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• W2031762637 abstract "Recent literature implicates a regulatory function of the juxtamembrane domain (JMD) in receptor tyrosine kinases. Mutations in the JMD of c-Kit and Flt3 are associated with gastrointestinal stromal tumors and acute myeloid leukemias, respectively. Additionally, autophosphorylated Tyr559 in the JMD of the colony stimulating factor-1 (CSF-1) receptor (CSF-1R) binds to Src family kinases (SFKs). To investigate SFK function in CSF-1 signaling we established stable 32D myeloid cell lines expressing CSF-1Rs with mutated SFK binding sites (Tyr559-TFI). Whereas binding to I562S was not significantly perturbed, Y559F and Y559D exhibited markedly decreased CSF-1-dependent SFK association. All JMD mutants retained intrinsic kinase activity, but Y559F, and less so Y559D, showed dramatically reduced CSF-1-induced autophosphorylation. CSF-1-mediated wild-type (WT)-CSF-1R phosphorylation was not markedly affected by SFK inhibition, indicating that lack of SFK binding is not responsible for diminished Y559F phosphorylation. Unexpectedly, cells expressing Y559F were hyperproliferative in response to CSF-1. Hyperproliferation correlated with prolonged activation of Akt, ERK, and Stat5 in the Y559F mutant. Consistent with a defect in receptor negative regulation, c-Cbl tyrosine phosphorylation and CSF-1R/c-Cbl co-association were almost undetectable in the Y559F mutant. Furthermore, Y559F underwent reduced multiubiquitination and delayed receptor internalization and degradation. In conclusion, we propose that Tyr559 is a switch residue that functions in kinase regulation, signal transduction and, indirectly, receptor down-regulation. These findings may have implications for the oncogenic conversion of c-Kit and Flt3 with JMD mutations. Recent literature implicates a regulatory function of the juxtamembrane domain (JMD) in receptor tyrosine kinases. Mutations in the JMD of c-Kit and Flt3 are associated with gastrointestinal stromal tumors and acute myeloid leukemias, respectively. Additionally, autophosphorylated Tyr559 in the JMD of the colony stimulating factor-1 (CSF-1) receptor (CSF-1R) binds to Src family kinases (SFKs). To investigate SFK function in CSF-1 signaling we established stable 32D myeloid cell lines expressing CSF-1Rs with mutated SFK binding sites (Tyr559-TFI). Whereas binding to I562S was not significantly perturbed, Y559F and Y559D exhibited markedly decreased CSF-1-dependent SFK association. All JMD mutants retained intrinsic kinase activity, but Y559F, and less so Y559D, showed dramatically reduced CSF-1-induced autophosphorylation. CSF-1-mediated wild-type (WT)-CSF-1R phosphorylation was not markedly affected by SFK inhibition, indicating that lack of SFK binding is not responsible for diminished Y559F phosphorylation. Unexpectedly, cells expressing Y559F were hyperproliferative in response to CSF-1. Hyperproliferation correlated with prolonged activation of Akt, ERK, and Stat5 in the Y559F mutant. Consistent with a defect in receptor negative regulation, c-Cbl tyrosine phosphorylation and CSF-1R/c-Cbl co-association were almost undetectable in the Y559F mutant. Furthermore, Y559F underwent reduced multiubiquitination and delayed receptor internalization and degradation. In conclusion, we propose that Tyr559 is a switch residue that functions in kinase regulation, signal transduction and, indirectly, receptor down-regulation. These findings may have implications for the oncogenic conversion of c-Kit and Flt3 with JMD mutations. Colony stimulating factor-1 (CSF-1) 1The abbreviations used are: CSF-1, colony stimulating