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• W1978531552 abstract "Dok-1 is an adaptor protein that is a substrate for Bcr-Abl and other tyrosine protein kinases. The presence of pleckstrin homology and phosphotyrosine binding domains as well as multiple tyrosine phosphorylation sites suggests that Dok-1 is involved in protein-protein and/or protein-lipid interactions. Here we show that stimulation of Mo7 hematopoietic cells with c-Kit ligand (KL) induces phosphatidylinositol (PI) 3-kinase-dependent tyrosine phosphorylation and membrane recruitment of Dok-1. Addition of the K-Ras membrane-targeting motif to Dok-1 generated a constitutively membrane-bound Dok-1 protein whose tyrosine phosphorylation was independent of PI 3-kinase. Membrane localization of Dok-1 was required for its ability to function as a negative regulator of cell proliferation. Additional experiments revealed that Dok-1 associated with the juxtamembrane region and C-terminal tail of c-Kit. Lyn promoted phosphorylation of c-Kit and association of c-Kit and Dok-1. Both Lyn and Tec were capable of phosphorylating Dok-1. However, the use of primary bone marrow mast cells from normal and Lyn-deficient mice demonstrated that Lyn is required for KL-dependent Dok-1 tyrosine phosphorylation. Taken together, these data indicate that activation of PI 3-kinase by KL promotes binding of the Dok pleckstrin homology domain and Dok-1 recruitment to the plasma membrane where Dok-1 is phosphorylated by Src and/or Tec family kinases. Dok-1 is an adaptor protein that is a substrate for Bcr-Abl and other tyrosine protein kinases. The presence of pleckstrin homology and phosphotyrosine binding domains as well as multiple tyrosine phosphorylation sites suggests that Dok-1 is involved in protein-protein and/or protein-lipid interactions. Here we show that stimulation of Mo7 hematopoietic cells with c-Kit ligand (KL) induces phosphatidylinositol (PI) 3-kinase-dependent tyrosine phosphorylation and membrane recruitment of Dok-1. Addition of the K-Ras membrane-targeting motif to Dok-1 generated a constitutively membrane-bound Dok-1 protein whose tyrosine phosphorylation was independent of PI 3-kinase. Membrane localization of Dok-1 was required for its ability to function as a negative regulator of cell proliferation. Additional experiments revealed that Dok-1 associated with the juxtamembrane region and C-terminal tail of c-Kit. Lyn promoted phosphorylation of c-Kit and association of c-Kit and Dok-1. Both Lyn and Tec were capable of phosphorylating Dok-1. However, the use of primary bone marrow mast cells from normal and Lyn-deficient mice demonstrated that Lyn is required for KL-dependent Dok-1 tyrosine phosphorylation. Taken together, these data indicate that activation of PI 3-kinase by KL promotes binding of the Dok pleckstrin homology domain and Dok-1 recruitment to the plasma membrane where Dok-1 is phosphorylated by Src and/or Tec family kinases. In patients with chronic myelogenous leukemia, thec-abl gene is translocated from chromosome 9 to chromosome 22 to produce a hybrid bcr-abl gene (1.de Klein A. van Kessel A.G. Grosveld G. Bartram C.R. Hagemeijer A. Bootsma D. Spurr N.K. Heisterkamp N. Groffen J. Stephenson J.R. Nature. 1982; 300: 765-767Crossref PubMed Scopus (1059) Google Scholar, 2.Heisterkamp N. Stephenson J.R. Groffen J. Hansen P.F. de Klein A. Bartram C.R. Grosveld G. Nature. 1983; 306: 239-242Crossref PubMed Scopus (628) Google Scholar, 3.Shtivelman E. Lifshitz B. Gale R.P. Canaani E. Nature. 1985; 315: 550-554Crossref PubMed Scopus (1270) Google Scholar). The fusion protein that results from this translocation, p210Bcr-Abl, encodes a tyrosine kinase whose activation results in phosphorylation of a number of cellular proteins including SHIP1, SHIP2, Cbl, Lyn, SHC, and Dok-1 (4.Wisniewski D. Strife A. Swendeman S. Erdjument-Bromage H. Geromanos S. Kavanaugh W.M. Tempst P. Clarkson B. Blood. 1999; 93: 2707-2720Crossref PubMed Google Scholar). These proteins are also tyrosine-phosphorylated when cells are stimulated with c-Kit ligand (KL), 1The abbreviations used are: KLKit ligandPIphosphatidylinositolPHpleckstrin homologyGSTglutathioneS-transferaseIMDMIscove's modified Dulbecco's mediumILinterleukinGAPGTPase-activating proteinMAPmitogen-activated proteinBMMCbone marrow mast cellsa growth factor that is critical for normal hematopoiesis. KL binding to the c-Kit receptor results in dimerization and autophosphorylation of c-Kit and phosphorylation of PI 3-kinase, Tec, phospholipase Cγ, and Vav in addition to the signaling proteins listed above (5.Tang B. Mano H. Yi T. Ihle J.N. Mol. Cell. Biol. 1994; 14: 8432-8437Crossref PubMed Scopus (88) Google Scholar, 6.Price D.J. Rivnay B. Avraham H. Biochem. Biophys. Res. Commun. 