HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 16, 13732-13738, April 19, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/16/13732    most recent M200277200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liang, X. Articles by Resh, M. D. Search for Related Content PubMed PubMed Citation Articles by Liang, X. Articles by Resh, M. D. Social Bookmarking                      What's this?

Phosphatidylinositol 3-Kinase and Src Family Kinases Are Required for Phosphorylation and Membrane Recruitment of Dok-1 in c-Kit Signaling^*

Xiquan Liang, David Wisniewski^§, Annabel Strife^§, Shivakrupa^¶, Bayard Clarkson^§, and Marilyn D. Resh

From the  Cell Biology Program and the ^§ Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 and the ^¶ Basic Research Laboratory and Laboratory of Immunobiology, Division of Basic Sciences, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702

Received for publication, January 10, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

In patients with chronic myelogenous leukemia, the c-abl gene is translocated from chromosome 9 to chromosome 22 to produce a hybrid bcr-abl gene (1-3). 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). These proteins are also tyrosine-phosphorylated when cells are stimulated with c-Kit ligand (KL),¹ a 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 C, and Vav in addition to the signaling proteins listed above (5-7).

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, 9). 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, 11). 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).

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, 21). 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 (5-7). 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). 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).

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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

cDNA Constructs-- 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 PHDok-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 p85 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).

Immunoprecipitation and Antibodies-- Mo7 cells, BMMC, or COS-1 cells were lysed in lysis buffer containing 50 mM Tris-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). 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), 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), 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).

Cell Culture and DNA Transfection-- 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.

Cell Fractionation-- 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).

Immune Complex Kinase Assays-- 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 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM MnCl₂, 10 mM MgCl₂, and 10 µCi of [-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 Binding Assays-- 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.

[³H]Thymidine Incorporation Assay-- COS-1 cells were transfected with either wild type Dok-1, membrane-targeted wild type Dok-1 (Dok-1-KRas), PHDok-1, or membrane-targeted PHDok-1 (PHDok-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.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Tyrosine Phosphorylation of Dok-1 Is Inhibited by the PI 3-Kinase Inhibitor Wortmannin-- Previous studies have established that Dok-1 is tyrosine-phosphorylated in KL-stimulated Mo7 cells (8). 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.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Wortmannin inhibits KL-stimulated tyrosine phosphorylation of Dok-1. Mo7 cells were deprived of IL-3 overnight and then treated with and without 100 nM wortmannin for 1 h followed by activation with 100 ng/ml KL. The cell lysate was immunoprecipitated with either anti-Dok-1 antibody or anti-c-Kit antibody, blotted with anti-Tyr(P) (pTyr)antibody, and stripped and reprobed with anti-Dok-1 or anti-c-Kit antibody, respectively. Each experiment was performed four times with similar results.

Dok-1 Is Recruited to the Membrane upon c-Kit Stimulation-- 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.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Subcellular distribution of Dok-1 upon stimulation with KL. Mo7 cells were deprived of IL-3 overnight and treated with or without 100 nM wortmannin for 1 h followed by activation with 100 ng/ml KL. Cells were homogenized in hypotonic buffer and fractionated into S100 (S) and P100 (P) fractions. A, fractions were immunoprecipitated with anti-Dok-1 antibody, blotted with anti-Tyr(P) (pTyr) antibody, and then reprobed with anti-Dok-1 antibody. B, wild type and PH mutant Dok-1 constructs were transfected into Mo7 cells by electroporation. 18 h later the cells were fractionated into S100 (S) and P100 (P) fractions followed by Western blotting using anti-Dok-1 antibody. Each experiment was performed three times with similar results.

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 PH mutant Dok-1 were transfected into Mo7 cells by electroporation. As depicted in Fig. 2B, the PHDok-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.

Membrane Binding of Dok-1 Is Required for Tyrosine Phosphorylation-- 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 PH 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 PHDok-1 mutant was localized to the cytosolic fraction (S100) and was not phosphorylated. In contrast the PHDok-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.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Membrane-targeted PHDok-1-KRas is constitutively tyrosine-phosphorylated in COS-1 cells. COS-1 cells were transfected with either empty vector, PHDok-1, PHDok-1-KRas, or wild type Dok-1, respectively. A, cell lysates either were immunoprecipitated with anti-Dok-1 and Western blotted with anti-Tyr(P) (pTyr) antibodies or were directly Western blotted with anti-Dok-1 antibody. B, cells were lysed in hypotonic buffer, fractionated into S100 (S) and P100 (P) fractions, and then subjected to SDS-PAGE and Western blotting with anti-Dok-1 antibody. The experiments were performed in triplicate.