factor-1; CSF-1R, colony stimulating factor-1 receptor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; Flt3, Fms-like tyrosine kinase 3; GST, glutathione S-transferase; JMD, juxtamembrane domain; KI, kinase insert; PDGFR, platelet-derived growth factor receptor; RTK, receptor tyrosine kinase; SFK, Src family kinase; SH2, Src homology 2; SHC, Src homology and collagen; SHIP, SH2-domain containing inositol 5′-phosphatase; TCL, total cell lysate; WT, wild-type; IL, interleukin; Stat, signal transducers and activators of transcription; ECD, extracellular domain; HRP, horseradish peroxidase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. is an essential factor for monocyte/macrophage cell proliferation, differentiation, and survival (1Stanley E.R. Berg K.L. Einstein D.B. Lee P.S. Pixley F.J. Wang Y. Yeung Y.G. Mol. Reprod. Dev. 1997; 46: 4-10Crossref PubMed Scopus (352) Google Scholar, 2Hamilton J.A. J. Leukocyte Biol. 1997; 62: 145-155Crossref PubMed Scopus (165) Google Scholar, 3Rohrschneider L.R. Bourette R.P. Lioubin M.N. Algate P.A. Myles G.M. Carlberg K. Mol. Reprod. Dev. 1997; 46: 96-103Crossref PubMed Scopus (59) Google Scholar). Mice producing an inactive form of CSF-1 (Csf(op)/Csf(op)) exhibit osteopetrosis because of a decrease in osteoclasts as well as reduced numbers of monocytes and macrophages (4Felix R. Cecchini M.G. Fleisch H. Endocrinology. 1990; 127: 2592-2594Crossref PubMed Scopus (401) Google Scholar, 5Wiktor-Jedrzejczak W. Bartocci A. Ferrante Jr, A.W. Ahmed-Ansari A. Sell K.W. Pollard J.W. Stanley E.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4828-4832Crossref PubMed Scopus (887) Google Scholar, 6Yoshida H. Hayashi S. Kunisada T. Ogawa M. Nishikawa S. Okamura H. Sudo T. Shultz L.D. Nature. 1990; 345: 442-444Crossref PubMed Scopus (1525) Google Scholar). They also have lower body weights, defects in reproduction, and a shorter lifespan. CSF-1 functions through a receptor tyrosine kinase (RTK), the CSF-1 receptor (CSF-1R), expressed on the surface of target cells. Recently, CSF-1R knockout mice were found to have a similar, but more severe phenotype, compared with the op/op mice, indicating that the CSF-1R is the main mediator of CSF-1 signaling (7Dai 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 (836) Google Scholar). CSF-1R is a member of the Class III RTK family, which also includes c-Kit, Flt3/Flk2, PDGFRα, and PDGFRβ (8Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3144) Google Scholar). These receptors are characterized by five immunoglobulin-like regions in the extracellular ligand-binding portion, a single spanning transmembrane region, a juxtamembrane domain (JMD), a kinase domain interrupted by a kinase insert (KI), and a carboxyl-terminal domain. Upon ligand binding, CSF-1R homodimerizes and autophosphorylates on six tyrosines in the cytoplasmic portion of the receptor. Tyr807 is located in the activation loop of the kinase domain (9Bourette R.P. Rohrschneider L.R. Growth Factors. 2000; 17: 155-166Crossref PubMed Scopus (107) Google Scholar) and its phosphorylation is important for kinase activity (10Davis J.N. Rock C.O. Cheng M. Watson J.B. Ashmun R.A. Kirk H. Kay R.J. Roussel M.F. Mol. Cell. Biol. 1997; 17: 7398-7406Crossref PubMed Scopus (37) Google Scholar). The remaining tyrosines serve as binding sites for proteins containing Src homology 2 (SH2) binding domains. Three sites are found in the KI: Grb2/Mona (Tyr697) (11van der Geer P. Hunter T. EMBO J. 1993; 12: 5161-5172Crossref PubMed Scopus (124) Google Scholar, 12Bourette R.P. Arnaud S. Myles G.M. Blanchet J.P. Rohrschneider L.R. Mouchiroud G. EMBO J. 1998; 17: 7273-7281Crossref PubMed Scopus (90) Google Scholar), p85 subunit of phosphatidylinositol 3-kinase (Tyr721) (13Reedijk M. Liu X. van der Geer P. Letwin K. Waterfield M.D. Hunter T. Pawson T. EMBO J. 1992; 11: 1365-1372Crossref PubMed Scopus (178) Google Scholar), and Stat1 (Tyr706) (14Novak U. Nice E. Hamilton J.A. Paradiso L. Oncogene. 