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Dok-R Binds c-Abl and Regulates Abl Kinase Activity and Mediates Cytoskeletal Reorganization

2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês

10.1074/jbc.m301339200

ISSN

1083-351X

Autores

Zubin Master, Jennifer M. Tran, Aseem Bishnoi, Stephen H. Chen, John M.L. Ebos, Paul Van Slyke, Robert S. Kerbel, Daniel Dumont,

Tópico(s)

Melanoma and MAPK Pathways

Resumo

Dok-R, also known as Dok-2/FRIP, belongs to the DOK family of signaling molecules that become tyrosinephosphorylated by several different receptor and cytoplasmic tyrosine kinases. Tyrosine phosphorylation of DOK proteins establishes high affinity binding sites for other signaling molecules leading to activation of a signaling cascade. Here we show that Dok-R associates with c-Abl directly via a constitutive SH3-mediated interaction and that this binding requires a PMMP motif in the proline-rich tail of Dok-R. The Dok-R-Abl interaction is further enhanced by an active c-Abl kinase, which requires the presence of its SH2 domain. Interaction of Dok-R with c-Abl also results in an increase in c-Abl tyrosine phosphorylation and kinase activity. Furthermore, we demonstrate that this increase in kinase activity correlates with a concomitant increase in c-Abl-mediated biological activity as measured by the formation of actin microspikes. Our data are the first to demonstrate that Dok-R and c-Abl interact in both a constitutive and inducible fashion and that Dok-R influences the intracellular kinase and biological activity of c-Abl. Dok-R, also known as Dok-2/FRIP, belongs to the DOK family of signaling molecules that become tyrosinephosphorylated by several different receptor and cytoplasmic tyrosine kinases. Tyrosine phosphorylation of DOK proteins establishes high affinity binding sites for other signaling molecules leading to activation of a signaling cascade. Here we show that Dok-R associates with c-Abl directly via a constitutive SH3-mediated interaction and that this binding requires a PMMP motif in the proline-rich tail of Dok-R. The Dok-R-Abl interaction is further enhanced by an active c-Abl kinase, which requires the presence of its SH2 domain. Interaction of Dok-R with c-Abl also results in an increase in c-Abl tyrosine phosphorylation and kinase activity. Furthermore, we demonstrate that this increase in kinase activity correlates with a concomitant increase in c-Abl-mediated biological activity as measured by the formation of actin microspikes. Our data are the first to demonstrate that Dok-R and c-Abl interact in both a constitutive and inducible fashion and that Dok-R influences the intracellular kinase and biological activity of c-Abl. Activation of receptor and cytoplasmic tyrosine kinases (TK) 1The abbreviations used are: TK, tyrosine kinase; HA, hemagglutinin; WT, wild type; KD, kinase dead; GST, glutathione S-transferase; HEK, human embryonic kidney; PRR, proline- and tyrosine-rich; DOK, Downstream Of Kinase; SH, Src homology domain; PTB, phosphotyrosine binding domain; EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase. induces tyrosine phosphorylation of target proteins that initiates a series of protein-protein interactions (1Madhani H.D. Cell. 2001; 106: 9-11Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). One of the many targets of TK-mediated signaling pathways is the actin cytoskeleton (2Jamora C. Fuchs E. Nat. Cell Biol. 2002; 4: E101-E108Crossref PubMed Scopus (492) Google Scholar). Actin cytoskeletal rearrangements are important fundamental events that occur during a variety of cellular processes such as adhesion, spreading, migration, proliferation, and differentiation (2Jamora C. Fuchs E. Nat. Cell Biol. 2002; 4: E101-E108Crossref PubMed Scopus (492) Google Scholar). Reorganization of the actin cytoskeleton involves cross-talk between a multitude of different signaling pathways involving kinases, integrins, and