From the Department of Human Genetics and Molecular
Biology Program, the
Cell Biology Program, and the
Molecular Pharmacology and Therapeutics
Program, Memorial Sloan-Kettering Cancer Center, New York, New York
10021, the § Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 11724, and the ¶ Program in Molecular and
Cellular Biology, State University of New York,
Stony Brook, New York 11794
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ABSTRACT |
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Chronic myelogenous leukemia (CML) is a disease characterized by the presence of p210bcr-abl, a chimeric protein with tyrosine kinase activity. Substrates for p210bcr-abl are likely to be involved in the pathogenesis of CML. Here we describe the purification, cDNA cloning, and characterization of a 56-kDa tyrosine phosphorylated protein, p56dok-2 (Dok-2), from p210bcr-abl expressing cells. The human dok-2 cDNA encodes a 412-amino acid protein with a predicted N-terminal pleckstrin homology domain as well as several other features of a signaling molecule, including 13 potential tyrosine phosphorylation sites, six PXXP motifs, and the ability to bind to p120RasGAP. Dok-2 was shown to be 35% identical to p62dok-1, a recently identified RasGAP binding protein from CML cells, and analysis of the expressed sequence tag data base revealed the presence of at least four additional proteins containing a Dok homology sequence motif. Dok mRNAs were primarily expressed in tissues of hematopoietic origin. These findings strongly suggest that a family of Dok-related proteins exists that bind to RasGAP and may mediate the effects of p210bcr-abl in CML.
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INTRODUCTION |
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Human chronic myelogenous leukemia (CML)1 is a myeloproliferative disease (reviewed in Ref. 1) characterized by the presence of a chromosomal translocation known as the Philadelphia chromosome. Cells from CML patients contain a t(9;22) translocation in which the 5' exons of the bcr (breakpoint cluster) gene on chromosome 22 are fused to the c-abl proto-oncogene on chromosome 9 (2). The most common fusion generates the chimeric protein p210bcr-abl responsible for CML. Introduction of p210bcr-abl constructs into transgenic mice has been shown to cause CML-like myelo-proliferative disease (3). Thus, it is generally accepted that Bcr-Abl fusion proteins are causative agents for human leukemias.
The normal functions of Bcr and c-Abl are not known. c-Abl encodes a tyrosine-protein kinase whose activity is down-regulated or inhibited in normal cells. Deregulation of c-Abl tyrosine kinase activity can occur when negative regulatory sequences within c-Abl are removed and the truncated c-Abl is fused with heterologous proteins (4, 5). For example, in CML, Bcr sequences are fused to the second exon of Abl, resulting in activation of the chimeric p210bcr-abl tyrosine kinase.
There is ample evidence indicating that enhanced tyrosine kinase activity is required for transformation by Bcr-Abl in vitro and disease development in vivo (4, 5). Thus, a key goal is to identify the critical intracellular target proteins phosphorylated by p210bcr-abl. To identify potential substrates relevant for transformation by p210bcr-abl, Clarkson and co-workers examined tyrosine phosphorylation patterns in primary chronic phase CML blasts (6). Several tyrosine phosphorylated proteins were apparent in the early blast subpopulations derived from the marrows of CML patients but not normal donors. Recently, one of these Tyr(P) proteins, p62dok, was purified, and its gene was cloned (7). p62dok binds to RasGAP and exhibits additional features of a signaling protein, including an N-terminal PH domain and clusters of PXXP motifs (7, 8). These studies also detected a second Tyr(P) protein, p56, which exhibited increased Tyr(P) levels in primary CML cells and in CML cell lines (6). Here, we describe the purification, cDNA cloning, and characterization of p56 and show that it is a member of a Dok family of RasGAP binding proteins.