1996; 13: 2607-2613PubMed Google Scholar), the c-Cbl binding site is in the COOH terminus (Tyr974) (15Mancini A. Koch A. Wilms R. Tamura T. J. Biol. Chem. 2002; 277: 14635-14640Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 16Wilhelmsen K. Burkhalter S. van der Geer P. Oncogene. 2002; 21: 1079-1089Crossref PubMed Scopus (66) Google Scholar), and the Src family kinase (SFK) binding site is in the JMD (Y559) (17Alonso G. Koegl M. Mazurenko N. Courtneidge S.A. J. Biol. Chem. 1995; 270: 9840-9848Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). These molecules further propagate the CSF-1 signal through activation of Ras/ERK, phosphatidylinositol 3-kinase/Akt, and STAT proteins. Two additional autophosphorylation sites have been described for v-fms (18Mancini A. Niedenthal R. Joos H. Koch A. Trouliaris S. Niemann H. Tamura T. Oncogene. 1997; 15: 1565-1572Crossref PubMed Scopus (44) Google Scholar, 19Joos H. Trouliaris S. Helftenbein G. Niemann H. Tamura T. J. Biol. Chem. 1996; 271: 24476-24481Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), the viral counterpart of the CSF-1R, but these have not been fully characterized for the CSF-1R. Precisely how the signal from the CSF-1R is transduced to these downstream targets and how they transmit the signal to the nucleus is not well understood. Following ligand binding, the CSF-1R is rapidly internalized and degraded. This process begins with multiubiquitination of the CSF-1R mediated by c-Cbl (20Lee 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 (252) Google Scholar), an E3-type ubiquitin ligase (21Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar). CSF-1R presumably then migrates to clathrin-coated pits, is internalized through clathrin-mediated endocytosis, sorted into lysosomes, and degraded, as has been shown for other RTKs (22Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (259) Google Scholar). c-Cbl appears to be important for both receptor internalization and degradation; c-Cbl overexpression leads to an increase in EGFR internalization (23Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar, 24Soubeyran P. Kowanetz K. Szymkiewicz I. Langdon W.Y. Dikic I. Nature. 2002; 416: 183-187Crossref PubMed Scopus (489) Google Scholar), whereas c-Cbl–/– macrophages show a delay in CSF-1R internalization (20Lee 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 (252) Google Scholar). c-Cbl may signal internalization through its interaction with CIN85, Cbl interacting protein of 85 kDa, an adapter molecule, and endophilin, a regulator of clathrin-mediated endocytosis (24Soubeyran P. Kowanetz K. Szymkiewicz I. Langdon W.Y. Dikic I. Nature. 2002; 416: 183-187Crossref PubMed Scopus (489) Google Scholar, 25Petrelli A. Gilestro G.F. Lanzardo S. Comoglio P.M. Migone N. Giordano S. Nature. 2002; 416: 187-190Crossref PubMed Scopus (380) Google Scholar). Recently, we showed that SFKs are activated by CSF-1 and serve as alternate activators of the Ras/ERK and phosphatidylinositol 3-kinase/Akt pathways in cells expressing a CSF-1R mutant lacking the KI (26Lee A.W. States D.J. Mol. Cell. Biol. 2000; 20: 6779-6798Crossref PubMed Scopus (79) Google Scholar). Several RTKs, including the CSF-1R (27Courtneidge S.A. Dhand R. Pilat D. Twamley G.M. Waterfield M.D. Roussel M.F. EMBO J. 1993; 12: 943-950Crossref PubMed Scopus (204) Google Scholar), PDGFRβ (28Kypta R.M. Goldberg Y. Ulug E.T. Courtneidge S.A. Cell. 1990; 62: 481-492Abstract Full Text PDF PubMed Scopus (480) Google Scholar), c-Kit (29Linnekin D. DeBerry C.S. Mou S. J. Biol. Chem. 1997; 272: 27450-27455Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), and EGFR (30Osherov N. Levitzki A. Eur. J. Biochem. 