Membrane-targeted Dok-1-KRas Is Hyperphosphorylated, and Its Phosphorylation Is PI 3-Kinase-independent-- 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 p85, 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 p85 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.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Membrane-targeted Dok-1-KRas is hyperphosphorylated, and its tyrosine phosphorylation is insensitive to a dominant negative mutant of PI 3-kinase. A, COS-1 cells were transfected with wild type Dok-1 or Dok-1-KRas alone, or they were cotransfected with a dominant negative mutant of PI 3-kinase (p85). The cell lysates were immunoprecipitated with anti-Dok-1 antibody, blotted with anti-Tyr(P) (pTyr) antibody, and then reprobed with anti-Dok-1 antibody. Expression of the mutant p85 was confirmed by direct Western blotting using anti-p85 antibody. Levels of endogenous p85 were below the detection limits of this Western blot. B, S100 and P100 fractions of COS cells transfected with Dok-1 were subjected to SDS-PAGE and Western blotting with anti-Dok-1 antibody. The data are representative of four independent experiments.

Expression of the Constitutively Membrane-targeted Dok-1 Inhibits Cell Proliferation-- Dok-1 has been reported to be a negative regulator of cell growth (17). To determine the significance of membrane localization for Dok-1 function, COS-1 cells were transfected with wild type Dok-1, Dok-1-KRas, PHDok-1, or PHDok-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 PHDok-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.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Membrane localization of Dok-1 is required for inhibition of cell proliferation. A, COS-1 cells were transfected for 24 h with either empty vector, wild type Dok-1, Dok-1-KRas, PHDok-1, or PHDok-1-KRas, and then [3H]thymidine was added for 16 h. Cells were harvested on a fiber filter, and 3H radioactivity was determined. B, expression levels of each construct were similar. The experiments were performed three times.

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 FcRIIB receptor promotes Dok-1 phosphorylation and association with RasGAP (22). 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). It is likely that association of Dok-1 with other downstream effector proteins mediates cell growth inhibition.

Dok-1, Lyn, and Tec Form Complexes with c-Kit-- 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). 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). 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.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Dok-1 coimmunoprecipitates with c-Kit and associates with the juxtamembrane region and C-terminal tail. A, Mo7 cells were deprived of IL-3 overnight and then stimulated with 100 ng/ml KL for 5 min. Cell lysates were immunoprecipitated with anti-Dok-1, anti-Lyn, or anti-Tec antibodies, blotted with anti-Tyr(P) (pTyr) antibody, and reprobed with anti-c-Kit, anti-Dok-1, anti-Lyn, or anti-Tec antibodies, respectively. B, purified GST fusion proteins encoding the juxtamembrane region (JXM), kinase region 1 (K1), kinase insert (KI), and C-terminal tail (C-tail) of c-Kit were incubated for 20 min with Lyn immunoprecipitates from Mo7 cells in the presence of [-32P]ATP and kinase assay buffer. Aliquots of the reaction mixture were analyzed by SDS-PAGE and either phosphorimaging or Western blotting with anti-GST antibody. C, 5 µg of the above GST or GST fusion proteins previously treated with Lyn kinase were incubated with KL-treated Mo7 cell lysates for 2 h at 4 °C in the presence of glutathione-Sepharose 4B. The beads were washed and analyzed by SDS-PAGE, followed by Western blotting with anti-Tyr(P) antibody. A sample containing immunoprecipitated Dok-1 was run alongside as a control. The data depicted are representative of three independent experiments.

Dok-1 Interacts with the Juxtamembrane Region and C-terminal Tail of c-Kit-- 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. 6B the 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).

