The Src-like Adaptor Protein 2 Regulates Colony-stimulating Factor-1 Receptor Signaling and Down-regulation

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 also as a negative regulator of RTK signaling S. G. A. Dixit V.M. S.A. A. Biol. 1998; 8: Full Text Full Text PDF PubMed Scopus Google Scholar, G. P. S. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). Both SLAP and SLAP-2 are in and cell SLAP-2 is also in myeloid cell such as and M.P. McGlade C.J. Oncogene. 2003; 22: PubMed Scopus Google Scholar). A by P. T. A. H. J.A. J. 2006; PubMed Scopus Google Scholar) demonstrated that SLAP-2 is in murine bone marrow-derived and a role for SLAP-2 in CSF-1 signaling. Here we that SLAP-2 plays a role in c-Cbl recruitment to activated CSF-1 and down-regulation of CSF-1R signaling by promoting and degradation of activated and SLAP-2 and SLAP-2 have been previously (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). SLAP-2 contains a 2 to has a the residue to and in the a was such that lacks the carboxyl-terminal SLAP-2 proteins as previously (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 and the by and for in of FD-Fms Cell Cell and myeloid cells expressing (FD-Fms cell by R. Rohrschneider FD-Fms cells in with fetal bovine and cell a of was by cells in and for FD-Fms cells with CSF-1, of by with phosphate-buffered and in with for in and for and in 1 with 1 and of FD-Fms cell by was in with and with the and SLAP-2. and was by a and used for of FD-Fms cells. FD-Fms cells in 3 of with from in for min and a was The cells in viral and and was to with and and cells for cells cell and as to of WT, and SLAP-2 protein of FD-Fms cell WT, and in with and in with and of by E. in by on 3 of to and with to with cells from the by with and of bone marrow-derived from the bone of bone was in with fetal bovine 5 of and 5 of for 2 cells cells and in with and of for in bone marrow-derived of CSF-1 for h and by with and for in with cells in and protein used for as and in with and 1 by for min and by with of protein with protein for with and protein protein as and with for in 1 of with and and proteins by for 5 min in proteins by on to and in for a of min to the addition of by enhanced In and proteins in and on In binding with FD-Fms cell with with of proteins for min proteins in and by with to of proteins. the was from by in 3 glutathione in in from the FD-Fms cell to and to The in with for 1 h followed by with in with with with in for 1 with for 5 min and 1 with was SLAP-2 was as previously (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). of SLAP-2 was used in and a of was used for The and for a of and (CSF-1R) was used 2 for of receptor (CSF-1R) and receptor and protein A to used to primary and to the and used for from cells with phosphate-buffered and in 1 of 1 and 1 a cells as by cells to by the addition of 5 to a of for min The the was The from the was in a by the for min The the was in in 1 The was to of cells for with cells on for 5 min and with with in 3 1 for 1 h cells with PBS, for 5 min in FBS, in PBS, and in 1 with 1 and proteins by of with of with and in by CSF-1R. SLAP-2 with c-Cbl and CSF-1R in and in FD-Fms the role of SLAP-2 in CSF-1R signaling, we from bone marrow-derived cell and that tyrosine-phosphorylated proteins with SLAP-2 CSF-1 stimulation with The tyrosine-phosphorylated protein of was as c-Cbl the association of SLAP-2 with c-Cbl was observed in cells was enhanced with A association SLAP-2 and c-Cbl has also been observed in primary (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 that with the CSF-1R was also observed was to its A tyrosine residues and on the activated CSF-1R was to the CSF-1R that was with SLAP-2 to a of and the SLAP-2 upon of the SLAP-2 the role of SLAP-2 in CSF-1R signaling, we the myeloid cell which murine CSF-1R L.R. D. Mol. Cell. Biol. PubMed Scopus Google Scholar, R.P. K. Rohrschneider L.R. Cell Growth 1995; Google Scholar). of SLAP-2 from of cells with to the and of SLAP-2 that have previously been in hematopoietic cell and (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). SLAP-2 was with tyrosine-phosphorylated proteins of and CSF-1 stimulation the as c-Cbl and that with SLAP-2 to CSF-1R stimulation was to the the we to demonstrate that the CSF-1R is with SLAP-2 in FD-Fms cells which of SLAP-2 mediate its association with c-Cbl and CSF-1R, from FD-Fms cells. with tyrosine-phosphorylated proteins of observed in SLAP-2 and the of c-Cbl and CSF-1R, Similarly, a of the SLAP also to c-Cbl and the CSF-1R. In SLAP-2 association with the receptor to SLAP binding to CSF-1R was by CSF-1 stimulation In addition, c-Cbl binding to both and was by CSF-1 stimulation A which lacks the carboxyl-terminal region was to with c-Cbl, with that the region of SLAP-2 mediates its association with c-Cbl (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, M.P. McGlade C.J. Oncogene. 