Characterization of a Myeloid Tyrosine Phosphatase, Lyp, and Its Role in the Bcr-Abl Signal Transduction Pathway

The Bcr-Abl protein-tyrosine kinase is implicated in the development of chronic myeloid leukemia. The potential role of protein-tyrosine phosphatase in the regulation of Bcr-Abl signaling was explored. First, expression patterns of tyrosine phosphatases in leukemic cell lines were investigated using degenerate primers for reverse transcription-PCR followed by cloning and sequencing of the cDNA. Distinct patterns of distribution of phosphatase were found in erythroid and myeloid leukemic cell lines. Whereas some phosphatases were ubiquitously expressed, others were limited to specific cell types. Surprisingly, a previously cloned “lymphocyte-specific” phosphatase, Lyp, was frequently detected in a number of myeloid cell lines as well as normal granulocytes and monocytes. Lyp was localized to the cytosol, and overexpression of Lyp caused reduction in the phosphorylation levels of multiple proteins in KCL22 chronic myeloid leukemia blast cells including Cbl, Bcr-Abl, Erk1/2, and CrkL. Co-expression of Lyp and Bcr-Abl in Cos-7 cells resulted in decreased levels of Bcr-Abl, Grb2, and Myc. Overexpression of Lyp markedly suppressed anchorage-independent clonal growth of KCL22 cells. Taken together, the data suggest that Lyp may play an antagonistic role in signaling by the Bcr-Abl fusion protein. The Bcr-Abl protein-tyrosine kinase is implicated in the development of chronic myeloid leukemia. The potential role of protein-tyrosine phosphatase in the regulation of Bcr-Abl signaling was explored. First, expression patterns of tyrosine phosphatases in leukemic cell lines were investigated using degenerate primers for reverse transcription-PCR followed by cloning and sequencing of the cDNA. Distinct patterns of distribution of phosphatase were found in erythroid and myeloid leukemic cell lines. Whereas some phosphatases were ubiquitously expressed, others were limited to specific cell types. Surprisingly, a previously cloned “lymphocyte-specific” phosphatase, Lyp, was frequently detected in a number of myeloid cell lines as well as normal granulocytes and monocytes. Lyp was localized to the cytosol, and overexpression of Lyp caused reduction in the phosphorylation levels of multiple proteins in KCL22 chronic myeloid leukemia blast cells including Cbl, Bcr-Abl, Erk1/2, and CrkL. Co-expression of Lyp and Bcr-Abl in Cos-7 cells resulted in decreased levels of Bcr-Abl, Grb2, and Myc. Overexpression of Lyp markedly suppressed anchorage-independent clonal growth of KCL22 cells. Taken together, the data suggest that Lyp may play an antagonistic role in signaling by the Bcr-Abl fusion protein. In Philadelphia chromosome-positive human chronic myeloid leukemia (CML), 1The abbreviations used are: CML, chronic myeloid leukemia; PTPase, protein-tyrosine phosphatase; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; SHIP, Src homology 2 domain containing inositol phosphatase; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription.1The abbreviations used are: CML, chronic myeloid leukemia; PTPase, protein-tyrosine phosphatase; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; SHIP, Src homology 2 domain containing inositol phosphatase; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription. malignant transformation is mediated by a constitutively active tyrosine kinase Bcr-Abl (1Lugo T.G. Pendergast A.M. Muller A.J. Witte O.N. Science. 1990; 247: 1079-1082Crossref PubMed Scopus (1112) Google Scholar, 2Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1908) Google Scholar). The abl gene product is a 145-kDa protein encoding a non-receptor tyrosine kinase (3Laneuville P. Semin. Immunol. 