Global Effects of BCR/ABL and TEL/PDGFRβ Expression on the Proteome and Phosphoproteome
2004; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês
10.1074/jbc.m410598200
ISSN1083-351X
AutoresRichard D. Unwin, David Sternberg, Yuning Lu, Andrew Pierce, D. Gary Gilliland, Anthony D. Whetton,
Tópico(s)Multiple Myeloma Research and Treatments
ResumoMany leukemic oncogenes form as a consequence of gene fusions or mutation that result in the activation or overexpression of a tyrosine kinase. To identify commonalities and differences in the action of two such kinases, breakpoint cluster region (BCR)/ABL and TEL/PDGFRβ, two-dimensional gel electrophoresis was employed to characterize their effects on the proteome. While both oncogenes affected expression of specific proteins, few common effects were observed. A number of proteins whose expression is altered by BCR/ABL, including gelsolin and stathmin, are related to cytoskeletal function whereas no such changes were seen in TEL/PDGFRβ-transfected cells. Treatment of cells with the kinase inhibitor STI571 for 4-h reversed changes in expression of some of these cytoskeletal proteins. Correspondingly, BCR/ABL-transfected cells were less responsive to chemotactic and chemokinetic stimuli than non-transfected cells and TEL/PDGFRβ-transfected Ba/F3 cells. Decreased motile response was reversed by a 16-h treatment with STI571. A phosphoprotein-specific gel stain was used to identify TEL/PDGFRβ and BCR/ABL-mediated changes in the phosphoproteome. These included changes on Crkl, Ras-GAP-binding protein 1, and for BCR/ABL, cytoskeletal proteins such as tubulin, and Nedd5. Decreased phosphorylation of Rho-GTPase dissociation inhibitor (Rho GDI) was also observed in BCR/ABL-transfected cells. This results in the activation of the Rho pathway, and treatment of cells with Y27632, an inhibitor of Rho kinase, inhibited DNA synthesis in BCR/ABL-transfected Ba/F3 cells but not TEL/PDGFRβ-expressing cells. Expression of a dominant-negative RhoA inhibited both DNA synthesis and transwell migration, demonstrating the significance of this pathway in BCR/ABL-mediated transformation. Many leukemic oncogenes form as a consequence of gene fusions or mutation that result in the activation or overexpression of a tyrosine kinase. To identify commonalities and differences in the action of two such kinases, breakpoint cluster region (BCR)/ABL and TEL/PDGFRβ, two-dimensional gel electrophoresis was employed to characterize their effects on the proteome. While both oncogenes affected expression of specific proteins, few common effects were observed. A number of proteins whose expression is altered by BCR/ABL, including gelsolin and stathmin, are related to cytoskeletal function whereas no such changes were seen in TEL/PDGFRβ-transfected cells. Treatment of cells with the kinase inhibitor STI571 for 4-h reversed changes in expression of some of these cytoskeletal proteins. Correspondingly, BCR/ABL-transfected cells were less responsive to chemotactic and chemokinetic stimuli than non-transfected cells and TEL/PDGFRβ-transfected Ba/F3 cells. Decreased motile response was reversed by a 16-h treatment with STI571. A phosphoprotein-specific gel stain was used to identify TEL/PDGFRβ and BCR/ABL-mediated changes in the phosphoproteome. These included changes on Crkl, Ras-GAP-binding protein 1, and for BCR/ABL, cytoskeletal proteins such as tubulin, and Nedd5. Decreased phosphorylation of Rho-GTPase dissociation inhibitor (Rho GDI) was also observed in BCR/ABL-transfected cells. This results