The reciprocal t(8;13) chromosome translocation results in a fusion gene (FUS) in which the N-terminal half of the zinc finger protein ZNF198 is combined with the cytoplasmic domain of the fibroblast growth factor receptor-1 (FGFR1). Expression of FUS is suggested to provide growth-promoting activity to myeloid cells similar to the activity of hematopoietic cytokine receptors. This study determined the specificity of FUS to activate signal transduction pathways. Because no tumor cell line expressing FUS was available, the mode of FUS action was identified in cells transiently and stably transfected with an expression vector for FUS. FUS acted as a constitutively active protein-tyrosine kinase and mediated phosphorylation of STAT1, 3, and 5 but not STAT4 and 6. The same specificity but lower activity was determined for normal FGFR1. STAT activation by FUS, similar to that by interleukin-6-type cytokines, promoted STAT-specific induction of genes. The functionality of FUS, as well as the relative recruitment of STAT isoforms, was determined by the dimerizing function of the zinc finger domain. Replacement of the ZNF198 portion by the Bcr portion as present in the t(8;22) translocation shifted the signaling toward a more prominent STAT5 activation. This study documents that both gene partners forming the fusion oncogene define the activity and the signaling specificity of the protein-tyrosine kinase of FGFR1. The reciprocal t(8;13) chromosome translocation results in a fusion gene (FUS) in which the N-terminal half of the zinc finger protein ZNF198 is combined with the cytoplasmic domain of the fibroblast growth factor receptor-1 (FGFR1). Expression of FUS is suggested to provide growth-promoting activity to myeloid cells similar to the activity of hematopoietic cytokine receptors. This study determined the specificity of FUS to activate signal transduction pathways. Because no tumor cell line expressing FUS was available, the mode of FUS action was identified in cells transiently and stably transfected with an expression vector for FUS. FUS acted as a constitutively active protein-tyrosine kinase and mediated phosphorylation of STAT1, 3, and 5 but not STAT4 and 6. The same specificity but lower activity was determined for normal FGFR1. STAT activation by FUS, similar to that by interleukin-6-type cytokines, promoted STAT-specific induction of genes. The functionality of FUS, as well as the relative recruitment of STAT isoforms, was determined by the dimerizing function of the zinc finger domain. Replacement of the ZNF198 portion by the Bcr portion as present in the t(8;22) translocation shifted the signaling toward a more prominent STAT5 activation. This study documents that both gene partners forming the fusion oncogene define the activity and the signaling specificity of the protein-tyrosine kinase of FGFR1. myeloid proliferative disease chloramphenicol acetyl transferase fibroblast growth factor fibroblast growth factor receptor-1 green fluorescent protein interleukin leukemia inhibitory factor oncostatin M proline-rich domain suppressor of cytokine signal signal transducers and activators of transcription zinc finger protein 198 extracellular signal-regulated kinase fast protein liquid chromatography epidermal growth factor EGF receptor Reciprocal chromosomal translocations in specific types of leukemia have consistently led to the isolation of genes important for the oncogenic process (1Rabbitts T.H. Science. 1994; 372: 143-149Google Scholar). An atypical chronic form of myeloproliferative disease (MPD)1 was described some years ago (2Abruzzo L.V. Jaffe E.S. Cotelingam J.D. Whang-Peng J. Del Duca V. Medeiros L.J. Am. J. Surg. Pathol. 1992; 16: 236-245Crossref PubMed Scopus (105) Google Scholar) that is associated with T-cell leukemia/lymphoma and peripheral blood eosinophilia. Cytogenetic analysis of bone marrow aspirated from these patients showed a consistent reciprocal chromosome translocation t(8;13)(p11;q12). In some cases this rearrangement was the only cytogenetic abnormality. In our initial studies we identified the position of the translocation breakpoints using fluorescentin situ hybridization (3Kempski H. MacDonald D. Michalski A.J. Roberts T. Goldman J.M. Cross C.P. Cowell J.K. Cancer. 1995; 12: 283-287Google Scholar) and then used somatic cell hybrids to clearly define the location of the breakpoints on both chromosomes (4Still I.H. Chernova O. Hurd D. Stone R.M. Cowell J.K. Blood. 1997; 90: 3136-3141Crossref PubMed Google Scholar, 5Chernova O. Still I.H. Kalaycio M. Hoeltge G. Cowell J.K. Genes Chromosomes Cancer. 