RESUMO
The Janus family of tyrosine kinases (JAK) plays an essential role in development and in coupling cytokine receptors to downstream intracellular signaling events. A t(9;12)(p24;p13) chromosomal translocation in a T cell childhood acute lymphoblastic leukemia patient was characterized and shown to fuse the 3' portion of JAK2 to the 5' region of TEL, a gene encoding a member of the ETS transcription factor family. The TEL-JAK2 fusion protein includes the catalytic domain of JAK2 and the TEL-specific oligomerization domain. TEL-induced oligomerization of TEL-JAK2 resulted in the constitutive activation of its tyrosine kinase activity and conferred cytokine-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line.
Assuntos
Leucemia-Linfoma de Células T do Adulto/metabolismo , Proteínas do Leite , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Sequência de Aminoácidos , Animais , Sequência de Bases , Biopolímeros , Divisão Celular , Linhagem Celular , Pré-Escolar , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Interleucina-3/fisiologia , Janus Quinase 2 , Leucemia-Linfoma de Células T do Adulto/genética , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Fosforilação , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-ets , Fator de Transcrição STAT5 , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Translocação Genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The t(5;12) translocation, associated with chronic myelomonocytic leukemia, generates a novel gene encoding a protein, TEL-PDGF beta R, composed of the 154 amino-terminal amino acids of the transcription factor TEL and the transmembrane and intracellular part of the PDGF beta-receptor (PDGF beta R). TEL also occurs as a tumor-associated fusion partner for the tyrosine kinases c-ABL, JAK2 and TRK-C. Previous studies have demonstrated growth promoting activity of TEL-PDGF beta R and also indicated that the TEL moiety activates the tyrosine kinase of the PDGF beta R through the formation of TEL-PDGF beta R oligomers. We demonstrate that tyrosine phosphorylation of the fusion protein can be attenuated through overexpression of the TEL part of TEL-PDGF beta R, suggesting a strategy for antagonizing the signaling of TEL-PDGF beta R, and other TEL-fusion proteins containing tyrosine kinase domains. Comparison of BaF/3 cell lines expressing TEL-PDGF beta R and ligand-stimulated PDGF beta R revealed that only TEL-PDGF beta R expression conferred IL-3-independent growth, suggesting differences in signaling capacity of the two proteins. Finally, tyrosine residues 17 and 27 in TEL-PDGF beta R was identified as autophosphorylation sites in TEL-PDGF beta R.
Assuntos
Leucemia Mielomonocítica Crônica/genética , Proteínas de Fusão Oncogênica/genética , Animais , Sequência de Bases , Células COS , Primers do DNA , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Proteínas de Fusão Oncogênica/metabolismo , Fenilalanina/genética , Fosforilação , Células Tumorais Cultivadas , Tirosina/genética , Tirosina/metabolismoRESUMO
The Src-like adaptor protein (SLAP) belongs to the subfamily of adapter proteins that negatively regulate cellular signalling initiated by tyrosine kinases. SLAP has a unique, myristylated N-terminus, followed by SH3 and SH2 domains with high homology to Src family tyrosine kinases (SFK) and a unique C-terminal tail, which is important for c-Cbl binding. We have previously shown that SLAP negatively regulates platelet-derived growth factor (PDGF)-induced mitogenesis in fibroblasts and we now report that it regulates F-actin assembly for dorsal ruffles formation. c-Cbl mediated SLAP inhibition towards actin remodelling. Moreover, SLAP enhanced PDGF-induced c-Cbl phosphorylation by SFK. In contrast, SLAP mitogenic inhibition was not mediated by c-Cbl, but it rather involved a competitive mechanism with SFK for PDGF-receptor (PDGFR) association and mitogenic signalling. Accordingly, phosphorylation of the Src mitogenic substrates Stat3 and Shc were reduced by SLAP. Thus, we concluded that SLAP regulates PDGFR signalling by two independent mechanisms: a competitive mechanism for PDGF-induced Src mitogenic signalling and a non-competitive mechanism for dorsal ruffles formation mediated by c-Cbl.