1999; 259: 611-616Crossref PubMed Scopus (17) Google Scholar, 7.Linnekin D. DeBerry C.S. Mou S. J. Biol. Chem. 1997; 272: 27450-27455Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Kit ligand phosphatidylinositol pleckstrin homology glutathioneS-transferase Iscove's modified Dulbecco's medium interleukin GTPase-activating protein mitogen-activated protein bone marrow mast cells We have been studying the role of Dok-1 in normal and malignant cell signaling. Dok-1 is tyrosine-phosphorylated in response to a variety of growth factors including platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, granulocyte-macrophage colony-stimulated factor, interleukin-3 (IL-3), and KL. Cloning of the Dok-1 cDNA revealed that the overall structure of Dok-1 is similar to insulin receptor substrate-1 (IRS-1), which harbors an N-terminal pleckstrin homology (PH) domain, a central phosphotyrosine binding domain, and a C-terminal tail containing multiple tyrosine phosphorylation sites (8.Carpino N. Wisniewski D. Strife A. Marshak D. Kobayashi R. Stillman B. Clarkson B. Cell. 1997; 88: 197-204Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 9.Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-211Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). The PH domain of Dok-1 is thought to mediate protein interaction with the plasma membrane possibly by binding to phospholipids. The phosphotyrosine binding domain of Dok-1 is thought to mediate protein-protein interactions by binding to phosphotyrosine-containing motifs with the sequence of NPXpY (10.Zhou M.M. Ravichandran K.S. Olejniczak E.F. Petros A.M. Meadows R.P. Sattler M. Harlan J.E. Wade W.S. Burakoff S.J. Fesik S.W. Nature. 1995; 378: 584-592Crossref PubMed Scopus (324) Google Scholar, 11.Eck M.J. Dhe-Paganon S. Trub T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). The multiple tyrosine residues at the C-terminal region are phosphorylated in response to various growth factors. When phosphorylated they act as docking sites for SH2-containing proteins such as p120RasGAP and NCK (12.Noguchi T. Matozaki T. Inagaki K. Tsuda M. Fukunaga K. Kitamura Y. Kitamura T. Shii K. Yamanashi Y. Kasuga M. EMBO J. 1999; 18: 1748-1760Crossref PubMed Scopus (104) Google Scholar). Analysis of Dok-1 −/− mice reveals that Dok-1 is a negative regulator of cell proliferation. Cells derived from Dok −/− mice hyperproliferate in response to a number of cytokines and growth factors including KL (17.Yamanashi Y. Tamura T. Kanamori T. Yamane H. Nariuchi H. Yamamoto T. Baltimore D. Genes Dev. 2000; 14: 11-16PubMed Google Scholar, 21.Di Cristofano A. Niki M. Zhao M. Karnell F.G. Clarkson B. Pear W.S. Van Aelst L. Pandolfi P-P. J. Exp. Med. 2001; 194: 275-284Crossref PubMed Scopus (98) Google Scholar). However, the mechanism responsible for the hyperproliferative effect has not yet been elucidated. Moreover, the kinase(s) that phosphorylates Dok-1 in c-Kit-mediated signaling has not been identified. Lyn (a Src family kinase) and Tec (a Tec family kinase) have been reported to be activated upon c-Kit activation (5.Tang B. Mano H. Yi T. Ihle J.N. Mol. Cell. Biol. 1994; 14: 8432-8437Crossref PubMed Scopus (88) Google Scholar, 6.Price D.J. Rivnay B. Avraham H. Biochem. Biophys. Res. Commun. 1999; 259: 611-616Crossref PubMed Scopus (17) Google Scholar, 7.Linnekin D. DeBerry C.S. Mou S. J. Biol. Chem. 1997; 272: 27450-27455Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Tec forms a trimolecular complex with Dok-1 and Lyn in KL-stimulated cells, and activation of Tec and phosphorylation of Dok-1 have been shown to require PI 3-kinase activity (13.van Dijk T.B. van Den Akker E. Amelsvoort M.P. Mano H. Lowenberg B. von Lindern M. Blood. 