1995; 7: 255-266Crossref PubMed Scopus (74) Google Scholar). The protein contains three src homology domains, SH1 with tyrosine kinase function, and SH2 and SH3 involved in protein-protein interaction. The enzymatic activity of Abl can be regulated through protein-binding domains and stimulated by growth factors and DNA damage (4Plattner R. Kadlec L. DeMali K.A. Kazlauskas A. Pendergast A.M. Genes Dev. 1999; 13: 2400-2411Crossref PubMed Scopus (367) Google Scholar, 5Ito Y. Pandey P. Mishra N. Kumar S. Narula N. Kharbanda S. Saxena S. Kufe D. Mol. Cell. Biol. 2001; 21: 6233-6242Crossref PubMed Scopus (115) Google Scholar). The Philadelphia chromosome results from a reciprocal translocation of the abl on chromosome 9 transposing to chromosome 22 in the break cluster region (bcr) gene (6Nowell P.C. Hungerford D.A. Science. 1960; 132: 1497Google Scholar, 7Rowley J.D. Nature. 1973; 243: 290-293Crossref PubMed Scopus (3309) Google Scholar). Interleukin 3-dependent Ba/F3 cells infected with the fusion gene bcr-abl become growth factor-independent and tumorigenic in nude mice (8Daley G.Q. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9312-9316Crossref PubMed Scopus (506) Google Scholar). The aberrant tyrosine phosphorylation levels of Bcr-Abl activate a series of signaling pathways, and a multitude of proteins exhibit a marked increase in their level of phosphorylation including Bcr-Abl itself, CrkL (an adaptor protein), and phosphatidylinositol 3-kinase (9Deininger M.W. Goldman J.M. Melo J.V. Blood. 2000; 96: 3343-3356Crossref PubMed Google Scholar). Cellular Cbl is a 120-kDa cytoplasmic protein that is ubiquitously expressed with high levels in hematopoietic cells. Cbl can be phosphorylated in response to activation by a variety of growth factors including epidermal growth factor, platelet-derived growth factor, erythropoietin, as well as granulocyte-macrophage colony-stimulating factor (10Odai H. Sasaki K. Iwamatsu A. Hanazono Y. Tanaka T. Mitani K. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 10800-10805Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 11Barber D.L. Mason J.M. Fukazawa T. Reedquist K.A. Druker B.J. Band H. D'Andrea A.D. Blood. 1997; 89: 3166-3174Crossref PubMed Google Scholar, 12Levkowitz G. Klapper L.N. Tzahar E. Freywald A. Sela M. Yarden Y. Oncogene. 1996; 12: 1117-1125PubMed Google Scholar). Tyrosine phosphorylation of Cbl is increased in Bcr-Abl transformed cells (13Salgia R. Sattler M. Pisick E. Li J.L. Griffin J.D. Exp. Hematol. 1996; 24: 310-313PubMed Google Scholar). Interaction between Cbl and CrkL is tyrosine phosphorylation-dependent, and the complex has been implicated in Bcr-Abl mediated transformation (14Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar). Protein-tyrosine phosphatases (PTPase) counter the activity of tyrosine kinases by removing phosphate groups from proteins that have been phosphorylated on tyrosyl residues. Whereas protein-tyrosine kinases have been intensely studied over the past decade, the significance of PTPase has just started to be recognized (15Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar). PTPases are involved in the regulation of cellular proliferation and differentiation, as well as cell death (16Fischer E.H. Adv. Enzyme Regul. 1999; 39: 359-369Crossref PubMed Scopus (60) Google Scholar, 17Li L. Dixon J.E. Semin. Immunol. 2000; 12: 75-84Crossref PubMed Scopus (147) Google Scholar). CD45 is one of the better studied hematopietic PTPases and is a key regulator of lymphocyte functions (18Alexander D.R. Semin. Immunol. 