RESUMO
The TEL/PDGFbetaR gene, which encodes a fusion protein containing the ETS-family member TEL fused to the protein-tyrosine kinase domain of the platelet-derived growth factor receptor-beta (PDGFbetaR), confers interleukin 3 (IL-3)-independent growth on Ba/F3 hematopoietic cells. TEL/PDGFbetaR mutants have been generated that contain tyrosine-to-phenylalanine (Tyr-->Phe) substitutions at phosphorylation sites present in the native PDGFbetaR to assess the role of these sites in cell transformation by TEL/PDGFbetaR. Similar to previous findings in a murine bone marrow transplantation model, full transformation of Ba/F3 cells to IL-3-independent survival and proliferation required the TEL/PDGFbetaR juxtamembrane and carboxy terminal phosphorylation sites. In contrast to previous reports concerning comparable mutants in the native PDGFbetaR, each of the TEL/PDGFbetaR mutants is fully active as a protein-tyrosine kinase. Expression of the TEL/PDGFbetaR fusion protein causes hyperphosphorylation and activation of signal transducer and activator of transcription (STAT5), and this activation of STAT5 requires the juxtamembrane Tyr579 and Tyr581 in the TEL/PDGFbetaR fusion. Hyperphosphosphorylation of phospholipase Cgamma (PLCgamma) and the p85 subunit of phosphatidylinositol 3-kinase (PI3K) requires the carboxy terminal tyrosine residues of TEL/PDGFbetaR. Thus, full transformation of Ba/F3 cells by TEL/PDGFbetaR requires engagement of PI3K and PLCgamma and activation of STAT5. Taken together with the growth properties of cells transformed by the TEL/PDGFbetaR variants, these findings indicate that a minimal combination of these signaling intermediates contributes to hematopoietic transformation by the wild-type TEL/PDGFbetaR fusion. (Blood. 2001;98:3390-3397)
Assuntos
Proteínas de Ligação a DNA/fisiologia , Leucemia Mielomonocítica Crônica/patologia , Proteínas do Leite , Proteínas de Fusão Oncogênica/fisiologia , Transdução de Sinais , Transativadores/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , Leucemia Mielomonocítica Crônica/genética , Camundongos , Mutação , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT5 , Relação Estrutura-AtividadeRESUMO
STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an expression cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an alpha (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4(+) T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19(+) B cells and CD14(+) monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.
Assuntos
Quimiocinas CC/química , Quimiocinas CX3C/química , Quimiocinas CXC/química , Quimiocinas CXC/genética , Clonagem Molecular/métodos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Receptores de Citocinas/metabolismo , Receptores Acoplados a Proteínas G , Receptores Imunológicos , Receptores Virais , Sequência de Aminoácidos , Southern Blotting , Linhagem Celular , Membrana Celular/genética , Membrana Celular/imunologia , Membrana Celular/metabolismo , Quimiocina CXCL16 , Quimiocinas CXC/biossíntese , Quimiocinas CXC/fisiologia , DNA Complementar/isolamento & purificação , Glicosilação , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Ligantes , Ativação Linfocitária , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , RNA/biossíntese , Receptores CXCR6 , Receptores de Quimiocinas , Receptores Depuradores , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de Proteína , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , TransfecçãoRESUMO
OBJECTIVE: TEL/PDGFbetaR is a tyrosine kinase fusion protein associated with the pathogenesis of chronic myelomonocytic leukemia. The following experiments were undertaken to understand the mechanisms whereby TEL/PDGFbetaR transforms cells. MATERIALS AND METHODS: Activation of JAK and STAT proteins was studied in an interleukin 3 (IL-3)-dependent cell line, Ba/F3, transformed to IL-3 independence by TEL/PDGFbetaR. RESULTS: TEL/PDGFbetaR activates STAT1 and STAT5 in transformed Ba/F3 cells through a JAK-independent pathway. Activation of STAT proteins requires the kinase activity of TEL/PDGFbetaR. JAK1, JAK2, JAK3, and TYK2 are not phosphorylated by TEL/PDGFbetaR. However, TEL/PDGFbetaR can phosphorylate STAT5 in transiently transfected COS cells, suggesting that TEL/PDGFbetaR may itself be the kinase involved in tyrosine phosphorylation of STAT proteins. In contrast, native PDGFbetaR stimulated by PDGF ligand does not activate STAT proteins to a significant degree in this hematopoietic context. STAT1 and STAT5 also are activated by TEL/ABL and TEL/JAK2 fusion proteins associated with human leukemia. CONCLUSIONS: STAT activation may be a common mechanism of transformation by leukemogenic tyrosine kinase fusion proteins.