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J Biol Chem, Vol. 273, Issue 9, 4827-4830, February 27, 1998

COMMUNICATION
Molecular Cloning and Characterization of p56^dok-2 Defines a New Family of RasGAP-binding Proteins^*

Antonio Di Cristofano, Nick Carpino^§¶, Nicolas Dunant^**, Gayle Friedland, Ryuji Kobayashi^§, Annabel Strife, David Wisniewski, Bayard Clarkson, Pier Paolo Pandolfi, and Marilyn D. Resh^§§

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

	ABSTRACT

Top Abstract Introduction Materials & Methods Results & Discussion References

Chronic myelogenous leukemia (CML) is a disease characterized by the presence of p210^bcr-abl, a chimeric protein with tyrosine kinase activity. Substrates for p210^bcr-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, p56^dok-2 (Dok-2), from p210^bcr-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 p120^RasGAP. Dok-2 was shown to be 35% identical to p62^dok-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 p210^bcr-abl in CML.

	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 p210^bcr-abl responsible for CML. Introduction of p210^bcr-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 p210^bcr-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 p210^bcr-abl. To identify potential substrates relevant for transformation by p210^bcr-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, p62^dok, was purified, and its gene was cloned (7). p62^dok 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.

	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 p210^bcr-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.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

To identify potential substrates for p210^bcr-abl, lysates were prepared from cell lines expressing p210^bcr-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 p210^bcr-abl but not in the parent cell line. Several of these Tyr(P) protein bands have been identified, including p62^dok, Cbl, Crkyl, p190^RhoGAP, 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.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Identification of p56 as a Tyr(P) GAP binding protein. A, tyrosine phosphorylated proteins in Mo7 and Mo7/p210 cells. Lysates of Mo7 (M) or Mo7/p210 (P) cells (50 µg protein/lane) were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Tyr(P) antibodies. An arrow indicates tyrosine phosphorylated p56. p62dok migrates as a doublet above p56. B, identification of a GAP-associated, tyrosine phosphorylated p56 protein. The indicated antibodies were used in immunoprecipitations from lysates of either Mo7 or Mo7/p210 cells. Following SDS-PAGE and transfer to nitrocellulose, the blot was probed with anti-Tyr(P) antibodies. The position of p56 is indicated by an arrow. C, co-precipitation of labeled p56 with GAP. The indicated antibodies were used in immunoprecipitations from lysates of [32P]orthophosphate-labeled Mo7/p210 cells. An arrow indicates the position of the GAP-associated p56 protein. NS, nonimmune serum. D, isolated p56 analyzed by two-dimensional (2D) gel electrophoresis. Anti-GAP antibodies were used to precipitate p56 from lysates of [32P]orthophosphate-labeled Mo7/p210 cells (lane 2 in C). Immune complexes were resolved by SDS-PAGE, the labeled protein migrating at 56 kDa was eluted from the gel and analyzed by two-dimensional electrophoresis. A similar pattern was obtained when the 56-kDa band from lane 3 of C was analyzed.

Our strategy was motivated by the recent identification of another Tyr(P) protein, p62^dok, in p210^bcr-abl expressing cells. p62^dok is a substrate for Bcr-Abl and binds to p120^RasGAP (7, 8). Lysates from Mo7p210 cells were immunoprecipitated with anti-GAP antibody followed by immunoblotting with anti-Tyr(P) antibody. In addition to p62^dok, 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 p62^dok, 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.

View larger version (70K): [in this window] [in a new window]   Fig. 2.   p56dok (Dok-2) and the Dok family of proteins. A, deduced amino acid sequences of human and mouse p56dok (Dok-2). Boxes highlight the PH domain, the DKH region, and the putative phosphotyrosine-containing motifs responsible for binding GAP SH2 domains. Underlined sequences correspond to the four peptide sequences determined from purified p56dok and used to design degenerate primers for RT-PCR. B, schematic representation of the structure of human p56. The PH domain and the DKH are indicated. PXXP motifs are indicated by P, and tyrosine residues are indicated by Y. The phosphotyrosine residues predicted to bind to RasGAP SH2 domains are highlighted by asterisks. C, aligned DKH regions. DKH regions of p62dok (Dok-1), p56dok (Dok-2), and expressed sequence tags AA275205, AA082651, T09328, and N26446 (Dok-3, -4, -5, and -6) are shown aligned. Dok-3 and Dok-4 contain an additional 30 amino acids upstream of the DKH region with homology to Dok-1 and Dok-2.

Further analysis of the expressed sequence tag data base revealed the presence of four potential additional members of a Dok family as depicted in Fig. 2C. In particular, extensive sequence homology was noted within 50 amino acids of the central core region of the six Dok proteins, prompting us to dub this region a "Dok homology" (DKH) sequence motif. The DKH motif also exhibits limited homology to a short region in IRS-2, as detected by a blast search, and IRS-1, as noted by Yamanashi and Baltimore (8). We propose the following nomenclature for the Dok family: Dok-1 to denote p62^dok (7, 8), Dok-2 to denote p56^dok, and Dok-3, -4, -5, and -6 to denote additional Dok family members.

Dok family mRNA expression was determined by Northern blot analysis of poly(A)⁺ RNA from human and mouse tissues. As depicted in Fig. 3, the expression patterns of p56 and p62 were coincident; both transcripts were highly expressed in cells and tissues of hematopoietic origin, as well as lung.

View larger version (75K): [in this window] [in a new window]   Fig. 3.   Gene expression patterns of the Dok family. Northern blot of dok-2 and dok-1 in various tissues and hematopoietic tissues. 1.9-kilobase (Kb) transcripts for dok-2 and dok-1 were detected; the identity of the upper band in some of the samples is not known.

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 p62^dok and p210^bcr-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.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Dok-2 is tyrosine phosphorylated by Bcr-Abl and binds to GAP. A, tyrosine phosphorylation of p56dok-2 and p62dok-1 by Bcr-Abl in vivo. pCMV5 expression vectors encoding either HA-tagged p56 (HA-p56) or p62 were transfected alone or in combination with a vector encoding Bcr-Abl into COS cells. Immunoprecipitations (IP) were conducted with either mAb12CA5 (anti-HA) or anti-p62 polyclonal antibody and analyzed by Western blotting using anti-HA or anti-p62 antibodies. The same blots were reprobed with mAb PY99 (anti-pTyr). B, binding of p56 and p62 to the SH2-SH3-SH2 domains of RasGAP. Lysates of the same cells as used for A were incubated with either GST or GST-RasGAP SH2-SH3-SH2 fusion protein (GST-GAP) coupled to glutathione-agarose. Bound proteins were analyzed by Western blotting. The same blot was reprobed with anti-HA, anti-p62, and anti-Tyr(P) antibodies. Top Panel, anti-HA; bottom panel, anti-p62.

Next we examined the interaction between Dok proteins and GAP. Lysates from transfected COS-1 cells were incubated with a GST fusion protein containing the SH2-SH3-SH2 domains of GAP or with GST alone. Immunoblotting with anti-HA or anti-p62 antibody was then performed. As depicted in Fig. 4B, Dok-2 bound to GST-GAP, but not GST, only when the cells were co-expressing Bcr-Abl. A similar result was obtained for Dok-1. Additional experiments confirmed that the interaction between Dok proteins and GAP was Tyr(P)-dependent (not shown). Thus, expression of Bcr-Abl results in tyrosine phosphorylation of Dok-1 and Dok-2 and association with GAP.

Conclusions-- The identification of p56^dok-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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* 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 GenBank^TM/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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