other adhesion molecules that target the actin cytoskeletal machinery. Many of these signaling proteins, including the integrin receptors and the cytosolic c-Abl tyrosine kinase, associate directly with the cytoskeleton. Tyrosine phosphorylation of docking and adapter proteins by receptor and cytosolic TKs promotes signal amplification by providing high affinity binding sites for numerous intracellular signaling molecules containing Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains. Protein-protein interactions can also occur in a non-inducible manner as seen with the constitutive interaction between Src homology 3 (SH3) domains binding proline-rich motifs. Pleckstrin homology (PH) domains on signaling proteins recognize phospholipids and assist in recruitment of the protein to the plasma membrane. In addition to their role in mediating protein interactions, many of these modular domains found in cytoplasmic TKs also serve to regulate the host kinase. Similar to Src family kinases, c-Abl kinase activity is tightly regulated through intramolecular interactions involving its amino-terminal SH3 domain-containing region (3Cicchetti P. Mayer B.J. Thiel G. Baltimore D. 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Moreover, the Bcr-Abl fusion protein resulting from a reciprocal translocation between the Bcr and c-Abl genes in chronic myelogenous leukemia contains increased intracellular kinase activity and leads to a polarized distribution of the oncoprotein in migrating cells (12Skourides P.A. Perera S.A. Ren R. Oncogene. 1999; 18: 1165-1176Crossref PubMed Scopus (31) Google Scholar). Cell adhesion through integrins also regulates the tyrosine phosphorylation of the cytoskeletal protein paxillin by c-Abl (13Lewis J.M. Schwartz M.A. J. Biol. Chem. 1998; 273: 14225-14230Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In this manner, Abl kinases provide a direct link between TK-mediated signaling and the actin cytoskeleton. In addition to a role for c-Abl in actin reorganization, several adapter and docking proteins have also been shown to be instrumental in regulating actin dynamics. These include Nck/Grb4 (14Smith J.M. Katz S. Mayer B.J. J. Biol. Chem. 1999; 274: 27956-27962Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), the Crk family of adapter proteins (15Feller S.M. Knudsen B. Hanafusa H. EMBO J. 1994; 13: 2341-2351Crossref PubMed Scopus (326) Google Scholar, 16Feller S.M. Ren R. Hanafusa H. Baltimore D. Trends Biochem. Sci. 1994; 19: 453-458Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 17Ren R. Ye Z.S. Baltimore D. Genes Dev. 1994; 8: 783-795Crossref PubMed Scopus (291) Google Scholar), as well as an emerging class of docking molecules known as the Downstream Of Kinase (DOK) protein family (18Hosooka T. Noguchi T. Nagai H. Horikawa T. Matozaki T. Ichihashi M. Kasuga M. Mol. Cell. Biol. 2002; 21: 5437-5546Crossref Scopus (29) Google Scholar, 19Cong F. Yuan B. Goff S.P. Mol. Cell. Biol. 1999; 19: 8314-8325Crossref PubMed Scopus (97) Google Scholar, 20Noguchi 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, 21Bhat A. Johnson K.J. Oda T. Corbin A.S. Druker B.J. J. Biol. Chem. 1998; 273: 32360-32368Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 22Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-211Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 23Di Cristofano A. Carpino N. Dunant N. Friedland G. Kobayashi R. Strife A. Wisniewski D. Clarkson B. Pandolfi P.P. Resh M.D. J. Biol. Chem. 1998; 273: 4827-4830Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The DOK family includes p62 (Dok-1), Dok-R (Dok-2/FRIP), Dok-L (Dok-3), Dok-4, Dok-5, and possibly IRS-3 (19Cong F. Yuan B. Goff S.P. Mol. Cell. Biol. 1999; 19: 8314-8325Crossref PubMed Scopus (97) Google Scholar, 22Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-211Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 24Lemay S. Davidson D. Latour S. Veillette A. Mol. Cell. Biol. 2000; 20: 2743-2754Crossref PubMed Scopus (156) Google Scholar, 25Nelms K. Snow A.L. Hu-Li J. Paul W.E. Immunity. 