in the activation of the Rho pathway, and treatment of cells with Y27632, an inhibitor of Rho kinase, inhibited DNA synthesis in BCR/ABL-transfected Ba/F3 cells but not TEL/PDGFRβ-expressing cells. Expression of a dominant-negative RhoA inhibited both DNA synthesis and transwell migration, demonstrating the significance of this pathway in BCR/ABL-mediated transformation. Chronic myeloid leukemia (CML) 1The abbreviations used are: CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; PDGFR, platelet-derived growth factor receptor; IL, interleukin; MGG, May-Grunwald-Giemsa; IPG, immobilized pH gradient; MALDI, matrix-assisted laser desorption/ionization; SDF-1, stromal cell-derived factor; LPA, lysophosphatidic acid; GDI, GDP dissociation inhibitor; GAP, GTPase-activating protein; TTL, tubulin-tyrosine ligase; Rho GDI, Rho GDP dissociation inhibitor; PCNA, proliferating cell nuclear antigen; Arp, actin-related protein; STAT, signal transducer and activator of transcription; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; MAP, mitogen-activated protein. is a disease with a characteristic t(9:22) chromosomal translocation giving rise to the Philadelphia chromosome (1Cortes J.E. Talpaz M. Kantarjian H. Am. J. Med. 1996; 100: 555-570Abstract Full Text PDF PubMed Scopus (131) Google Scholar). This translocation results in the juxtaposition of the BCR (breakpoint cluster region) gene and the c-ABL oncogene resulting in the constitutive expression of a BCR/ABL fusion oncoprotein (2Ben-Neriah Y. Daley G.Q. Mes-Masson A.M. Witte O.N. Baltimore D. Science. 1986; 233: 212-214Crossref PubMed Scopus (679) Google Scholar). Another chimeric leukemogenic oncogene product, TEL/PDGFRβ, isolated from chronic myelomonocytic leukemia (CMML) patients bearing a t(5, 12) translocation, also has constitutive tyrosine kinase activity. The transforming ability of BCR/ABL and TEL/PDGFRβ resides in their protein-tyrosine kinase activity. A number of signaling proteins activated by BCR/ABL have been identified including Ras, STAT5, protein kinase C, and phosphatidylinositol (PI) 3-kinase (3Tauchi T. Boswell H.S. Leibowitz D. Broxmeyer H.E. J. Exp. 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Blood. 2000; 96: 3343-3356Crossref PubMed Google Scholar. TEL/PDGFRβ can also activate signaling proteins such as PI 3-kinase, STAT1, STAT5, and stress-activated protein kinase (10Dierov J. Xu Q. Dierova R. Carroll M. Blood. 2002; 99: 1758-1765Crossref PubMed Scopus (30) Google Scholar, 11Wilbanks A.M. Mahajan S. Frank D.A. Druker B.J. Gilliland D.G. Carroll M. Exp. Hematol. 2000; 28: 584-593Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12Carroll M. Tomasson M.H. Barker G.F. Golub T.R. Gilliland D.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14845-14850Crossref PubMed Scopus (246) Google Scholar). As with BCR/ABL, Myc is an essential element in transformation by TEL/PDGFRβ (13Bourgeade M.F. Defachelles A.S. Cayre Y.E. Blood. 1998; 91: 3333-3339Crossref PubMed Google Scholar). Recently, an inhibitor of the BCR/ABL protein-tyrosine kinase activity has shown great promise for the treatment of CML. STI571 (also known as Imatinib mesylate or Gleevec™) can inhibit BCR/ABL kinase activity both in vitro and in intact cells. STI571 can inhibit proliferation and induce apoptosis in BCR/ABL- and TEL/PDGFRβ-expressing cells (14Carroll M. Ohno-Jones S. Tamura S. Buchdunger E. Zimmermann J. Lydon N.B. Gilliland D.G. Druker B.J. Blood. 