Novel signal transduction pathways in constitutive active mutants of receptor tyrosine kianses

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Constitutive active mutants of receptor tyrosine kinases such as c-Kit and FLT3 have been identified as the frequent genetic abnormalities in acute myeloid leukemia, which serve as possible therapeutic targets. We have made a series of single Tyr-Phe mutants of c-Kit and FLT3, either wild type or active mutants, and analysed the critical signal transduction pathways which lead to cell function and oncogenic transformation. By analyzing the Tyr-Phe mutations of each 22 tyrosine residue of c-Kit, we have identified that Tyr567, Tyr569, and Tyr719 are the critical tyrosine residues which regulated the chemotactic function of c-Kit. Tyr567 and Tyr719 activates Src family kinases (SFK) and PI3K respectively, which cooperatively regulate the c-Kit/SCF mediated chemotaxis. By analyzing Gab2 (-/-) mast cells, we find that Gab2 is required for SCF-evoked proliferation, activation of Rac/JNK, and Ras. In wild type c-Kit, Tyr567 mediates SFK binding, and SFK activity was required for Gab2 tyrosyl phosphorylation and association with Shp-2. Thus, we find that Gab2, which is the downstream signaling intermediate of Tyr567, regulates c-Kit/SCF-mediated cellular function. In constitutive active mutants, STAT3/5 is highly activated compared to wild type. The mechanism of this aberrant activation has not been clarified. As FLT3 has three consensus STAT3 binding motifs in cytoplasmic domain, we have analysed the involvement of these tyrosine residues in the activation of STAT3 by FLT3 Asp835Val. In FLT3 Asp835Val, the Tyr-Phe conversion of these three tyrosine residues diminished, but not completely abolished the STAT3 activation. This result suggests that in constitutive active mutants of receptor tyrosine kinases, the aberrant STAT activation still partially depends on the binding to the consensus motif sequence, but also on the surrogate activation pathways such as src familily kinase pathways, which may be mediated by the juxtamembrane region and Gab2.