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Src Family Kinases Phosphorylate the Bcr-Abl SH3-SH2 Region and Modulate Bcr-Abl Transforming Activity *

Src Family Kinases Phosphorylate the Bcr-Abl SH3-SH2 Region and Modulate Bcr-Abl Transforming... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 41, pp. 30907–30916, October 13, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Src Family Kinases Phosphorylate the Bcr-Abl SH3-SH2 Region and Modulate Bcr-Abl Transforming Activity Received for publication, June 20, 2006, and in revised form, August 7, 2006 Published, JBC Papers in Press, August 15, 2006, DOI 10.1074/jbc.M605902200 ‡1 ‡1 § ‡ ‡ ¶ Malcolm A. Meyn III , Matthew B. Wilson , Fadi A. Abdi , Nathalie Fahey , Anthony P. Schiavone , Jiong Wu , ‡2 James M. Hochrein , John R. Engen , and Thomas E. Smithgall From the Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, § ¶ Pittsburgh, Pennsylvania 15261, Applied Biosystems, Framingham, Massachusetts 01701, Cell Signaling Technology, Beverly, Massachusetts 01915, and the Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131 Bcr-Abl is the oncogenic protein-tyrosine kinase responsible (2, 3). The Ph translocation results in the expression of Bcr-Abl, for chronic myelogenous leukemia. Recently, we observed that a 210-kDa oncogenic fusion protein with constitutive protein- inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, tyrosine kinase activity. Bcr-Abl transforms hematopoietic cell and Fyn) induces growth arrest and apoptosis in Bcr-Abl-trans- lines (4) and bone marrow cells (5) in culture and produces a formed cells, suggesting that cell transformation by Bcr-Abl CML-like myeloproliferative disease in mice (6, 7), directly involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, implicating this chimeric protein-tyrosine kinase in disease H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075– onset. 8088). Here, we report the unexpected observation that Hck, Bcr-Abl phosphorylates a broad range of cellular proteins, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of affecting many signaling pathways linked to the growth, differ- Bcr-Abl. Seven phosphorylation sites were identified by matrix- entiation, and survival of hematopoietic progenitors (3). For assisted laser desorption ionization time-of-flight mass spec- example, the Grb2-Sos guanine nucleotide exchange factor 89 134 trometry: Tyr and Tyr in the Abl-derived SH3 domain; complex binds to phospho-Tyr in the Bcr-derived region, 147 158 191 204 Tyr in the SH3-SH2 connector; and Tyr , Tyr , Tyr , contributing to the activation of Ras (8, 9). Bcr-Abl also acti- 234 89 and Tyr in the SH2 domain. SH3 domain Tyr , the most vates Ras via Shc, an adapter protein that couples the receptors prominent phosphorylation site in vitro, was strongly phospho- for many growth factors and cytokines to the Grb2-Sos com- rylated in chronic myelogenous leukemia cells in a Src family plex (10). In addition, Bcr-Abl associates with and phospho- kinase-dependent manner. Substitution of the SH3-SH2 tyro- rylates the CrkL adapter (11), stimulates phosphatidylinositol sine phosphorylation sites with phenylalanine substantially 3-kinase/Akt survival signaling by direct interaction with the reduced Bcr-Abl-mediated transformation of TF-1 myeloid p85 subunit of phosphatidylinositol 3-kinase (12), and activates cells to cytokine independence. The positions of these tyrosines several Stat transcription factors (13, 14). All of these signaling in the crystal structure of the c-Abl core and the transformation pathways involve components with SH2 and SH3 domains and defect of the corresponding Bcr-Abl mutants together suggest are dependent upon interactions with phosphotyrosine or that phosphorylation of the SH3-SH2 region by Src family polyproline docking sites, respectively. kinases impacts Bcr-Abl protein conformation and signaling. Despite its intrinsic protein-tyrosine kinase activity, several studies have established that Bcr-Abl interacts with other tyro- sine kinases, including members of the Fps/Fes (15), Jak (16), The cytogenetic hallmark of chronic myelogenous leukemia and Src (17–20) kinase families. Hallek and co-workers (20) (CML) is the Philadelphia (Ph) chromosome (1), which results originally showed that Bcr-Abl forms complexes with the Src from a reciprocal translocation between the c-abl proto-onco- family members Hck and Lyn in several Ph-positive cell lines gene on chromosome 9 and the bcr locus on chromosome 22 and is phosphorylated by Hck at Bcr Tyr (19), which links Bcr-Abl to Grb2-Sos as described above (8). Interestingly, Hck activation does not require Bcr-Abl kinase activity (19), sug- * This work was supported by National Institutes of Health Grant CA101828 (to T. E. S.) and Grants GM70590 and RR016480 (to J. R. E.) and by National gesting that Bcr-Abl may stimulate Src family kinases through Science Foundation Research Experiences for Undergraduates Site Award displacement of inhibitory intramolecular interactions (21). 0243735 (to N. F.). The costs of publication of this article were defrayed in This idea is supported by subsequent work showing that the part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to SH3 and SH2 domains of Hck directly associate with Bcr-Abl indicate this fact. (17). Furthermore, kinase-defective Hck blocks transformation Both authors contributed equally to this work. of myeloid leukemia cells to cytokine independence by Bcr-Abl To whom correspondence should be addressed: Dept. of Molecular Genet- ics and Biochemistry, University of Pittsburgh School of Medicine, E1240 (17), whereas pharmacological inhibitors selective for Src fam- Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15261. Tel.: 412- ily kinases induce apoptosis in the CML cell lines Meg-01 and 648-9495; Fax: 412-624-1401; E-mail: [email protected]. K-562 (18). The effects of these compounds correlate with The abbreviations used are: CML, chronic myelogenous leukemia; Ph, Phil- adelphia; Stat, signal transducer and activator of transcription; SH, Src down-regulation of both Stat5 and Erk (extracellular signal- homology; GST, glutathione S-transferase; MS, mass spectrometry; MALDI- regulated kinase) activation, suggesting that Src family kinases TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tan- may couple Bcr-Abl to certain downstream signaling pathways dem mass spectrometry; ACTH, adrenocorticotropic hormone; GM-CSF, granulocyte-macrophage colony-stimulating factor. (18). OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30907 This is an Open Access article under the CC BY license. Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases Although Bcr-Abl interacts directly with Hck, Lyn, and other phorylation reactions (50 l) were conducted in 50 mM HEPES Src family kinases, the molecular mechanisms and functional (pH 7.4) containing 10 mM MgCl and 500 M ATP for 30 min consequences of binding in terms of Bcr-Abl signaling and at 30 °C and quenched by freezing in a dry ice/ethanol bath. oncogenesis are not understood. In this study, we demonstrate Aliquots of each reaction were analyzed by immunoblotting that Bcr-Abl binds multiple members of the Src family through with anti-GST or anti-phosphotyrosine (PY99) monoclonal the Abl-derived SH3-SH2 regulatory region. Unexpectedly, antibodies (Santa Cruz Biotechnology, Inc.). Hck, Lyn, and Fyn phosphorylated the Bcr-Abl SH3-SH2 region Hexahistidine-Bcr-Abl Fusion Protein Purification—The 57 224 at multiple tyrosine residues. The most prominent of these is Bcr-Abl SH3-SH2 coding region (Gly –Thr ; human c-Abl SH3 domain Tyr , which was also phosphorylated in CML numbering) was subcloned into the pET-14b expression vector cells in a Src family kinase-dependent manner. Substitution of (Novagen) to allow addition of an N-terminal hexahistidine tag, Tyr and other SH3-SH2 phosphorylation sites with pheny- yielding His -Abl32. BL21(DE3)pLysS bacterial cells were lalanine reduced Bcr-Abl transforming activity without altering transformed with the vector, and His -Abl32 protein expres- phosphorylation of the Abl kinase domain activation loop. sion was induced with isopropyl -D-thiogalactopyranoside. These data show that Src family kinase-mediated phosphoryl- His -Abl32 was purified from clarified cell extracts using a ation of tyrosine residues located in the SH3-SH2 region is crit- HiTrap metal-chelating column charged with Ni (GE ical for Bcr-Abl function, offering a possible mechanistic expla- Healthcare), and phosphorylated with recombinant Hck, Lyn, nation for the remarkable efficacy of Src-selective and dual Src/ and Fyn as described above for the GST-Abl32 protein. Abl inhibitors against CML (18, 22, 23). Full-length kinase-dead p210 Bcr-Abl was expressed in Sf9 insect cells and purified as follows. A hexahistidine tag was MATERIALS AND METHODS added to the C-terminal coding region of a kinase-defective Binding Assays—Interaction of glutathione S-transferase mutant of Bcr-Abl (15) using a PCR-based approach and sub- (GST)-fused human c-Abl proteins with Src family kinases was cloned into the baculovirus transfer vector pVL1393 (BD Bio- conducted in Sf9 insect cells as described previously (17). For sciences). A recombinant baculovirus was created using the binding assays, Sf9 cells (2.5  10 ) were co-infected with each transfer vector and BaculoGold DNA according to the suppli- of the GST-Abl baculoviruses (or GST as a negative control) er’s protocol. Spinner cultures of Sf9 cells (1 liter, 2 10 cells/ and wild-type Hck, Lyn, or Fyn baculovirus. Forty-eight hours ml) were infected with 100 ml of high titer virus and harvested after infection, the cells were lysed in Hck lysis buffer (17), and 72 h later. Cells were lysed in 20 mM Tris-HCl (pH 8.3) contain- GST fusion proteins were precipitated with glutathione-agar- ing 10% glycerol, 5 mM -mercaptoethanol, 20 mM imidazole, ose beads. The precipitates were washed with radioimmune and 500 mM NaCl. The protein was purified in one step using a precipitation assay buffer as described previously (17), and HiTrap metal-chelating column. The Bcr-Abl-positive frac- bound proteins were eluted in SDS-PAGE sample buffer. Pro- tions were identified by SDS-PAGE and pooled, and the con- teins were resolved on SDS-polyacrylamide gels and trans- centration of Bcr-Abl was quantitated by densitometry of a ferred to polyvinylidene difluoride membranes, and associated Coomassie Blue-stained gel. Phosphorylation reactions with Hck, Lyn, and Fyn were visualized by immunoblotting with Src family kinases were conducted as described above for the kinase isoform-specific polyclonal antibodies (Santa Cruz Bio- GST-Abl fusion proteins and contained 8 pmol of recombi- technology, Inc.). The amount of precipitated GST-Abl fusion nant Bcr-Abl p210-KR. protein present in each reaction was determined by immuno- Mass Spectrometry (MS)—Recombinant His -Abl32 and blotting with anti-GST antibodies (Santa Cruz Biotechnology, full-length kinase-dead Bcr-Abl (p210-KR) were phosphoryla- Inc.). Equivalent expression of each Src family kinase was ted in vitro with purified Src kinases, and aliquots of the phos- verified by immunoblotting the clarified cell lysates. Immu- phorylation reactions were digested overnight with trypsin at a noreactive bands were visualized with alkaline phosphatase- 1:25 trypsin/protein ratio. The His -Abl32 peptides were conjugated goat anti-rabbit secondary antibodies with mixed with matrix (-cyano-4-hydroxycinnamic acid), spotted 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazo- onto a MALDI target, and analyzed directly by MALDI- lium as colorimetric substrate (SouthernBiotech). TOF-MS (Applied Biosystems 4700 proteomics analyzer). GST-Bcr-Abl Fusion Protein Purification and in Vitro Kinase The area under the isotope distribution for the unphospho- 57 126 Assay—The Bcr-Abl SH3 domain (Gly –Ser ), SH2 domain rylated and phosphorylated forms of each tryptic peptide 121 224 (Ser –Thr ), and SH3-SH2 coding regions were subcloned was determined and used to estimate the relative extent of into the bacterial expression vector pGEX-2T, and the corre- phosphorylation. sponding GST fusion proteins were expressed in Escherichia Tryptic fragments derived from full-length Bcr-Abl p210-KR coli and purified with glutathione-agarose beads (17). The were separated by reverse-phase capillary high pressure liquid recombinant proteins were dialyzed overnight against 20 mM chromatography (Dionex Ultimate Nano-LC system). Column HEPES (pH 7.4) and 1 mM -mercaptoethanol, and protein fractions were mixed on-line with MALDI matrix and automat- concentrations were determined by densitometry of Coomas- ically spotted onto a MALDI plate at 20-s intervals with a sie Blue-stained SDS-polyacrylamide gels relative to a bovine Dionex Probot robot (144 spots total). Peaks for phosphopep- serum albumin standard. Each purified GST fusion protein or tides and their unphosphorylated counterparts were identified GST alone (380 pmol) was incubated with recombinant puri- in the MALDI-TOF spectra based on their m/z ratios, and the fied Hck-YEEI (24), Lyn (Upstate), or Fyn (Upstate). The molar sequence of each peptide was verified by tandem MS (MS/MS) ratio of substrate to kinase was at least 20:1 in each case. Phos- analysis when possible. The accuracy of the m/z measurements 30908 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 •OCTOBER 13, 2006 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases was 0.1 Da. An internal calibrant consisting