1997; 21: 160-165Crossref Scopus (11) Google Scholar). The 8p11 translocation breakpoint was subsequently shown to interrupt the FGFR1 gene, and in all of the patients reported so far, these breakpoints cluster within intron 8. The chromosome breakpoint in 13q12 was reported by several groups to involve a zinc finger-containing gene, ZNF198 (also called RAMP8 and FIM), where the breakpoint is consistently located in intron 17. Despite some discrepancies in early reports (6Smedley D. Hamoudi R. Clark J. Warren W. Abdul-Rauf M. Somers G. Venter D. Fagan K. Cooper J. PubMed Scopus Google Scholar, J. Jaffe E.S. Stone R. PubMed Scopus Google the of the ZNF198 gene and the of the fusion gene I.H. Cowell J.K. Blood. PubMed Google Scholar, J. V. M. G. M. D. D. PubMed Scopus Google which that the gene from an fusion of the ZNF198 zinc finger and proline-rich domain with the domain the kinase of FGFR1. ZNF198 is a gene and is to a protein with a of J. Jaffe E.S. Stone R. PubMed Scopus Google Scholar, I.H. Cowell J.K. Blood. PubMed Google Scholar, J. V. M. G. M. D. D. PubMed Scopus Google Scholar, D. Goldman J.M. Cross PubMed Scopus Google Scholar). of ZNF198 the zinc finger and a within the portion of the protein and an domain the of the The zinc finger is in that is of a transcription Despite these the function of this protein is FGFR1 is a receptor protein-tyrosine kinase to the fibroblast growth factor receptor Cancer. Scholar). fusion of the cytoplasmic kinase domain to the the is to signaling for the oncogenic in myeloid cells D. Cross PubMed Scopus Google Scholar). expression of FUS in cells the growth-promoting activity by the cells with V. G. D. R. D. J. PubMed Scopus Google Scholar) Blood. PubMed Google Scholar). The same cells an signaling that the and protein kinase Blood. PubMed Google Scholar, K. M. G. R. Goldman J.M. Cross Blood. PubMed Scopus Google Scholar). The signaling specificity of the fusion kinase on the function of FGFR1 Cancer. Scholar, PubMed Google Scholar). the of signaling not the FGFR1 the the fusion kinase as a cytoplasmic to some The analysis of fusion kinase that the cytoplasmic domain of the of the kinase to in cells that and and G. V. D. 21: PubMed Scopus Google Scholar). The activity of FGFR1 as of to the activity by of the N-terminal fusion partners J. V. M. G. M. D. D. PubMed Scopus Google Scholar, V. G. D. R. D. J. PubMed Scopus Google Scholar, K. M. G. R. Goldman J.M. Cross Blood. PubMed Scopus Google Scholar, T. R. M. T. Genes Chromosomes Cancer. PubMed Scopus Google Scholar) Because the expression of in cells some of the associated with the action of hematopoietic cytokine we the of signaling that is by the fusion kinase and this signaling is by the specificity of of genes. using cell we have identified a signaling that is with that of The expression for ZNF198 and FGFR1 described in The ZNF198 was from a bone marrow and (FUS) was from a (4Still I.H. Chernova O. Hurd D. Stone R.M. Cowell J.K. Blood. 1997; 90: 3136-3141Crossref PubMed Google Scholar). The the The from of these was to and then the vector using and Still I.H. Cowell J.K. Scholar). The FGFR1 gene was from using a FGFR1 and FGFR1 and then of the the portion was by using the and the The FGFR1 portion was using the and and of the FUS of the zinc and the was by the fusion of ZNF198 with FGFR1 using The ZNF198 gene, which the portion of ZNF198 that is present in FUS, was using and and the vector The as described by K. M. G. R. Goldman J.M. Cross Blood. PubMed Scopus Google was using and the in with the FGFR1 and the FGFR1 The with the and the expression of the by The expression used for the in the vector J. D. T. M. H. PubMed Scopus Google of and of D. Science. PubMed Scopus Google Scholar) J. PubMed Google and D. PubMed Scopus Google Scholar) in the vector by The for and in the and STAT1, and in the vector J. H. J. 1995; PubMed Scopus Google Scholar, J. H. H. J. PubMed Scopus Google Scholar). The gene the the and the growth and J. D. H. J. 1995; PubMed Scopus Google Scholar). and cells in and and cells transfected using the J. D. T. M. H. PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar) and and cells with was by with the expression vector for fluorescent protein and by analysis as described J. D. T. M. H. PubMed Scopus Google Scholar). signaling transfected and a the cells with in the of of kinase as in the to the on the signal by the induction of genes was the of from to the cells within the with activity was determined in cell and as described J. D. T. M. H. PubMed Scopus Google Scholar). cells stably expressing FUS, by the cells with the expression vector for these by in the of for to The of cells of stably transfected using the of cell and on of the and phosphorylation by and where The of expression in the by of of cell of on The to of the on for of J. PubMed Scopus Google Scholar). This the of of the process of of the same The with to STAT1, the of FGFR1 the N-terminal half of ZNF198 Still I.H. Cowell J.K. 