2000; 96: 3406-3413Crossref PubMed Google Scholar). Other studies have documented a role for Src family kinases in Dok-1 phosphorylation. For example, Lck is required for CD2-mediated phosphorylation of Dok-1 in JcaM1.6 cells, and Src, Fyn, and Lck can phosphorylate Dok-1 in COS-7 cells (14.Nemorin J.G. Duplay P. J. Biol. Chem. 2000; 275: 14590-14597Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In this study, we examine the mechanisms involved in tyrosine phosphorylation, membrane recruitment, and signal transduction by Dok-1 during KL stimulation. Here we report that Dok-1 becomes tyrosine-phosphorylated and recruited to the membrane in a PI 3-kinase-dependent manner when Mo7 hematopoietic cells are stimulated with KL. Removal of the PH domain of Dok-1 results in loss of membrane localization and phosphorylation. However, Dok-1 phosphorylation can be restored by replacing the PH domain with the membrane-targeting motif of K-Ras, implying that membrane localization is required for Dok-1 phosphorylation. We demonstrate that Dok-1 associates with the juxtamembrane region and C-terminal tail of c-Kit. Moreover, we show that Lyn is necessary for KL-stimulated tyrosine phosphorylation of Dok-1 during c-Kit signal transduction. Constitutively membrane-targeted Dok proteins were generated by attaching the K-Ras tail (farnesyl/polybasic) to the C terminus of wild type Dok-1 or the ΔPHDok-1 mutant. The following primers were used for PCR and cloning: 5′-CGG AAT TCG CCA CC ATG GGC TAC CCA TAC GAC GTC CCA GAC TAC GCT GAC GGA GCA GTG ATG GAA GGG CCG-3′; 5′-CGG AAT TCG CCA CC ATG GGC TAC CCA TAC GAC GTC CCA GAC TAC GCT AAA GGC AGC TGG ACT CTG-3′; 5′-AA CTG CAG TCA CAT AAT TAC ACA CTT TGT CTT TGA CTT CTT TTT CTT CTT TTT ACC ATC TTT GCT GGT AGA GCC CTC TGA CTT GAC-3′. The Δp85 gene was kindly provided by Dr. Kurt Ballmer-Hofer (Villigen, Paul Scherrer Institute, Switzerland). BMMC from normal and Lyn-deficient mice and GST fusion proteins of c-Kit receptor were kind gifts from Dr. Diana Linnekin (Frederick Cancer Research and Development Center, Frederick, MD). pcDNA3-c-Kit was a generous gift from Dr. Hava Avraham (Harvard Medical School, Boston, MA). PME18s-WTTec was generously provided by Dr. Leslie J. Berg (University of Massachusetts Medical School, Worcester, MA) and was subcloned into pcDNA3.1 at the EcoRI site. The kinase-inactive form of Fyn (K299M) was prepared previously by Dr. Wouter van't Hof in our laboratory (15.van't Hof W. Resh M.D. J. Cell Biol. 1999; 145: 377-389Crossref PubMed Scopus (104) Google Scholar). Mo7 cells, BMMC, or COS-1 cells were lysed in lysis buffer containing 50 mmTris-HCl (pH 8.0), 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 1 mm Na3VO4, 10 mm NaF, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, and 0.5 μg/ml aprotinin. COS-1 cells expressing membrane-targeted Dok-1 were lysed in radioimmune precipitation buffer (15.van't Hof W. Resh M.D. J. Cell Biol. 1999; 145: 377-389Crossref PubMed Scopus (104) Google Scholar). Prior to immunoprecipitation cell lysates were clarified by centrifugation at 100,000 × g for 15 min. The antibodies used in this study were affinity-purified rabbit anti-Dok-1 (8.Carpino N. Wisniewski D. Strife A. Marshak D. Kobayashi R. Stillman B. Clarkson B. Cell. 1997; 88: 197-204Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar), rabbit anti-Tec raised against a synthetic peptide corresponding to amino acid residues 165–182 (KRRPPPPIPPEEENTEEI), rabbit anti-SHIP1 (a gift from Dr. W. M. Kavanaugh, Chiron Corporation, Emeryville, CA), rabbit anti-SHIP2 prepared as described (4.Wisniewski D. Strife A. Swendeman S. Erdjument-Bromage H. Geromanos S. Kavanaugh W.M. Tempst P. Clarkson B. Blood. 1999; 93: 2707-2720Crossref PubMed Google Scholar), rabbit anti-Lyn, rabbit anti-c-Kit, mouse anti-Dok-1, anti-Tyr(P) 99 (Santa Cruz Biotechnology, CA), rabbit anti-p85 (Upstate Biotechnology, Lake Placid, NY), mouse anti-Lyn, and mouse anti-SHC (Transduction Laboratories). Western blots were detected with ECL reagents (Amersham Biosciences). The Mo7 megakaryoblastic cell line was maintained in Iscove's modified Dulbecco's medium (IMDM) containing 20% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT) supplemented with 10 ng/ml human rIL-3 (BD PharMingen) at 37 °C and 5% CO2. BMMC were maintained in IMDM containing 10% fetal bovine serum and murine 30 ng/ml rIL-3. Mo7 cells were transfected by electroporation with 15–30 μg of plasmid DNA. Mo7 cells (20 × 106/300 μl) were pulsed at 126 V and 1700 μF using an ECM 600 (BTX) electroporator and were collected 18 h after transfection. For stimulation with c-Kit ligand Mo7 cells or BMMC were washed free of growth factor and starved overnight at 37 °C in IMDM containing 1% fetal bovine serum. Cells were harvested by centrifugation, resuspended in IMDM + 1% serum at a