2000; 12: 349-359Crossref PubMed Scopus (122) Google Scholar). Other hematopoietic PTPases, such as SHP-1, are involved in cytokine receptor signaling (19Kim H. Baumann H. Mol. Cell. Biol. 1999; 18: 5326-5338Crossref Scopus (148) Google Scholar). PTPases display high sequence homology in their catalytic domain (20Andersen J.N. Mortensen O.H. Peters G.H. Drake P.G. Iversen L.F. Olsen O.H. Jansen P.G. Andersen H.S. Tonks N.K. Moller N.P. Mol. Cell. Biol. 2001; 21: 7117-7136Crossref PubMed Scopus (588) Google Scholar), which allows the identification of PTPase family members by using degenerate primers in RT-PCR amplification. We have employed this method to investigate the expression pattern of PTPase in leukemic cells. Degenerate PTPase primers were designed and used to amplify different PTPases from RNA isolated from leukemic cell lines. PCR products were cloned, and individual clones were identified by DNA sequencing. Specific PTPase expression patterns were obtained for each cell line. Lyp, a PTPase that was previously shown to be expressed in lymphoid cells (21Cohen S. Dadi H. Shaoul E. Sharfe N. Roifman C.M. Blood. 1999; 93: 2013-2024Crossref PubMed Google Scholar), was identified in this study as one of the major PTPases in myeloid leukemic cells. Lyp was localized to the cytoplasm in the KCL22 CML cell line. Overexpression of Lyp in KCL22 CML cells caused reduction of total cellular phosphorylation levels of proteins. Of particular interest, phosphorylation of Cbl and Bcr-Abl markedly decreased in these cells, and this was associated with markedly decreased levels of Bcr-Abl. Molecules that are substrates of Bcr-Abl, such as CrkL and Erk1/2, also had a decrease in their phosphorylation levels, and amounts of the Grb2 and Myc proteins decreased. Anchorage-independent clonal growth in soft agar markedly decreased in KCL22 cells overexpressing Lyp. Our study suggests a novel mechanism for Bcr-Abl regulation. Cell Culture, Transfection, and CML Patient Samples—Cell lines were purchased from ATCC (Manassas, VA) except for the following: ML-1 cells were a gift from Dr. M. Kastan (The Johns Hopkins University, Baltimore, MD). Kasumi-1 and Kasumi-3 cells were established by Dr. H. Asou (Hiroshima University, Hiroshima, Japan). Adherent and suspension cells were grown in Dulbecco's modified Eagle's medium and RPMI, respectively, supplemented with 10% fetal bovine serum. Transfection of KCL22 cells (5 × 107) was performed by electroporation at 340 V with 20 μg of plasmid in RPMI containing 50% fetal calf serum. Transfection of Cos-7 cells was carried out using LipofectAMINE 2000 (Invitrogen) over 4 h according to the manufacturer's protocol. Proteins and RNA were prepared from the bone marrow of CML patients after their informed consent. Reverse Transcription and Polymerase Chain Reaction—Two μg of total RNA isolated with TRIzol reagent was reverse-transcribed with Superscript II and random primers according to the manufacturer's protocol (Invitrogen). PCR consisted of 22–30 cycles of denaturation at 95 °C for 1 min, annealing at 62–64 °C for 1 min, and extension at 72 °C for 1 min. With degenerate primers, the annealing cycles were modified by starting the annealing process at 37 °C and heating to 72 °C within 2 min. Primers used are: β-actin-specific, 5′-TACATGGCTGGGGTGTTGAA-3′, 5′-AAGAGAGGCATCCTCACCCT-3′; Lyp-specific, 5′-TGGCCTCCAAGTGGTACCAG-3′, 5′-CATCGGCAAGAAAGAAGGAC-3′. Degenerate primers for PTPase were deduced from amino acid sequences: ACKCCNGCNSWRCARTG (upper strand) and AGYGAYTAYATHAAYGC (lower strand). PCR products amplified from degenerate primers were cloned into pBluescript for sequencing. DNA sequences were compared with the NCBI data base using the BLAST program. Separation of Peripheral Blood Cells—Polymorphonuclear cells (neutrophils) were isolated from anticoagulated blood using polymorphonuclear neutrophil solution (Robbins Scientific, Sunnyville, CA) according to the one-step density gradient centrifugation method. Briefly, whole blood was layered over polymorphonuclear neutrophil solution and centrifuged for 25 min at 500 × g. Mononuclear cells and neutrophils were separated into two distinct bands, whereas erythrocytes pelleted to the bottom of the tube. Neutrophils were obtained from the lower band and were washed twice with serum-free medium. Monocytes were separated from the mononuclear cell fraction by adherence to plastic cell culture dishes. CD3+ cells were isolated from the mononuclear fraction by FACStar flow cytometer (BD Biosciences) using monoclonal murine antibodies against CD3 conjugated to fluorescein isothiocyanate and phosphatidylethanolamine, respectively (Dako, Carpinteria, CA). Hybridization with an Internal Oligonucleotide—Gel-separated PCR products were blotted onto nylon membrane (Amersham Biosciences) by capillary transfer in 20× SSC. Prehybridization and hybridization were performed at 42 °C in solution The were used for hybridization Lyp-specific, were with using the was detected by with followed by were with fluorescent protein at h with 20 in solution for min at 37 washed with three and a fluorescent The data that are shown are of multiple RNA was reverse-transcribed as PCR was performed on PCR a total of 25 and consisted of of 500 of Lyp and 1 of were for each cDNA. of Lyp was to were with 1 and containing and 1 each of and 1 of was with CA) at 4 °C for 2 h and with protein at 4 °C for 1 The were washed with and in the onto a 10% antibodies were used followed by (Amersham of cells were in agar containing fetal bovine and RPMI on of agar in was and after 2 of results of dishes. of Protein-tyrosine in Cell distribution of PTPase in myeloid cells was by RT-PCR using degenerate PTPase primers and cloning of individual protein-tyrosine individual clones were from three myeloid cell and The of a number of clones a of the expression pattern of PTPase in human myeloid leukemic cells and may the to a The cells are cells, the ML-1 cells are and the cells are CD45 was the expressed PTPase, and of the clones isolated from and respectively In cells, CD45 and the PTPase were with expressed in the number of clones of a total of was the PTPase in ML-1 cells In between and different PTPases were identified cell with the in expression different cell a distinct of and be identified in cells. and were found in cells. and were identified in ML-1 cells, whereas and Lyp were in three cell of phosphatase expression in myeloid leukemic cell number of PCR products amplified from degenerate PTPase primers were cloned into pBluescript for sequencing. DNA sequences were compared with the NCBI using the BLAST number of PCR products amplified from degenerate PTPase primers were cloned into pBluescript for sequencing. DNA sequences were compared with the NCBI using the BLAST in a of has been as a PTPase (21Cohen S. Dadi H. Shaoul E. Sharfe N. Roifman C.M. Blood. 1999; 93: 2013-2024Crossref PubMed Google were to to be one of the frequently expressed PTPases in the three myeloid cell lines used in this study Lyp of the clones in cells and of clones in and ML-1 cells. studied Lyp expression in a of human leukemic cell lines as well as in normal blood using RT-PCR with In the myeloid cell high levels of Lyp in and ML-1 as well as and KCL22 The expression was with that in the In Lyp was expressed in Kasumi-1 the fusion protein), and Kasumi-3 cells We also a series of cell lines and found expression of Lyp isolated different of normal human blood cells to Lyp We found that Lyp was expressed at a level in neutrophils and as compared with CD3+ of Lyp in blood cells. of human blood cells were separated and by RT-PCR using polymorphonuclear blood mononuclear cells, cells CD3 blood CD3 The of the Lyp phosphatase was The Cos-7 and KCL22 CML myeloid blast cells were with an expression of Lyp to and by h after We found that Lyp was cytoplasmic of Lyp in Lyp phosphatase has previously been shown to with and the tyrosine phosphorylation of Cbl in (21Cohen S. Dadi H. Shaoul E. Sharfe N. Roifman C.M. Blood. 