of a mixture of mutagenesis kit (Stratagene). To create the 6YF mutant, the des-Arg-bradykinin, angiotensin I, Glu-fibrinopeptide B, and Bcr-Abl 7YF mutant was used as the template, and oligonucleo- ACTH-(18–39) was added to each MALDI spot prior to tides encoding Tyr were used to revert this site from Phe to analysis. Tyr. These mutants and wild-type Bcr-Abl were also subcloned Coexpression of Src Family Kinases and Bcr-Abl in Sf9 Cells— into the pMSCV-neo retroviral vector. Sf9 insect cells were co-infected with full-length kinase-dead Transformation of TF-1 Cells with Bcr-Abl Retroviruses— Bcr-Abl and Hck, Lyn, or Fyn baculovirus as described previ- The human granulocyte-macrophage colony-stimulating factor ously (25). Cells were lysed 48 h later in 1.0 ml of ice-cold radio- (GM-CSF)-dependent myeloid leukemia cell line TF-1 (27) was immune precipitation assay buffer, and Bcr-Abl was immuno- obtained from American Type Culture Collection and grown in precipitated with 4 g of anti-c-Abl monoclonal antibody 8E9 RPMI 1640 medium supplemented with 10% fetal bovine (Pharmingen) and 25 l of protein G-Sepharose (50:50 (w/v) serum, 50 g/ml gentamycin, and 1 ng/ml recombinant human slurry; GE Healthcare) by rotation overnight at 4 °C. Precipi- GM-CSF. To make retroviral stocks, 293T cells were cotrans- tated proteins were collected by centrifugation, washed with fected with each retroviral construct and an amphotropic pack- radioimmune precipitation assay buffer, and eluted in SDS- aging vector as described (28, 29). TF-1 cells (10 ) were incu- PAGE sample buffer. Proteins were resolved by SDS-PAGE; bated with 5 ml of viral supernatant in the presence of 4 g/ml transferred to polyvinylidene difluoride membranes; and Polybrene and centrifuged at 2400 rpm for 3 h at room temper- immunoblotted with antibodies specific for Bcr phos- ature to enhance infection (17, 28). Cell populations were pho-Tyr (Cellular Signaling Technology), c-Abl phospho- selected with 800 g/ml G418 for 10–14 days. To analyze wild- Tyr (Cellular Signaling Technology), c-Abl phospho- type and mutant Bcr-Abl protein expression and phosphoryla- Tyr (Cellular Signaling and BIOSOURCE), and c-Abl tion status, 10 cells were collected by centrifugation, washed protein (Pharmingen). once with phosphate-buffered saline, and lysed in ice-cold Generation of Phospho-specific Antibodies to Abl Phospho- radioimmune precipitation assay buffer supplemented with Tyr —Phospho-specific antibodies were raised in rabbits the protease inhibitors aprotinin (25 g/ml), leupeptin (25 against a phosphopeptide corresponding to the Abl Tyr phos- g/ml), and phenylmethylsulfonyl fluoride (1 mM) and the phorylation site. The sequence of the peptide antigen was phosphatase inhibitors NaF (10 mM) and Na VO (1 mM). 3 4 LFVALpYDFVASGDN. The antiserum was first purified by Clarified lysates were resolved by SDS-PAGE, transferred to protein A chromatography, and nonspecific antibodies were polyvinylidene difluoride membranes, and blotted with anti- removed by passing the protein A eluate through a second col- bodies specific for Abl protein and site-specific phosphoryl- umn on which the corresponding unphosphorylated peptide ation as described above. GM-CSF-independent cell prolif- was immobilized. The eluate from this column was further eration was assessed using the CellTiter-Blue cell viability purified on a column containing the original phosphopeptide. assay (Promega Corp.). Cells were washed and seeded in To test antibody specificity, recombinant His -Abl32 pro- 5-ml cultures at 10 /ml in the absence of GM-CSF. For each tein was phosphorylated with Hck as described above, time point, 100 l of each culture was withdrawn, mixed resolved by SDS-PAGE, and immunoblotted with the anti-Abl with 20 l of the assay reagent in a 96-well plate, and incu- phospho-Tyr antibody at 1:1000 dilution. This experiment bated at 37 °C for 120 min prior to reading the fluorescence was repeated with purified His -Abl32 protein in which Tyr (excitation at 560 nm and emission at 590 nm) on a Molec- was changed to Phe. The antibody was also used to probe Bcr- ular Dynamics SpectraMax Gemini XS fluorometric plate Abl Tyr phosphorylation in the CML cell lines Meg-01 and reader. All time points were assayed in triplicate, and the K-562 by immunoblotting as described previously (18). To entire experiment was repeated three times. demonstrate the requirement for Src family kinases in the phos- phorylation event, cell lines were treated with the Src family RESULTS kinase-selective inhibitor A-419259 overnight prior to lysis and Src Family Kinases Phosphorylate the Bcr-Abl SH3-SH2 immunoblotting as described (18, 26). Src family kinase activity Region at Multiple Tyrosines—Previously, we found that the Src was determined by immunoblotting with the anti-Src phospho- family kinase Hck binds directly to the SH3-SH2 region, kinase Tyr antibody (BIOSOURCE), which reacts with the phos- domain, and C-terminal region of Bcr-Abl in vitro (17). In the phorylated activation loop of all members of the Src kinase present study, we extended these findings to include Lyn and family (26). Fyn, which are also present in Bcr-Abl-transformed myeloid Mutagenesis of Bcr-Abl SH3-SH2 Phosphorylation Sites—A progenitor cells. Each Src family member was coexpressed in 1121-bp oligonucleotide spanning the SH3-SH2 region of Bcr- Sf9 insect cells along with a series of GST fusion proteins Abl containing seven tyrosine-to-phenylalanine substitutions encompassing the Abl-derived portion of Bcr-Abl (Fig. 1A). corresponding to the phosphorylation sites shown in Fig. 2 The fusion proteins were precipitated and analyzed for bound (7YF mutant) and flanked by unique restriction sites was com- Src family kinases by immunoblotting. Hck, Lyn, and Fyn mercially synthesized (DNA 2.0 Inc.). The SH3-SH2 7YF oligo- exhibited a nearly identical pattern of binding involving the nucleotide was swapped for the corresponding region of wild- type Bcr-Abl, and the resulting full-length Bcr-Abl 7YF coding SH3-SH2, kinase, and C-terminal regions of Abl (Fig. 1B). No region was subcloned into the retroviral vector pMSCV-neo association was observed with GST alone. This result shows (Clontech). The Y89F single mutant and a 6YF add-back that Src family kinases expressed in myeloid cells associate with mutant were created using the QuikChange site-directed Bcr-Abl by a common mechanism. OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30909 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases of GST was observed, indicating that the phosphorylation sites local- ize to the Abl-derived portion of each fusion protein. We next identified the sites of Src family kinase-mediated Abl SH3- SH2 phosphorylation by MALDI- TOF-MS. To eliminate interference from the GST moiety, the SH3-SH2 region was re-expressed with a hexahistidine tag at the N terminus (His -Abl32), purified, and incu- bated in vitro with a catalytic amount of recombinant Hck, Lyn, or Fyn. As shown in Fig. 2A, all three Src kinases strongly phosphorylated the purified His -Abl32 protein. Each phosphorylation reaction was digested overnight with trypsin, and the resulting peptides were analyzed by MALDI-TOF-MS. Of 10 possible tyrosines in the His -Abl32 pro- tein, seven were reproducibly phos- 89 134 phorylated: Tyr and Tyr in the SH3 domain; Tyr in the SH3-SH2 158 191 connector; and Tyr , Tyr , 204 234 Tyr , and Tyr in the SH2 domain. The extent of phosphoryl- ation of each peptide (estimated from the ratios of the peak intensi- ties for the phosphopeptides and FIGURE 1. Bcr-Abl interacts with Hck, Lyn, and Fyn through common mechanisms. A, recombinant bacu- their unphosphorylated counter- loviruses were constructed to express the Bcr-Abl regions shown as GST fusion proteins: the SH3-SH2 region 57 224 215 489 (Gly –Thr ; 32); the kinase domain (Tyr –Ile ; Kin); and the C-terminal region as a series of four fusion parts) is presented in Fig. 2A. Tyr 480 638 639 813 801 993 994 1130 proteins encompassing Pro –Gly (CT1), Arg –Leu (CT2), Ile –Ala (CT3), and Gly –Arg (CT4). was phosphorylated to very high Numbering is based on the human c-Abl sequence. B, the recombinant GST-Abl fusion proteins shown in A and GST alone as a negative control were coexpressed with Hck, Lyn, or Fyn in Sf9 insect cells. Fusion proteins were stoichiometry by all three Src family precipitated from clarified cell extracts using glutathione-agarose beads and washed, and associated Src family members. Interestingly, Hck phos- kinases were visualized by immunoblotting. The recovery of each GST fusion protein was verified by immuno- phorylated several other sites to blotting an aliquot of the precipitate with anti-GST antibodies (data not shown). Equivalent expression of each Src family member was verified by immunoblotting the cell lysates with Src family kinase-specific antibodies higher stoichiometry relative to Lyn (not shown). C, recombinant GST-Abl SH3 domain, GST-Abl SH2 domain, and GST-Abl32 fusion proteins and or Fyn. Whether this represents GST alone were purified from bacteria and incubated in the absence (No kinase) or presence of recombinant true differences in substrate speci- Hck, Lyn, or Fyn as described under “Materials and Methods.” Aliquots of each reaction were separated by SDS-PAGE, and phosphotyrosine ( P-Tyr) content was determined by immunoblotting (upper panels). The posi- ficity among the Src kinase isoforms tions of GST and each GST-Abl fusion protein are indicated on the right (arrows). Replicate filters were probed or simply differences in the specific with anti-GST antibodies to confirm equal amounts of purified protein in each reaction (lower panels). Each experiment was repeated at least twice with comparable results. activities of the purified kinase preparations will require further investigation. Note that Tyr in 186 193 The immunoblots from the binding assays shown in Fig. 1 the Abl SH3 domain and Tyr and Tyr in the SH2 domain were then reprobed with anti-phosphotyrosine antibodies. were not detectably phosphorylated, indicative of selectivity for Very strong phosphorylation of the Abl SH3-SH2 region was specific Tyr sites (data not shown). observed upon coexpression with Hck, Lyn, and Fyn (data not The positionsofthesenovelSrcfamilykinasetyrosinephospho- shown). To confirm this finding, we purified the GST-Abl32 rylation sites within the crystal structure of the c-Abl core region fusion protein and tested it as a substrate for recombinant Hck, (30) are shown in Fig. 2B. Intriguingly, four of these tyrosine resi- 89 134 158 191 Lyn, and Fyn in vitro. As shown in Fig. 1C, catalytic amounts of dues (Tyr and Tyr in the SH3 domain and Tyr and Tyr all three Src family kinases strongly phosphorylated the Bcr-Abl in the SH2 domain) lie along the interface between the SH3-SH2 SH3-SH2 region under these conditions. Lower levels of phos- region and the kinase domain. Because c-Abl kinase activity is phorylation were observed with equimolar amounts of GST- down-regulated in part through tight docking of the SH3-SH2 Abl SH2 and GST-Abl SH3 domain fusion proteins, suggesting “clamp” onto the back of the kinase domain, phosphorylation of that tethering of the domains is required for proper recognition residues along this interface could influence kinase activity, even in and phosphotransfer by Src family kinases. No phosphorylation the context of Bcr-Abl (see “Discussion”). 30910 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 •OCTOBER 13, 2006 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases FIGURE 3. Src family kinases phosphorylate full-length Bcr-Abl in the SH3- SH2 regulatory region. A, full-length kinase-dead Bcr-Abl was expressed as a C-terminal hexahistidine fusion protein in Sf9 insect cells, purified, and incu- bated in the presence () or absence () of recombinant Hck in vitro. Aliquots of each reaction were immunoblotted with anti-phosphotyrosine ( P-Tyr) and anti-Abl antibodies. The remainder of the reaction was digested with trypsin, and tryptic peptides were resolved by reverse-phase liquid chromatography and analyzed by MALDI-TOF-MS. B, shown are the mass spectra of the Tyr tryptic peptide (upper panel) and its Hck-phosphorylated counterpart (lower panel). This peptide is derived from the Bcr-Abl SH3-SH2 connector region and has a predicted mass of 1225.59 Da, whereas the phosphorylated form of the peptide has a predicted mass of 1305.55 Da (arrows). The amino acid sequences of both peptides were confirmed in MS/MS experiments (see Fig. 4). FIGURE 2. Src family kinases phosphorylate the Bcr-Abl SH3-SH2 tag on its C terminus. This form of Bcr-Abl cannot undergo auto- region at multiple tyrosine residues. A, the Abl SH3-SH2 region was phosphorylation, allowing for clear characterization of Src fam- expressed in E. coli as an N-terminal hexahistidine fusion protein (His - Abl32), purified, and incubated in vitro in the absence (control (Con)) or ily kinase-dependent phosphorylation events. Purified kinase- presence of recombinant Hck, Lyn, or Fyn. Aliquots of each reaction were dead Bcr-Abl (p210-KR) was incubated either alone or with a immunoblotted with anti-phosphotyrosine antibodies ( P-Tyr) or with an catalytic amount of recombinant Hck, and an aliquot of the phos- antibody to the histidine tag to confirm equal amounts of the Abl protein in each reaction (inset). The His -Abl32 protein is indicated by the phorylation reaction was analyzed by anti-phosphotyrosine im- arrows. The remainder of each reaction was digested