2003; 22: PubMed Scopus Google Scholar). form of SLAP-2 also a in CSF-1R binding suggesting that region also SLAP-2 binding to the A form of SLAP-2 with an in the SH2 domain to CSF-1R suggesting that the SLAP-2 SH2 domain is required for receptor association in the of growth factor of observed that of the SH2 domain of the an on CSF-1R binding when in cells P. T. A. H. J.A. J. 2006; PubMed Scopus Google Scholar). the association SLAP-2 and CSF-1R is CSF-1R was from FD-Fms cell and by and to The was with and with to shown in to a the for CSF-1R as well as Subsequent of the with that the with the CSF-1R signal and that SLAP-2 associates with CSF-1R both and stimulation SLAP-2 upon CSF-1 SLAP-2 was from of FD-Fms cells we observed in addition to the SLAP in these cells. we and P. T. A. H. J.A. J. 2006; PubMed Scopus Google Scholar) that SLAP-2 is on residues when tyrosine been the SLAP-2 observed CSF-1 stimulation is also SLAP-2 was from a cell to SLAP-2 and with A tyrosine-phosphorylated that with the form of SLAP-2 was suggesting that SLAP-2 is tyrosine-phosphorylated upon activation of the CSF-1R. SLAP-2 with the was and by a in these results that SLAP-2 tyrosine-phosphorylated upon CSF-1 SLAP-2 c-Cbl and of the the role of SLAP-2 in CSF-1R signaling and we FD-Fms cell expressing SLAP-2 of SLAP-2 that myristoylation and the SH2 domain cell WT, and with CSF-1 the kinetics of receptor and and for CSF-1R and activated CSF-1R In cells SLAP-2, a in the receptor and the of CSF-1R in the was observed CSF-1 stimulation with In in cells expressing SLAP-2 a of the activated receptor in the as with cells expressing the SLAP-2 was observed results that SLAP-2 receptor of a dominant form of SLAP-2 with down-regulation of the activated The E3 ligase c-Cbl associates with the CSF-1 receptor upon stimulation and is a to receptor ubiquitination and the of SLAP-2 on the association c-Cbl and CSF-1R, the receptor was from and SLAP-2 and cell and stimulation with with that of SLAP-2 the on the association of CSF-1R and c-Cbl 1 min of CSF-1 in cells expressing SLAP-2 a in c-Cbl was observed the that SLAP-2 plays a role in c-Cbl mediated down-regulation of the CSF-1R. c-Cbl has been demonstrated to important for ubiquitination of the CSF-1R (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). the SH2 of SLAP-2 c-Cbl is from the CSF-1 we its expression CSF-1R ubiquitination. FD-Fms cell WT, and with CSF-1 and CSF-1R was and with an CSF-1R ubiquitination was observed by 3 and ubiquitinated by min of SLAP-2 to ubiquitination of CSF-1R 3 min down-regulation and In cells expressing SLAP-2 the of CSF-1R ubiquitination was the in to and SLAP-2 cells the observed in ubiquitination of CSF-1R correlated with changes in receptor and degradation we FD-Fms cell with CSF-1 as and with proteins and the of CSF-1R was by All the cell a of CSF-1R with the and degradation of activated in cells SLAP-2 was a of CSF-1R the cell 1 suggesting that of the receptor was impaired In addition, a 5 an in the cell of CSF-1R was observed min in the SLAP-2 in the and cells cell receptor was is with a in which expression of SLAP-2 with of the receptor to the and promotes the of the CSF-1R. in FD-Fms SLAP-2 cells with factor as cells with a L.R. D. Mol. Cell. Biol. PubMed Scopus Google Scholar). in the of CSF-1 results in differentiation a complex and cells as by and a of differentiation on FD-Fms cells in L.R. D. Mol. Cell. Biol. PubMed Scopus Google Scholar). that expression of SLAP-2 to interfere with CSF-1R down-regulation we CSF-1-mediated differentiation of FD-Fms cells. Growth of the cell SLAP-2, and in changes in differentiation and 3 of in CSF-1, SLAP-2 expression CSF-1-mediated differentiation suggesting that SLAP-2 the of CSF-1 in with cells expressing SLAP-2 a in the of cells that in to CSF-1, as was by cells and of The of cells was for the SLAP-2 cell in to the was with the in cells such that the of cells that growth in CSF-1 was for the SLAP-2 cell with the The SLAP-2 of and cells as the cell results that with SLAP-2 function CSF-1 signaling and that SLAP-2 as a negative regulator of CSF-1R. SLAP-2 CSF-1R which signaling events cells expressing the and of SLAP-2 with Cell to in the of downstream CSF-1R changes in the of activated was suggesting that the changes by SLAP-2 expression to of the activity of these activation of of which in leads to the and activation of family R.P. Rohrschneider L.R. Growth Factors. 