concentration of 10 × 106/ml, and stimulated with human or mouse c-Kit ligand (R&D Systems, Minneapolis, MN) at a concentration of 100 ng/ml for 5 min at 37 °C. For treatments with wortmannin (Sigma) or Src family kinase inhibitor PP2 (Calbiochem, La Jolla, CA), Mo7 cells were first starved overnight and then incubated with various concentrations of drugs for 1 h before the cells were stimulated with human c-Kit ligand. Cells were then pelleted, lysed, and processed for immunoprecipitation and Western blot analysis. COS-1 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C and 5% CO2. Cells were transfected with LipofectAMINE 2000 (Invitrogen) as described by the manufacturer. For stimulation with c-Kit ligand, COS-1 cells first were starved overnight in Dulbecco's modified Eagle's medium containing 1% fetal bovine serum and then were stimulated with 200 ng/ml c-Kit ligand for 10 min at 37 °C. Cells were resuspended in hypotonic buffer, lysed by homogenization with 30 strokes of a Dounce homogenizer, and centrifuged at 100,000 × g for 1 h to obtain a cytosolic fraction (S100) and a membrane fraction (P100) as described (16.Alland L. Peseckis S.M. Atherton R.E. Berthiaume L. Resh M.D. J. Biol. Chem. 1994; 269: 16701-16705Abstract Full Text PDF PubMed Google Scholar). Mo7 cell lysates were immunoprecipitated with either polyclonal anti-Lyn, anti-c-Kit, or anti-Tec antibodies. Immune complexes were washed with lysis buffer and resuspended in 30 μl of kinase buffer containing 20 mmTris-HCl (pH 7.4), 150 mm NaCl, 10 mmMnCl2, 10 mm MgCl2, and 10 μCi of [γ-32P]ATP. Recombinant Dok-1 (2 μg) was used as substrate for Lyn and Tec kinase assays. The reaction mixtures were incubated at 30 °C for 20 min, and reactions were terminated by the addition of SDS sample buffer. GST fusion proteins of juxtamembrane, kinase 1, kinase insert, and C-terminal tail domains of c-Kit were purified using glutathione-Sepharose 4B as described by the manufacturer (Amersham Biosciences). The protein concentration of the various GST fusion proteins was determined using the Bradford method (Bio-Rad). Phosphorylated GST fusion proteins were generated by incubating 5 μg of GST fusion proteins in kinase assay buffer with Lyn immunoprecipitates from Mo7 cell lysates. Lyn immunoprecipitates were removed by centrifugation. The supernatant containing phosphorylated GST fusion proteins was then added to KL-treated Mo7 cell lysates in the presence of 12 μl of glutathione-Sepharose 4B beads. Unphosphorylated GST fusion proteins were used as control. The mixture was gently agitated at 4 °C for 2 h, and the beads were washed three times with Mo7 lysis buffer. The samples were boiled for 5 min in SDS sample buffer and analyzed by SDS-PAGE. COS-1 cells were transfected with either wild type Dok-1, membrane-targeted wild type Dok-1 (Dok-1-KRas), ΔPHDok-1, or membrane-targeted ΔPHDok-1 (ΔPHDok-1-KRas) for 24 h and then trypsinized and seeded in triplicate in 96-well plates with a cell concentration of 5 × 104 cells/0.2 ml/well. 1 μCi of [3H]thymidine (PerkinElmer Life Sciences) was added to each well, and the cells were incubated for 16 h at 37 °C and 5% CO2. Cells were harvested, and 3H radioactivity was measured in a scintillation counter. Previous studies have established that Dok-1 is tyrosine-phosphorylated in KL-stimulated Mo7 cells (8.Carpino N. Wisniewski D. Strife A. Marshak D. Kobayashi R. Stillman B. Clarkson B. Cell. 1997; 88: 197-204Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Because Dok-1 contains a PH domain, and many PH domains bind to the products of PI 3-kinase, we tested whether PI 3-kinase was involved in a KL-mediated signaling pathway leading to Dok-1 phosphorylation. Mo7 cells were starved overnight and then incubated in the presence or absence of 100 nm wortmannin for 1 h prior to stimulation with KL. As indicated in Fig. 1, Dok-1 was tyrosine-phosphorylated in response to KL. Addition of wortmannin inhibited KL-stimulated phosphorylation of Dok-1 by >90%. Wortmannin had no effect on the level of tyrosine phosphorylation of the c-Kit. Western blotting with anti-Dok-1 or anti-c-Kit antibodies revealed the presence of the same amount of Dok-1 and c-Kit in each sample. These data suggest that KL-stimulated tyrosine phosphorylation of Dok-1 is PI 3-kinase-dependent. The products of PI 3-kinase enzymatic activity (PI(3,4)P2 and PI(3,4,5)P3) form a binding site in the plasma membrane for proteins