overnight with tryp- munoblotting. As shown in Fig. 3, p210-KR was strongly phospho- sin, and the extent of tyrosine phosphorylation of each resulting peptide rylated in the presence of Hck. The phosphorylated p210-KR was determined by MALDI-TOF-MS. Blue bars, Hck; green bars, Lyn; yellow bars, Fyn. MS analysis was performed in duplicate, and the ratio of the protein was then digested with trypsin, and the resulting peptides mean peak intensities S.D. is shown. B, Src phosphorylation sites map to were separated by liquid chromatography. Each column fraction the SH3-SH2 region in the c-Abl crystal structure. The crystal structure of the c-Abl core is shown (Protein Data Bank code 1OPK) (30), with Src family was spotted onto a MALDI plate, and a total of 144 spectra were kinase phosphorylation sites numbered. SH3 domain residues are shown collected and analyzed. Using a peak picking routine, we were 134 89 in red (Tyr and Tyr ); the SH3-SH2 connector residue is shown in gray 147 158 191 able to identify ions corresponding to six of the phosphoty- (Tyr ); and the SH2 domain residues are shown in blue (Tyr , Tyr , 204 234 Tyr , and Tyr ). The SH2-kinase linker is shown in orange, and the rosine-containing peptides originally observed in the smaller kinase domain is rendered in violet. The location of bound myristic acid is 361 His -Abl32 construct (Fig. 2). Fig. 3B presents representative shown in green (Myr). The C-terminal lobe residue Tyr , which forms an aromatic stacking interaction with the SH2 domain residue Tyr , is also spectra for the tryptic peptides containing Tyr , a phosphoryla- shown. The three tyrosines that were not detectably phosphorylated tion site found in the SH3-SH2 connector. The peak at 1225.59 Da 112 186 193 (Tyr , Tyr , and Tyr ) are indicated in parentheses. corresponds to the Tyr tryptic peptide (HSWYHGPVSR), and the peak at 1305.55 Da corresponds to its phosphorylated coun- Hck Phosphorylates Full-length Bcr-Abl in the SH3-SH2 Re- terpart (HSWpYHGPVSR). Note the expected 80-Da shift in gion—We next investigated whether Src family kinases phospho- molecular mass corresponding to the covalent addition of a phos- rylate the Abl SH3-SH2 region within the context of the full-length phate group by Hck. The sequences of these peptides were con- Bcr-Abl protein. Full-length kinase-dead p210 Bcr-Abl was ex- firmed in subsequent MS/MS experiments, which are presented in pressed in Sf9 insect cells and purified by virtue of a hexahistidine Fig. 4. OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30911 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases The complete results of the MALDI-TOF-MS analysis of the fied and confirmed by MS/MS sequencing. A mass peak corre- Hck-phosphorylated, full-length Bcr-Abl p210-KR protein are sponding to the Tyr tryptic phosphopeptide (VYHYR) could summarized in Table 1. In addition to Tyr , mass peaks cor- not be found, despite strong phosphorylation of this site by Hck 134 158 responding to the Tyr and Tyr tryptic phosphopeptides in the His -Abl32 protein (Fig. 2). However, the unphospho- (along with their unphosphorylated counterparts) were identi- rylated form of the Tyr peptide was also absent, suggesting possible loss during chromatogra- phy due to the small size and hydro- phobic nature of this peptide. Candidate peaks for both the phos- phorylated and unphosphorylated 89 204 peptides containing Tyr , Tyr , and Tyr were detected, although low abundance prevented sequence confirmation by MS/MS analysis. The Tyr peptide is much longer in the context of full-length Bcr-Abl relative to His -Abl32, where it is directly adjacent to the hexahisti- dine tag; this may account for its low abundance. We also surveyed the full-length Bcr-Abl data set for phosphorylated tryptic peptides corresponding to reported sites of c-Abl and Bcr-Abl autophosphorylation and c-Abl transphosphorylation by Src family kinases, including Tyr in the SH2-kinase linker, Tyr in the kinase domain activation loop, and Tyr in the Bcr region (Table 1). Tryptic phosphopeptides corre- 177 412 sponding to Tyr and Tyr were detected (as were their unphospho- rylated counterparts), and their amino acid sequences and phospho- rylation states were confirmed by MS/MS sequencing. In contrast, the tryptic peptide containing Tyr or FIGURE 4. Confirmation of phosphorylation of Bcr-Abl SH3-SH2 connector peptides. Parent ions correspond- its phosphorylated counterpart ing to the Bcr-Abl SH3-SH2 connector tryptic peptide (HSWYHGPVSR) as well as its phosphorylated counterpart were subjected to collision-induced dissociation, and the product spectra for the unphosphorylated (A) and phos- could not be located, despite strong phorylated (B) forms are shown. Mass differences between peaks comprising the y ion series correspond to the reactivity of this site with a phos- partial amino acid sequences shown at the top. The y ion series in the phosphorylated peptide (B) is shifted by80 pho-specific antibody (see below). Da beginning with the y ion, indicating phosphorylation of the tyrosine. In the b ion series, the b ,b ,b ,b , and b 7 5 6 7 8 9 ions are similarly shifted. For simplicity, the identities of other ions present are not indicated. This is likely due to the limitations TABLE 1 Hck phosphorylates SH3-SH2 sites in full-length Bcr-Abl Tyrosine phosphopeptides Unphosphorylated peptides Residue Location Predicted mass Observed mass Predicted mass Observed mass Da Da Tyr SH3 3606.86 3606.67 3526.88 ND 134 b b Tyr SH3 2183.03 2183.01 2103.05 2103.05 147 b b Tyr SH3-SH2 connector 1305.57 1305.55 1225.59 1225.59 158 b b Tyr SH2 1990.99 1990.97 1911.01 1911.01 191 c Tyr SH2 817.35 ND 737.35 ND Tyr SH2 1020.45 1020.56 940.47 ND Tyr SH2 3162.59 3162.41 3082.61 ND 177 b b Tyr Bcr (Grb2 binding) 2701.21 2701.23 2621.23 2621.18 Tyr SH2-kinase linker 1661.77 ND 1581.79 1581.92 412 b b Tyr Activation loop 1516.66 1516.68 1436.68 1436.68 a 177 Numbering was based on the c-Abl crystal structure (30), except for Bcr-derived Tyr . Peptide identity was confirmed by MS/MS sequencing. Not detected. 30912 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 •OCTOBER 13, 2006 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases FIGURE 6. Phosphorylation of Bcr-Abl SH3 domain Tyr by Src family kinases in CML cells. A, validation of the anti-Abl phospho-Tyr antibody. The wild-type (WT) and Y89F mutant Abl SH3-SH2 regions were expressed in E. coli as N-terminal hexahistidine fusion proteins, purified, and incubated in vitro in the absence or presence of recombinant Hck as indicated. Aliquots of each reaction were immunoblotted with the anti-Abl phospho-Tyr anti- body ( pY89), a general anti-phosphotyrosine antibody ( pTyr), or with an anti- body to the histidine tag to confirm equal amounts of the Abl protein in each reaction (His). B, Bcr-Abl Tyr is phosphorylated in CML cell lines. Lysates from the Ph-positive CML cell lines K-562 and Meg-01 were probed with the anti- Abl phospho-Tyr antibody (upper panels) or with an anti-Abl antibody to verify Bcr-Abl protein expression (lower panels). Bcr-Abl bands are indicated by the arrows. Similar blots from Ph-negative TF-1 myeloid leukemia cells are included as a negative control. C, phosphorylation of multiple Bcr-Abl tyro- sine sites requires Src family kinase activity. Meg-01 CML cells were treated FIGURE 5. Src family kinases phosphorylate full-length Bcr-Abl in the overnight with the indicated micromolar concentrations of the Src family SH2-kinase linker, activation loop, and Grb2-binding sites. A, full-length kinase inhibitor A-419259. Cell lysates were probed with phospho-specific 89 177 kinase-dead Bcr-Abl was expressed in Sf9 insect cells either alone (control antibodies against Abl phospho-Tyr , Bcr phospho-Tyr ( pY177), Abl phos- 245 412 (Con)) or together with Hck, Lyn, or Fyn (left panels). Active Bcr-Abl was pho-Tyr ( pY245), and Abl phospho-Tyr ( pY412). Lysates were also expressed alone as a positive control (right panels). Bcr-Abl proteins were probed for Src family kinase autophosphorylation with the anti-Src phospho- immunoprecipitated using an anti-Abl antibody and immunoblotted with Tyr antibody (Src pY418) and for Bcr-Abl protein levels. Each experiment phospho-specific antibodies for the Grb2-binding site (Bcr phospho-Tyr was repeated at least twice with comparable results. ( pY177)), the Abl SH2-kinase linker (phospho-Tyr ( pY245)), the activation loop tyrosine (phospho-Tyr ( pY412)), and the Bcr-Abl protein. Each exper- Abl. For reference, the locations of Tyr in the SH2-kinase linker iment was repeated at least twice with comparable results. B, the location of 245 412 the linker (Tyr ) and activation loop (Tyr ) tyrosine residues are indicated and Tyr in the activation loop are mapped onto the crystal on the crystal structure of the c-Abl core (30); domains are colored as 177 structure of the c-Abl core in Fig. 5B. described in the legend to Fig. 2. Bcr-Abl Tyr is found in the N-terminal Src Family Kinase-dependent Phosphorylation of the Bcr-Abl Bcr-derived portion of the protein and is not present in this structure. SH3 Domain in CML Cells—We next investigated whether of separation and detection of the large number of Bcr-Abl phosphorylation of the Bcr-Abl SH3-SH2 region by Src family peptides. kinases occurs in the context of CML cells. For these experi- To complement the MS analysis of Bcr-Abl tyrosine phospho- ments, we focused on Tyr , the SH3 site most strongly phos- rylation, we also used an immunoblot strategy with phospho-spe- phorylated by Hck, Lyn, and Fyn in vitro (Fig. 2). To probe Tyr cific antibodies. For these experiments, full-length kinase-dead phosphorylation in cells, we raised a phospho-specific antibody Bcr-Abl was expressed alone or together with Hck, Lyn, or Fyn in against this site and validated it using the recombinant His - Sf9cells.ActiveBcr-Ablwasalsoexpressedalonetogaugeautophos- Abl32 protein originally used to map the phosphorylation sites phorylation. Bcr-Abl proteins were then immunoprecipitated and by MS. As shown in Fig. 6A, the anti-Abl phospho-Tyr antibody probed with antibodies specific for phosphorylation of Bcr Tyr , did not recognize the unphosphorylated His -Abl32 protein. 245 412 Tyr , and Tyr . As shown in Fig. 5A, strong phosphorylation of However, incubation of the protein with Hck prior to immuno- all three sites by Hck, Lyn, and Fyn was observed using this blotting resulted in a strong signal with the antibody. The experi- approach, suggesting that these sites are transphosphorylated by ment was then repeated with a mutant form of His -Abl32in Src family kinases in full-length Bcr-Abl. Note that all three sites which Tyr was replaced with Phe (Y89F mutant). This mutant were also strongly autophosphorylated within kinase-active Bcr- form of His -Abl32 did not react with the anti-Abl phospho- OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30913 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases Tyr antibody following incubation with Hck, indicating that the antibody is specific for Bcr-Abl phospho-Tyr . A replicate filter with the same four samples was then probed with a general anti-phosphotyrosine antibody. In this case, immunoreac- tivity was reduced but not eliminated with the Y89F form of His -Abl32 following phosphorylation by Hck in compar- ison with the wild-type protein. This observation agrees with the MS showing that Tyr is the preferred site of phospho- rylation for Src family kinases in this protein (Fig. 2). Using the anti-Abl phospho-Tyr antibody, we next investi- gated the phosphorylation of Tyr in the CML-derived cell lines K-562 and Meg-01. Our previous work has shown that these two cell lines are very sensitive to Src family kinase inhib- itors in terms of growth arrest and programmed cell death (18). Fig. 6B shows that lysates from both CML cell lines exhibited a band of 210 kDa that reacted strongly with the anti-Abl phos- pho-Tyr antibody, indicating that the SH3 domain of Bcr-Abl is phosphorylated at this site in cells. In contrast, the Ph-nega- tive leukemia cell line TF-1 showed no immunoreactivity with the anti-Abl phospho-Tyr antibody, consistent with the lack of Bcr-Abl expression in this cell line. As expected, control immunoblots showed expression of p210 Bcr-Abl protein in FIGURE 7. Tyrosine phosphorylation of the SH3-SH2 region is essential K-562 and Meg-01 cells, but not in TF-1 cells. forfullBcr-Ablbiologicalactivity.ThesevenBcr-AblSH3-SH2tyrosinephos- phorylation sites for Src family kinases identified by MS were replaced with To implicate Src family kinases in the phosphorylation of the Phe in full-length Bcr-Abl (7YF mutant). Tyr was restored in the context of Bcr-Abl SH3 domain in CML cells, we employed the Src family the 7YF mutant to create mutant 6YF. A single point mutant of Tyr was also created (Y89F). Each of these mutants, as well as wild-type (WT ) Bcr-Abl, were kinase inhibitor A-419259 (31). Previous studies by our group expressed in the human GM-CSF-dependent myeloid progenitor cell line TF-1 have shown that this compound blocks Src family kinase using recombinant retroviruses. A, GM-CSF-independent proliferation of activity in vitro in the low nanomolar range, but is at least 2 each cell line was monitored using the CellTiter-Blue cell viability assay as described under “Materials and Methods.” Cells infected with a green fluores- orders of magnitude less active against the Abl kinase domain cent protein retrovirus served as negative control (Con). The experiment was (18, 