1998; 9: 13-24Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 26Carpino 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, 27Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Members of the DOK family share a similar structure characterized by an amino-terminal PH domain followed by a central PTB domain and a proline and tyrosine-rich carboxyl-terminal tail. Using different receptor-mediated pathways, both Dok and Dok-R have been shown to be critical in cell motility via a phosphotyrosine-dependent association with Nck (18Hosooka T. Noguchi T. Nagai H. Horikawa T. Matozaki T. Ichihashi M. Kasuga M. Mol. Cell. Biol. 2002; 21: 5437-5546Crossref Scopus (29) Google Scholar, 20Noguchi 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, 28Master Z. Jones N. Tran J. Kerbel S. Dumont D.J. EMBO J. 2001; PubMed Google Scholar). Nck associates with a host of effector proteins involved in cytoskeletal rearrangement such as the serine/threonine kinase Pak (29Kiosses W.B. Daniels R.H. Otey C. Bokoch G.M. Schwartz M.A. J. Cell Biol. 1999; 147: 831-844Crossref PubMed Scopus (251) Google Scholar, 30Sells M.A. Boyd J.T. Chernoff J. J. Cell Biol. 1999; 145: 837-849Crossref PubMed Scopus (328) Google Scholar, 31Bokoch G.M. Wang Y. Bohl B.P. Sells M.A. Quilliam L.A. Knaus U.G. J. Biol. Chem. 1996; 271: 25746-25749Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar) and the cytoplasmic tyrosine kinase c-Abl (14Smith J.M. Katz S. Mayer B.J. J. Biol. Chem. 1999; 274: 27956-27962Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 17Ren R. Ye Z.S. Baltimore D. Genes Dev. 1994; 8: 783-795Crossref PubMed Scopus (291) Google Scholar). Additionally, both Dok and DokL are known to bind v-Abl and Bcr-Abl in a kinase activation-dependent manner (19Cong F. Yuan B. Goff S.P. Mol. Cell. Biol. 1999; 19: 8314-8325Crossref PubMed Scopus (97) Google Scholar, 21Bhat A. Johnson K.J. Oda T. Corbin A.S. Druker B.J. J. Biol. Chem. 1998; 273: 32360-32368Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 22Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-211Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 26Carpino 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). Since some members of the DOK family of proteins have been shown to bind different Abl kinases (19Cong F. Yuan B. Goff S.P. Mol. Cell. Biol. 1999; 19: 8314-8325Crossref PubMed Scopus (97) Google Scholar, 21Bhat A. Johnson K.J. Oda T. Corbin A.S. Druker B.J. J. Biol. Chem. 1998; 273: 32360-32368Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 22Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-211Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 26Carpino 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), we sought to determine if c-Abl can associate with Dok-R and if so, by what mechanism. In this article, we show that Dok-R directly associate with c-Abl via both a constitutive and phosphotyrosine-dependent manner. Dok-R constitutively associates with c-Abl via its SH3 domain, which requires a PMMP motif on Dok-R. In addition, we demonstrate that this binding is enhanced upon activation of the Abl kinase and is also mediated through the c-Abl SH2 domain. Upon engagement with Dok-R, c-Abl can utilize Dok-R as a substrate for tyrosine phosphorylation in vitro and within cells, which can be abrogated upon the addition of the pharmacological inhibitor of the c-Abl kinase, STI571. The association of Dok-R with c-Abl leads to an increase in c-Abl tyrosine phosphorylation and kinase activity. Importantly, expression of Dok-R also enhances c-Abl-dependent biological activity as measured by cytoskeletal rearrangements of condensed actin cell protrusions. These results are the first to demonstrate that Dok-R and c-Abl associate both constitutively and in a phosphotyrosine-dependent manner and suggest that a mechanism of processive binding between Dok-R and c-Abl may physiologically regulate the kinase activity of c-Abl. Plasmids—The plasmid encoding hemagglutinin (HA)-tagged Dok-R has been previously described (32Jones N. Dumont D.J. Curr. Biol. 1999; 9: 1057-1060Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 33Jones N. Dumont D.J. Oncogene. 