2000; 17: 155-166Crossref PubMed Scopus (111) Google Scholar, D. J. 1998; PubMed Scopus Google Scholar). Although have been demonstrated to involved in was shown to activated upon CSF-1 stimulation in FD-Fms cells H. J. M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, I. R.P. Rohrschneider L.R. O. J. Leukocyte Biol. 2003; PubMed Scopus Google Scholar). that SLAP-2 to interfere with CSF-1-mediated we the of to to the upon CSF-1 of cells expressing WT, of SLAP-2 by A in the of in the CSF-1 stimulation was observed in cells expressing SLAP-2 with cells In the cell expressing SLAP-2 an in in the 1 and 2 h CSF-1 stimulation with both and cell of has also been shown to upon CSF-1 stimulation of FD-Fms cells U. H. V. R. L. 1998; PubMed Scopus Google Scholar, Y.G. Wang Y. P.S. Stanley E.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and to a role in differentiation U. S. R.P. Rohrschneider L.R. W. L. Growth Factors. 1998; 15: PubMed Scopus Google Scholar). we the activation of with stimulation with of SLAP-2 the of proteins with cells cells expressing SLAP-2 and these that SLAP-2 functions to the activation of CSF-1 of activated is a in signaling from these for the protein c-Cbl is involved in CSF-1R and degradation (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). Here we that the adaptor protein SLAP-2 in demonstrate a role for SLAP-2 in c-Cbl-dependent down-regulation of CSF-1R. SLAP-2 is to in bone and has been implicated in CSF-1 signaling. and the of SLAP-2 differentiation of bone primary bone marrow-derived in to CSF-1 suggesting that SLAP-2 an important regulator in biological P. T. A. H. J.A. J. 2006; PubMed Scopus Google Scholar). to also an association of SLAP-2 with the CSF-1R a role in CSF-1R signaling. results that the SLAP-2, c-Cbl, and the CSF-1R in primary bone marrow-derived important for CSF-1R down-regulation the of signaling from the CSF-1R in myeloid show that expression of a dominant negative form of SLAP-2 in FD-Fms cells c-Cbl binding to CSF-1R stimulation and ubiquitination, and events to CSF-1R SLAP-2 has previously been shown to a negative regulator downstream of activated T-cell 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) and B-cell antigen (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). of a dominant negative form of SLAP-2 in cells TCR signaling (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). we have shown that with SLAP-2 function and down-regulation of activated CSF-1R signaling, and CSF-1-mediated differentiation of FD-Fms cells. the dominant negative SLAP-2 with c-Cbl and with the activated by c-Cbl from proteins and c-Cbl upon ligand stimulation of CSF-1R, SLAP-2 plays a role in c-Cbl to the site of activated the in an receptor ubiquitination and recruitment of proteins. a closely family has been and shown to with c-Cbl and TCR in an the of M.D. Dragone L.L. Weiss A. J. Cell Biol. 2005; 170: 285-294Crossref PubMed Scopus (63) Google Scholar). Similarly, results a role for SLAP-2 in a that c-Cbl and ubiquitination, with SLAP-2 function CSF-1R with which of endocytosis SLAP-2 the of SLAP-2 adaptor proteins within the context of antigen signaling in and RTK signaling in myeloid cells. in and in vivo binding that SLAP-2 associates with the CSF-1 receptor of ligand SLAP-2 associates with activated of the CD3 complex 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). that SLAP-2 is to RTK signaling in a from antigen and that the association unique of SLAP-2 binding to CSF-1R is to tyrosine of SLAP-2 CSF-1R which has been observed activation of the TCR, have as on SLAP-2 function 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). is to the of the SLAP-2 with the CSF-1R, as well as the of SLAP-2 tyrosine and their role in SLAP-2 also that of SLAP-2 which lacks the amino-terminal myristoylation on that myristoylation of SLAP-2 is for its of proteins is to association with and P.S. Immunol. 2000; PubMed Scopus Google Scholar). work that the SLAP-2 myristoylation site is for association with the (17Loreto M.P. Berry D.M. McGlade C.J. Mol. Cell. Biol. 2002; 22: 4241-4255Crossref PubMed Scopus (35) Google Scholar). are required to that SLAP-2 in in vivo and the role of in of SLAP-2. in have also demonstrated a of SLAP-2 on TCR signaling and CD3 in expression of SLAP-2 on signaling and CSF-1R in the role of SLAP-2 and c-Cbl in regulation of these of an that the myeloid cells used in have of SLAP-2 with to CSF-1 regulation that are SLAP is also in FD-Fms C. L. M. M. P. and C. J. we SLAP also a role in CSF-1R SLAP and SLAP-2 have both been demonstrated to function as of TCR signaling, to the TCR via a is the observed upon expression of the dominant negative SLAP-2 in to dominant on both SLAP and SLAP-2 of myeloid lineage cells from and the and functions of these Rohrschneider for the FD-Fms cell Stanley for CSF-1, and for the and cell also Berry for of the and of the McGlade for