26). A-419259 also induces growth suppression and apo- performed in triplicate, and the mean -fold increase in cell number  S.D. ptotic cell death in both the K-562 and Meg-01 CML cell lines, is shown at each time point. B, lysates from each of the cell lines shown in A were probed with phospho-specific antibodies against Bcr-Abl phospho-Tyr (pY89) but does not affect Ph-negative myeloid leukemia cells (18). To and phospho-Tyr (pY412). Replicate membranes were also probed with a gen- determine whether Src family kinase activity is required for eral anti-phosphotyrosine antibody (pTyr) and with anti-Abl antibodies to con- trol for Bcr-Abl expression. Blotting was performed on two separate infected cell phosphorylation of Bcr-Abl at Tyr , Meg-01 cells were treated populations and produced the same result in each case. with a range of A-419259 concentrations, followed by immu- noblotting with the anti-Abl phospho-Tyr antibody. As 89 89 shown in Fig. 6C, phosphorylation of Abl Tyr was blocked in Phosphorylation of Tyr and Other Sites in the SH3-SH2 a concentration-dependent manner, with IC  300 nM, and Region Is Required for Full Bcr-Abl Transforming Function—To phosphorylation of this site was completely blocked at 1 M. determine whether tyrosine phosphorylation of the SH3-SH2 This dose response for inhibition of Abl Tyr phosphorylation region is important for Bcr-Abl function, we engineered a closely paralleled suppression of Src family kinase autophospho- mutant form of Bcr-Abl (7YF) in which phenylalanine replaced rylation (Src phospho-Tyr immunoblot), strongly implicating each of the seven SH3-SH2 tyrosine phosphorylation sites for Src family kinases in the phosphorylation of the Bcr-Abl SH3 Src family kinases shown in Fig. 2 and Table 1. The 7YF mutant domain at Tyr in CML cells. Control blots showed equivalent was then compared with wild-type Bcr-Abl in terms of its abil- levels of Bcr-Abl protein in each sample. Similar results were ity to transform the human TF-1 myeloid cell line to cytokine obtained with K-562 cells (data not shown). independence. TF-1 cells require GM-CSF or interleukin-3 for The data presented in Fig. 5 and Table 1 show that Hck phos- growth and survival and undergo apoptosis following cytokine 177 245 412 phorylated Bcr-Abl at Bcr Tyr , Abl Tyr , and Abl Tyr in withdrawal (27); introduction of Bcr-Abl with a recombinant vitro, all of which are known sites of Bcr-Abl phosphorylation. retrovirus reverses the cytokine dependence. TF-1 cells were To evaluate whether Hck and other Src kinases contribute to infected with wild-type and 7YF mutant Bcr-Abl retroviruses, phosphorylation of these sites in CML cells, lysates from the and cell growth in the absence of GM-CSF was measured over 3 A-419259-treated Meg-01 cells were probed with phospho- days. Fig. 7A shows that the cytokine-independent proliferation specific antibodies directed against these sites. As shown in Fig. of TF-1 cells expressing the 7YF mutant was reduced by 50% 6C, A-419259 treatment also substantially reduced the phos- relative to cells expressing wild-type Bcr-Abl, providing evi- phorylation of these sites, suggesting that Src family kinases dence that phosphorylation of the Bcr-Abl SH3-SH2 region is may have a major role in maintaining the active conformation required for full transforming activity. of Bcr-Abl in CML cells (see “Discussion”). Very similar results Because Tyr is most prominent among the Src family were obtained with K-562 cells (data not shown). kinase phosphorylation sites in the Bcr-Abl SH3-SH2 region, we 30914 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 •OCTOBER 13, 2006 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases next investigated whether adding back this single phosphorylation of Bcr-Abl can be complemented by mutations predicted to site restores the biological activity of the 7YF mutant in the TF-1 disrupt SH3/linker interaction, consistent with the idea that the cell transformation assay. This mutant, termed 6YF, showed trans- SH3 domain still exerts some negative regulatory influence over forming activity intermediate to that observed with wild-type Bcr- Bcr-Abl tyrosine kinase activity. Phosphorylation of the SH3 89 134 Abl and the 7YF mutant. In addition, we created a Bcr-Abl point domain at Tyr or Tyr by Src family kinases as described here mutant in which Tyr alone was replaced with Phe (Y89F). This may have a similar destabilizing effect, as these sites come in close mutant also exhibited transforming activity intermediate to that of proximitytotheSH2-kinaselinker.Indeed,phosphorylationofthe wild-type Bcr-Abl and the 7YF mutant. Taken together, these Abl SH3-SH2 region by Hck caused dissociation of the SH2-kinase 89 4 results suggest that, although phosphorylation of Tyr is required linker as determined by hydrogen exchange MS. for full transforming activity, the other tyrosine phosphorylation In addition to the SH3 sites, several of the other tyrosine sites within the SH3-SH2 region are likely to contribute to Bcr-Abl residues phosphorylated by Src family kinases localize to the function in CML cells. interface between the SH3-SH2 regulatory clamp and the large Finally, we investigated the impact of SH3-SH2 phosphoryl- lobe of the c-Abl kinase domain (Fig. 2). Particularly interesting 158 158 ation site mutagenesis on Bcr-Abl at the biochemical level by is the SH2 domain residue Tyr . The aromatic rings of Tyr immunoblotting lysates from each of the transformed TF-1 cell and the kinase domain residue Tyr stack together in the populations with phospho-specific antibodies. As shown in Fig. down-regulated c-Abl structure, and the hydroxyl group of 7B, wild-type Bcr-Abl reacted strongly with the anti-Abl phos- Tyr forms a hydrogen bond with the backbone carbonyl 89 393 pho-Tyr antibody, indicating that this site is phosphorylated group of Asn of the kinase domain (30). Interestingly, muta- in TF-1 cells in a manner similar to that in the CML cell lines tion of Tyr to glutamate results in a nearly 4-fold stimulation (Fig. 6B). The immunoreactivity of the anti-Abl phospho-Tyr of c-Abl kinase activity compared with the wild type (35). Phos- antibody was sharply reduced in TF-1 cells expressing the Bcr- phorylation of this site by Src family kinases may also disrupt Abl 7YF and Y89F mutants, but was completely restored with kinase regulation of c-Abl and raises the possibility of a similar the Tyr add-back mutant, 6YF. In contrast, phosphorylation regulatory interaction in Bcr-Abl. of the Bcr-Abl activation loop (phospho-Tyr immunoblot) Other recent studies show that mutations outside of the cat- and the overall Bcr-Abl phosphotyrosine content (phospho- alytic domain can allosterically affect not only Bcr-Abl kinase Tyr immunoblot) were not remarkably changed between the activity but sensitivity to imatinib as well. For example, Azam wild-type and mutant Bcr-Abl proteins, supporting the idea et al. (36) used an unbiased random mutagenesis screen to that the Tyr-to-Phe substitutions in the SH3-SH2 region do not uncover novel mutations in Bcr-Abl that confer imatinib resist- simply disrupt the folding of the Bcr-Abl protein. Notably, all ance and mapped these residues onto the autoinhibited c-Abl four TF-1 cell populations expressed equivalent levels of Bcr- structure. These authors uncovered a striking correlation Abl protein (Abl immunoblot). between residues that impair c-Abl negative regulation and imatinib resistance of Bcr-Abl, again strongly suggesting that DISCUSSION mechanisms governing c-Abl autoinhibition are retained in This work provides the first evidence for tyrosine phospho- Bcr-Abl. These observations agree with previous work showing rylation of the Bcr-Abl SH3-SH2 regulatory region by Src fam- that imatinib favors the down-regulated conformation of the Abl ily kinases both in vitro and in CML cells. The possible impact catalytic cleft for binding (33, 37). Notably, several of these ima- of these phosphorylation events on Bcr-Abl function requires tinib resistance mutations localize to SH3, SH2, and kinase further consideration of c-Abl structure and regulation. domain residues that contribute to the negative regulatory inter- Although Bcr-Abl exhibits constitutive tyrosine kinase activity, face. In particular, Azam et al. (36) found that substitution of the Hantschel and Superti-Furga (32) have proposed that Bcr-Abl Bcr-Abl SH3 domain residue Tyr , which is strongly phosphoryl- may retain some of the regulatory features observed in the ated by Hck, Lyn, and Fyn in vitro (Fig. 2) and in CML cells (Fig. 6), recent x-ray crystal structures of the c-Abl core (see Fig. 2) (30, resulted in Bcr-Abl imatinib resistance in four independent iso- 33). These structures show that, in the down-regulated confor- lates. Phosphorylation of this site by Src family kinases may also mation, the c-Abl SH3 domain engages the polyproline type II stabilize the active conformation of Bcr-Abl and contribute to its helix formed by the SH2-kinase linker in an intramolecular transforming activity. The reduced transforming activity of the fashion, as is the case for c-Src and Hck (21). Unlike Src kinases, Y89F mutant in TF-1 cells also supports this view (Fig. 7). however, the SH2 domain docks onto the back of the C-termi- In addition to novel sites in the SH3-SH2 region, we found nal lobe of the Abl kinase domain. This interaction is stabilized that Hck, Lyn, and Fyn phosphorylate full-length Bcr-Abl at 177 245 412 by binding of the myristoylated N-terminal cap through a Tyr in the Bcr region and at Tyr and Tyr in the Abl unique pocket in the kinase domain (Fig. 2). Residues linking kinase domain (Fig. 5 and Table 1). Although phosphorylation the SH3 and SH2 domains form a rigid connector that dynam- of Bcr-Abl Tyr by Src family kinases was readily detected ically couples the SH3 and SH2 domains, which together pro- both in vitro and in CML cells using a phospho-specific anti- vide a regulatory clamp that allosterically holds the kinase body (Figs. 5 and 6), we were unable to locate this phosphopep- domain in the closed inactive state (30). Although the regula- tide (or its unphosphorylated counterpart) by MS. This dis- tory impact of myristoylation is lacking in Bcr-Abl, some evi- crepancy most likely reflects technical limitations of the dence suggests that SH3/linker interaction may be retained. For example, Smith et al. (34) showed that a transformation defect associated with mutations in the N-terminal coiled-coil region S. Chen, T. E. Smithgall, and J. R. Engen, unpublished data. OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30915 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases N., Batzer, A., Rabun, K. M., Der, C. J., Schlessinger, J., and Gishizky, M. L. MS-based approach. Experiments with the selective inhibitor (1993) Cell 75, 175–185 A-419259 strongly suggested that Src family kinases contribute 9. Puil, L., Liu, J., Gish, G., Mbamalu, G., Bowtell, D., Pelicci, P. G., Arling- to the phosphorylation of these sites in CML cells as well (Fig. haus, R., and Pawson, T. (1994) EMBO J. 13, 764–773 6). Previous work has shown that Hck phosphorylates Bcr 10. Goga, A., McLaughlin, J., Afar, D. E., Saffran, D. C., and Witte, O. N. (1995) Tyr (19), creating a docking site for the Grb2-Sos guanine Cell 82, 981–988 245 412 nucleotide exchanger for Ras (8, 9). Tyr and Tyr have been 11. Senechal, K., Halpern, J., and Sawyers, C. L. (1996) J. Biol. Chem. 271, established as sites of both c-Abl autophosphorylation (38) and 23255–23261 12. Skorski, T., Bellacosa, A., Nieborowska-Skorska, M. N., Majewski, M., transphosphorylation by Src family kinases (discussed below) Martinez, R., Choi, J. K., Trotta, R., Wlodarski, P., Perrotti, D., Chan, T. O., (39, 40), and phosphorylation of these sites strongly up-regu- Wasik, M. A., Tsichlis, P. N., and Calabretta, B. (1997) EMBO J. 16, lates c-Abl kinase activity (38). 6151–6161 Several studies have linked Src family kinases to c-Abl regu- 13. Carlesso, N., Frank, D. A., and Griffin, J. D. (1996) J. Exp. Med. 183, lation and signaling, which have important implications for 811–820 Bcr-Abl function and drug sensitivity. Plattner et al. (39) dem- 14. Ilaria, R. L., Jr., and Van Etten, R. A. (1996) J. Biol. Chem. 271, 31704–31710 15. Lionberger, J. M., and Smithgall, T. E. (2000) Cancer Res. 60, 1097–1103 onstrated that the kinase activity of c-Abl increases 10–20-fold 16. Xie, S., Wang, Y., Liu, J., Sun, T., Wilson, M. B., Smithgall, T. E., and in the presence of constitutively active v-Src in mouse Ba/F3 Arlinghaus, R. B. (2001) Oncogene 20, 6188–6195 hematopoietic cells and 10T1/2 fibroblasts. This increase in 17. Lionberger, J. M., Wilson, M. B., and Smithgall, T. E. (2000) J. Biol. Chem. c-Abl kinase activity directly correlates with phosphorylation of 275, 18581–18585 c-Abl by Src or Fyn. Dorey et al. (40) showed that active c-Src 18. Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. 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Y., Stapley, J., Gallick, G., Lin, H., Arlinghaus, R., and 7. Van Etten, R. A. (2002) Oncogene 21, 8643–8651 8. Pendergast, A. M., Quilliam, L. A., Cripe, L. D., Bassing, C. H., Dai, Z., Li, Talpaz, M. (2003) Blood 101, 690–698 30916 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 •OCTOBER 13, 2006 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Src Family Kinases Phosphorylate the Bcr-Abl SH3-SH2 Region and Modulate Bcr-Abl Transforming Activity *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2006 Elsevier Inc.