Tyrosine Phosphorylation of Dok-1 and c-Kit by Src Family Kinases-- The Src family member Lyn has been shown to be required for KL-mediated responses of mast cells and progenitor cells (25). 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.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Src family kinases mediate tyrosine phosphorylation of Dok-1. A, COS-1 cells were transfected with c-Kit alone or cotransfected with c-Kit and Lyn. Cells were starved overnight and stimulated with KL. Cell lysates were immunoprecipitated with anti-c-Kit antibody and analyzed by SDS-PAGE followed by Western blotting with anti-Tyr(P) (pTyr) antibody and reprobing with anti-c-Kit antibody. The tyrosine phosphorylation level of c-Kit was quantitated by normalizing the amount of [Tyr(P)]c-Kit to the total amount of c-Kit in each sample. B, COS-1 cells were transfected with Dok-1 alone or cotransfected with either Fyn or a kinase-inactive form of Fyn (FynK299M). Cell lysates were immunoprecipitated with anti-Dok-1 antibody and analyzed by SDS-PAGE followed by Western blotting with anti-Tyr(P) antibody and reprobing with anti-Dok-1 antibody. The tyrosine phosphorylation level of Dok-1 was quantitated. C, COS cells were transfected for 24 h with Dok alone or cotransfected with c-Kit in the presence or absence of wild type Fyn or the kinase-inactive Fyn mutant. The cells were starved overnight and then stimulated with KL. Cell lysates were immunoprecipitated with anti-Dok-1 antibody and analyzed by SDS-PAGE followed by sequential Western blotting with anti-Tyr(P) and anti-Dok-1 antibodies, respectively. The tyrosine phosphorylation level of c-Kit was quantitated. Bars where no error bar is shown reflect a standard deviation of the data of <8%. The experiments were performed four times with similar results.

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.

Tyrosine Phosphorylation of Dok-1 by Tec-- Both Lyn and Tec are expressed in KL-responsive Mo7 cells (6, 7). 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 [-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).

View larger version (35K): [in this window] [in a new window]   Fig. 8.   Phosphorylation of Dok-1 by Lyn and Tec. Mo7 cells were deprived of IL-3 overnight and incubated with 100 ng/ml KL for 5 min. Equal amounts of Mo7 cell lysates were immunoprecipitated with either anti-Lyn, anti-c-Kit, or anti-Tec antibodies. A, immune complexes formed with anti-Lyn, anti-c-Kit, or anti-Tec antibodies were incubated in the presence or absence of recombinant Dok-1 and [-32P]ATP in kinase assay buffer. The reaction mixture was analyzed by SDS-PAGE and either phosphorimaging or Western blotting with the indicated antibodies. B, autophosphorylation of Tec in vitro. Anti-Tec immunoprecipitates were incubated with [-32P]ATP and analyzed by SDS-PAGE and either phosphorimaging or Western blotting with anti-Tec antibody. The intensity on the phosphorimaging device was increased to visualize the Tec bands. C, COS-1 cells were transfected with Dok-1, Lyn, and/or Tec, and cell lysates were analyzed by immunoprecipitation and/or Western blotting with the indicated antibodies. The experiments were performed three times with similar results.

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). This is consistent with the ability of Src family kinases to phosphorylate and thereby activate Tec family kinases (18, 24). We conclude that both Lyn and Tec are capable of phosphorylating Dok-1.

Src Family Kinases Are Required for Tyrosine Phosphorylation of 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).

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Src family kinases are required for tyrosine phosphorylation of Dok-1. A, Mo7 cells were starved overnight and treated with PP2 for 1 h prior to a 5-min stimulation with KL. Cell lysates were immunoprecipitated with anti-Dok-1 antibody and analyzed by SDS-PAGE followed by sequential Western blotting with anti-Tyr(P) and anti-Dok-1 antibodies. The tyrosine phosphorylation level of c-Kit was quantitated. Bars where no error bar is shown reflect a standard deviation of the data of <8%. B, BMMC from normal and Lyn-deficient mice were stimulated with c-Kit. Cell lysates were immunoprecipitated with agarose-conjugated anti-Tyr(P) (pTyr) antibodies and blotted with anti-Dok-1 antibody. The total amount of Dok-1 in each sample was determined by blotting with anti-Dok-1 antibody. The experiments were performed twice with similar results.

Lyn has been shown to be an important contributor to KL-induced proliferation of primary hematopoietic cells (25). 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.

A Model for the Role of Dok-1 in c-Kit Signal Transduction-- 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). 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, 20), and Lyn, Tec, and Dok have been shown to form a complex in hematopoietic cells stimulated with KL (13). 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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant PO1 CA64593.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Cell Biology Program, Sloan-Kettering Inst. for Cancer Research, 1275 York Ave., Box 143, New York, NY 10021. Tel.: 212-639-2514; Fax: 212-717-3317; E-mail: m-resh@ski.mskcc.org.

