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

受体酪氨酸激酶的组成型活性突变体如c-Kit和FLT 3已被确定为急性髓系白血病中常见的遗传异常，其作为可能的治疗靶点。我们已经制备了一系列c-Kit和FLT 3的单一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的22个酪氨酸残基的Tyr-Phe突变，我们确定Tyr 567、Tyr 569和Tyr 719是调节c-Kit趋化功能的关键酪氨酸残基。Tyr 567和Tyr 719分别激活Src家族激酶（SFK）和PI 3 K，其协同调节c-Kit/SCF介导的趋化性。通过分析Gab 2（-/-）肥大细胞，我们发现Gab 2是SCF诱发的增殖、Rac/JNK和Ras激活所必需的。在野生型c-Kit中，Tyr 567介导SFK结合，并且SFK活性是Gab 2酪氨酰磷酸化和与Shp-2结合所需的。因此，我们发现，Gab 2，这是下游信号中间的Tyr 567，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，STAT 3/5被高度活化。这种异常激活的机制尚未阐明。由于FLT 3在胞质结构域中具有三个共有的STAT 3结合基序，我们分析了这些酪氨酸残基在FLT 3 Asp 835 Val激活STAT 3中的参与。在FLT 3 Asp 835 Val中，这三个酪氨酸残基的Tyr-Phe转化减少，但没有完全消除STAT 3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，异常STAT激活仍然部分依赖于与共有基序序列的结合，但也依赖于替代激活途径，如src家族激酶途径，其可能由质膜区域和Gab 2介导。

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.