| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
ARG is a widely expressed tyrosine kinase, while the function is not well understood. In the study, TEL/ARG was used to investigate the function of ARG, and it's role in leukemohenesis.
The TEL/ARG oncogene associated with AML is formed by the t(;12)(q25;p13) reciprocal translocation, fusing part of the TEL gene to the tyrosine kinase, c-ARG. I have previously demonstrated that expression of TEL/ARG in Ba/F3 cells results in prolonged viability and hyper-responsiveness to hematopoietic growth factors. In an effort to determine the contribution of various domains of the TEL/ARG oncoprotein to signaling and biological activity, a series of mutants of either the TEL or ARG component of TEL/ARG were generated. A deletion mutation of TEL was studied, lacking the PNT domain of TEL (ΔPNT). Three deletion mutants of ARG were generated, deleting either the SH3 domain (ΔSH3), the SH2 domain (ΔSH2) and the C-terminal domain (ΔC-term). In addition two point mutations were studied, Y314F, which muta
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tes the GRB2 binding site in TEL, and K317R, creating a kinase dead mutant of ARG. Sublines of Ba/F3 cells were then generated in which wild type or mutant TEL/ARG cDNA's were expressed under the control of a doxycycline-inducible promoter and analyzed for signaling defects, viability, and responsiveness to growth factors. Consistent with the results from other TEL-tyrosine kinase oncogenes, both signaling and biological effects were profoundly impaired in ΔPNT and K317R TEL/ARG mutants. The ability to support viability was moderately impaired in the Y314F mutant, while the ΔSH3 mutant was equivalent to wild type. However, each of those mutants lost the ability to induce hyper-responsiveness to IL-3, which is characteristic of the wild type oncogene. In contrast, the ΔSH2 oncogene promoted hyer-responsiveness to IL-3, but supported viability only minimally. Finally, all biological functions of the ΔC-term mutant were profoundly impaired, despite maintaining high levels of kinase activity. When expressed in CHO cells, wild type TEL/ARG induced the formation of fillopodia, in a fashion dependent on the C-terminal portion and intact kinase actifity, suggesting an important role for the C-terminal domain in this oncogene. Thus, the mutants described here have defined several critical domains within TEL/ARG necessary for function, and indicate that the signaling pathways necessary for viability, growth factor hyper-responsiveness and cytoskeletal reorganization are likely to be separate. Less
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