1997; 90: 4947-4952Crossref PubMed Google Scholar, 15Gambacorti-Passerini C. le Coutre P. Mologni L. Fanelli M. Bertazzoli C. Marchesi E. Di Nicola M. Biondi A. Corneo G.M. Belotti D. Pogliani E. Lydon N.B. Blood Cells Mol. Dis. 1997; 23: 380-394Crossref PubMed Scopus (302) Google Scholar). Clinical trials using STI571 have demonstrated impressive hematologic and cytogenetic responses in CML patients (16Kantarjian H.M. Cortes J.E. O'Brien S. Giles F. Garcia-Manero G. Faderl S. Thomas D. Jeha S. Rios M.B. Letvak L. Bochinski K. Arlinghaus R. Talpaz M. 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Microarray experiments on primary CML samples have shown that the expression of BCR/ABL can alter transcript levels of genes involved in a wide variety of cellular processes, with poor disease prognosis in chronic phase being associated with changes in DNA repair, cell cycle, and cell adhesion pathways, as well as STAT5 and Myc pathway targets. Whether these are directly attributable to BCR/ABL activity or secondary changes within the tumor was not determined (19Nowicki M.O. Pawlowski P. Fischer T. Hess G. Pawlowski T. Skorski T. Oncogene. 2003; 22: 3952-3963Crossref PubMed Scopus (102) Google Scholar). Studies using transfected cells plus screening for altered transcript levels by subtractive hybridization show alterations in genes involved in, for example, the MAP kinase (TOPK, or T-LAK cell-originated protein, a MAP kinase kinase), ubiquitination (HSPC150), or protein transport (NUP98 and RAN) pathways (20Salesse S. Verfaillie C.M. Mol. Cancer Ther. 2003; 2: 173-182Crossref PubMed Scopus (30) Google Scholar). A recent study using an inducible p210BCR/ABL system screened using cDNA microarrays revealed that BCR/ABL up-regulates a range in interferon-inducible genes, as well as transcription factors (STAT1, JUN), and cell growth and differentiation-related genes (PCNA, REL, Stathmin) (21Hakansson P. Segal D. Lassen C. Gullberg U. Morse 3rd, H.C. Fioretos T. Meltzer P.S. Exp. Hematol. 2004; 32: 476-482Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The situation with TEL/PDGFRβ-expressing cells remains relatively uninvestigated. BCR/ABL can also initiate key changes that occur only at the proteome level. However, this phenomenon has also not been explored for TEL/PDGFRβ. BCR/ABL is known to decrease expression of the Abl inhibitory proteins via a proteasome-mediated mechanism (22Dai Z. Quackenbush R.C. Courtney K.D. Grove M. Cortez D. Reuther G.W. Pendergast A.M. Genes Dev. 1998; 12: 1415-1424Crossref PubMed Scopus (104) Google Scholar). Other proteins whose expression alters with no apparent change in mRNA abundance as a consequence of BCR/ABL action include p53 (down-regulated as a result of post-translational-mediated MDM2 overexpression) (23Francis J.M. Heyworth C.M. Spooncer E. Pierce A. Dexter T.M. Whetton A.D. J. Biol. Chem. 2000; 275: 39137-39145Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 24Trotta R. Vignudelli T. Candini O. Intine R.V. Pecorari L. Guerzoni C. Santilli G. Byrom M.W. Goldoni S. Ford L.P. Caligiuri M.A. Maraia R.J. Perrotti D. Calabretta B. Cancer Cell. 2003; 3: 145-160Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) and C/EBPα, whose mRNA is bound by an inhibitory poly(rC)-binding protein hnRNP E2 (25Perrotti D. Calabretta B. Oncogene. 2002; 21: 8577-8583Crossref PubMed Scopus (50) Google Scholar). These proteins have important functions in hematopoietic cell survival and differentiation; thus, post-translational regulation of protein levels can be seen to have a role in transformation processes. A model system for comparing the transforming effects of oncogenes is the Ba/F3 murine cytokine-dependent cell line transformed with BCR/ABL and TEL/PDGFRβ, respectively (26Daley G.Q. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9312-9316Crossref PubMed Scopus (524) Google Scholar). We have used two-dimensional gel electrophoresis with the objective of comparing the effects of BCR/ABL and TEL/PDGFRβ on Ba/F3 cells by studying protein expression and phosphoprotein profiles with or without treatment with STI571. Very different outcomes are generated in Ba/F3 cells expressing these chimeric tyrosine kinases indicating pleiotropic mechanisms lead to or are associated with transformation by these 2 leukemogenic kinases. Cell Lines—Ba/F3 cells were transduced with either an empty MSCV (murine stem cell virus) retroviral vector or MSCV containing the BCR/ABL or TEL/PDGFRβ gene, respectively. The resultant Ba/F3-BCR/ABL or Ba/F3-TEL/PDGFRβ cells were maintained in suspension in culture in RPMI with 10% (v/v) fetal bovine serum. Ba/F3-MSCV cells were grown in RPMI with 10% (v/v) fetal bovine serum supplemented with 1 mg/ml G418 (Sigma) and 1 ng/ml IL-3 (R&D Systems, Minneapolis, MN). Prior to lysis, cells were either starved for 4 h in RPMI with 1% (w/v) bovine serum albumin (Sigma) and, where appropriate, 5 μm STI571, or were treated with 50 μm Y27632 (Merck Biosciences, Nottingham, UK) for 6 h. Cells were washed twice in ice-cold PBS and once in ice-cold 250 mm sucrose with 0.4 mm sodium orthovanadate (Sigma), and cell pellets were stored at –80 °C until use. Further transduction with Rho N19 was performed as described previously (27Pierce A. Owen-Lynch P.J. Spooncer E. Dexter T.M. Whetton A.D. Oncogene. 1998; 17: 667-672Crossref PubMed Scopus (33) Google Scholar). Successfully transduced cells were selected for expression of green fluorescent protein marker using a flow cytometer (BD Biosciences). Cells were stained using May-Grunwald-Giemsa (MGG) stain as described previously (27Pierce A. Owen-Lynch P.J. Spooncer E. Dexter T.M. Whetton A.D. Oncogene. 1998; 17: 667-672Crossref PubMed Scopus (33) Google Scholar). Two-dimensional Gel Electrophoresis—Cells were lysed in 9 m urea, 2 m thiourea, 4% (w/v) CHAPS, 1% (w/v) dithiothreitol, and 2% IPG buffers (Amersham Biosciences, Little Chalfont, UK), and protein concentration was determined using the Bio-Rad modified Bradford protein assay. For silver-stained gels, 100 μg of protein (1 mg for gels used for spot identification) was loaded by in-gel rehydration onto 24 cm, pH 3–10 nl IPG strips (Amersham Biosciences) in a total volume of 450 μl of lysis buffer with a trace of Orange G (Sigma). For ProQ® Diamond (Molecular Probes, Leiden, The Netherlands)-stained gels, 500 μg of protein were loaded onto 18-cm pH 4–7 IPG strips (Amersham Biosciences) in a 350-μl final volume. Strips were rehydrated at room temperature overnight, transferred to a Multiphor II apparatus (Amersham Biosciences), and protein focused over 2 days for a total of 115 kV h. Second dimension separation was carried out on 10%T SDS-PAGE gels with 4%T stacking gel using a Hoeffer vertical electrophoresis system (Amersham Biosciences) at 18 mA/gel overnight until the dye front reached the end of the gel. Detailed protocols can be found at www.lrf.umist.ac.uk. Silver Staining—Analytical gels were stained using a silver staining kit from OWL separation systems (Portsmouth, NH) employing a modified protocol, as described in Ref. 28Unwin R.D. Craven R.A. Harnden P. Hanrahan S. Totty N. Knowles M. Eardley I. Selby P.J. Banks R.E. Proteomics. 