2 Regulates Colony-stimulating Factor-1 Receptor Signaling and Down-regulation" @default.
• W2009284265 cites W1514353293 @default.
• W2009284265 cites W1526569781 @default.
• W2009284265 cites W1551174843 @default.
• W2009284265 cites W1572814792 @default.
• W2009284265 cites W1624856187 @default.
• W2009284265 cites W1639582946 @default.
• W2009284265 cites W1966048621 @default.
• W2009284265 cites W1966290418 @default.
• W2009284265 cites W1967658924 @default.
• W2009284265 cites W1978358848 @default.
• W2009284265 cites W1979032866 @default.
• W2009284265 cites W1998635509 @default.
• W2009284265 cites W2000757945 @default.
• W2009284265 cites W2001201225 @default.
• W2009284265 cites W2012771591 @default.
• W2009284265 cites W2017923612 @default.
• W2009284265 cites W2018088003 @default.
• W2009284265 cites W2018529431 @default.
• W2009284265 cites W2027533965 @default.
• W2009284265 cites W2031603575 @default.
• W2009284265 cites W2044153794 @default.
• W2009284265 cites W2046259235 @default.
• W2009284265 cites W2062853094 @default.
• W2009284265 cites W2082706608 @default.
• W2009284265 cites W2094534216 @default.
• W2009284265 cites W2115790755 @default.
• W2009284265 cites W2133188024 @default.
• W2009284265 cites W2140924980 @default.
• W2009284265 cites W2147066049 @default.
• W2009284265 cites W2171164575 @default.
• W2009284265 cites W2180005201 @default.
• W2009284265 cites W2315802016 @default.
• W2009284265 cites W32737249 @default.
• W2009284265 cites W4232940172 @default.
• W2009284265 cites W73223339 @default.
• W2009284265 doi "https://doi.org/10.1074/jbc.m701182200" @default.
• W2009284265 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17353186" @default.
• W2009284265 hasPublicationYear "2007" @default.
• W2009284265 type Work @default.
• W2009284265 sameAs 2009284265 @default.
• W2009284265 citedByCount "28" @default.
• W2009284265 countsByYear W20092842652012 @default.
• W2009284265 countsByYear W20092842652013 @default.
• W2009284265 countsByYear W20092842652015 @default.
• W2009284265 countsByYear W20092842652016 @default.
• W2009284265 countsByYear W20092842652017 @default.
• W2009284265 countsByYear W20092842652018 @default.
• W2009284265 countsByYear W20092842652019 @default.
• W2009284265 countsByYear W20092842652020 @default.
• W2009284265 countsByYear W20092842652021 @default.
• W2009284265 crossrefType "journal-article" @default.
• W2009284265 hasAuthorship W2009284265A5005172437 @default.
• W2009284265 hasAuthorship W2009284265A5018232373 @default.
• W2009284265 hasAuthorship W2009284265A5018488437 @default.
• W2009284265 hasAuthorship W2009284265A5032495270 @default.
• W2009284265 hasAuthorship W2009284265A5037450756 @default.
• W2009284265 hasBestOaLocation W20092842651 @default.
• W2009284265 hasConcept C108636557 @default.
• W2009284265 hasConcept C170493617 @default.
• W2009284265 hasConcept C183786373 @default.
• W2009284265 hasConcept C185592680 @default.
• W2009284265 hasConcept C55493867 @default.
• W2009284265 hasConcept C62478195 @default.
• W2009284265 hasConcept C86803240 @default.
• W2009284265 hasConcept C95444343 @default.
• W2009284265 hasConceptScore W2009284265C108636557 @default.
• W2009284265 hasConceptScore W2009284265C170493617 @default.
• W2009284265 hasConceptScore W2009284265C183786373 @default.
• W2009284265 hasConceptScore W2009284265C185592680 @default.
• W2009284265 hasConceptScore W2009284265C55493867 @default.
• W2009284265 hasConceptScore W2009284265C62478195 @default.
• W2009284265 hasConceptScore W2009284265C86803240 @default.
• W2009284265 hasConceptScore W2009284265C95444343 @default.
• W2009284265 hasIssue "25" @default.
• W2009284265 hasLocation W20092842651 @default.
• W2009284265 hasOpenAccess W2009284265 @default.
• W2009284265 hasPrimaryLocation W20092842651 @default.
• W2009284265 hasRelatedWork W1482523072 @default.
• W2009284265 hasRelatedWork W1975510716 @default.
• W2009284265 hasRelatedWork W1978760124 @default.
• W2009284265 hasRelatedWork W1981419744 @default.
• W2009284265 hasRelatedWork W2058432650 @default.
• W2009284265 hasRelatedWork W2144354735 @default.
• W2009284265 hasRelatedWork W2160536323 @default.
• W2009284265 hasRelatedWork W2169932670 @default.
• W2009284265 hasRelatedWork W2346314153 @default.
• W2009284265 hasRelatedWork W2809080492 @default.
• W2009284265 hasVolume "282" @default.

• next