1998; 17: 1097-1108Crossref PubMed Scopus (134) Google Scholar). HA-Dok-RΔPRR, HA-Dok-RΔC-PRR were engineered by PCR-based site-directed mutagenesis where TAG stop codons were placed nucleotides 815 and 1010 within HA-Dok-R, respectively, as previously described elsewhere (32Jones N. Dumont D.J. Curr. Biol. 1999; 9: 1057-1060Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). HA-Dok-RP279A, HA-Dok-RP286A, HA-Dok-RPMMP, HA-Dok-RPESP, HA-Dok-RPGAP, where proline residues were mutated to alanine, in wild-type Dok-R were engineered by site-directed mutagenesis (32Jones N. Dumont D.J. Curr. Biol. 1999; 9: 1057-1060Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Mammalian PECE expression vector containing wild-type c-Abl was generously provided by Dr. Rob Rottapel. Bacterial GST fusion of Dok-RΔPH has been previously described in (33Jones N. Dumont D.J. Oncogene. 1998; 17: 1097-1108Crossref PubMed Scopus (134) Google Scholar). Bacterial GST-SH3 fusion proteins of c-Abl, Vav-C, Spectrin and Lck were a kind gift from Dr. Jane McGlade and FLAG epitope-tagged c-AblWT, c-AblKD (kinase defective), c-AblΔSH2 in mammalian retroviral expression plasmid pMSCV-hpg were a kind gift from Drs. Pamela Woodring and Tony Hunter (8Woodring P.J. Hunter T. Wang J.Y. J. Biol. Chem. 2001; 276: 27104-27110Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Full-length recombinant c-Abl (rAbl) with six amino-terminal histidine tags was purified using nickel-agarose (Upstate Biotechnology). Cell Culture and Stable Cell Lines—HEK293 and HEK293T human kidney epithelial cell lines and COS-1 monkey fibroblasts (American Type Culture Collection, ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin (Invitrogen). HEK293-Vector, HEK293-Dok-R, and HEK293-Dok-RΔPRR stable cell lines were previously described elsewhere (28Master Z. Jones N. Tran J. Kerbel S. Dumont D.J. EMBO J. 2001; PubMed Google Scholar). All cell transient transfections were performed with 5 or 10 μg of the appropriate plasmids by lipofectamine (Invitrogen) according to the manufacturer's instructions. Clones for the stable HEK293 cell lines were maintained in media described above supplemented with 500 μg/ml zeocin (Invitrogen) (28Master Z. Jones N. Tran J. Kerbel S. Dumont D.J. EMBO J. 2001; PubMed Google Scholar). The IL-3-dependent murine myeloid cell line 32D and 32D-p210 (stably transfected with p210Bcr-Abl) were a kind gift from Dr. Kerbel's laboratory and were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, and 10% WEHI-3B-conditioned medium as source of IL-3. EGF Cell Stimulations—COS-1 cells were serum-starved (0% fetal bovine serum) for 18 h and stimulated with 100 ng/ml EGF (Upstate Biotechnology) and incubated for 10 min at 37 °C and then harvested (34Jones N. Master Z. Jones J. Bouchard D. Gunji Y. Sasaki H. Daly R. Alitalo K. Dumont D.J. J. Biol. Chem. 1999; 274: 30896-30905Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). STI571 Incubations—STI571 was provided by Novartis Inc., Basel, Switzerland. Lyophilized STI571 was dissolved in 100% Me2SO (Sigma) at 10 mm stocks and stored at –20 °C. 32D and 32D-p210 cell treatments were carried out using 0.25 μm STI571 or Me2SO as control for 24 h. 293 stable cell lines were treated for 24 h using 1.5 μm STI571 or Me2SO as control. The proliferation rates of 32D and 32D-p210 cells using a range of STI571 concentrations was previously determined in which 0.25 μm STI571 is not toxic to cells (35Ebos J.M. Tran J. Master Z. Dumont D. Melo J.V. Buchdunger E. Kerbel R.S. Mol. Cancer Res. 2002; 1: 89-95PubMed