1994; 225: 1047-1053Crossref PubMed Scopus (266) Google Scholar), activate SFKs, but it remains unclear what role these proteins play in RTK signaling. SFKs have been reported to activate the Ras/ERK, phosphatidylinositol 3-kinase/Akt, and STAT pathways (26Lee A.W. States D.J. Mol. Cell. Biol. 2000; 20: 6779-6798Crossref PubMed Scopus (79) Google Scholar, 31Bondzi C. Litz J. Dent P. Krystal G.W. Cell Growth Differ. 2000; 11: 305-314PubMed Google Scholar, 32Marks D.C. Csar X.F. Wilson N.J. Novak U. Ward A.C. Kanagasundarum V. Hoffmann B.W. Hamilton J.A. Mol. Cell. Biol. Res. Commun. 1999; 1: 144-152Crossref PubMed Scopus (32) Google Scholar, 33Wang Y.Z. Wharton W. Garcia R. Kraker A. Jove R. Pledger W.J. Oncogene. 2000; 19: 2075-2085Crossref PubMed Scopus (100) Google Scholar) as well as play a role in growth factor-induced cell cycle progression and c-myc transcription (34Courtneidge S.A. Biochem. Soc. Trans. 2002; 30: 11-17Crossref PubMed Scopus (66) Google Scholar, 35DeMali K.A. Godwin S.L. Soltoff S.P. Kazlauskas A. Exp. Cell Res. 1999; 253: 271-279Crossref PubMed Scopus (51) Google Scholar). However, it has also been suggested that SFKs have a negative regulatory role in growth factor signaling, through reduction of Akt activity (36Baran C.P. Tridandapani S. Helgason C.D. Humphries R.K. Krystal G. Marsh C.B. J. Biol. Chem. 2003; 278: 38628-38636Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) or enhancement of c-Cbl activity (37Rosenkranz S. Ikuno Y. Leong F.L. Klinghoffer R.A. Miyake S. Band H. Kazlauskas A. J. Biol. Chem. 2000; 275: 9620-9627Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To investigate the role of SFKs in CSF-1 signaling, Tyr559 has been mutated in previous studies to Ala or Phe. The Y559A mutant showed normal in vitro kinase activity as well as normal ligand-induced receptor internalization (38Myles G.M. Brandt C.S. Carlberg K. Rohrschneider L.R. Mol. Cell. Biol. 1994; 14: 4843-4854Crossref PubMed Scopus (27) Google Scholar). SFK binding was not investigated. The Y559F mutant exhibited decreased SFK binding, receptor phosphorylation and activity, and Stat3 phosphorylation (32Marks D.C. Csar X.F. Wilson N.J. Novak U. Ward A.C. Kanagasundarum V. Hoffmann B.W. Hamilton J.A. Mol. Cell. Biol. Res. Commun. 1999; 1: 144-152Crossref PubMed Scopus (32) Google Scholar). It is puzzling that mutation of the same residue to different amino acids should have such a different effect. In addition to providing binding sites for interacting proteins, the JMD of RTKs may play a role in regulation of kinase function. Duplications, deletions, and point mutations in the JMD of Flt3 and c-Kit are commonly found in acute myeloid leukemias and gastrointestinal stromal tumors, respectively, causing constitutive receptor activation (39Scheijen B. Griffin J.D. Oncogene. 2002; 21: 3314-3333Crossref PubMed Scopus (142) Google Scholar). Several lines of evidence support an autoinhibitory role of the JMD in RTKs. In vitro peptide binding studies demonstrate that unphosphorylated c-Kit JMD can inhibit its own kinase activity (40Chan P.M. Ilangumaran S. La Rose J. Chakrabartty A. Rottapel R. Mol. Cell. Biol. 2003; 23: 3067-3078Crossref PubMed Scopus (133) Google Scholar). X-ray crystal studies of the inactive, non-type III RTKs, Eph (41Wybenga-Groot L.E. Baskin B. Ong S.H. Tong J. Pawson T. Sicheri F. Cell. 2001; 106: 745-757Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), and MuSK (42Till J.H. Becerra M. Watty A. Lu Y. Ma Y. Neubert T.A. Burden S.J. Hubbard S.R. Structure (Lond.). 