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MATERIALS AND METHODS |
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Cell Culture and DNA Transfections-- The Mo7 megakaryoblastic cell line (9) and a derivative of Mo7 that expresses p210bcr-abl, Mo7/p210 were maintained as described (6, 7). Transfection of the cDNAs encoding HAp56, p62, or Bcr-Abl subcloned into pCMV5 expression vectors was carried out as described (10).
Immunoprecipitations and Western Blot Analysis-- Cells were radiolabeled, and cell lysates were prepared, standardized for equal protein concentration (750 µg of protein in a volume of 500 µl of lysis buffer), and immunoprecipitated as described (10). The following antibodies were used for immunoprecipitations: anti-p120 RasGAP mAb B4F8 (Santa Cruz Biotechnology) and polyclonal serum (Santa Cruz Biotechnology), anti-phosphotyrosine mAb PY20 (ICN Bio-medicals), and anti-p62dok polyclonal antibody (14). For Western blotting, mAb PY99 (anti-Tyr(P), Santa Cruz Biotechnology), mAb 12CA5, and anti-p62dok were used. SDS-PAGE and Western transfer were done according to standard protocols. Immunoblotting primary antibody was anti-phosphotyrosine rabbit polyclonal antibody B5 (11). Secondary antibody was horseradish peroxidase-linked sheep anti-mouse Fab2 or sheep anti-rabbit Fab2 (Amersham Corp.).
Plasmid Constructs-- The sequence encoding HA-tagged p56 was generated by PCR using a 5' primer introducing a sequence coding for the HA epitope between the first and the second codon of the open reading frame of a human p56 cDNA. The PCR product was subcloned into pCMV5 as an EcoRI/XbaI fragment. The cDNAs encoding p62 or Bcr-Abl were also subcloned into pCMV5.
Two-dimensional Gel Electrophoresis-- Two-dimensional gel electrophoresis was carried out essentially as described (12, 13). For the experiment illustrated in Fig. 1B, the labeled band representing p56 was excised from the polyacrylamide gel, eluted overnight, and processed for two-dimensional gel electrophoresis.
Purification of p56-- 10 liters of Mo7/p210 cells were lysed as described (7), and nuclei were removed by centrifugation. The lysate was adjusted to 3 M urea, 50 mM Tris-HCl, pH 8.3, 25 mM NaCl, 5 mM EDTA, 1 mM EGTA, 0.5% Triton X-100 (Buffer A) and loaded onto a Q Sepharose HP (Pharmacia) column (19 × 5 cm) previously equilibrated in Buffer A. After extensive washing, the bound proteins were eluted from the column with a linear gradient of 25-600 mM NaCl in Buffer A. Fractions were collected and assayed for the presence of p56 by anti-Tyr(P) Western blotting. Fractions containing p56 were pooled, adjusted to 50 mM acetic acid, pH 4.6, 100 mM NaCl, 5 mM EDTA, 3 M urea (Buffer B), and loaded onto a SP ToyoPearl (TosoHaas) column equilibrated in Buffer B. Bound proteins were eluted with a linear gradient of 100-800 mM NaCl in Buffer B. Fractions containing p56 were pooled, total protein was acetone precipitated, and precipitated protein was resuspended and dialyzed as described. Immunoaffinity purification on an anti-phosphotyrosine antibody column (4G10-Sepharose, Upstate Biotechnology Inc.) was performed as described (7). Eluted material was resuspended in gel sample buffer and resolved by two-dimensional gel electrophoresis. The peptide sequence of p56 was obtained as described (14).
Northern Blotting-- Human and mouse tissue poly(A)+ RNA blots (CLONTECH) were hybridized with radiolabeled to human and mouse p56 cDNA probes according to the manufacturer's instructions.