Google Scholar). Cellular toxicity of HEK293 stable cell was measured by [3H]thymidine incorporation and trypan blue staining after 24 h treatment with 1.5, 2, and 3 μm concentrations of STI571. No significant cellular toxicity was observed (data not shown). Antibodies Used for Immunoprecipitations, Immunofluorescence, and Western Blotting—Commercially available antibodies used were as follows: polyclonal anti-c-Abl (K12) antibody (Santa Cruz Biotechnology); monoclonal anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology); monoclonal anti-HA clone 12CA5 (Roche Applied Science); anti-EGFR (Transduction Laboratories), anti-Bcr antibody (Oncogene Inc.); monoclonal anti-Actin (Sigma); anti-paxillin clone 5H11 (Upstate Biotechnology). The anti-Dok-R antibodies used have been previously described (33Jones N. Dumont D.J. Oncogene. 1998; 17: 1097-1108Crossref PubMed Scopus (134) Google Scholar). GST mixing experiments, coimmunoprecipitation experiments, and Western blotting procedures has been previously described (33Jones N. Dumont D.J. Oncogene. 1998; 17: 1097-1108Crossref PubMed Scopus (134) Google Scholar, 34Jones N. Master Z. Jones J. Bouchard D. Gunji Y. Sasaki H. Daly R. Alitalo K. Dumont D.J. J. Biol. Chem. 1999; 274: 30896-30905Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Immunofluorescence and Confocal Analysis of Cell Protrusion Assay—For actin staining of membrane protrusions, HEK293-Vector, HEK293-Dok-R, and HEK293-Dok-RΔPRR cells were seeded on glass coverslips and transiently transfected with PECE-c-Abl or empty vector by LipofectAMINE and then serum-starved for 18 h at 37 °C before harvesting. Cells were fixed in 4% paraformaldehyde for 20 min and permeabilized in phosphate-buffered saline containing 1% Nonidet P40 (Sigma) for 10 min. Samples were blocked in 5% goat serum (Jackson ImmunoResearch Laboratories Inc.) and then incubated with antibodies to actin (1:500), c-Abl (1:500), or Dok-R (1:2000) for 2 h at room temperature. Samples were washed and incubated with secondary antibodies conjugated to fluorescein isothiocyanate (FITC) (1:100) or rhodamine (1:100) for 1 h at room temperature (28Master Z. Jones N. Tran J. Kerbel S. Dumont D.J. EMBO J. 2001; PubMed Google Scholar). Slides were mounted using Aquapolymount (Polysciences Inc.) and visualized using Zeiss Axiovert 100 m laser scanning microscope (LSM) confocal microscopy. Analyses were performed using LSM510 (version 2.3) scanning software (Zeiss), and images were captured at ×630 magnification. Actin cell protrusion counts were performed at this magnification on the Zeiss Axiovert laser scanning microscope. For cell protrusion analysis, we scored cells either as having or not having protrusions per field with an average of 10 fields. We also scored cells by counting the average number of protrusions per cell per sample with sample sizes equaling 60 or more. A Student's t test was used to determine the statistical significance with a 95% confidence interval between all STI571 versus Me2SO treatments and between certain cell lines. All experiments were repeated at least twice with at least two replicates per experiment. In Vitro c-Abl Kinase Assays—Cell lysates were immunoprecipitated with anti-Abl antibodies for 2 h at 4 °C. Immunoprecipitates were washed 3× in PLC lysis buffer (without sodium fluoride or sodium pyrophosphate) (33Jones N. Dumont D.J. Oncogene. 