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0021-9258
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1083-351X
DOI
10.1074/jbc.m605902200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 41, pp. 30907–30916, October 13, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Src Family Kinases Phosphorylate the Bcr-Abl SH3-SH2 Region and Modulate Bcr-Abl Transforming Activity Received for publication, June 20, 2006, and in revised form, August 7, 2006 Published, JBC Papers in Press, August 15, 2006, DOI 10.1074/jbc.M605902200 ‡1 ‡1 § ‡ ‡ ¶ Malcolm A. Meyn III , Matthew B. Wilson , Fadi A. Abdi , Nathalie Fahey , Anthony P. Schiavone , Jiong Wu , ‡2 James M. Hochrein , John R. Engen , and Thomas E. Smithgall From the Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, § ¶ Pittsburgh, Pennsylvania 15261, Applied Biosystems, Framingham, Massachusetts 01701, Cell Signaling Technology, Beverly, Massachusetts 01915, and the Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131 Bcr-Abl is the oncogenic protein-tyrosine kinase responsible (2, 3). The Ph translocation results in the expression of Bcr-Abl, for chronic myelogenous leukemia. Recently, we observed that a 210-kDa oncogenic fusion protein with constitutive protein- inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, tyrosine kinase activity. Bcr-Abl transforms hematopoietic cell and Fyn) induces growth arrest and apoptosis in Bcr-Abl-trans- lines (4) and bone marrow cells (5) in culture and produces a formed cells, suggesting that cell transformation by Bcr-Abl CML-like myeloproliferative disease in mice (6, 7), directly involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, implicating this chimeric protein-tyrosine kinase in disease H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075– onset. 8088). Here, we report the unexpected observation that Hck, Bcr-Abl phosphorylates a broad range of cellular proteins, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of affecting many signaling pathways linked to the growth, differ- Bcr-Abl. Seven phosphorylation sites were identified by matrix- entiation, and survival of hematopoietic progenitors (3). For assisted laser desorption ionization time-of-flight mass spec- example, the Grb2-Sos guanine nucleotide exchange factor 89 134 trometry: Tyr and Tyr in the Abl-derived SH3 domain; complex binds to phospho-Tyr in the Bcr-derived region, 147 158 191 204 Tyr in the SH3-SH2 connector; and Tyr , Tyr , Tyr , contributing to the activation of Ras (8, 9). Bcr-Abl also acti- 234 89 and Tyr in the SH2 domain. SH3 domain Tyr , the most vates Ras via Shc, an adapter protein that couples the receptors prominent phosphorylation site in vitro, was strongly phospho- for many growth factors and cytokines to the Grb2-Sos com- rylated in chronic myelogenous leukemia cells in a Src family plex (10). In addition, Bcr-Abl associates with and phospho- kinase-dependent manner. Substitution of the SH3-SH2 tyro- rylates the CrkL adapter (11), stimulates phosphatidylinositol sine phosphorylation sites with phenylalanine substantially 3-kinase/Akt survival signaling by direct interaction with the reduced Bcr-Abl-mediated transformation of TF-1 myeloid p85 subunit of phosphatidylinositol 3-kinase (12), and activates cells to cytokine independence. The positions of these tyrosines several Stat transcription factors (13, 14). All of these signaling in the crystal structure of the c-Abl core and the transformation pathways involve components with SH2 and SH3 domains and defect of the corresponding Bcr-Abl mutants together suggest are dependent upon interactions with phosphotyrosine or that phosphorylation of the SH3-SH2 region by Src family polyproline docking sites, respectively. kinases impacts Bcr-Abl protein conformation and signaling. Despite its intrinsic protein-tyrosine kinase activity, several studies have established that Bcr-Abl interacts with other tyro- sine kinases, including members of the Fps/Fes (15), Jak (16), The cytogenetic hallmark of chronic myelogenous leukemia and Src (17–20) kinase families. Hallek and co-workers (20) (CML) is the Philadelphia (Ph) chromosome (1), which results originally showed that Bcr-Abl forms complexes with the Src from a reciprocal translocation between the c-abl proto-onco- family members Hck and Lyn in several Ph-positive cell lines gene on chromosome 9 and the bcr locus on chromosome 22 and is phosphorylated by Hck at Bcr Tyr (19), which links Bcr-Abl to Grb2-Sos as described above (8). Interestingly, Hck activation does not require Bcr-Abl kinase activity (19), sug- * This work was supported by National Institutes of Health Grant CA101828 (to T. E. S.) and Grants GM70590 and RR016480 (to J. R. E.) and by National gesting that Bcr-Abl may stimulate Src family kinases through Science Foundation Research Experiences for Undergraduates Site Award displacement of inhibitory intramolecular interactions (21). 0243735 (to N. F.). The costs of publication of this article were defrayed in This idea is supported by subsequent work showing that the part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to SH3 and SH2 domains of Hck directly associate with Bcr-Abl indicate this fact. (17). Furthermore, kinase-defective Hck blocks transformation Both authors contributed equally to this work. of myeloid leukemia cells to cytokine independence by Bcr-Abl To whom correspondence should be addressed: Dept. of Molecular Genet- ics and Biochemistry, University of Pittsburgh School of Medicine, E1240 (17), whereas pharmacological inhibitors selective for Src fam- Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15261. Tel.: 412- ily kinases induce apoptosis in the CML cell lines Meg-01 and 648-9495; Fax: 412-624-1401; E-mail: [email protected]. K-562 (18). The effects of these compounds correlate with The abbreviations used are: CML, chronic myelogenous leukemia; Ph, Phil- adelphia; Stat, signal transducer and activator of transcription; SH, Src down-regulation of both Stat5 and Erk (extracellular signal- homology; GST, glutathione S-transferase; MS, mass spectrometry; MALDI- regulated kinase) activation, suggesting that Src family kinases TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tan- may couple Bcr-Abl to certain downstream signaling pathways dem mass spectrometry; ACTH, adrenocorticotropic hormone; GM-CSF, granulocyte-macrophage colony-stimulating factor. (18). OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30907 This is an Open Access article under the CC BY license. Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases Although Bcr-Abl interacts directly with Hck, Lyn, and other phorylation reactions (50 l) were conducted in 50 mM HEPES Src family kinases, the molecular mechanisms and functional (pH 7.4) containing 10 mM MgCl and 500 M ATP for 30 min consequences of binding in terms of Bcr-Abl signaling and at 30 °C and quenched by freezing in a dry ice/ethanol bath. oncogenesis are not understood. In this study, we demonstrate Aliquots of each reaction were analyzed by immunoblotting that Bcr-Abl binds multiple members of the Src family through with anti-GST or anti-phosphotyrosine (PY99) monoclonal the Abl-derived SH3-SH2 regulatory region. Unexpectedly, antibodies (Santa Cruz Biotechnology, Inc.). Hck, Lyn, and Fyn phosphorylated the Bcr-Abl SH3-SH2 region Hexahistidine-Bcr-Abl Fusion Protein Purification—The 57 224 at multiple tyrosine residues. The most prominent of these is Bcr-Abl SH3-SH2 coding region (Gly –Thr ; human c-Abl SH3 domain Tyr , which was also phosphorylated in CML numbering) was subcloned into the pET-14b expression vector cells in a Src family kinase-dependent manner. Substitution of (Novagen) to allow addition of an N-terminal hexahistidine tag, Tyr and other SH3-SH2 phosphorylation sites with pheny- yielding His -Abl32. BL21(DE3)pLysS bacterial cells were lalanine reduced Bcr-Abl transforming activity without altering transformed with the vector, and His -Abl32 protein expres- phosphorylation of the Abl kinase domain activation loop. sion was induced with isopropyl -D-thiogalactopyranoside. These data show that Src family kinase-mediated phosphoryl- His -Abl32 was purified from clarified cell extracts using a ation of tyrosine residues located in the SH3-SH2 region is crit- HiTrap metal-chelating column charged with Ni (GE ical for Bcr-Abl function, offering a possible mechanistic expla- Healthcare), and phosphorylated with recombinant Hck, Lyn, nation for the remarkable efficacy of Src-selective and dual Src/ and Fyn as described above for the GST-Abl32 protein. Abl inhibitors against CML (18, 22, 23). Full-length kinase-dead p210 Bcr-Abl was expressed in Sf9 insect cells and purified as follows. A hexahistidine tag was MATERIALS AND METHODS added to the C-terminal coding region of a kinase-defective Binding Assays—Interaction of glutathione S-transferase mutant of Bcr-Abl (15) using a PCR-based approach and sub- (GST)-fused human c-Abl proteins with Src family kinases was cloned into the baculovirus transfer vector pVL1393 (BD Bio- conducted in Sf9 insect cells as described previously (17). For sciences). A recombinant baculovirus was created using the binding assays, Sf9 cells (2.5  10 ) were co-infected with each transfer vector and BaculoGold DNA according to the suppli- of the GST-Abl baculoviruses (or GST as a negative control) er’s protocol. Spinner cultures of Sf9 cells (1 liter, 2 10 cells/ and wild-type Hck, Lyn, or Fyn baculovirus. Forty-eight hours ml) were infected with 100 ml of high titer virus and harvested after infection, the cells were lysed in Hck lysis buffer (17), and 72 h later. Cells were lysed in 20 mM Tris-HCl (pH 8.3) contain- GST fusion proteins were precipitated with glutathione-agar- ing 10% glycerol, 5 mM -mercaptoethanol, 20 mM imidazole, ose beads. The precipitates were washed with radioimmune and 500 mM NaCl. The protein was purified in one step using a precipitation assay buffer as described previously (17), and HiTrap metal-chelating column. The Bcr-Abl-positive frac- bound proteins were eluted in SDS-PAGE sample buffer. Pro- tions were identified by SDS-PAGE and pooled, and the con- teins were resolved on SDS-polyacrylamide gels and trans- centration of Bcr-Abl was quantitated by densitometry of a ferred to polyvinylidene difluoride membranes, and associated Coomassie Blue-stained gel. Phosphorylation reactions with Hck, Lyn, and Fyn were visualized by immunoblotting with Src family kinases were conducted as described above for the kinase isoform-specific polyclonal antibodies (Santa Cruz Bio- GST-Abl fusion proteins and contained 8 pmol of recombi- technology, Inc.). The amount of precipitated GST-Abl fusion nant Bcr-Abl p210-KR. protein present in each reaction was determined by immuno- Mass Spectrometry (MS)—Recombinant His -Abl32 and blotting with anti-GST antibodies (Santa Cruz Biotechnology, full-length kinase-dead Bcr-Abl (p210-KR) were phosphoryla- Inc.). Equivalent expression of each Src family kinase was ted in vitro with purified Src kinases, and aliquots of the phos- verified by immunoblotting the clarified cell lysates. Immu- phorylation reactions were digested overnight with trypsin at a noreactive bands were visualized with alkaline phosphatase- 1:25 trypsin/protein ratio. The His -Abl32 peptides were conjugated goat anti-rabbit secondary antibodies with mixed with matrix (-cyano-4-hydroxycinnamic acid), spotted 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazo- onto a MALDI target, and analyzed directly by MALDI- lium as colorimetric substrate (SouthernBiotech). TOF-MS (Applied Biosystems 4700 proteomics analyzer). GST-Bcr-Abl Fusion Protein Purification and in Vitro Kinase The area under the isotope distribution for the unphospho- 57 126 Assay—The Bcr-Abl SH3 domain (Gly –Ser ), SH2 domain rylated and phosphorylated forms of each tryptic peptide 121 224 (Ser –Thr ), and SH3-SH2 coding regions were subcloned was determined and used to estimate the relative extent of into the bacterial expression vector pGEX-2T, and the corre- phosphorylation. sponding GST fusion proteins were expressed in Escherichia Tryptic fragments derived from full-length Bcr-Abl p210-KR coli and purified with glutathione-agarose beads (17). The were separated by reverse-phase capillary high pressure liquid recombinant proteins were dialyzed overnight against 20 mM chromatography (Dionex Ultimate Nano-LC system). Column HEPES (pH 7.4) and 1 mM -mercaptoethanol, and protein fractions were mixed on-line with MALDI matrix and automat- concentrations were determined by densitometry of Coomas- ically spotted onto a MALDI plate at 20-s intervals with a sie Blue-stained SDS-polyacrylamide gels relative to a bovine Dionex Probot robot (144 spots total). Peaks for phosphopep- serum albumin standard. Each purified GST fusion protein or tides and their unphosphorylated counterparts were identified GST alone (380 pmol) was incubated with recombinant puri- in the MALDI-TOF spectra based on their m/z ratios, and the fied Hck-YEEI (24), Lyn (Upstate), or Fyn (Upstate). The molar sequence of each peptide was verified by tandem MS (MS/MS) ratio of substrate to kinase was at least 20:1 in each case. Phos- analysis when possible. The accuracy of the m/z measurements 30908 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 •OCTOBER 13, 2006 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases was 0.1 Da. An internal calibrant consisting of a mixture of mutagenesis kit (Stratagene). To create the 6YF mutant, the des-Arg-bradykinin, angiotensin I, Glu-fibrinopeptide B, and Bcr-Abl 7YF mutant was used as the template, and