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PubMed Scopus Google of cells stably expressing FUS, of cells transfected with the expression vector for FUS, by in of cells and to a of to the of the expression cell from the cells and and of by for and The position of the is a and fluorescent of the of was and is with the fluorescent of cells the of and FUS and FGFR1 is by the of FUS on and we cell stably expressing FUS. these several important the of cells to expression of FUS which for to showed prominent FUS and expression as by in the by fluorescent with of the the of and cells in the In cells transfected with the expression vector for FGFR1 and for protein This suggested that the expression of the active FUS kinase with growth The in was by the expression of the green fluorescent cells present to the and in a of the this we that expression of FUS was and the of with expression the cells that more of the from these cells The expression of FUS was with of to in from to In all of the of to an the same in cells expressing normal as shown in a we to that the fusion kinase in all cells and the transfected of we in expressing to expressing FUS by of for kinase expression by to we expression of FUS in and initial the FUS expression in these cells to the of a we to the The cell in of kinase expression with the for and for The expression of as using the the with the with cells a of FUS expression with expression of the cells more cell with to the similar of with prominent and of cell was in the expressing FUS but not in the expressing FGFR1 the ZNF198 in stably transfected cells to V. 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PubMed Scopus Google Scholar) and that the N-terminal portion the cytoplasmic and the The transfected FUS is a constitutively active protein-tyrosine kinase that a of and of the of FUS expression is the phosphorylation and activation of the STAT The STAT specificity of this action was determined in the cell stably expressing FUS and of not The cells showed a constitutively phosphorylation of FUS 5 The same cells a phosphorylation of STAT1, and STAT5 5 The expression of STAT4 and STAT was in and activation by FUS not the action of FUS on with that of an we the of a of the cells with as a The of cytokine in cells is with receptor the only of the for cell of cells not an phosphorylation of that the in cells 5 the activation of was 5 was no of active in cells that the activation of this was by FUS. was not on and STAT5 in that FUS a of signaling the In as well as the of was a for activation of the we used the of a with In both and to a that that of cells 5 This that FUS, was to a the activation of the associated with the action of J. 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An that the to the by the signal of using of FUS and as a normal we not of FUS to the of ZNF198 in the using analysis of the we that FUS and ZNF198 to in the phosphorylation of ZNF198 ZNF198 in the gene we a of ZNF198 on the FUS action on transcription the cytoplasmic of the function of ZNF198 and to with FUS, we a of This the to which the of protein from as a was to the the to as a cytoplasmic protein and to have a on FUS action the function of the N-terminal half of ZNF198 but the that the of this function is by the prominent of and of the ZNF198 of FUS a FGFR1 kinase activity by the ZNF198 zinc finger domain. This activity was to the in hematopoietic cells D. Cross PubMed Scopus Google Scholar). Because a of have to in FGFR1 we the of the specificity of the kinase identified as oncogenic of FGFR1 K. M. G. R. Goldman J.M. Cross Blood. PubMed Scopus Google Scholar, T. R. M. T. Genes Chromosomes Cancer. PubMed Scopus Google Scholar). that the N-terminal portion of Bcr is the of a PubMed Scopus Google we signaling activity of FUS and that in An expression vector for was The expression of this kinase in transfected cells was with that of the FUS was that not to a as for FUS. The relative of to similar the expression FUS was more in the phosphorylation of The recruitment of signaling was a lower phosphorylation of STAT1, and with FUS but a phosphorylation of STAT5 to FUS. The was the of activation of STAT-specific gene was as as FUS in the but only the to of that of FUS. In FGFR1 was as fusion kinase in expression of the gene This that the not only activation of the kinase but determined to some the of the kinase function toward signal transduction pathways. chromosome of the of (1Rabbitts T.H. Science. 1994; 372: 143-149Google Scholar). In cases these specific for a of leukemia and as have used as The of these is to activation of genes that to the D. Abdul-Rauf M. Cooper J. Cross PubMed Scopus Google Scholar). of these genes fusion which for the activation of specific that to of the normal for growth and in the tumor This is the with FUS (2Abruzzo L.V. Jaffe E.S. Cotelingam J.D. Whang-Peng J. Del Duca V. Medeiros L.J. Am. J. Surg. Pathol. 