1998; 17: 1097-1108Crossref PubMed Scopus (134) Google Scholar) and 2× in kinase buffer (2 mm MnCl2 + 50 mm HEPES pH 7.5 + 10 mm MgCl2 + 0.2 mm dithiothreitol) and then incubated with 4 μg of GST-Dok-RΔPH as substrate including 40 μCi of [γ-32P]ATP (Amersham Biosciences), 20 μm ATP (Amersham Biosciences) for 30 min at 30 °C (28Master Z. Jones N. Tran J. Kerbel S. Dumont D.J. EMBO J. 2001; PubMed Google Scholar). Kinase reactions were stopped by the addition of 2× SDS-containing sample buffer and boiled for 10 min. c-Abl immunoprecipitates were electrophoresed and half the gel was used to resolve c-Abl expression by Western analysis using anti-c-Abl antibodies while the other half was dried and exposed to phosphorimager analysis and quantification (ImageQuant). The representative values of c-Abl kinase activity were reflected as the value of GST-Dok-RΔPH phosphorylation over the amount of immunoprecipitated c-Abl in each of the samples. All experiments were performed twice or more times with similar results. The graph represents the average of the experiments ± S.D. c-Abl and Bcr-Abl Constitutively Associate with Dok-R, Which Is Enhanced Upon Abl Activation—The association of Dok-R with several tyrosine-phosphorylated proteins such as epidermal growth factor (EGF) receptor (EGFR), ErbB2, and Tie2/Tek is mediated via its PTB domain (33Jones N. Dumont D.J. Oncogene. 1998; 17: 1097-1108Crossref PubMed Scopus (134) Google Scholar, 36Dankort D. Jeyabalan N. Jones N. Dumont D.J. Muller W.J. J. Biol. Chem. 2001; 19;276: 38921-38928Abstract Full Text Full Text PDF Scopus (68) Google Scholar, 37Kim I. Kim H.G. So J.-N. Kim J.H. Kwak H.J. Koh G.Y. Circ. Res. 2000; 86: 24-29Crossref PubMed Scopus (521) Google Scholar). Originally, Dok-R, and its related member Dok, were identified as phosphoproteins migrating at relative molecular masses of 56 and 62 kDa, respectively, from cells expressing Bcr-Abl (22Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-211Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 23Di Cristofano A. Carpino N. Dunant N. Friedland G. Kobayashi R. Strife A. Wisniewski D. Clarkson B. Pandolfi P.P. Resh M.D. J. Biol. Chem. 1998; 273: 4827-4830Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 38Druker B. Okuda K. Matulonis U. Salgia R. Roberts T. Griffin J.D. Blood. 1992; 79: 2215-2220Crossref PubMed Google Scholar). However, since the nature of the interaction between Abl and Dok-R has yet to be resolved, we set out to determine whether Dok-R could associate with c-Abl and if so, to define the mechanism behind this interaction. COS-1 cells transfected with HA-tagged Dok-R (HA-Dok-R) were either left untreated or stimulated with EGF, which is known to activate c-Abl (6Plattner R. Kadlec L. DeMali K.A. Kazlauskas A. Pendergast A.M. Genes Dev. 1999; 13: 2400-2411Crossref PubMed Scopus (373) Google Scholar). As previously demonstrated, Dok-R becomes tyrosine-phosphorylated upon EGF stimulation and can coimmunoprecipitate tyrosine-phosphorylated EGFR (Fig. 1A, below) (32Jones N. Dumont D.J. Curr. Biol. 1999; 9: 1057-1060Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Dok-R immunoprecipitates contained c-Abl in both untreated and EGF-stimulated COS-1 cells and similarly, c-Abl immunoprecipitates contained Dok-R (Fig. 1A, top). These results suggest that Dok-R and c-Abl may associate in a constitutive manner. To determine if Abl kinase activity enhances its constitutive association with Dok-R, we chose a system that was independent of EGF stimulation to activate c-Abl. We treated murine parental 32D cells and 32D-p210Bcr-Abl stable cells, where Bcr-Abl is activated (39Avanzi G.C. Lista P. Giovinazzo B. Miniero R. Saglio G. Benetton G. Coda R. Cattoretti G. Pegoraro L. Br. J. Haematol. 1988; 69: 359-366Crossref PubMed Scopus (256) Google Scholar, 40Daley G.Q. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9312-9316Crossref PubMed Scopus (510) Google Scholar), with either the small molecule Abl inhibitor STI571 or Me2SO as a control. Dok-R, endogenous to these cells, was found to be tyrosine-phosphorylated in the 32D-p210Bcr-Abl stable cell line and could bind p210Bcr-Abl in the presence or absence of STI571 treatment (Fig. 1B, top). Interestingly, the addition of STI571 to 32D-p210Bcr-Abl cells resulted in diminished tyrosine phosphorylation of Dok-R and dramatically reduced coimmunoprecipitation of phosphorylated Bcr-Abl (Fig. 1B, top). Similar treatments of STI571 to 32D and 