Analysis of oncogenic signal transduction and target genes by constitutive active mutations of c-Kit and Flt3

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1. Flt3 mutation specific target genesMicrochip analysis revealed that Flt3 internal tandem duplication (Flt3-ITD) mutation specifically induced STAT3/5 target genes such as pim-2, SOCS3 and CIS. These genes were also induced by constitutive active, c-Kit mutations. Pim-2 was.highly expressed in the leukemic cells form AML patients compared to normal bone marrow cells, which suggested that STAT3/5-pim2 was the common oncogenic signaling pathway in AML. Flt3-ITD specifically suppressed myeloid-specific transcription factors such as C/EBPalpha and PU.1, which suggested that Flt3-ITD is involved in the differentiation block in AML.2. Gytoplasmic tyrosine residues involved in the activation and siganl transduction of receptor tyrosine kinasesWe analyzed effects of phenylalanine conversion of 22 tyrosine residues in the cytoplasmic domain of c-Kit of Flt3 on receptor activation, down stream signal transduction and cell function. In the c-Kit kinase domain mutant, Phe-substitution of Tyr719 abolished the binding of p85 PI3-kinase subunit, the kinase activity of receptor itself and the proliferative activity. In the Flt3 kinase domain mutant, Phe-substitution of Tyr845, 892 and 922 suppressed the kinase activity, downstream signal transduction such as Erk1/2 and STAT3/5, factor-independent growth and survival. These results suggested that specific tyrosine residues criticallyregulated the oncogemc acivation of kinase domain muta its of receptor tyrosine kinases.