RT-PCR and cDNA Library Screening-- mRNA was isolated from Mo7p210 cells and reverse transcribed into cDNA using the mRNA Capture kit and Titan kit from Boehringer Mannheim. Two rounds of nested PCR were performed using degenerate primers based on the peptide sequences VIRLSDXLRVAEAGGEASSPRDTSAFFL and QSRPCMEENELYSSAVTVGPHK. The resulting 200-bp PCR product was radiolabeled and used to screen a Mo7p210 cDNA library (a kind gift from Dr. Gerald Krystal, The Terry Fox Laboratory, University of British Columbia, Vancouver, BC, Canada). To obtain a mouse Dok-2 clone, a mouse macrophage cDNA library was screened with a 210-bp fragment obtained by low stringency RT-PCR using oligonucleotides corresponding to the coding regions of mouse p62dok. Positive plaques were rescreened until purified. The full-length cDNAs were sequenced on both strands by the dideoxy chain termination method.
RasGAP Binding Assay-- 5 µg of GST or GST-RasGAP SH2-SH3-SH2 fusion protein (Santa Cruz Biotechnology) immobilized on glutathione-agarose beads were incubated with cell lysate for 2 h at 4 °C. Beads were washed three times with buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 2.5 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 0.5 µg/ml leupeptin, and 0.1 µg/ml aprotinin and analyzed by Western blotting.
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RESULTS AND DISCUSSION |
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To identify potential substrates for p210bcr-abl, lysates were prepared from cell lines expressing p210bcr-abl and immunoblotted with anti-Tyr(P) antibody (Fig. 1A). As described previously (6, 7), a number of prominent bands are evident in cells expressing p210bcr-abl but not in the parent cell line. Several of these Tyr(P) protein bands have been identified, including p62dok, Cbl, Crkyl, p190RhoGAP, SHIP, Shc, and the p85 subunit of phosphatidylinositol 3-kinase. The band migrating at 56 kDa had not been identified, prompting us to initiate further characterization and purification.
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Our strategy was motivated by the recent identification of another Tyr(P) protein, p62dok, in p210bcr-abl expressing cells. p62dok is a substrate for Bcr-Abl and binds to p120RasGAP (7, 8). Lysates from Mo7p210 cells were immunoprecipitated with anti-GAP antibody followed by immunoblotting with anti-Tyr(P) antibody. In addition to p62dok, a band at 56 kDa that contained Tyr(P) was also evident (Fig. 1B). To further analyze p56, cells were radiolabeled with [32P]phosphate, and lysates were immunoprecipitated with anti-GAP antibody (Fig. 1C). The 56-kDa band from these immunoprecipitates was subjected to two-dimensional gel electrophoresis. A series of spots migrating at 56 kDa was evident, and this pattern was used for identification of p56 during purification (Fig. 1D).
A combination of ion exchange and anti-Tyr(P) affinity chromatography was used to purify p56 from Mo7p210 cells, and the final products were separated by two-dimensional gel electrophoresis. Individual spots were excised from the gel and digested with protease, and peptide fragments were sequenced. Degenerate oligonucleotide primers were designed based on two of the peptide sequences. Two rounds of nested RT-PCR were performed, resulting in a 200-bp PCR product that was used as a probe to screen a cDNA library; cDNA clones were isolated as described under "Materials and Methods."
Identification of a Dok Family-- The predicted amino acid sequence from the longest human cDNA clone is illustrated in Fig. 2A. The cDNA codes for a 412-amino acid protein with a calculated molecular mass of 45,548 Da and contains all of the peptides identified by microsequencing of p56. In vitro translation of p56 cDNA in a rabbit reticulocyte lysate yielded a doublet of approximately 53 and 56 kDa, suggesting that the p56 clone encodes a full-length cDNA. Analysis of the p56 sequence yielded several striking results (Fig. 2B). First, a profile search against the Prosite data base detected a potential pleckstrin homology (PH) domain at the N terminus. Second, p56 contains 13 potential tyrosine phosphorylation sites, six PXXP motifs, and two YXXPXD motifs (predicted RasGAP SH2 domain binding sites (15)). Although the sequence of human p56 contains a glycine at position 2, p56 is unlikely to be N-myristoylated because it contains a negatively charged residue at position 3 and lacks a conserved Ser/Thr residue at position 6 (16). Third, a blast search revealed significant homology to p62dok, a protein that also contains a predicted PH domain at its N terminus. Overall, there is 34.8% identity between the two proteins, with the N-terminal PH domains and the central cores exhibiting the greatest similarity. The homology between p56 and p62 accounts for our ability to independently isolate the mouse homolog of p56dok, using primers from the mouse p62dok sequence. The sequence of mouse p56 is also illustrated in Fig. 2A; the mouse and human p56 proteins are 72.1% identical.
