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

受体酪氨酸激酶组成型活性突变体的新信号转导途径

基本信息

批准号：
18591058
负责人：
MIZUKI Masao
金额：
$ 2.49万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

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.

受体酪氨酸激酶（如c-Kit和FLT3）的组成型活性突变体已被确定为急性髓性白血病中常见的遗传异常，可作为可能的治疗靶点。我们制作了一系列c-Kit和FLT3的单Tyr-Phe突变体，无论是野生型还是活性突变体，并分析了导致细胞功能和致癌转化的关键信号转导途径。通过分析c-Kit各22个酪氨酸残基的tyrl - phe突变，我们发现Tyr567、Tyr569和Tyr719是调节c-Kit趋化功能的关键酪氨酸残基。Tyr567和Tyr719分别激活Src家族激酶（SFK）和PI3K，它们共同调节c-Kit/SCF介导的趋化性。通过分析Gab2（-/-）肥大细胞，我们发现Gab2是scf诱导的增殖、Rac/JNK和Ras的激活所必需的。在野生型c-Kit中，Tyr567介导SFK结合，SFK活性是Gab2酪氨酸磷酸化和与Shp-2结合所必需的。因此，我们发现Gab2， Tyr567的下游信号中间体，调节c-Kit/ scf介导的细胞功能。在组成型活性突变体中，与野生型相比，STAT3/5被高度激活。这种异常激活的机制尚不清楚。由于FLT3在细胞质域有三个一致的STAT3结合基序，我们分析了这些酪氨酸残基在FLT3 Asp835Val激活STAT3中的作用。在FLT3 Asp835Val中，这三个酪氨酸残基的tyrl - phe转化减少，但没有完全消除STAT3的激活。这一结果表明，在受体酪氨酸激酶的组成型活性突变体中，STAT的异常激活仍然部分依赖于与共识基序序列的结合，但也依赖于替代激活途径，如src家族激酶途径，这些途径可能由近膜区和Gab2介导。