2003; 3: 1620-1632Crossref PubMed Scopus (214) Google Scholar. Preparative grade gels were stained with the mass spectrometry-compatible silver stain of Shevchenko et al. (29Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7883) Google Scholar). ProQ® Diamond and Coomassie Blue Staining—Gel staining with ProQ® Diamond was carried out according to the manufacturer's instructions. Briefly, gels were fixed in 50% (v/v) methanol, 10% (w/v) trichloroacetic acid overnight, followed by a second fix for 1 h, washed in water 4× 15 min, and then stained with ProQ® Diamond for 4 h in the dark. Gels were then destained with 50 mm sodium acetate (pH 4.0) with 4% (v/v) acetonitrile for 2 × 1 h, and a third wash overnight. Gels were imaged on a Typhoon™ 8600 scanner (Amersham Biosciences) using 532-nm excitation and 610-nm emission filters, with photomultiplier tube voltage set at 600 V. Selected gels were subsequently stained with Coomassie Blue to visualize the pattern of total protein and determine the specificity of the ProQ® Diamond stain. Gels were washed in water for 30 min, then stained in 10% (w/v) ammonium sulfate/2% (v/v) phosphoric acid with 0.1% Coomassie Blue G (Sigma) for 48 h. Staining solution was made up at least 24 h before use and diluted 4 parts stain to 1 part methanol before use. The gels were destained briefly in 50% (v/v) methanol and scanned on a Molecular Imager FX (Bio-Rad). Gels were stained with ProQ® Diamond, imaged, and stored until analysis had been completed. Spots were then excised from these gels using as a template a 1:1 scale image of the gel. Following cutting, gels were rescanned to ensure that the correct spot had been excised. Image Analysis—All gel analysis was performed using Progenesis (Non-linear Dynamics, Newcastle, UK) software. Changes in spot intensity were deemed significant where the average normalized volume altered by greater than 1.5-fold between samples, with p < 0.05 from a Student's t test on 3 or more replicate gels. Spot-normalized volume defines the volume of a given spot as a percentage of the total volume of all spots in the gel. In-gel Digestion and Protein Identification—Spots excised from silver-stained gels were first destained using a 50:50 mixture of 30 mm potassium III ferricyanide and 100 mm sodium thiosulfate. All spots were washed twice in water, then equilibrated three times with 25 mm ammonium bicarbonate, and dried by three washes in acetonitrile. Dried gel pieces were rehydrated in 25 ng/μl trypsin (Promega, Southampton, UK) in 25 mm ammonium bicarbonate on ice for 20 min. A further 20 μl of 25 mm ammonium bicarbonate was added to prevent drying out, and the gel was incubated at 37 °C overnight. Supernatant was then dried to around 2 μl in a SpeedVac centrifuge. Remaining peptides were extracted from the gel piece by addition of 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and sonication for 15 min. Extracted peptides were added to the concentrated supernatant and dried in a SpeedVac centrifuge. Peptides were reconstituted in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and spotted along with 0.5 μl of 10 mg/ml α-cyano 4-hydroxycinnamic acid (CHCA, Sigma) in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid onto a MALDI target for analysis using a reflectron MALDI-ToF mass spectrometer (M@LDI, Micromass, Manchester). Spectra were internally calibrated using trypsin autolysis fragments at m/z 842.509 or 2211.104. Tandem MS experiments were performed with an ABI QSTAR-Pulsar XL mass spectrometer (ABI/Sciex, Thornhill, Ontario, Canada) using an on-line liquid chromatography system (Dionex, Amsterdam, Netherlands). Dried peptides were reconstituted in 2% (v/v) acetonitrile, 0.1% (v/v) formic acid and were separated in a C18 reverse phase column on-line with the Q-STAR using a gradient of 2–60% acetonitrile over 15 min. Protein identities were obtained by searching against either the SWISS-PROT or NCBI non-redundant data bases using MASCOT (Matrix Science, London). Western Blotting—To confirm changes in the expression of cytoskeletal/motility-associated proteins, 20 μg of cell lysate was separated on a 10% one-dimensional SDS-PAGE gel (this was increased to 50 μg of lysate on a 15% SDS-PAGE gel for stathmin detection), and transferred to a nitrocellulose membrane using an Electro-Blot apparatus (Web Scientific, Crewe, UK) in 192 mm glycine, 25 mm Tris, 20% (v/v) methanol at 100 V for 45 min. Membranes were blocked in PBS with 0.1% Tween-20 (PBS-T) with 5% (w/v) dried nonfat milk (Marvel) at 4 °C overnight. The membrane was then washed once in PBS-T, and incubated with primary antibodies for 1 h as follows; anti-stathmin rabbit polyclonal antibody (30Gavet O. Ozon S. Manceau V. Lawler S. Curmi P. Sobel A. J. Cell Sci. 1998; 111: 3333-3346Crossref PubMed Google Scholar) (a gift from E. Sobel, INSERM, Paris) at 1:10,000, rabbit anti-Arp2 (Chemicon International, Chandlers ford, UK) at 1:1,000, and rabbit anti-gelsolin serum (31Azuma T. Witke W. Stossel T.P. Hartwig J.H. Kwiatkowski D.J. EMBO J. 1998; 17: 1362-1370Crossref PubMed Scopus (236) Google Scholar) (a gift from D. Kwiatkowski, Harvard Medical School, Boston, MA) at 1:10,000. Membranes were washed three times, incubated in peroxidase-conjugated mouse anti-rabbit Ig (Amersham Biosciences) at 1:10,000 for 1 h, and washed four times. All antibody dilutions were in PBS-T with 1% w/v milk. All washes were performed with PBS-T. Detection was carried out using Supersignal West Pico chemiluminescent substrate (Pierce), and signal detected on Kodak x-ray (Sigma) film. All blots were subsequently stained with Coomassie Blue to ensure equal loading. Chemotaxis and Cell Motility Assay—The migration of Ba/F3 cells in response to agonists was assessed using a 24-well transwell plate (Costar, Corning, New York). These consisted of two wells separated by a membrane containing 5-micron pores. Cells (1–2 × 105 in 100 μl) were placed in the top well, and agonists were added to the top and/or bottom wells (bottom well volume 600 μl) in Fishers medium plus 20% (v/v) batch-tested horse serum. After 6–8 h of incubation at 37 °C in a 5% CO2 humidified incubator, viable cells in the lower well were counted using trypan blue (Sigma). In no experiment was cell viability less than 98% in either top or bottom well after incubation. [3H]Thymidine Incorporation Assay for Cell Proliferation—[3H]thymidine incorporation assays were performed as described previously (32Pierce A. Whetton A.D. Owen-Lynch P.J. Tavernier J. Spooncer E. Dexter T.M. Heyworth C.M. J. Cell Sci. 1998; 111: 815-823Crossref PubMed Google Scholar). BCR/ABL- and TEL/PDGFRβ-transfected Ba/F3 Cells Display Different Changes in Protein Expression That Are Partially Reversed by STI571—Ba/F3 cells expressing either BCR/ABL or TEL/PDGFRβ exhibit a number of behavioral changes. Most notable among these, is that transduced cells no longer require the presence of growth factor (IL-3) for their survival or proliferation (12Carroll M. Tomasson M.H. Barker G.F. Golub T.R. Gilliland D.