2002; 10: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) as well as type III RTKs, Flt3 (43Griffith J. Black J. Faerman C. Swenson L. Wynn M. Lu F. Lippke J. Saxena K. Mol. Cell. 2004; 13: 169-178Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar), and c-Kit (44Mol C.D. Dougan D.R. Schneider T.R. Skene R.J. Kraus M.L. Scheibe D.N. Snell G.P. Zou H. Sang B. Wilson K.P. J. Biol. Chem. 2004; 279: 31655-31663Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar) reveal that there is an intimate relationship between the JMD and the kinase domain. For example, in c-Kit, the closest relative to the CSF-1R, the JMD forms a hairpin that is partially buried in the interface between the NH2- and COOH-terminal kinase lobes. This alters the position of a helix important for catalytic function (αC), prevents the activation loop from extending into an active conformation, blocks nucleotide binding, and allows Tyr823 to bind as a pseudosubstrate. It is easy to see how phosphorylation of the JMD tyrosines (Tyr568/Tyr570) can induce a conformational change that will disrupt some of the tight interactions between the JMD and kinase domain. In support of this possibility, the active c-Kit structure shows a vastly different conformation compared with the inactive structure (45Mol C.D. Lim K.B. Sridhar V. Zou H. Chien E.Y.T. Sang B. Nowakowski J. Kassel D.B. Cronin C.N. McRee D.E. J. Biol. Chem. 2003; 278: 31461-31464Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). To more fully elucidate the role of the JMD in CSF-1R signaling, we focused on the SFK binding motif. Stable IL-3-dependent 32Dcl23 cell lines expressing JMD CSF-1R mutants were established. In agreement with previous findings (17Alonso G. Koegl M. Mazurenko N. Courtneidge S.A. J. Biol. Chem. 1995; 270: 9840-9848Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 32Marks D.C. Csar X.F. Wilson N.J. Novak U. Ward A.C. Kanagasundarum V. Hoffmann B.W. Hamilton J.A. Mol. Cell. Biol. Res. Commun. 1999; 1: 144-152Crossref PubMed Scopus (32) Google Scholar), mutation of Tyr559 significantly reduced SFK binding. We also found that the Y559F mutant exhibited substantially decreased CSF-1-induced tyrosine phosphorylation, suggesting it may have a similar role as proposed for Tyr568/Tyr570 of c-Kit. Despite this decrease in receptor autophosphorylation, cells expressing Y559F displayed a CSF-1-dependent hyperproliferative phenotype. Y559F showed a marked reduction in c-Cbl binding, resulting in diminished CSF-1-induced c-Cbl tyrosine phosphorylation. Consistently, the Y559F mutant showed decreased CSF-1-induced multiubiquitination of Y559F and delayed receptor degradation. Consequently, Y559F and to a lesser extent Y559D, which exhibits an intermediate phenotype compared with the WT and Y559F receptors, transduced a low, but persistent activation of down-stream signaling pathways, including Akt, ERK, and Stat5. Thus, hyperproliferation occurred as a result of the disruption of the balance between positive and negative signals. In summary, our studies support the model that the JMD of the CSF-1R has a dual role: autoinhibition in the unliganded state, and upon ligand binding and autophosphorylation, provision of docking sites for interacting proteins. Antibodies and Reagents—Cell culture reagents were from Invitrogen (Gaithersburg, MD) and Sigma. Recombinant human CSF-1 was a gift of the Genetics Institute (Cambridge, MA). Recombinant mouse IL-3 was purchased