with PH domains. We tested whether activation of Mo7 cells with KL promoted recruitment of Dok-1 to the membrane via a PI 3-kinase-dependent mechanism. Mo7 cells were starved overnight and treated with or without wortmannin for 1 h prior to stimulation with KL. Cells were lysed in hypotonic buffer and separated into cytosolic (S100) and membrane (P100) fractions by differential ultracentrifugation. As indicated in Fig. 2A, in the absence of KL stimulation little or no [Tyr(P)]Dok-1 was observed. However, when Mo7 cells were stimulated with KL most (70%) of the [Tyr(P)]Dok-1 was localized to the membrane fraction (P100). Western blotting with anti-Dok-1 antibody revealed that there was no significant change in the distribution of total Dok-1 before and after KL stimulation, suggesting that only a small amount of Dok-1 was phosphorylated upon KL stimulation. To test whether the PH domain of Dok-1 was required for membrane association, a precise deletion of the PH domain was generated within Dok-1. The wild type and ΔPH mutant Dok-1 were transfected into Mo7 cells by electroporation. As depicted in Fig. 2B, the ΔPHDok-1 mutant was localized nearly exclusively in the S100 fraction, whereas wild type Dok-1 was evenly distributed in the S100 and P100 fractions. These data support a model in which activation of PI 3-kinase by KL generates a binding site for the Dok-1 PH domain that serves to recruit Dok-1 to the plasma membrane. The next set of experiments was designed to determine whether tyrosine phosphorylation of Dok-1 precedes or follows membrane binding. To distinguish between these two possibilities, we generated a chimeric Dok-1 protein that was constitutively targeted to the plasma membrane. This was accomplished by fusing the membrane-targeting motif of K-Ras (farnesyl + polybasic) to the C terminus of wild type and ΔPH mutant Dok-1, respectively. Because the efficiency of transfection in Mo7 cells was very low the proteins were expressed in transfected COS-1 cells. As depicted in Fig. 3, wild type Dok-1 was evenly distributed in the S100 and P100 fractions and was tyrosine-phosphorylated (most likely by endogenous tyrosine kinases). The ΔPHDok-1 mutant was localized to the cytosolic fraction (S100) and was not phosphorylated. In contrast the ΔPHDok-1 mutant with the K-Ras tail was primarily associated with the membrane fraction (P100) and was constitutively tyrosine-phosphorylated. These results indicate that the PH domain of Dok-1 is required for membrane localization and phosphorylation and that membrane binding is required for Dok-1 tyrosine phosphorylation. We next tested whether the addition of a K-Ras tail to full-length Dok-1 could bypass the requirement for PI 3-kinase for Dok-1 tyrosine phosphorylation. COS-1 cells were transfected with wild type Dok-1 or Dok-1-KRas and empty vector or Δp85, a dominant negative mutant of PI 3-kinase. Cell lysates were immunoprecipitated with anti-Dok-1 antibody and analyzed by SDS-PAGE, followed by Western blotting with anti-Tyr(P) and anti-Dok-1 antibodies. Compared with wild type Dok-1, Dok-1-KRas was hyperphosphorylated, with an approximately 2-fold enhancement of Tyr(P) levels per unit of protein (Fig. 4A). Phosphorylation of the wild type Dok-1 was inhibited by expression of a dominant negative mutant of PI 3-kinase. In contrast, the tyrosine phosphorylation of Dok-1-KRas was insensitive to Δp85 expression. The Dok-1-KRas construct was nearly exclusively localized to the membrane fraction (Fig. 4B). These results indicate that constitutive targeting of Dok-1 to the membrane via a K-Ras tail results in PI 3-kinase-independent hyperphosphorylation of Dok-1. Dok-1 has been reported to be a negative regulator of cell growth (17.Yamanashi Y. Tamura T. Kanamori T. Yamane H. Nariuchi H. Yamamoto T. Baltimore D. Genes Dev. 2000; 14: 11-16PubMed Google Scholar). To determine the significance of membrane localization for Dok-1 function, COS-1 cells were transfected with wild type Dok-1, Dok-1-KRas, ΔPHDok-1, or ΔPHDok-1-KRas, and [3H]thymidine incorporation was measured. As depicted in Fig. 5A, expression of wild type Dok-1 inhibited cell growth by ∼22%. The constitutively membrane-bound Dok-1-KRas or ΔPHDok-1-KRas exhibited a greater negative effect on cell proliferation with ∼45% reduction in [3H]thymidine incorporation. Western blotting with anti-Dok-1 antibody revealed that the expression level for each construct was the same (Fig. 5B). These data suggest that membrane localization of Dok-1 is required for negative regulation of cell proliferation. The mechanism by which membrane translocation contributes to the ability of Dok-1 to inhibit cell proliferation is not yet known. In B cells, binding of Dok-1 to the membrane-bound FcγRIIB receptor promotes Dok-1 phosphorylation and association with RasGAP (22.Tamir I. Stolpa J.C. Helgason C.D. Nakamura K. Bruhns P. Daeron M. Cambier J.C. Immunity. 