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（例如C-KIT和FLT3）的组成型活性突变体已被鉴定为急性髓样白血病中频繁的遗传异常，它们作为可能的治疗靶点。我们已经制作了一系列C-KIT和FLT3的Tyr-Phe突变体，无论是野生型还是活动突变体，并分析了导致细胞功能和致癌转化的临界信号转导途径。通过分析C-KIT的每个22个酪氨酸残基的Tyr-Phe突变，我们已经确定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，它们协同调节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诱发的增殖，RAC/JNK的激活和RAS所必需的。在野生型C-KIT中，Tyr567介导SFK结合，而SFK活性是GAB2酪酶磷酸化和与SHP-2的关联所必需的。因此，我们发现GAB2是Tyr567的下游信号传导中间体，它调节C-KIT/SCF介导的细胞功能。在本构主动突变体中，与野生型相比，STAT3/5高度激活。这种异常激活的机制尚未澄清。由于FLT3在细胞质结构域中具有三个共识STAT3结合基序，因此我们分析了这些酪氨酸残基在FLT3 ASP835VAL激活STAT3中的参与。在FLT3 ASP835VAL中，这三个酪氨酸残基的Tyr-Phe转化减少了，但并未完全消除STAT3激活。该结果表明，在受体酪氨酸激酶的组成型活性突变体中，异常的统计激活仍然部分取决于与共有基序序列的结合，但也取决于替代激活途径，例如SRC族激酶途径，例如近距离区域和GAB2。