oligonucleo- ACTH-(18–39) was added to each MALDI spot prior to tides encoding Tyr were used to revert this site from Phe to analysis. Tyr. These mutants and wild-type Bcr-Abl were also subcloned Coexpression of Src Family Kinases and Bcr-Abl in Sf9 Cells— into the pMSCV-neo retroviral vector. Sf9 insect cells were co-infected with full-length kinase-dead Transformation of TF-1 Cells with Bcr-Abl Retroviruses— Bcr-Abl and Hck, Lyn, or Fyn baculovirus as described previ- The human granulocyte-macrophage colony-stimulating factor ously (25). Cells were lysed 48 h later in 1.0 ml of ice-cold radio- (GM-CSF)-dependent myeloid leukemia cell line TF-1 (27) was immune precipitation assay buffer, and Bcr-Abl was immuno- obtained from American Type Culture Collection and grown in precipitated with 4 g of anti-c-Abl monoclonal antibody 8E9 RPMI 1640 medium supplemented with 10% fetal bovine (Pharmingen) and 25 l of protein G-Sepharose (50:50 (w/v) serum, 50 g/ml gentamycin, and 1 ng/ml recombinant human slurry; GE Healthcare) by rotation overnight at 4 °C. Precipi- GM-CSF. To make retroviral stocks, 293T cells were cotrans- tated proteins were collected by centrifugation, washed with fected with each retroviral construct and an amphotropic pack- radioimmune precipitation assay buffer, and eluted in SDS- aging vector as described (28, 29). TF-1 cells (10 ) were incu- PAGE sample buffer. Proteins were resolved by SDS-PAGE; bated with 5 ml of viral supernatant in the presence of 4 g/ml transferred to polyvinylidene difluoride membranes; and Polybrene and centrifuged at 2400 rpm for 3 h at room temper- immunoblotted with antibodies specific for Bcr phos- ature to enhance infection (17, 28). Cell populations were pho-Tyr (Cellular Signaling Technology), c-Abl phospho- selected with 800 g/ml G418 for 10–14 days. To analyze wild- Tyr (Cellular Signaling Technology), c-Abl phospho- type and mutant Bcr-Abl protein expression and phosphoryla- Tyr (Cellular Signaling and BIOSOURCE), and c-Abl tion status, 10 cells were collected by centrifugation, washed protein (Pharmingen). once with phosphate-buffered saline, and lysed in ice-cold Generation of Phospho-specific Antibodies to Abl Phospho- radioimmune precipitation assay buffer supplemented with Tyr —Phospho-specific antibodies were raised in rabbits the protease inhibitors aprotinin (25 g/ml), leupeptin (25 against a phosphopeptide corresponding to the Abl Tyr phos- g/ml), and phenylmethylsulfonyl fluoride (1 mM) and the phorylation site. The sequence of the peptide antigen was phosphatase inhibitors NaF (10 mM) and Na VO (1 mM). 3 4 LFVALpYDFVASGDN. The antiserum was first purified by Clarified lysates were resolved by SDS-PAGE, transferred to protein A chromatography, and nonspecific antibodies were polyvinylidene difluoride membranes, and blotted with anti- removed by passing the protein A eluate through a second col- bodies specific for Abl protein and site-specific phosphoryl- umn on which the corresponding unphosphorylated peptide ation as described above. GM-CSF-independent cell prolif- was immobilized. The eluate from this column was further eration was assessed using the CellTiter-Blue cell viability purified on a column containing the original phosphopeptide. assay (Promega Corp.). Cells were washed and seeded in To test antibody specificity, recombinant His -Abl32 pro- 5-ml cultures at 10 /ml in the absence of GM-CSF. For each tein was phosphorylated with Hck as described above, time point, 100 l of each culture was withdrawn, mixed resolved by SDS-PAGE, and immunoblotted with the anti-Abl with 20 l of the assay reagent in a 96-well plate, and incu- phospho-Tyr antibody at 1:1000 dilution. This experiment bated at 37 °C for 120 min prior to reading the fluorescence was repeated with purified His -Abl32 protein in which Tyr (excitation at 560 nm and emission at 590 nm) on a Molec- was changed to Phe. The antibody was also used to probe Bcr- ular Dynamics SpectraMax Gemini XS fluorometric plate Abl Tyr phosphorylation in the CML cell lines Meg-01 and reader. All time points were assayed in triplicate, and the K-562 by immunoblotting as described previously (18). To entire experiment was repeated three times. demonstrate the requirement for Src family kinases in the phos- phorylation event, cell lines were treated with the Src family RESULTS kinase-selective inhibitor A-419259 overnight prior to lysis and Src Family Kinases Phosphorylate the Bcr-Abl SH3-SH2 immunoblotting as described (18, 26). Src family kinase activity Region at Multiple Tyrosines—Previously, we found that the Src was determined by immunoblotting with the anti-Src phospho- family kinase Hck binds directly to the SH3-SH2 region, kinase Tyr antibody (BIOSOURCE), which reacts with the phos- domain, and C-terminal region of Bcr-Abl in vitro (17). In the phorylated activation loop of all members of the Src kinase present study, we extended these findings to include Lyn and family (26). Fyn, which are also present in Bcr-Abl-transformed myeloid Mutagenesis of Bcr-Abl SH3-SH2 Phosphorylation Sites—A progenitor cells. Each Src family member was coexpressed in 1121-bp oligonucleotide spanning the SH3-SH2 region of Bcr- Sf9 insect cells along with a series of GST fusion proteins Abl containing seven tyrosine-to-phenylalanine substitutions encompassing the Abl-derived portion of Bcr-Abl (Fig. 1A). corresponding to the phosphorylation sites shown in Fig. 2 The fusion proteins were precipitated and analyzed for bound (7YF mutant) and flanked by unique restriction sites was com- Src family kinases by immunoblotting. Hck, Lyn, and Fyn mercially synthesized (DNA 2.0 Inc.). The SH3-SH2 7YF oligo- exhibited a nearly identical pattern of binding involving the nucleotide was swapped for the corresponding region of wild- type Bcr-Abl, and the resulting full-length Bcr-Abl 7YF coding SH3-SH2, kinase, and C-terminal regions of Abl (Fig. 1B). No region was subcloned into the retroviral vector pMSCV-neo association was observed with GST alone. This result shows (Clontech). The Y89F single mutant and a 6YF add-back that Src family kinases expressed in myeloid cells associate with mutant were created using the QuikChange site-directed Bcr-Abl by a common mechanism. OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30909 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases of GST was observed, indicating that the phosphorylation sites local- ize to the Abl-derived portion of each fusion protein. We next identified the sites of Src family kinase-mediated Abl SH3- SH2 phosphorylation by MALDI- TOF-MS. To eliminate interference from the GST moiety, the SH3-SH2 region was re-expressed with a hexahistidine tag at the N terminus (His -Abl32), purified, and incu- bated in vitro with a catalytic amount of recombinant Hck, Lyn, or Fyn. As shown in Fig. 2A, all three Src kinases strongly phosphorylated the purified His -Abl32 protein. Each phosphorylation reaction was digested overnight with trypsin, and the resulting peptides were analyzed by MALDI-TOF-MS. Of 10 possible tyrosines in the His -Abl32 pro- tein, seven were reproducibly phos- 89 134 phorylated: Tyr and Tyr in the SH3 domain; Tyr in the SH3-SH2 158 191 connector; and Tyr , Tyr , 204 234 Tyr , and Tyr in the SH2 domain. The extent of phosphoryl- ation of each peptide (estimated from the ratios of the peak intensi- ties for the phosphopeptides and FIGURE 1. Bcr-Abl interacts with Hck, Lyn, and Fyn through common mechanisms. A, recombinant bacu- their unphosphorylated counter- loviruses were constructed to express the Bcr-Abl regions shown as GST fusion proteins: the SH3-SH2 region 57 224 215 489 (Gly –Thr ; 32); the kinase domain (Tyr –Ile ; Kin); and the C-terminal region as a series of four fusion parts) is presented in Fig. 2A. Tyr 480 638 639 813 801 993 994 1130 proteins encompassing Pro –Gly (CT1), Arg –Leu (CT2), Ile –Ala (CT3), and Gly –Arg (CT4). was phosphorylated to very high Numbering is based on the human c-Abl sequence. B, the recombinant GST-Abl fusion proteins shown in A and GST alone as a negative control were coexpressed with Hck, Lyn, or Fyn in Sf9 insect cells. Fusion proteins were stoichiometry by all three Src family precipitated from clarified cell extracts using glutathione-agarose beads and washed, and associated Src family members. Interestingly, Hck phos- kinases were visualized by immunoblotting. The recovery of each GST fusion protein was verified by immuno- phorylated several other sites to blotting an aliquot of the precipitate with anti-GST antibodies (data not shown). Equivalent expression of each Src family member was verified by immunoblotting the cell lysates with Src family kinase-specific antibodies higher stoichiometry relative to Lyn (not shown). C, recombinant GST-Abl SH3 domain, GST-Abl SH2 domain, and GST-Abl32 fusion proteins and or Fyn. Whether this represents GST alone were purified from bacteria and incubated in the absence (No kinase) or presence of recombinant true differences in substrate speci- Hck, Lyn, or Fyn as described under “Materials and Methods.” Aliquots of each reaction were separated by SDS-PAGE, and phosphotyrosine ( P-Tyr) content was determined by immunoblotting (upper panels). The posi- ficity among the Src kinase isoforms tions of GST and each GST-Abl fusion protein are indicated on the right (arrows). Replicate filters were probed or simply differences in the specific with anti-GST antibodies to confirm equal amounts of purified protein in each reaction (lower panels). Each experiment was repeated at least twice with comparable results. activities of the purified kinase preparations will require further investigation. Note that Tyr in 186 193 The immunoblots from the binding assays shown in Fig. 1 the Abl SH3 domain and Tyr and Tyr in the SH2 domain were then reprobed with anti-phosphotyrosine antibodies. were not detectably phosphorylated, indicative of selectivity for Very strong phosphorylation of the Abl SH3-SH2 region was specific Tyr sites (data not shown). observed upon coexpression with Hck, Lyn, and Fyn (data not The positionsofthesenovelSrcfamilykinasetyrosinephospho- shown). To confirm this finding, we purified the GST-Abl32 rylation sites within the crystal structure of the c-Abl core region fusion protein and tested it as a substrate for recombinant Hck, (30) are shown in Fig. 2B. Intriguingly, four of these tyrosine resi- 89 134 158 191 Lyn, and Fyn in vitro. As shown in Fig. 1C, catalytic amounts of dues (Tyr and Tyr in the SH3 domain and Tyr and Tyr all three Src family kinases strongly phosphorylated the Bcr-Abl in the SH2 domain) lie along the interface between the SH3-SH2 SH3-SH2 region under these conditions. Lower levels of phos- region and the kinase domain. Because c-Abl kinase activity is phorylation were observed with equimolar amounts of GST- down-regulated in part through tight docking of the SH3-SH2 Abl SH2 and GST-Abl SH3 domain fusion proteins, suggesting “clamp” onto the back of the kinase domain, phosphorylation of that tethering of the domains is required for proper recognition residues along this interface could influence kinase activity, even in and phosphotransfer by Src family kinases. No phosphorylation the context of Bcr-Abl (see “Discussion”). 30910 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 •OCTOBER 13, 2006 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases FIGURE 3. Src family kinases phosphorylate full-length Bcr-Abl in the SH3- SH2 regulatory region. A, full-length kinase-dead Bcr-Abl was expressed as a C-terminal hexahistidine fusion protein in Sf9 insect cells, purified, and incu- bated in the presence () or absence () of recombinant Hck in vitro. Aliquots of each reaction were immunoblotted with anti-phosphotyrosine ( P-Tyr) and anti-Abl antibodies. The remainder of the reaction was digested with trypsin, and tryptic peptides were resolved by reverse-phase liquid chromatography and analyzed by MALDI-TOF-MS. B, shown are the mass spectra of the Tyr tryptic peptide (upper panel) and its Hck-phosphorylated counterpart (lower panel). This peptide is derived from the Bcr-Abl SH3-SH2 connector region and has a predicted mass of 1225.59 Da, whereas the phosphorylated form of the peptide has a predicted mass of 1305.55 Da (arrows). The amino acid sequences of both peptides were confirmed in MS/MS experiments (see Fig. 4). FIGURE 2. Src family kinases phosphorylate the Bcr-Abl SH3-SH2 tag on its C terminus. This form of Bcr-Abl cannot undergo auto- region at multiple tyrosine residues. A, the Abl SH3-SH2 region was phosphorylation, allowing for clear characterization of Src fam- expressed in E. coli as an N-terminal hexahistidine fusion protein (His - Abl32), purified, and incubated in vitro in the absence (control (Con)) or ily kinase-dependent phosphorylation events. Purified kinase- presence of recombinant Hck, Lyn, or Fyn. Aliquots of each reaction were dead Bcr-Abl (p210-KR) was incubated either alone or with a immunoblotted with anti-phosphotyrosine antibodies ( P-Tyr) or with an catalytic amount of recombinant Hck, and an aliquot of the phos- antibody to the histidine tag to confirm equal amounts of the Abl protein in each reaction (inset). The His -Abl32 protein is indicated by the phorylation reaction was analyzed by anti-phosphotyrosine im- arrows. The remainder of each reaction was digested overnight with tryp- munoblotting. As shown in Fig. 3, p210-KR was strongly phospho- sin, and the extent of tyrosine phosphorylation of each resulting peptide rylated in the presence of Hck. The phosphorylated p210-KR was determined by MALDI-TOF-MS. Blue bars, Hck; green