1992; 16: 236-245Crossref PubMed Scopus (105) Google which identified in a form of no reported that this several have all of which in the activation of the FGFR1 kinase domain 16: PubMed Scopus Google Scholar). no tumor cell this rearrangement available, which to study the function of FUS in the same cell in which was cells have used in studies for fusion cells and not a analysis of the signaling function of the kinase V. G. D. R. D. J. PubMed Scopus Google Scholar, Blood. PubMed Google Scholar, K. M. G. R. Goldman J.M. Cross Blood. PubMed Scopus Google Scholar). In the of a cell for this we have used a of well cell that have to the signaling of FUS to more these studies that FUS signaling STAT was more prominent reported by K. M. G. R. Goldman J.M. Cross Blood. PubMed Scopus Google Scholar, G. V. D. 21: PubMed Scopus Google Scholar, D. Abdul-Rauf M. Cooper J. Cross PubMed Scopus Google Scholar). The the STAT activation to that of the normal FGFR1 as well as cytokine which the STAT to that the STAT signaling by FUS is similar in specificity to of cytokines, in to FUS to function in a similar to of the FGFR1 kinase in FUS, in FUS showed phosphorylation of as well as a to activate the same genes. Despite the of phosphorylation of in FUS from some oncogenic as in to activate STAT4 and a action for this kinase J. D. T. M. H. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The FUS and both cytokine and FGFR1 in to the phosphorylation of is to the that FUS is no to the and so a to This of in to the oncogenic activity of FUS. The of FUS, with cytokine in by the that activity of FUS, that of but that of is not by the the is important to that FUS is in the we have the of FUS expression to that in the is not and of the of cells from determined as The of FUS to a signaling is not to cells expressing as in transiently transfected of stably transfected which of FUS, prominent phosphorylation of and 5 cells transfected with of FUS expression showed activation of genes a is In cases of of FUS, the transfected cells within a the cells that have for expression and in myeloid cells the a FUS that In of the transfected of FUS was a to the to the of FUS as was in of transfected cells and in of cells Expression of FUS and which results in the of cells that form in the This is with that for where the kinase which is PubMed Scopus Google Scholar). of the using in cells clearly in the 5 which in the kinase not that FUS expression cell by The is within some of the cells is an active of the FUS protein that is determined by in ZNF198 This that cells have an that ZNF198 as of cytoplasmic and the that for In the of FUS, the some of the inhibitory activity of FUS The of the process to studies have using of that the zinc finger domain as a to activate the kinase (6Smedley D. Hamoudi R. Clark J. Warren W. Abdul-Rauf M. Somers G. Venter D. Fagan K. Cooper J. PubMed Scopus Google Scholar, J. V. M. G. M. D. D. PubMed Scopus Google Scholar, V. G. D. R. D. J. PubMed Scopus Google Scholar). analysis we have of the fusion gene and that this is the zinc but not the is with a Blood. PubMed Google Scholar) that the for the of FUS. In the by Blood. PubMed Google the fusion kinase used was the fusion kinase in is that of this fusion protein function we have clearly that the is for in the of the fusion The of normal and in the same hematopoietic cell is the that the normal function of gene we have shown that FUS and ZNF198 form and that this results in the phosphorylation of the of ZNF198 in the the of FUS. the of this for is not the of FUS, as a of this not in of FUS. we have shown that of FUS activity to a signaling from the FUS. groups have reported in some cases of this the FGFR1 kinase domain is with the Bcr gene that is associated with the fusion kinase in as well as the fusion kinase gene associated with a rearrangement J. V. M. G. M. D. D. PubMed Scopus Google Scholar, D. Cross PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). fusion genes to activation of the kinase function Bcr is with we have the N-terminal half of Bcr protein for the ZNF198 zinc finger to and activate the This to FUS, is not as in our in phosphorylation of in the and and this to a form of the disease from the reports from the patients with the of the fusion kinase gene described so The for the mode of action of FUS that the FGFR1 which is to the of the FUS to the which is for the oncogenic in the expressing of the for this from V. G. D. R. D. J. PubMed Scopus Google Scholar, Blood. PubMed Google Scholar, D. Abdul-Rauf M. Cooper J. Cross PubMed Scopus Google in cells as and we have that the fusion protein is in the the this same protein in the several In stably transfected the protein to the cell and in within the the of the FUS protein is located in the is not to the cytoplasmic FUS for the process V. G. D. R. D. J. PubMed Scopus Google Scholar). that the FUS protein is in the in cells stably expressing the oncogenic by the action of FUS located in the The of fusion gene with and the analysis of to provide more and the for cytokines, for for R. for the expression for and M. and for and for for the to the and for
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