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Tyrosine Phosphorylation of Dok-2 and Binding to GAP Are Increased in Bcr-Abl Expressing Cells-- The interactions among Dok-2, Bcr-Abl, and GAP were studied using transient expression in COS-1 cells. dok-2 cDNA was tagged with an influenza HA epitope and transfected with or without bcr-abl cDNA. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-Tyr(P) antibody. A doublet that migrated at 56/58 kDa was apparent on the blot from cells transfected with HA-tagged Dok-2, whereas cells co-transfected with dok-2 and bcr-abl exhibited at least four bands in the 56-60-kDa region (Fig. 4A). Immunoblotting with anti-HA antibody confirmed that equivalent levels of Dok-2 were expressed in each sample. Parallel experiments were performed with Dok-1. Co-expression of p62dok and p210bcr-abl resulted in an additional Tyr(P) form of p62, as has been previously reported for Mo7p210 cells (7). These data imply that expression of Bcr-Abl induces additional tyrosine phosphorylation on Dok-2, consistent with the hypothesis that Dok-2 is a direct or indirect substrate for Bcr-Abl.
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Conclusions-- The identification of p56dok-2 in this manuscript has served to define a new family of Dok proteins. Data base searches have revealed the presence of at least six Dok family members, each containing a 50-amino acid Dok homology motif. Our studies of Dok-1 and Dok-2 strongly suggest that one function of Dok proteins is to bind to GAP. More structural and functional characterization of Doks 3-6 will be required to extend this conclusion to other Dok family members. It is tempting to speculate that Dok association regulates GAP activity toward Ras and serves as a mediator of Bcr-Abl signaling. There are numerous reports documenting that Bcr-Abl activates Ras (17, 18), and co-expression of the catalytic domain of GAP with Bcr-Abl leads to cellular transformation (19). However, it is important to consider that only a small percentage of the total Dok protein is associated with GAP, and it is therefore likely that Dok proteins interact with additional signaling molecules. Future studies will be directed toward elucidating the role of Dok proteins in signaling by Bcr-Abl and other oncogenic tyrosine-protein kinases.
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ACKNOWLEDGEMENTS |
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We thank Raisa Louft-Nisenbaum for technical assistance, Neena Sareen and Nick Bizios for help with the two-dimensional gels, Nora Poppito and Camille Walker for assistance with high pressure liquid chromatography peptide mapping and sequencing, Tony Rossamondo for anti-Tyr(P) antibody R5, and Melissa Ray for manuscript preparation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA 64593 and a grant from the United Leukemia Fund.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF034970 and AF035117.
** Supported by fellowships from the Roche Research Foundation and the Swiss National Science Foundation.
§§ Established Scientist of the American Heart Association. To whom correspondence should be addressed: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 143, New York, NY 10021. Tel.: 212-639-2514; Fax: 212-717-3317; E-mail: m-resh{at}ski.mskcc.org.
1 The abbreviations used are: CML, chronic myelogenous leukemia; GAP, GTPase-activating protein; GST, glutathione S-transferase; HA, hemagglutinin; PBS, phosphate-buffered saline; PAO, phenylarsine oxide; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; PCR, polymerase chain reaction; RT, reverse transcription; bp, base pair; PH, pleckstrin homology; DKH, Dok homology.
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REFERENCES |
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