from Pepro Tech, Inc. (Rocky Hill, NJ). SU6656 and MG-132 were purchased from Calbiochem (La Jolla, CA). Chloroquine and methylamine were obtained from Sigma. JMD peptides used in the competitive GST pull-down assay were synthesized by the University of Michigan Protein Structure Facility (Ann Arbor, MI). The JMD peptides, spanning residues 553–567 of the mouse CSF-1R, were synthesized with Tyr559 in the unphosphorylated or phosphorylated state. Peptides were dissolved in Me2SO. CSF-1R 422, 423, and 425 antibodies have been previously described (26Lee A.W. States D.J. Mol. Cell. Biol. 2000; 20: 6779-6798Crossref PubMed Scopus (79) Google Scholar) and the combination is referred to as anti-CSF-1R antibodies in the text. Anti-extracellular domain (ECD)-Fms (referred to as anti-ECD-Fms in the text) and anti-phosphotyrosine (Tyr(P)) (4G10) antibodies were purchased from Upstate (Lake Placid, NY); anti-Akt, Tyr(P) (PY99), ubiquitin, c-Cbl, Stat3, Stat5, GST, and Fyn antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); phospho-specific antibodies for CSF-1R, Akt, ERK, Stat3, and Stat5 were purchased from Cell Signaling Technology (Beverly, MA); anti-SHC antibodies were from Transduction Laboratories (San Diego, CA); anti-ERK and goat anti-mouse HRP antibodies were from Zymed Laboratories, Inc. (S. San Francisco, CA); donkey anti-rabbit HRP secondary antibody was from Amersham; and NeutrAvidin-HRP was from Pierce. Cell Culture and Transfections—IL-3-dependent murine 32Dcl23 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 5–10% WEHI-conditioned media as a source of IL-3. 32D cell lines expressing WT-CSF-1R have been previously described (46Lee A.W. Blood. 1999; 93: 537-553Crossref PubMed Google Scholar). The JMD CSF-1R mutants were constructed by replacing the internal StuI fragment of the murine CSF-1R cDNA with the corresponding fragment containing point mutations, produced using a two-step PCR method (47Lee A.W. Nienhuis A.W. J. Biol. Chem. 1992; 267: 16472-16483Abstract Full Text PDF PubMed Google Scholar). All PCR-amplified segments were sequenced in their entirety and on both strands. The CSF-1R mutant cDNAs were inserted into pMSCV-IRES-puro and transiently transfected into BOSC23 cells. Viral supernatants obtained after 48 h were used to transduce 32Dcl23 cells. Clones were selected using 1 μg/ml puromycin and screened by 125I-CSF-1 binding as described (26Lee A.W. States D.J. Mol. Cell. Biol. 2000; 20: 6779-6798Crossref PubMed Scopus (79) Google Scholar). JMD clones designated as (lo) expressed similar receptor levels as the WT (hi) clone, which was used as a control in all experiments except where indicated. JMD clones described as (hi) expressed higher levels (∼1.5–2-fold) of receptors compared with the WT (hi) clone. Unless otherwise indicated, JMD (lo) clones were used in all experiments. For clones of interest, RNA was extracted, reverse transcribed, and the sequence of the JMD verified. Protein Analysis—Except where noted, immunoprecipitation and immunoblotting were performed as described (26Lee A.W. States D.J. Mol. Cell. Biol. 2000; 20: 6779-6798Crossref PubMed Scopus (79) Google Scholar). Equal amounts of protein as determined by the Bio-Rad protein assay were loaded for each immunoblot of total cell lysate (TCL) as shown. In all cases, quantitation of receptor levels in receptor immunoprecipitations was carried out on duplicate samples subjected to the same processing. For most experiments, the anti-ECD-Fms antibody was used to immunoprecipitate the CSF-1R. Western blots of the receptor were probed sequentially with anti-ECD-Fms and then anti-CSF-1R (422/423/425) antibodies to