1999; 93: 2013-2024Crossref PubMed Google Scholar). Cbl has been to be a for tyrosine phosphorylation by the Bcr-Abl tyrosine kinase R. J. N. J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). were to Lyp have a role in CML cells, such as Lyp was into a and the Lyp expression and the were into KCL22 cells. cell lines were isolated by The of Lyp was by PCR for to 4 of to We found a of Lyp KCL22 with to the after 2 and of after 4 in the of of Lyp cellular proteins were isolated from 2 and 4 of and the total cell were for Lyp protein expression by Bcr-Abl is a major protein involved in the of at the Bcr-Abl protein levels in these cells. We found that after 4 of Bcr-Abl protein level is decreased by Lyp tyrosine a from the protein with an to overexpression of Lyp the level of tyrosine phosphorylation as compared with the cells. We a decrease in the level of protein tyrosine phosphorylation Lyp overexpression Of particular was the marked reduction of tyrosine phosphorylation for proteins of and We that these proteins were Cbl and Bcr-Abl, respectively, on their the was with antibodies against these two which that the proteins at and were Cbl and Bcr-Abl and of the KCL22 KCL22 cells that the of Cbl protein was by the of Lyp the was with an against that overexpression of Lyp resulted in a decrease in Bcr-Abl protein expression to was We also the expression of Lyp protein in the leukemic cells from patients with CML has a chronic a into the blast Lyp was expressed in these at levels, and was between the of expression of Lyp protein and of the CML were at Lyp RNA levels in CML patients by the number of was investigate the role of Lyp in the regulation of Bcr-Abl, Cos-7 cells with an expression for Bcr-Abl and cellular proteins were isolated and by The of an and Lyp the expression of the Lyp phosphatase in these cells with and Bcr-Abl and Bcr-Abl be detected from the Cos-7 cells with Bcr-Abl and the that amounts of protein were used results suggest that the expression of Lyp a decrease in Bcr-Abl protein of the signaling by Bcr-Abl a series of including between the adaptor protein Grb2 and the phosphorylated Bcr-Abl A.M. Li N. A. J. Cell. Full Text PDF PubMed Scopus Google Scholar), of Myc protein D. Kadlec L. Pendergast A.M. Mol. Cell Biol. 1995; PubMed Scopus Google Scholar), and phosphorylation of CrkL and We found that Lyp was expressed, levels of Bcr-Abl and Grb2 markedly and a reduction of Myc protein levels was a decrease of CrkL and phosphorylation in these cells The of Lyp overexpression on anchorage-independent clonal growth in soft agar of KCL22 CML cells was KCL22 in soft agar In KCL22 cells had markedly decreased clonal in number and of the that overexpression of Lyp the potential of KCL22 CML cells In this identified a number of PTPases in three human myeloid leukemic cell lines. Of and Lyp were the also as the is an membrane protein expressed on hematopoietic cells M. Y. H. J. Exp. PubMed Scopus Google Scholar, T. J. J.M. J. Cell Biol. 2001; PubMed Scopus Google Scholar). the activity of the Src family of kinases H. N. S. S. Li Blood. PubMed Scopus Google Scholar). CD45 has been shown to be expressed on hematopoietic and of were that was the PTPase detected in a major PTPase identified in cells, has been as an regulator of cell A.J. Tonks N.K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and has been to have a role in leukemia through an with Y. P. M. D. A. Salgia R. Griffin J.D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The major PTPase detected in the myeloid cells was Lyp. Lyp was identified as a PTPase (21Cohen S. Dadi H. Shaoul E. Sharfe N. Roifman C.M. Blood. 1999; 93: 2013-2024Crossref PubMed Google Scholar). We found that Lyp of the clones obtained from the cells, and a of Lyp expression was found in myeloid cell lines including cells and the cells. of Lyp can also be found in blood granulocytes and a potential of Lyp in myeloid cell Lyp has cytoplasmic expression and some in cells (21Cohen S. Dadi H. Shaoul E. Sharfe N. Roifman C.M. Blood. 