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14845-14850Crossref PubMed Scopus (246) Google Scholar, 26Daley G.Q. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9312-9316Crossref PubMed Scopus (524) Google Scholar). Protein from Ba/F3 cells transfected with an empty vector (MSCV), and cells transfected to express either BCR/ABL or TEL/PDGFRβ, (with or without treatment with STI571 for 4 h) were separated using a pH 3–10 immobilized pH gradient gel in the first dimension followed by SDS-PAGE. The spot patterns (from three gels) were analyzed to detect significant differences in levels of expression of specific proteins between the control Ba/F3 cells, Ba/F3-BCR/ABL cells and TEL/PDGFRβ-transfected Ba/F3 cells (Fig. 1). The latter two samples were also treated with STI571 and then compared with non-treated controls using two-dimensional electrophoresis (Fig. 1). The major changes in protein levels observed were quantified using Progenesis software. Proteins shown to alter in their expression according to the gel patterns were identified where possible using mass spectrometry, as shown in Table I.Table IIdentities of proteins whose expression is altered by expression of BCR/ABL or TEL/PDGFRβ, and/or treatment with STI571SpotIdentityMpISWISS-Prot/NCBIMean normalized spot volume ± S.E.Ba/F3Oncogene+STI571kDaProteins decreased by BCR/ABL expressionCB1GRP7870.55.01P200290.271 ± 0.0090.006 ± 0.0060.000 ± 0.000CB2Gelsolin83.35.72P130200.120 ± 0.0080.046 ± 0.0030.072 ± 0.011CB3Adseverin (Scinderin) (Gelsolin-like protein)80.35.64Q606040.072 ± 0.0090.000 ± 0.0000.059 ± 0.009CB4Adseverin (Scinderin) (Gelsolin-like protein)80.35.64Q606040.151 ± 0.0130.000 ± 0.0000.024 ± 0.014CB5Thimet oligopeptidase 178.05.72Q8K2D40.113 ± 0.0160.000 ± 0.0000.000 ± 0.000CB6Not identified———0.145 ± 0.0210.072 ± 0.0060.084 ± 0.010CB7Unknown protein—similar to Serpin EIA42.55.85Gi 128348910.219 ± 0.0030.045 ± 0.0000.025 ± 0.015CB8Arp244.76.30O151420.249 ± 0.0370.119 ± 0.0020.158 ± 0.005CB9Eukaryotic translation initiation factor 3, subunit 339.86.19Q91WK20.101 ± 0.0080.064 ± 0.0050.098 ± 0.002CB10Transaldolase37.46.57Q930920.128 ± 0.0030.060 ± 0.0060.069 ± 0.006CB11Unknown protein - similar to HSCARG34.46.37Gi 244319370.070 ± 0.0130.018 ± 0.0010.033 ± 0.004CB12Proteasome activator complex subunit 128.75.73P973710.111 ± 0.0180.079 ± 0.0030.103 ± 0.007CB13Proteasome subunit β-type 323.06.15Q9R1P10.112 ± 0.0140.018 ± 0.0050.025 ± 0.010CB14Phosphatidylethanolamine-binding protein20.75.19P702960.342 ± 0.0290.231 ± 0.0170.229 ± 0.013Proteins increased by BCR/ABL expressionB3Protein kinase C, δ77.57.20P288670.047 ± 0.0040.141 ± 0.0150.171 ± 0.015B4VEGF C precursor13.08.79P979530.000 ± 0.0000.088 ± 0.0110.033 ± 0.009B5Not identified———0.013 ± 0.0050.026 ± 0.0030.019 ± 0.003B6Not identified———0.013 ± 0.0010.039 ± 0.0010.026 ± 0.006B7Tropomyosin α3; isoform 232.84.68P211070.140 ± 0.0190.270 ± 0.0060.255 ± 0.030B8Tropomyosin α3; isoform 232.84.68P211070.140 ± 0.0130.322 ± 0.0190.129 ± 0.025B9Tropomyosin α3; isoform 232.84.68P211070.000 ± 0.0000.174 ± 0.0050.267 ± 0.011B11Not identified———0.000 ± 0.0000.083 ± 0.0010.058 ± 0.007B1214-3-3 protein epsilon (mouse)29.14.63P426550.040 ± 0.0020.168 ± 0.0120.133 ± 0.002B1314-3-3 protein zeta/delta (mouse)27.84.73P352150.040 ± 0.0020.197 ± 0.0230.155 ± 0.007B1414-3-3 protein zeta/delta (mouse)27.84.73P352150.066 ± 0.0040.342 ± 0.0500.163 ± 0.033B1514-3-3 protein beta/alpha (mouse) or 14-3-3 protein gamma (mouse)28.0/28.24.77/4.80Q9CQV8/P352140.061 ± 0.0080.289 ± 0.0320.358 ± 0.006B16Growth factor