eliminate possible differences in antibody recognition. For downstream substrate activation experiments, cells (2 × 106/ml) were starved for 3 h in a 24-well plate. Cells were left unstimulated or stimulated with 10 nm CSF-1 for the times indicated in the figures and then transferred to ice-cold Hanks' balanced salt solution containing 0.2 mm Na3VO4. After two washes, cells were lysed as described (26Lee A.W. States D.J. Mol. Cell. Biol. 2000; 20: 6779-6798Crossref PubMed Scopus (79) Google Scholar). For CSF-1R kinase assays, lysates from starved cells were first pre-cleared by incubation with rabbit serum-bound protein A-Sepharose for 45 min. Then, lysates were incubated with anti-ECD-Fms pre-bound to protein A-Sepharose for 6 h at 4 °C with rocking, followed by extensive washing of the immune complexes. The complexes were incubated for 20 min at 30 °C in kinase buffer (20 mm Hepes, 10 mm MnCl2, 1 mm dithiothreitol, 1 mm Na3VO4) containing 200 μm ATP. The kinase assay buffer was removed and the beads were washed once with HBS (25 mm Hepes, 150 mm NaCl, 1 mm dithiothreitol) supplemented with protease and phosphatase inhibitors. The reaction was stopped by addition of 2× Laemmli buffer and boiled. Proteins were separated using SDS-PAGE, transferred to polyvinylidene difluoride (Millipore) and immunoblotted as described in the figure legends. For CSF-1R/c-Cbl co-immunoprecipitation experiments, cells were either treated with methylamine or chloroquine, in combination with MG-132, for 1 h prior to CSF-1 stimulation at 37 °C, or cells were stimulated at 4 °C for 20 min. Lysis was with an equal volume of 2× TNE lysis buffer (20 mm Tris, pH 7.8, 300 mm NaCl, 2 mm EDTA, 0.2% Nonidet P-40) as previously described (48Ota J. Sato K. Kimura F. Wakimoto N. Nakamura Y. Nagata N. Suzu S. Yamada M. Shimamura S. Motoyoshi K. FEBS Lett. 2000; 466: 96-100Crossref PubMed Scopus (8) Google Scholar) and supplemented with protease and phosphatase inhibitors. 750 μg of cell lysate was incubated with anti-ECD-Fms or anti-c-Cbl antibodies followed by capture with protein A-Sepharose beads. After extensive washing, 2× Laemmli was added; the samples were boiled and analyzed by SDS-PAGE and immunoblotting. Quantitation of immunoblot results was carried out as described previously (26Lee A.W. States D.J. Mol. Cell. Biol. 2000; 20: 6779-6798Crossref PubMed Scopus (79) Google Scholar) by scanning multiple exposures with Adobe Photoshop 7.0 and quantitating band intensities with NIH Image 1.62 software. GST Pull-down Assay—GST pull-down assays were performed essentially as previously described (26Lee A.W. States D.J. Mol. Cell. Biol. 2000; 20: 6779-6798Crossref PubMed Scopus (79) Google Scholar). Pre-cleared 32D cell lysates were incubated with 5 μg of GST or GST-Fyn-SH2 bound to GSH-Sepharose (Amersham Biosciences) for 3 h at 4 °C with rocking followed by extensive washing of complexes. Bound proteins were eluted by boiling in 2× Laemmli. CSF-1R proteins were separated by SDS-PAGE and detected by immunoblotting with anti-ECD-Fms antibodies. Peptide competition assays were performed as described above except that, after preclearing, the lysates were incubated with Me2SO or the indicated amount of unphosphorylated or phosphorylated JMD peptide at 4 °C for 1 h before incubation with GST-Fyn-SH2. Biotinylation and Internalization Assay—After stimulation, cells were immediately placed on ice and washed multiple times with ice-cold phosphate-buffered saline, pH 7.8. Cells were incubated with 0.5 mg/ml NHS-PEO4-Biotin (Pierce) for 1 h at 4 °C to label the remaining cell surface proteins. The biotinylation reaction was stopped by incubating cells