2000; 12: 347-358Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). This results in formation of RasGDP and attenuation of the Ras/Raf/MAP kinase signaling pathway. However, recent studies using fibroblasts from Dok-1 −/− mice revealed that RasGAP binding is not required for negative regulation of cell proliferation by Dok-1 (23.Zhao M. Schmitz A.A.P. Yi Q. Di Cristofano A. Pandolfi P-P. Van Aelst L. J. Exp. Med. 2001; 194: 265-274Crossref PubMed Scopus (62) Google Scholar). It is likely that association of Dok-1 with other downstream effector proteins mediates cell growth inhibition. The next set of experiments was designed to identify the kinase(s) responsible for KL-stimulated phosphorylation of Dok-1. The likely candidates were c-Kit itself as well as Lyn and Tec kinases, which have been shown to interact with c-Kit. Mo7 cells were incubated in the presence or absence of KL, and cell lysates were immunoprecipitated with antibodies to Dok-1, Lyn, or Tec. Western blotting of anti-Dok-1 immunoprecipitates with anti-Tyr(P) antibody revealed the presence of a 145-kDa phosphoprotein that coprecipitated with Dok-1 with higher levels present in KL-stimulated cells. Reprobing of the Western blot with anti-c-Kit antibody revealed that the 145-kDa protein was c-Kit (Fig. 6A). c-Kit also associated with Lyn in a KL-independent manner as has previously been reported (7.Linnekin D. DeBerry C.S. Mou S. J. Biol. Chem. 1997; 272: 27450-27455Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Probing of anti-Tec immunoprecipitates with anti-Tyr(P) antibody revealed the presence of c-Kit in the KL-treated samples. The level of tyrosine phosphorylation of endogenous Tec was very low, and little tyrosine-phosphorylated Dok-1 was observed in these complexes. Two proteins with molecular masses of 62 and 70 kDa were detected when the blot was stripped and reprobed with anti-Tec antibody. These likely represent isoforms of Tec kinases, which have been reported to result from alternative splicing (18.Mano H. Yamashita Y. Sato K. Yazaki Y. Hirai H. Blood. 1995; 85: 343-350Crossref PubMed Google Scholar). Upon KL stimulation, tyrosine-phosphorylated SHC was detected in the anti-Tec immunoprecipitates (data not shown). These data indicate that c-Kit forms complexes with Dok-1, Lyn, and Tec. GST fusion pulldown experiments were performed to identify the region(s) of c-Kit that interacts with Dok-1. GST fusion proteins containing the juxtamembrane, kinase insert, and C-terminal tail regions as well as the first half of the kinase domain of c-Kit were purified from Escherichia coli. Because the interaction between Dok-1 and c-Kit was likely to be Tyr(P)-dependent, the fusion proteins were first phosphorylated by incubation with Lyn immunoprecipitates from Mo7 cells. As depicted in Fig. 6Bthe juxtamembrane region and C-terminal tail of c-Kit were phosphorylated. The fusion proteins were then incubated with lysates from KL-stimulated Mo7 cells, and the amount of Dok-1 that was bound was determined. As indicated in Fig. 6C only the phosphorylated juxtamembrane region and C-terminal tail of c-Kit associated with phosphorylated Dok-1. Phosphorylation of the GST fusion proteins was necessary for association with Dok-1 because no interaction occurred when the fusion proteins were not prephosphorylated by Lyn (data not shown). The Src family member Lyn has been shown to be required for KL-mediated responses of mast cells and progenitor cells (25.O'Laughlin-Bunner B. Radosevic N. Taylor M.L. Shivakrupa DeBerry C. Metcalfe D.D. Zhou M. Lowell C. Linnekin D. Blood. 