32D-p210 cells, were immunoprecipitated using anti-Bcr antibodies and immunoblotted for phosphotyrosine. Although more p210Bcr-Abl was immunoprecipitated in the 32D-p210 STI571-treated lane, there was a 2-fold decrease in p210Bcr-Abl tyrosine phosphorylation and co-precipitation of tyrosine-phosphorylated Dok-R (Fig. 1B, bottom). These results demonstrate that the Dok-R and Abl association does not require Abl kinase activity, but is enhanced upon activation of Abl. The SH3 Domain of c-Abl Mediates Binding to a PmmP266 Motif on Dok-R—The constitutive interaction between Dok-R and c-Abl led us to investigate the possibility that binding between these proteins may be mediated through the SH3 domain of c-Abl and one of the eight PXXP motifs found within the carboxyl tail of Dok-R. In order to bypass the requirement of stimulating cells with EGF, we utilized the property that overexpression of c-Abl in COS-1 cells leads to its kinase activation (41Wang J.Y. Oncogene Res. 1988; 3: 293-298PubMed Google Scholar). To further define the site of interaction between Dok-R and c-Abl, we introduced mutant forms of Dok-R with c-Abl in 293T or COS-1 cells (Fig. 2A). Under these conditions, we observed an increase in tyrosine phosphorylation of c-Abl upon coexpression with wild-type Dok-R (Fig. 2B). This enhanced phosphorylation was retained with a Dok-R mutant carrying a termination codon (at amino acid 321) midway through the proline-rich tail (Dok-RΔC-PRR), but was completely abolished with a Dok-R mutant completely lacking the proline- and tyrosine-rich tail (PRR) by placing a stop codon at amino acid 257 (Dok-RΔPRR) (Fig. 2B). Moreover, the immunoprecipitation of c-Abl lead to the coimmunoprecipitation of Dok-R and Dok-RΔC-PRR, but not with Dok-RΔPRR, indicating that the amino-terminal half of the PRR is responsible for Dok-R binding c-Abl. Additionally, inverse coimmunoprecipitation experiments resulted in similar findings in that Dok-R or Dok-RΔC-PRR could coimmunoprecipitate c-Abl whereas Dok-RΔPRR could not (data not shown). These results indicate that the c-Abl/Dok-R interaction requires the proline- and tyrosine-rich region (PRR) of Dok-R between amino acids 257 and 321. In order to further delineate the c-Abl binding site within the amino-terminal PRR of Dok-R, several different proline Dok-R mutants were engineered and co-transfected along with c-Abl (Fig. 2A). The different Dok-R mutants tested were able to coimmunoprecipitate c-Abl, however, the Dok-RAMMA mutant, where prolines 263 and 266 are engineered to alanine, displayed a decrease in c-Abl and Dok-R tyrosine phosphorylation (Fig. 2C). These results indicate that the constitutive interaction between c-Abl and Dok-R is at least partially mediated via a PmmP266 motif on Dok-R. To confirm that the c-Abl SH3 domain can associate with Dok-R through its PMMP motif, we performed several in vitro binding experiments using bacterially purified recombinant GST fusion proteins. Cell extracts prepared from c-Abl transfected cells were incubated with either GST-beads or with GST-Dok-RΔPH-beads. Bound proteins were subsequently resolved on SDS-PAGE and immunoblotted for c-Abl. c-Abl did not co-precipitate with GST alone, however, a distinct band representing c-Abl was seen in mixing experiments with GST-Dok-RΔPH (Fig. 2D). Moreover, the specificity of the SH3 domain of c-Abl for Dok-R was illustrated by in vitro binding experiments where cell extracts from HA-Dok-R-transfected cells were mixed with purified GST-SH3 domains of c-Abl, Vav, Spectrin, and Lck. Specifically, the c-Abl GST-SH3 protein, but not the others, could precipitate Dok-R (Fig. 2E). Participation of the Dok-R PMMP motif in associating c-Abl was further illustrated by in vitro binding experiments where all the Dok-R proteins tested could be precipitated by the SH3 domain of c-Abl, except for Dok-RΔPRR and Dok-RAMMA

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