receptor-bound protein 2; GRB225.25.72Q606310.026 ± 0.0000.073 ± 0.0060.023 ± 0.008B17Antioxidant protein 2 and isopentenyl-diphosphate δ-isomerase 124.7/26.35.72/5.79O08709/P580440.026 ± 0.0110.085 ± 0.0150.072 ± 0.005B18Putative lymphocyte G0/G1 switch protein211.18.55Q615850.000 ± 0.0000.615 ± 0.0200.438 ± 0.064B19Stathmin (oncoprotein 18)17.15.76P542270.002 ± 0.0020.013 ± 0.0020.016 ± 0.002B20Stathmin (oncoprotein 18)17.15.76P542270.013 ± 0.0010.047 ± 0.0000.039 ± 0.006SB5PCNA28.84.66P179180.210 ± 0.0710.512 ± 0.0530.298 ± 0.041Proteins increased by STI571 in BCR/ABL-expressing cellsBS4Single strand selective monofunctional uracil DNA glycosylase30.76.40Q6P5C50.145 ± 0.0030.159 ± 0.0080.067 ± 0.008Proteins decreased by TEL/PDGFRβ expressionCT1GRP7870.55.01P200290.271 ± 0.0090.081 ± 0.0030.007 ± 0.003CT25′,3′-Nucleotidase, cytosolic23.05.31Q9JM140.029 ± 0.0070.015 ± 0.0010.016 ± 0.001CT3Phosphatidylethanolamine-binding protein20.75.19P702960.392 ± 0.0210.219 ± 0.0030.243 ± 0.029Proteins increased by TEL/PDGFRβ expressionT1Follistatin-related protein 132.55.41Q623560.021 ± 0.0100.065 ± 0.0130.026 ± 0.003T2Tropomyosin α3; isoform 232.84.68P211070.140 ± 0.0190.212 ± 0.0220.209 ± 0.021T3Tropomyosin α3; isoform 232.84.68P211070.140 ± 0.0130.465 ± 0.0720.115 ± 0.004T4Not identified———0.100 ± 0.0410.516 ± 0.0690.206 ± 0.035T614-3-3 protein zeta/delta27.84.73P352150.040 ± 0.0020.405 ± 0.0620.296 ± 0.005T714-3-3 protein zeta/delta27.84.73P352150.066 ± 0.0040.380 ± 0.0710.186 ± 0.085T8NADH ubiquinone oxidoreductase 24-kDa subunit23.85.31Q9D6J60.001 ± 0.0000.014 ± 0.0040.003 ± 0.002ST1Not identified———0.067 ± 0.0270.308 ± 0.0380.128 ± 0.025ST2PCNA28.84.66P179180.210 ± 0.0710.427 ± 0.0420.258 ± 0.012ST3Not identified———0.238 ± 0.0240.402 ± 0.0130.272 ± 0.009 Open table in a new tab Ba/F3-BCR/ABL cells showed significant changes in 32 proteins compared with Ba/F3 cells. Several protein functional groups can be identified in the list of proteins that change as a consequence of BCR/ABL expression, for example signaling proteins such as 14-3-3 proteins and Grb2, metabolic enzymes, and cytoskeleton regulators (see Table I). TEL/PDGFRβ induced 13 significant changes in protein expression, and these showed some overlap to those seen with BCR/ABL. However there are a significantly fewer changes induced by TEL/PDGFRβ than by BCR/ABL: each tyrosine kinase, while inducing growth factor independence is not affecting the proteome in a similar fashion. BCR/ABL but Not TEL/PDGFRβ Induces Changes in Cytoskeletal Proteins and Regulators of the Motile Response— When compared with control Ba/F3 cells, changes in cytoskeletal-associated proteins such as decreased levels of gelsolin, adseverin, and actin-related protein 2 (Arp2), and increased expression of stathmin are seen in BCR/ABL expressing Ba/F3 cells but not TEL/PDGFRβ-transfected cells. Gelsolin and adseverin are closely related proteins that play a role in actin filament severing and capping, and are therefore closely involved in cell motility (33Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (328) Google Scholar). Arp2 is part of the Arp2/3 complex that is also associated with formation of short, branched actin filaments in the leading edge of motile cells (34Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Crossref PubMed Scopus (1060) Google Scholar). In contrast, tropomyosin, several forms of which were increased by both BCR/ABL and TEL/PDGFRβ in this study, has been shown to cause annealing of gels
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