1999; 93: 2013-2024Crossref PubMed Google Scholar). We in KCL22 CML cells that Lyp is localized to the may with Bcr-Abl and play a role in the signaling of this fusion protein. We found that overexpression of Lyp resulted in a decrease in the expression of Bcr-Abl as well as a reduction in the phosphorylation levels of The decrease in Cbl tyrosine phosphorylation is the of Lyp phosphatase activity of Bcr-Abl Cbl has been shown to be a of Lyp activity in cells containing Bcr-Abl (21Cohen S. Dadi H. Shaoul E. Sharfe N. Roifman C.M. Blood. 1999; 93: 2013-2024Crossref PubMed Google Scholar). In cells, Lyp cell receptor signaling by with and Cbl is a of Bcr-Abl tyrosine and of Bcr-Abl a reduction in the level of Cbl CML is caused in by activation of signaling by the aberrant tyrosine kinase activity of the Bcr-Abl fusion protein. active tyrosine kinase a number of signaling including Cbl, Grb2, and CrkL. The of Bcr-Abl with Cbl activation of the phosphatidylinositol 3-kinase (14Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar), and between Bcr-Abl with Grb2 can to the activation of of of these is for a of Grb2 D. Pendergast A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Bcr-Abl with in the SH2 domain can Bcr-Abl transformation A. J. Witte O.N. Cell. 1995; Full Text PDF PubMed Scopus Google and Etten R.A. Blood. 2000; 96: PubMed Google Scholar). Lyp has also been shown to with the adaptor Grb2 S. K. M. Exp. Hematol. Full Text Full Text PDF PubMed Scopus Google Scholar). In this Lyp, a Lyp, had a role in cell signaling regulation of of Grb2 with the cell In KCL22 CML cells, lower Grb2 expression levels were found Lyp was The SH3 domain of Grb2 with factor of and and the kinase of CML blast cell proliferation can be by of H. M. M. Blood. 2001; PubMed Scopus Google Scholar). the of Grb2 can be a of overexpression of Lyp, and of the complex to of and to the of the potential of KCL22 cells in the soft agar phosphorylation of CrkL may to the of potential of cells. tyrosine are phosphorylated in CrkL by Bcr-Abl, and in CrkL cell transformation and in and hematopoietic cells K. Druker Mol. Cell. Biol. 18: PubMed Scopus Google Scholar). a major of CML has on the aberrant tyrosine kinase activity of Bcr-Abl and the of tyrosine levels of tyrosine phosphorylation are by the of kinases and Bcr-Abl can with and In some Bcr-Abl these and these phosphatases can Bcr-Abl, in a decrease in Bcr-Abl kinase activity G. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Bcr-Abl the expression of PTPase, which in Bcr-Abl, in the of Grb2 and of activity A.J. A.M. Tonks N.K. Mol. Cell. Biol. 18: PubMed Scopus Google Scholar). Bcr-Abl has been shown to with and to the expression of SHIP, an inositol phosphatase that the phosphatidylinositol 3-kinase signaling T. R. D. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Bcr-Abl can the expression of phosphatase by the of as well as the of the protein M. Salgia R. G. S. J.L. Griffin J.D. Oncogene. 1997; PubMed Scopus Google Scholar). the activity of the phosphatidylinositol 3-kinase signaling of this regulator by Bcr-Abl can in increased myeloid as was in mice Sasaki T. A. A. J.M. Genes Dev. 1999; 13: PubMed Scopus Google Scholar). In that an of a phosphatase such as with Bcr-Abl, the Bcr-Abl kinase activity in the expression of the fusion protein G. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A.J. A.M. Tonks N.K. Mol. Cell. Biol. 18: PubMed Scopus Google Scholar). Our study to the of a phosphatase Bcr-Abl signaling of the levels of Bcr-Abl protein. The mechanism by which this has to be We to Bcr-Abl was by Lyp. The results of these that Bcr-Abl was by of Lyp In cells, the a complex with Bcr-Abl, the of Bcr-Abl Cell 2000; Google Scholar). with the of can Bcr-Abl protein Cell 2000; Google Scholar). overexpression of Lyp the between the and Bcr-Abl complex and Bcr-Abl In that Lyp is expressed in the myeloid cell and Lyp overexpression in KCL22 cells Bcr-Abl and Grb2 as well as of Cbl, and Overexpression of Lyp potential of KCL22 cells. Our data suggest that Lyp may as a

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