Assuntos
Actinas/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Ligação Competitiva , Fibroblastos/citologia , Fibroblastos/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Mitose/fisiologia , Células NIH 3T3 , Fosforilação , Mutação Puntual , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
The cytoplasmic tyrosine kinase Src has been implicated in signal transduction induced by growth factors and integrins. Src also shows oncogenic activity when deregulated. Accumulating evidence indicates that the tyrosine kinase Abl is an important substrate for Src signalling in normal cells. Here we show that Abl is also required for Src-induced transformation of mouse fibroblasts. Abl does not mediate tyrosine phosphorylation of Stat3 and Shc, two important regulators of Src oncogenic activity. In contrast, Abl controls the activation of the small GTPase Rac for oncogenic signalling and active Rac partly rescued Src transformation in cells with inactive Abl. Moreover, Abl mediates Src-induced extracellular regulated kinase 5 (ERK5) activation to drive cell transformation. Finally, we find that Abl/Rac and Abl/ERK5 pathways also operate in human MCF7 and BT549 breast cancer cells, where neoplastic transformation depends on Src-like activities. Therefore, Abl is an important regulator of Src oncogenic activity both in mouse fibroblasts and in human cancer cells. Targeting these Abl-dependent signalling cascades may be of therapeutic value in breast cancers where Src-like function is important.
Assuntos
Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células 3T3 BALB/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células Cultivadas , Ativação Enzimática , Fibroblastos/citologia , Humanos , Camundongos , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Células NIH 3T3/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Fator de Transcrição STAT3/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Tirosina/metabolismoRESUMO
The cytoplasmic tyrosine kinases of the Src family (SFK) play important roles in cell responses induced by growth factors, including cell growth, survival and migration. Here, we review how SFK participate in PDGF (platelet-derived growth factor) receptor signalling leading to DNA synthesis and actin assembly. Furthermore, evidence for a spatial compartmentalization of SFK signalling is also discussed.
Assuntos
Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Modelos BiológicosRESUMO
TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Oncogênicas/fisiologia , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Repressoras , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Divisão Celular , Sequência Conservada , Proteínas de Ligação a DNA/química , Drosophila , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Oncogênicas/química , Proteínas Proto-Oncogênicas c-ets , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Receptores do Fator de Crescimento Derivado de Plaquetas/química , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The involvement of the cytokine signaling pathway in oncogenesis has long been postulated. Recently, rearrangements of the gene encoding the tyrosine Janus kinase 2 (JAK2) have been reported in human leukemias indicating a direct JAK-signal transduction and activator of transcription (STAT)-mediated leukemic process. The leukemia-associated TEL-JAK2 fusion protein is formed by the oligomerization domain of the translocated ets leukemia (TEL) protein fused to the catalytic domain of JAK2. TEL-mediated oligomerization results in a constitutive tyrosine kinase activity that, in turn, is able to confer growth factor independence to the murine hematopoietic interleukin-3 (IL-3)-dependent Ba/F3 cell line. Results of the present study indicate that fusion proteins containing the oligomerization domain of TEL and the tyrosine kinase domains of Jak1, Jak2, JAK3, or TYK2 share similar properties and are able to efficiently substitute for the survival and mitogenic signals controlled by IL-3, without concomitant activation of the IL-3 receptor. Electrophoretic mobility shift assays demonstrated Stat5 as the only activated Stat factor in TEL-Jak2- and TEL-Jak1-expressing cells, whereas other Stats, namely Stat1 and Stat3, could be detected in TEL-JAK3-, TEL-TYK2-, and also in TEL-ABL-expressing Ba/F3 cells. High levels of expression of the Stat5-target genes pim-1, osm, and Cis were observed in all the cytokine-independent cell lines. Furthermore, the expression of a dominant negative form of Stat5A markedly interfered with the growth factor independence process mediated by TEL-Jak2 in Ba/F3 cells. Because the BCR-ABL and TEL-PDGFbetaR oncoproteins also activate Stat5, activation of this factor should be a crucial step in activated tyrosine kinase-mediated leukemogenesis. (Blood. 2000;95:2076-2083)