with stop buffer (phosphate-buffered saline, pH 7.8, containing 10 mm glycine) for 10 min at 4 °C. Cells were washed again with stop buffer and then lysed with 1× lysis buffer supplemented with protease and phosphatase inhibitors. CSF-1Rs were immunoprecipitated from cell lysates. Immune complexes were sequentially blotted with anti-CSF-1R antibodies, to determine receptor degradation, and with NeutrAvidin-HRP, to detect CSF-1Rs remaining on the cell surface after stimulation. Cell Proliferation Studies—Cells were washed multiple times (1× with RPMI, 2% fetal bovine serum, and 2 times with Hanks' balanced salt solution) before seeding at 7.5 × 104 cells/ml in 96-well plates in RPMI, 10% fetal bovine serum. Unless otherwise indicated, 10 nm CSF-1 or 5 ng/ml recombinant murine IL-3 were added to each well. Cells were split 1:10 every 2 days into fresh media supplemented with the appropriate growth factor and studies were carried out for 6 days. Living cells were counted every 24 h using trypan blue exclusion and a hemocytometer. Additionally, an MTS assay (Cell Titer 96 AQueous Cell Proliferation Assay; Promega, Madison, WI) was performed every day according to the manufacturer's instructions, to test for metabolically active cells. For SU6656 growth studies, cells were pretreated with SU6656 for 1 h before addition of CSF-1. MTS activity was analyzed after 48 h. CSF-1R Modeling—The JMD and kinase domain of the mouse CSF-1R were modeled on the autoinhibited FLT3 structure (Protein Data Bank code 1RJB) using SWISS-MODEL (version 36.0003) (49Peitsch M.C. Bio/Technology. 1995; 13: 658-660Crossref Scopus (115) Google Scholar, 50Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9589) Google Scholar, 51Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4506) Google Scholar). Statistical Analysis—p values were calculated using the Student's t test (2-sided). Mutation of Tyr559 Significantly Reduces SFK Association with CSF-1R—To investigate the role of SFK in CSF-1 signaling, we performed a mutagenesis study of the SFK binding site in the JMD of the CSF-1R (Fig. 1A). Investigations of the PDGF, Eph, and MuSK receptors point to an additional, autoinhibitory role for the JMD (41Wybenga-Groot L.E. Baskin B. Ong S.H. Tong J. Pawson T. Sicheri F. Cell. 2001; 106: 745-757Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 42Till J.H. Becerra M. Watty A. Lu Y. Ma Y. Neubert T.A. Burden S.J. Hubbard S.R. Structure (Lond.). 2002; 10: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 52Irusta P.M. DiMaio D. EMBO J. 1998; 17: 6912-6923Crossref PubMed Scopus (57) Google Scholar, 53Irusta P.M. Luo Y. Bakht O. Lai C.C. Smith S.O. DiMaio D. J. Biol. Chem. 2002; 277: 38627-38634Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 54Baxter R.M. Secrist J.P. Vaillancourt R.R. Kazlauskas A. J. Biol. Chem. 1998; 273: 17050-17055Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Taking into account this possibility, we made three point mutants. A Tyr to Phe substitution at residue 559 is predicted to eliminate binding by the SH2 domain of SFKs but at the same time may lock the receptor in an inactive conformation, a possibility proposed for analogous mutations in the Eph receptor (55Binns K.L. Taylor P.P. Sicheri F. Pawson T. Holland S.J. Mol. Cell. Biol. 2000; 20: 4791-4805Crossref PubMed Scopus (158) Google Scholar). A Tyr" @default.
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• W2031762637 title "A Juxtamembrane Tyrosine in the Colony Stimulating Factor-1 Receptor Regulates Ligand-induced Src Association, Receptor Kinase Function, and Down-regulation" @default.
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