Published, JBC Papers in Press, February 1, 2002, DOI 10.1074/jbc.M200277200

	ABBREVIATIONS

The abbreviations used are: KL, Kit ligand; PI, phosphatidylinositol; PH, pleckstrin homology; GST, glutathione S-transferase; IMDM, Iscove's modified Dulbecco's medium; IL, interleukin; GAP, GTPase-activating protein; MAP, mitogen-activated protein; BMMC, bone marrow mast cells.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				V. G. Karur, C. A. Lowell, P. Besmer, V. Agosti, and D. M. Wojchowski Lyn kinase promotes erythroblast expansion and late-stage development Blood, September 1, 2006; 108(5): 1524 - 1532. [Abstract] [Full Text] [PDF]

				Y. Niu, F. Roy, F. Saltel, C. Andrieu-Soler, W. Dong, A.-L. Chantegrel, R. Accardi, A. Thepot, N. Foiselle, M. Tommasino, et al. A nuclear export signal and phosphorylation regulate dok1 subcellular localization and functions. Mol. Cell. Biol., June 1, 2006; 26(11): 4288 - 4301. [Abstract] [Full Text] [PDF]

				R. Biswas, D. Stein, and E. R. Stanley Drosophila Dok is required for embryonic dorsal closure Development, January 15, 2006; 133(2): 217 - 227. [Abstract] [Full Text] [PDF]

				S. Itoh, S. Lemay, M. Osawa, W. Che, Y. Duan, A. Tompkins, P. S. Brookes, S.-S. Sheu, and J.-i. Abe Mitochondrial Dok-4 Recruits Src Kinase and Regulates NF-{kappa}B Activation in Endothelial Cells J. Biol. Chem., July 15, 2005; 280(28): 26383 - 26396. [Abstract] [Full Text] [PDF]

				J. Lennartsson, T. Jelacic, D. Linnekin, and R. Shivakrupa Normal and Oncogenic Forms of the Receptor Tyrosine Kinase Kit Stem Cells, January 1, 2005; 23(1): 16 - 43. [Abstract] [Full Text] [PDF]

				S. Lee, C. Andrieu, F. Saltel, O. Destaing, J. Auclair, V. Pouchkine, J. Michelon, B. Salaun, R. Kobayashi, P. Jurdic, et al. I{kappa}B kinase {beta} phosphorylates Dok1 serines in response to TNF, IL-1, or {gamma} radiation PNAS, December 14, 2004; 101(50): 17416 - 17421. [Abstract] [Full Text] [PDF]

				R. J. Crowder, H. Enomoto, M. Yang, E. M. Johnson Jr., and J. Milbrandt Dok-6, a Novel p62 Dok Family Member, Promotes Ret-mediated Neurite Outgrowth J. Biol. Chem., October 1, 2004; 279(40): 42072 - 42081. [Abstract] [Full Text] [PDF]

				A. Bedirian, C. Baldwin, J.-i. Abe, T. Takano, and S. Lemay Pleckstrin Homology and Phosphotyrosine-binding Domain-dependent Membrane Association and Tyrosine Phosphorylation of Dok-4, an Inhibitory Adapter Molecule Expressed in Epithelial Cells J. Biol. Chem., April 30, 2004; 279(18): 19335 - 19349. [Abstract] [Full Text] [PDF]

				N. Jones, S. H. Chen, C. Sturk, Z. Master, J. Tran, R. S. Kerbel, and D. J. Dumont A Unique Autophosphorylation Site on Tie2/Tek Mediates Dok-R Phosphotyrosine Binding Domain Binding and Function Mol. Cell. Biol., April 15, 2003; 23(8): 2658 - 2668. [Abstract] [Full Text] [PDF]

				O. W. Lindwasser and M. D. Resh Myristoylation as a target for inhibiting HIV assembly: Unsaturated fatty acids block viral budding PNAS, October 1, 2002; 99(20): 13037 - 13042. [Abstract] [Full Text] [PDF]

				T. Takada, T. Noguchi, K. Inagaki, T. Hosooka, K. Fukunaga, T. Yamao, W. Ogawa, T. Matozaki, and M. Kasuga Induction of Apoptosis by Stomach Cancer-associated Protein-tyrosine Phosphatase-1 J. Biol. Chem., September 6, 2002; 277(37): 34359 - 34366. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/16/13732    most recent M200277200v1 Alert me when this article is cited