2001; 98: 343-350Crossref PubMed Scopus (81) Google Scholar). Because Lyn constitutively associates with c-Kit in Mo7 cells, we tested whether Lyn is necessary for c-Kit to achieve maximum tyrosine phosphorylation and activation. c-Kit was expressed alone or was coexpressed with Lyn in COS-1 cells, and c-Kit tyrosine phosphorylation was examined upon stimulation with c-Kit ligand. As depicted in Fig. 7A, upon KL stimulation tyrosine phosphorylation of c-Kit was detected. However, in the presence of Lyn, KL stimulation of c-Kit phosphorylation increased ∼10-fold, indicating that Src family kinases promote maximal phosphorylation of c-Kit. Because Src family kinases (especially Lyn) are activated in response to KL, we tested whether Dok-1 is phosphorylated by Src family kinases. Dok-1 was expressed alone or coexpressed with the Src family kinase Fyn, and tyrosine phosphorylation of Dok-1 was examined. As depicted in Fig. 7B, tyrosine phosphorylation of Dok-1 increased ∼20-fold when wild type Fyn was coexpressed, whereas expression of a kinase-inactive mutant of Fyn mutant (K299M) had no effect on Dok-1 tyrosine phosphorylation. To study the role of Src family kinases in the c-Kit pathway, we attempted to reconstitute the KL signaling system in COS-1 cells. As depicted in Fig. 7C, when COS-1 cells were transfected with Dok-1 alone Dok-1 was phosphorylated to low levels by endogenous kinases, and addition of KL had no effect. KL stimulation of COS-1 cells expressing Dok-1 and c-Kit resulted in a 2-fold increase in the tyrosine phosphorylation of Dok-1 compared with cells without KL stimulation. However, when COS-1 cells were cotransfected with Dok-1, c-Kit, and Fyn the tyrosine phosphorylation of Dok-1 increased 10-fold, and stimulation with KL resulted in an additional 2-fold increase. Moreover, the Dok-1·c-Kit receptor complex was only detected when Fyn was present (data not shown). When the kinase-inactive mutant of Fyn was expressed in place of wild type Fyn, tyrosine phosphorylation of Dok-1 dramatically decreased. These data strongly suggest that Src family kinases mediate tyrosine phosphorylation of Dok-1. Both Lyn and Tec are expressed in KL-responsive Mo7 cells (6.Price D.J. Rivnay B. Avraham H. Biochem. Biophys. Res. Commun. 1999; 259: 611-616Crossref PubMed Scopus (17) Google Scholar, 7.Linnekin D. DeBerry C.S. Mou S. J. Biol. Chem. 1997; 272: 27450-27455Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The abilities of Lyn and Tec kinases to phosphorylate Dok-1 were compared directly as follows. Mo7 cells were starved overnight and then stimulated with KL. Lyn and Tec kinases were immunoprecipitated from the same amount of cell lysate. In vitro kinase assays were performed using recombinant Dok-1 as substrate in the presence of [γ-32P]ATP. As shown in Fig. 8A, Lyn was constitutively active in Mo7 cells, and incubation with recombinant Dok-1 resulted in robust phosphorylation of Dok-1. The ∼140-kDa phosphoprotein detected in Lyn immune complex kinase assays was identified as c-Kit because it comigrated with the c-Kit band in anti-c-Kit immunoprecipitates (data not shown). No detectable phosphorylation of recombinant Dok-1 by Tec was observed. Tec protein was clearly present in the anti-Tec immunoprecipitate (Fig. 8, A and B), but the autophosphorylation activity of Tec kinase was only detectable when the intensity on the phosphorimaging device was greatly increased (20-fold) (Fig. 8B). Others have also observed that Tec autophosphorylation activity is relatively weak (20.Yang W.-C. Ghiotto M. Barbarat B. Olive D. J. Biol. Chem. 1999; 274: 607-617Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). It is possible that the inability to detect phosphorylated Dok-1 in anti-Tec immunoprecipitates was due to low levels of Tec kinase in Mo7 cells. Experiments designed to increase the level of Tec were not feasible in Mo7 cells because of low transfection efficiencies combined with a loss of KL responsiveness following electroporation. We therefore expressed Tec in COS-1 cells and directly compared the ability of Tec and Lyn kinases to phosphorylate Dok-1 in vivo. As depicted in Fig. 8C both Tec and Lyn were capable of phosphorylating Dok-1 to equivalent extents. Cotransfection of both Tec and Lyn resulted in a 2-fold increase in Dok-1 phosphorylation compared with Tec alone. A similar effect was recently noted by van Dijk et al. (13.van Dijk T.B. van Den Akker E. Amelsvoort M.P. Mano H. Lowenberg B. von Lindern M. Blood. 2000; 96: 3406-3413Crossref PubMed Google Scholar). This is consistent with the ability of Src family kinases to phosphorylate and thereby activate Tec family kinases (18.Mano H. Yamashita Y. Sato K. Yazaki Y. Hirai H. Blood. 