bars, Lyn; yellow bars, Fyn. MS analysis was performed in duplicate, and the ratio of the protein was then digested with trypsin, and the resulting peptides mean peak intensities S.D. is shown. B, Src phosphorylation sites map to were separated by liquid chromatography. Each column fraction the SH3-SH2 region in the c-Abl crystal structure. The crystal structure of the c-Abl core is shown (Protein Data Bank code 1OPK) (30), with Src family was spotted onto a MALDI plate, and a total of 144 spectra were kinase phosphorylation sites numbered. SH3 domain residues are shown collected and analyzed. Using a peak picking routine, we were 134 89 in red (Tyr and Tyr ); the SH3-SH2 connector residue is shown in gray 147 158 191 able to identify ions corresponding to six of the phosphoty- (Tyr ); and the SH2 domain residues are shown in blue (Tyr , Tyr , 204 234 Tyr , and Tyr ). The SH2-kinase linker is shown in orange, and the rosine-containing peptides originally observed in the smaller kinase domain is rendered in violet. The location of bound myristic acid is 361 His -Abl32 construct (Fig. 2). Fig. 3B presents representative shown in green (Myr). The C-terminal lobe residue Tyr , which forms an aromatic stacking interaction with the SH2 domain residue Tyr , is also spectra for the tryptic peptides containing Tyr , a phosphoryla- shown. The three tyrosines that were not detectably phosphorylated tion site found in the SH3-SH2 connector. The peak at 1225.59 Da 112 186 193 (Tyr , Tyr , and Tyr ) are indicated in parentheses. corresponds to the Tyr tryptic peptide (HSWYHGPVSR), and the peak at 1305.55 Da corresponds to its phosphorylated coun- Hck Phosphorylates Full-length Bcr-Abl in the SH3-SH2 Re- terpart (HSWpYHGPVSR). Note the expected 80-Da shift in gion—We next investigated whether Src family kinases phospho- molecular mass corresponding to the covalent addition of a phos- rylate the Abl SH3-SH2 region within the context of the full-length phate group by Hck. The sequences of these peptides were con- Bcr-Abl protein. Full-length kinase-dead p210 Bcr-Abl was ex- firmed in subsequent MS/MS experiments, which are presented in pressed in Sf9 insect cells and purified by virtue of a hexahistidine Fig. 4. OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30911 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases The complete results of the MALDI-TOF-MS analysis of the fied and confirmed by MS/MS sequencing. A mass peak corre- Hck-phosphorylated, full-length Bcr-Abl p210-KR protein are sponding to the Tyr tryptic phosphopeptide (VYHYR) could summarized in Table 1. In addition to Tyr , mass peaks cor- not be found, despite strong phosphorylation of this site by Hck 134 158 responding to the Tyr and Tyr tryptic phosphopeptides in the His -Abl32 protein (Fig. 2). However, the unphospho- (along with their unphosphorylated counterparts) were identi- rylated form of the Tyr peptide was also absent, suggesting possible loss during chromatogra- phy due to the small size and hydro- phobic nature of this peptide. Candidate peaks for both the phos- phorylated and unphosphorylated 89 204 peptides containing Tyr , Tyr , and Tyr were detected, although low abundance prevented sequence confirmation by MS/MS analysis. The Tyr peptide is much longer in the context of full-length Bcr-Abl relative to His -Abl32, where it is directly adjacent to the hexahisti- dine tag; this may account for its low abundance. We also surveyed the full-length Bcr-Abl data set for phosphorylated tryptic peptides corresponding to reported sites of c-Abl and Bcr-Abl autophosphorylation and c-Abl transphosphorylation by Src family kinases, including Tyr in the SH2-kinase linker, Tyr in the kinase domain activation loop, and Tyr in the Bcr region (Table 1). Tryptic phosphopeptides corre- 177 412 sponding to Tyr and Tyr were detected (as were their unphospho- rylated counterparts), and their amino acid sequences and phospho- rylation states were confirmed by MS/MS sequencing. In contrast, the tryptic peptide containing Tyr or FIGURE 4. Confirmation of phosphorylation of Bcr-Abl SH3-SH2 connector peptides. Parent ions correspond- its phosphorylated counterpart ing to the Bcr-Abl SH3-SH2 connector tryptic peptide (HSWYHGPVSR) as well as its phosphorylated counterpart were subjected to collision-induced dissociation, and the product spectra for the unphosphorylated (A) and phos- could not be located, despite strong phorylated (B) forms are shown. Mass differences between peaks comprising the y ion series correspond to the reactivity of this site with a phos- partial amino acid sequences shown at the top. The y ion series in the phosphorylated peptide (B) is shifted by80 pho-specific antibody (see below). Da beginning with the y ion, indicating phosphorylation of the tyrosine. In the b ion series, the b ,b ,b ,b , and b 7 5 6 7 8 9 ions are similarly shifted. For simplicity, the identities of other ions present are not indicated. This is likely due to the limitations TABLE 1 Hck phosphorylates SH3-SH2 sites in full-length Bcr-Abl Tyrosine phosphopeptides Unphosphorylated peptides Residue Location Predicted mass Observed mass Predicted mass Observed mass Da Da Tyr SH3 3606.86 3606.67 3526.88 ND 134 b b Tyr SH3 2183.03 2183.01 2103.05 2103.05 147 b b Tyr SH3-SH2 connector 1305.57 1305.55 1225.59 1225.59 158 b b Tyr SH2 1990.99 1990.97 1911.01 1911.01 191 c Tyr SH2 817.35 ND 737.35 ND Tyr SH2 1020.45 1020.56 940.47 ND Tyr SH2 3162.59 3162.41 3082.61 ND 177 b b Tyr Bcr (Grb2 binding) 2701.21 2701.23 2621.23 2621.18 Tyr SH2-kinase linker 1661.77 ND 1581.79 1581.92 412 b b Tyr Activation loop 1516.66 1516.68 1436.68 1436.68 a 177 Numbering was based on the c-Abl crystal structure (30), except for Bcr-derived Tyr . Peptide identity was confirmed by MS/MS sequencing. Not detected. 30912 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 •OCTOBER 13, 2006 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases FIGURE 6. Phosphorylation of Bcr-Abl SH3 domain Tyr by Src family kinases in CML cells. A, validation of the anti-Abl phospho-Tyr antibody. The wild-type (WT) and Y89F mutant Abl SH3-SH2 regions were expressed in E. coli as N-terminal hexahistidine fusion proteins, purified, and incubated in vitro in the absence or presence of recombinant Hck as indicated. Aliquots of each reaction were immunoblotted with the anti-Abl phospho-Tyr anti- body ( pY89), a general anti-phosphotyrosine antibody ( pTyr), or with an anti- body to the histidine tag to confirm equal amounts of the Abl protein in each reaction (His). B, Bcr-Abl Tyr is phosphorylated in CML cell lines. Lysates from the Ph-positive CML cell lines K-562 and Meg-01 were probed with the anti- Abl phospho-Tyr antibody (upper panels) or with an anti-Abl antibody to verify Bcr-Abl protein expression (lower panels). Bcr-Abl bands are indicated by the arrows. Similar blots from Ph-negative TF-1 myeloid leukemia cells are included as a negative control. C, phosphorylation of multiple Bcr-Abl tyro- sine sites requires Src family kinase activity. Meg-01 CML cells were treated FIGURE 5. Src family kinases phosphorylate full-length Bcr-Abl in the overnight with the indicated micromolar concentrations of the Src family SH2-kinase linker, activation loop, and Grb2-binding sites. A, full-length kinase inhibitor A-419259. Cell lysates were probed with phospho-specific 89 177 kinase-dead Bcr-Abl was expressed in Sf9 insect cells either alone (control antibodies against Abl phospho-Tyr , Bcr phospho-Tyr ( pY177), Abl phos- 245 412 (Con)) or together with Hck, Lyn, or Fyn (left panels). Active Bcr-Abl was pho-Tyr ( pY245), and Abl phospho-Tyr ( pY412). Lysates were also expressed alone as a positive control (right panels). Bcr-Abl proteins were probed for Src family kinase autophosphorylation with the anti-Src phospho- immunoprecipitated using an anti-Abl antibody and immunoblotted with Tyr antibody (Src pY418) and for Bcr-Abl protein levels. Each experiment phospho-specific antibodies for the Grb2-binding site (Bcr phospho-Tyr was repeated at least twice with comparable results. ( pY177)), the Abl SH2-kinase linker (phospho-Tyr ( pY245)), the activation loop tyrosine (phospho-Tyr ( pY412)), and the Bcr-Abl protein. Each exper- Abl. For reference, the locations of Tyr in the SH2-kinase linker iment was repeated at least twice with comparable results. B, the location of 245 412 the linker (Tyr ) and activation loop (Tyr ) tyrosine residues are indicated and Tyr in the activation loop are mapped onto the crystal on the crystal structure of the c-Abl core (30); domains are colored as 177 structure of the c-Abl core in Fig. 5B. described in the legend to Fig. 2. Bcr-Abl Tyr is found in the N-terminal Src Family Kinase-dependent Phosphorylation of the Bcr-Abl Bcr-derived portion of the protein and is not present in this structure. SH3 Domain in CML Cells—We next investigated whether of separation and detection of the large number of Bcr-Abl phosphorylation of the Bcr-Abl SH3-SH2 region by Src family peptides. kinases occurs in the context of CML cells. For these experi- To complement the MS analysis of Bcr-Abl tyrosine phospho- ments, we focused on Tyr , the SH3 site most strongly phos- rylation, we also used an immunoblot strategy with phospho-spe- phorylated by Hck, Lyn, and Fyn in vitro (Fig. 2). To probe Tyr cific antibodies. For these experiments, full-length kinase-dead phosphorylation in cells, we raised a phospho-specific antibody Bcr-Abl was expressed alone or together with Hck, Lyn, or Fyn in against this site and validated it using the recombinant His - Sf9cells.ActiveBcr-Ablwasalsoexpressedalonetogaugeautophos- Abl32 protein originally used to map the phosphorylation sites phorylation. Bcr-Abl proteins were then immunoprecipitated and by MS. As shown in Fig. 6A, the anti-Abl phospho-Tyr antibody probed with antibodies specific for phosphorylation of Bcr Tyr , did not recognize the unphosphorylated His -Abl32 protein. 245 412 Tyr , and Tyr . As shown in Fig. 5A, strong phosphorylation of However, incubation of the protein with Hck prior to immuno- all three sites by Hck, Lyn, and Fyn was observed using this blotting resulted in a strong signal with the antibody. The experi- approach, suggesting that these sites are transphosphorylated by ment was then repeated with a mutant form of His -Abl32in Src family kinases in full-length Bcr-Abl. Note that all three sites which Tyr was replaced with Phe (Y89F mutant). This mutant were also strongly autophosphorylated within kinase-active Bcr- form of His -Abl32 did not react with the anti-Abl phospho- OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30913 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases Tyr antibody following incubation with Hck, indicating that the antibody is specific for Bcr-Abl phospho-Tyr . A replicate filter with the same four samples was then probed with a general anti-phosphotyrosine antibody. In this case, immunoreac- tivity was reduced but not eliminated with the Y89F form of His -Abl32 following phosphorylation by Hck in compar- ison with the wild-type protein. This observation agrees with the MS showing that Tyr is the preferred site of phospho- rylation for Src family kinases in this protein (Fig. 2). Using the anti-Abl phospho-Tyr antibody, we next investi- gated the phosphorylation of Tyr in the CML-derived cell lines K-562 and Meg-01. Our previous work has shown that these two cell lines are very sensitive to Src family kinase inhib- itors in terms of growth arrest and programmed cell death (18). Fig. 6B shows that lysates from both CML cell lines exhibited a band of 210 kDa that reacted strongly with the anti-Abl phos- pho-Tyr antibody, indicating that the SH3 domain of Bcr-Abl is phosphorylated at this site in cells. In contrast, the Ph-nega- tive leukemia cell line TF-1 showed no immunoreactivity with the anti-Abl phospho-Tyr antibody, consistent with the lack of Bcr-Abl expression in this cell line. As expected, control immunoblots showed expression of p210 Bcr-Abl protein in FIGURE 7. Tyrosine phosphorylation of the SH3-SH2 region is essential K-562 and Meg-01 cells, but not in TF-1 cells. forfullBcr-Ablbiologicalactivity.ThesevenBcr-AblSH3-SH2tyrosinephos- phorylation sites for Src family kinases identified by MS were replaced with To implicate Src family kinases in the phosphorylation of the Phe in full-length Bcr-Abl (7YF mutant). Tyr was restored in the context of Bcr-Abl SH3 domain in CML cells, we employed the Src family the 7YF mutant to create mutant 6YF. A single point mutant of Tyr was also created (Y89F). Each of these mutants, as well as wild-type (WT ) Bcr-Abl, were kinase inhibitor A-419259 (31). Previous studies by our group expressed in the human GM-CSF-dependent myeloid progenitor cell line TF-1 have shown that this compound blocks Src family kinase using recombinant retroviruses. A, GM-CSF-independent proliferation of activity in vitro in the low nanomolar range, but is at least 2 each cell line was monitored using the CellTiter-Blue cell viability assay as described under “Materials and Methods.” Cells infected with a green fluores- orders of magnitude less active against the Abl kinase domain cent protein retrovirus served as negative control (Con). The experiment was (18, 26). A-419259 also induces growth suppression and apo- performed in triplicate, and the mean -fold increase in cell number  S.D. ptotic cell death in both the K-562 and Meg-01 CML cell lines, is shown at each time point. B, lysates from each of the cell lines shown in A were probed with phospho-specific antibodies against Bcr-Abl phospho-Tyr (pY89) but