1995; 85: 343-350Crossref PubMed Google Scholar, 24.Wahl M.I. Fluckiger A.C. Kato R.M. Park H. Witte O.N. Rawlings D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11526-11533Crossref PubMed Scopus (109) Google Scholar). We conclude that both Lyn and Tec are capable of phosphorylating Dok-1. To further determine the role of Src family kinases in mediating Dok-1 tyrosine phosphorylation, Mo7 cells were starved overnight and incubated with various concentrations of Src family kinase inhibitor PP2 for 1 h prior to stimulation with KL. As depicted in Fig. 9A, inhibition of KL-stimulated tyrosine phosphorylation of Dok-1 was dose-dependent. Tyrosine phosphorylation of Dok-1 was completely inhibited by 5 μm PP2. As a control the inactive analog PP3 had no effect on tyrosine phosphorylation of Dok-1 (data not shown). Lyn has been shown to be an important contributor to KL-induced proliferation of primary hematopoietic cells (25.O'Laughlin-Bunner B. Radosevic N. Taylor M.L. Shivakrupa DeBerry C. Metcalfe D.D. Zhou M. Lowell C. Linnekin D. Blood. 2001; 98: 343-350Crossref PubMed Scopus (81) Google Scholar). We therefore determined the requirement for Lyn in Dok-1 tyrosine phosphorylation during c-Kit signaling. Primary BMMC isolated from normal and Lyn-deficient mice were starved overnight and then stimulated with KL. Phosphorylated Dok-1 was immunoprecipitated with agarose-conjugated anti-Tyr(P) antibodies and blotted with anti-Dok-1 antibody. As depicted in Fig. 9B, upon c-Kit stimulation tyrosine phosphorylation of Dok-1 was increased ∼2-fold in primary normal BMMC but not in Lyn-deficient BMMC, indicating that Lyn is required for KL-dependent tyrosine phosphorylation of Dok-1. Western blotting with anti-Dok-1 antibody revealed the presence of the same amount of Dok-1 in each sample. In conclusion, the data presented in this manuscript support the following model. Binding of KL to c-Kit results in activation of PI 3-kinase and generation of inositol phospholipids at the plasma membrane that serve as binding sites for PH domains. Dok-1 and Tec are recruited to the plasma membrane via their respective PH domains. In addition, Dok-1 binds to the juxtamembrane region and C-terminal tail of c-Kit, placing it in close proximity to membrane-bound c-Kit-associated Lyn. Several lines of evidence indicate that Lyn plays a critical role in c-Kit signaling. A recent study reported that Lyn is required for an optimal response to KL-mediated cell proliferation and chemotaxis in primary hematopoietic progenitor cells and mast cells (25.O'Laughlin-Bunner B. Radosevic N. Taylor M.L. Shivakrupa DeBerry C. Metcalfe D.D. Zhou M. Lowell C. Linnekin D. Blood. 2001; 98: 343-350Crossref PubMed Scopus (81) Google Scholar). Here we show that Lyn is required for association of Dok-1 and c-Kit most likely because Lyn phosphorylates sites on c-Kit that promote Dok-1 binding. Moreover, Lyn is required for tyrosine phosphorylation of Dok-1 in KL-stimulated Mo7 cells and BMMC. This may occur by direct phosphorylation of Dok-1 by Lyn. Alternatively, or in parallel, Lyn phosphorylation of Tec would result in Tec activation and phosphorylation of Dok-1 by Tec. Other studies in B and T cells have revealed that Tec can phosphorylate Dok-1 (19.Yoshida K. Yamashita Y. Miyazato A. Ohya K. Kitanaka A. Ikeda U. Shimada K. Yamanaka T. Ozawa K. Mano H. J. Biol. Chem. 2000; 275: 24945-24952Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 20.Yang W.-C. Ghiotto M. Barbarat B. Olive D. J. Biol. Chem. 1999; 274: 607-617Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and Lyn, Tec, and Dok have been shown to form a complex in hematopoietic cells stimulated with KL (13.van Dijk T.B. van Den Akker E. Amelsvoort M.P. Mano H. Lowenberg B. von Lindern M. Blood. 2000; 96: 3406-3413Crossref PubMed Google Scholar). Regardless of which kinase(s) phosphorylates Dok-1, it is clear that membrane localization of Dok-1 is essential for its tyrosine phosphorylation during c-Kit signaling. Identification of Dok-1 tyrosine phosphorylation sites will ultimately be important for understanding the function of Dok-1 as an adaptor protein in c-Kit-mediated signal transduction. We thank Raisa Louft-Nisenbaum, Chong-Yuan Liu, and Carol Lambek for technical support, Dr. Steve Swendeman for helpful advice, and Debra Alston for secretarial support. We also thank Drs. Kurt Ballmer-Hofer, Diana Linnekin, Leslie Berg, Hava Abraham, and Mike Kavanaugh for generous gifts of reagents." @default.
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