does not affect Ph-negative myeloid leukemia cells (18). To and phospho-Tyr (pY412). Replicate membranes were also probed with a gen- determine whether Src family kinase activity is required for eral anti-phosphotyrosine antibody (pTyr) and with anti-Abl antibodies to con- trol for Bcr-Abl expression. Blotting was performed on two separate infected cell phosphorylation of Bcr-Abl at Tyr , Meg-01 cells were treated populations and produced the same result in each case. with a range of A-419259 concentrations, followed by immu- noblotting with the anti-Abl phospho-Tyr antibody. As 89 89 shown in Fig. 6C, phosphorylation of Abl Tyr was blocked in Phosphorylation of Tyr and Other Sites in the SH3-SH2 a concentration-dependent manner, with IC  300 nM, and Region Is Required for Full Bcr-Abl Transforming Function—To phosphorylation of this site was completely blocked at 1 M. determine whether tyrosine phosphorylation of the SH3-SH2 This dose response for inhibition of Abl Tyr phosphorylation region is important for Bcr-Abl function, we engineered a closely paralleled suppression of Src family kinase autophospho- mutant form of Bcr-Abl (7YF) in which phenylalanine replaced rylation (Src phospho-Tyr immunoblot), strongly implicating each of the seven SH3-SH2 tyrosine phosphorylation sites for Src family kinases in the phosphorylation of the Bcr-Abl SH3 Src family kinases shown in Fig. 2 and Table 1. The 7YF mutant domain at Tyr in CML cells. Control blots showed equivalent was then compared with wild-type Bcr-Abl in terms of its abil- levels of Bcr-Abl protein in each sample. Similar results were ity to transform the human TF-1 myeloid cell line to cytokine obtained with K-562 cells (data not shown). independence. TF-1 cells require GM-CSF or interleukin-3 for The data presented in Fig. 5 and Table 1 show that Hck phos- growth and survival and undergo apoptosis following cytokine 177 245 412 phorylated Bcr-Abl at Bcr Tyr , Abl Tyr , and Abl Tyr in withdrawal (27); introduction of Bcr-Abl with a recombinant vitro, all of which are known sites of Bcr-Abl phosphorylation. retrovirus reverses the cytokine dependence. TF-1 cells were To evaluate whether Hck and other Src kinases contribute to infected with wild-type and 7YF mutant Bcr-Abl retroviruses, phosphorylation of these sites in CML cells, lysates from the and cell growth in the absence of GM-CSF was measured over 3 A-419259-treated Meg-01 cells were probed with phospho- days. Fig. 7A shows that the cytokine-independent proliferation specific antibodies directed against these sites. As shown in Fig. of TF-1 cells expressing the 7YF mutant was reduced by 50% 6C, A-419259 treatment also substantially reduced the phos- relative to cells expressing wild-type Bcr-Abl, providing evi- phorylation of these sites, suggesting that Src family kinases dence that phosphorylation of the Bcr-Abl SH3-SH2 region is may have a major role in maintaining the active conformation required for full transforming activity. of Bcr-Abl in CML cells (see “Discussion”). Very similar results Because Tyr is most prominent among the Src family were obtained with K-562 cells (data not shown). kinase phosphorylation sites in the Bcr-Abl SH3-SH2 region, we 30914 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 •OCTOBER 13, 2006 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases next investigated whether adding back this single phosphorylation of Bcr-Abl can be complemented by mutations predicted to site restores the biological activity of the 7YF mutant in the TF-1 disrupt SH3/linker interaction, consistent with the idea that the cell transformation assay. This mutant, termed 6YF, showed trans- SH3 domain still exerts some negative regulatory influence over forming activity intermediate to that observed with wild-type Bcr- Bcr-Abl tyrosine kinase activity. Phosphorylation of the SH3 89 134 Abl and the 7YF mutant. In addition, we created a Bcr-Abl point domain at Tyr or Tyr by Src family kinases as described here mutant in which Tyr alone was replaced with Phe (Y89F). This may have a similar destabilizing effect, as these sites come in close mutant also exhibited transforming activity intermediate to that of proximitytotheSH2-kinaselinker.Indeed,phosphorylationofthe wild-type Bcr-Abl and the 7YF mutant. Taken together, these Abl SH3-SH2 region by Hck caused dissociation of the SH2-kinase 89 4 results suggest that, although phosphorylation of Tyr is required linker as determined by hydrogen exchange MS. for full transforming activity, the other tyrosine phosphorylation In addition to the SH3 sites, several of the other tyrosine sites within the SH3-SH2 region are likely to contribute to Bcr-Abl residues phosphorylated by Src family kinases localize to the function in CML cells. interface between the SH3-SH2 regulatory clamp and the large Finally, we investigated the impact of SH3-SH2 phosphoryl- lobe of the c-Abl kinase domain (Fig. 2). Particularly interesting 158 158 ation site mutagenesis on Bcr-Abl at the biochemical level by is the SH2 domain residue Tyr . The aromatic rings of Tyr immunoblotting lysates from each of the transformed TF-1 cell and the kinase domain residue Tyr stack together in the populations with phospho-specific antibodies. As shown in Fig. down-regulated c-Abl structure, and the hydroxyl group of 7B, wild-type Bcr-Abl reacted strongly with the anti-Abl phos- Tyr forms a hydrogen bond with the backbone carbonyl 89 393 pho-Tyr antibody, indicating that this site is phosphorylated group of Asn of the kinase domain (30). Interestingly, muta- in TF-1 cells in a manner similar to that in the CML cell lines tion of Tyr to glutamate results in a nearly 4-fold stimulation (Fig. 6B). The immunoreactivity of the anti-Abl phospho-Tyr of c-Abl kinase activity compared with the wild type (35). Phos- antibody was sharply reduced in TF-1 cells expressing the Bcr- phorylation of this site by Src family kinases may also disrupt Abl 7YF and Y89F mutants, but was completely restored with kinase regulation of c-Abl and raises the possibility of a similar the Tyr add-back mutant, 6YF. In contrast, phosphorylation regulatory interaction in Bcr-Abl. of the Bcr-Abl activation loop (phospho-Tyr immunoblot) Other recent studies show that mutations outside of the cat- and the overall Bcr-Abl phosphotyrosine content (phospho- alytic domain can allosterically affect not only Bcr-Abl kinase Tyr immunoblot) were not remarkably changed between the activity but sensitivity to imatinib as well. For example, Azam wild-type and mutant Bcr-Abl proteins, supporting the idea et al. (36) used an unbiased random mutagenesis screen to that the Tyr-to-Phe substitutions in the SH3-SH2 region do not uncover novel mutations in Bcr-Abl that confer imatinib resist- simply disrupt the folding of the Bcr-Abl protein. Notably, all ance and mapped these residues onto the autoinhibited c-Abl four TF-1 cell populations expressed equivalent levels of Bcr- structure. These authors uncovered a striking correlation Abl protein (Abl immunoblot). between residues that impair c-Abl negative regulation and imatinib resistance of Bcr-Abl, again strongly suggesting that DISCUSSION mechanisms governing c-Abl autoinhibition are retained in This work provides the first evidence for tyrosine phospho- Bcr-Abl. These observations agree with previous work showing rylation of the Bcr-Abl SH3-SH2 regulatory region by Src fam- that imatinib favors the down-regulated conformation of the Abl ily kinases both in vitro and in CML cells. The possible impact catalytic cleft for binding (33, 37). Notably, several of these ima- of these phosphorylation events on Bcr-Abl function requires tinib resistance mutations localize to SH3, SH2, and kinase further consideration of c-Abl structure and regulation. domain residues that contribute to the negative regulatory inter- Although Bcr-Abl exhibits constitutive tyrosine kinase activity, face. In particular, Azam et al. (36) found that substitution of the Hantschel and Superti-Furga (32) have proposed that Bcr-Abl Bcr-Abl SH3 domain residue Tyr , which is strongly phosphoryl- may retain some of the regulatory features observed in the ated by Hck, Lyn, and Fyn in vitro (Fig. 2) and in CML cells (Fig. 6), recent x-ray crystal structures of the c-Abl core (see Fig. 2) (30, resulted in Bcr-Abl imatinib resistance in four independent iso- 33). These structures show that, in the down-regulated confor- lates. Phosphorylation of this site by Src family kinases may also mation, the c-Abl SH3 domain engages the polyproline type II stabilize the active conformation of Bcr-Abl and contribute to its helix formed by the SH2-kinase linker in an intramolecular transforming activity. The reduced transforming activity of the fashion, as is the case for c-Src and Hck (21). Unlike Src kinases, Y89F mutant in TF-1 cells also supports this view (Fig. 7). however, the SH2 domain docks onto the back of the C-termi- In addition to novel sites in the SH3-SH2 region, we found nal lobe of the Abl kinase domain. This interaction is stabilized that Hck, Lyn, and Fyn phosphorylate full-length Bcr-Abl at 177 245 412 by binding of the myristoylated N-terminal cap through a Tyr in the Bcr region and at Tyr and Tyr in the Abl unique pocket in the kinase domain (Fig. 2). Residues linking kinase domain (Fig. 5 and Table 1). Although phosphorylation the SH3 and SH2 domains form a rigid connector that dynam- of Bcr-Abl Tyr by Src family kinases was readily detected ically couples the SH3 and SH2 domains, which together pro- both in vitro and in CML cells using a phospho-specific anti- vide a regulatory clamp that allosterically holds the kinase body (Figs. 5 and 6), we were unable to locate this phosphopep- domain in the closed inactive state (30). Although the regula- tide (or its unphosphorylated counterpart) by MS. This dis- tory impact of myristoylation is lacking in Bcr-Abl, some evi- crepancy most likely reflects technical limitations of the dence suggests that SH3/linker interaction may be retained. For example, Smith et al. (34) showed that a transformation defect associated with mutations in the N-terminal coiled-coil region S. Chen, T. E. Smithgall, and J. R. Engen, unpublished data. OCTOBER 13, 2006• VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30915 Phosphorylation of Bcr-Abl SH3-SH2 by Src Family Kinases N., Batzer, A., Rabun, K. M., Der, C. J., Schlessinger, J., and Gishizky, M. L. MS-based approach. Experiments with the selective inhibitor (1993) Cell 75, 175–185 A-419259 strongly suggested that Src family kinases contribute 9. Puil, L., Liu, J., Gish, G., Mbamalu, G., Bowtell, D., Pelicci, P. G., Arling- to the phosphorylation of these sites in CML cells as well (Fig. haus, R., and Pawson, T. (1994) EMBO J. 13, 764–773 6). Previous work has shown that Hck phosphorylates Bcr 10. Goga, A., McLaughlin, J., Afar, D. E., Saffran, D. C., and Witte, O. N. (1995) Tyr (19), creating a docking site for the Grb2-Sos guanine Cell 82, 981–988 245 412 nucleotide exchanger for Ras (8, 9). Tyr and Tyr have been 11. Senechal, K., Halpern, J., and Sawyers, C. L. (1996) J. Biol. Chem. 271, established as sites of both c-Abl autophosphorylation (38) and 23255–23261 12. Skorski, T., Bellacosa, A., Nieborowska-Skorska, M. N., Majewski, M., transphosphorylation by Src family kinases (discussed below) Martinez, R., Choi, J. K., Trotta, R., Wlodarski, P., Perrotti, D., Chan, T. O., (39, 40), and phosphorylation of these sites strongly up-regu- Wasik, M. A., Tsichlis, P. N., and Calabretta, B. (1997) EMBO J. 16, lates c-Abl kinase activity (38). 6151–6161 Several studies have linked Src family kinases to c-Abl regu- 13. Carlesso, N., Frank, D. A., and Griffin, J. D. (1996) J. Exp. Med. 183, lation and signaling, which have important implications for 811–820 Bcr-Abl function and drug sensitivity. Plattner et al. (39) dem- 14. Ilaria, R. L., Jr., and Van Etten, R. A. (1996) J. Biol. Chem. 271, 31704–31710 15. Lionberger, J. M., and Smithgall, T. E. (2000) Cancer Res. 60, 1097–1103 onstrated that the kinase activity of c-Abl increases 10–20-fold 16. Xie, S., Wang, Y., Liu, J., Sun, T., Wilson, M. B., Smithgall, T. E., and in the presence of constitutively active v-Src in mouse Ba/F3 Arlinghaus, R. B. (2001) Oncogene 20, 6188–6195 hematopoietic cells and 10T1/2 fibroblasts. This increase in 17. Lionberger, J. M., Wilson, M. B., and Smithgall, T. E. (2000) J. Biol. Chem. c-Abl kinase activity directly correlates with phosphorylation of 275, 18581–18585 c-Abl by Src or Fyn. Dorey et al. (40) showed that active c-Src 18. Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. can phosphorylate the c-Abl activation loop at Tyr and (2002) Oncogene 21, 8075–8088 19. Warmuth, M., Bergmann, M., Priess, A., Hauslmann, K., Emmerich, B., enhance the ability of c-Abl to phosphorylate the downstream and Hallek, M. (1997) J. Biol. Chem. 272, 33260–33270 substrate c-Jun. A similar study provided evidence that c-Src 412 245 20. Danhauser-Riedl, S., Warmuth, M., Druker, B. J., Emmerich, B., and phosphorylates c-Abl at both Tyr and Tyr in NIH 3T3 Hallek, M. (1996) Cancer Res. 56, 3589–3596 cells and that dual phosphorylation by c-Src is required for 21. Boggon, T. J., and Eck, M. J. (2004) Oncogene 23, 7918–7927 c-Abl function (41). Consistent with this report, Brasher and 22. Shah, N. P., Tran, C., Lee, F. Y., Chen, P., Norris, D., and Sawyers, C. L. Van Etten (38) proposed that phosphorylation of Tyr (2004) Science 305, 399–401 23. O’Hare, T., Corbin, A. S., and Druker, B. J. (2006) Curr. Opin. Genet. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Oct 13, 2006

References