Mechanisms underlying constitutive activation and oncogenic potential of mutant c-kit receptor tyrosine kinase

c-kit受体酪氨酸激酶（KIT）在胚胎发育、造血和恶性转化中起重要作用。我们发现，两个点突变，Val 559->Gly（G559）突变的质膜结构域和Asp 814->瓦尔（V814）突变的激酶结构域，导致KIT的组成和致癌激活。Tyrophostines是一类酪氨酸激酶抑制剂，通过竞争底物或通过ATP结合。当KIT-WT、KIT-G559和KIT-V814表达载体转染到胚肾细胞系293 T中并用增加剂量的tyrophostin AG 1296处理时，KIT-G559蛋白的激酶活性比KIT-WT更有效地被抑制，而KIT-V814的激酶活性完全不受影响。当这些KIT基因被引入小鼠白细胞介素（IL）-3-d ...更多信息 Ba/F3、Ba/F3-KIT-WT细胞系具有干细胞因子（SCF）依赖性，而Ba/F3-KIT-G559和Ba/F3-KIT-V814细胞系不具有SCF依赖性。AG 1296以剂量依赖性方式抑制Ba/F3-KIT-G559的因子非依赖性生长和Ba/F3-KIT-WT的SCF依赖性生长，但效力不同。AG 1296对Ba/F3-KIT-V814的因子非依赖性生长或Ba/F3-mock的IL-3依赖性生长没有影响。AG 1296对Ba/F3-KIT-G559中MAP激酶活性的抑制作用强于Ba/F3-KIT-WT，对KIT-G559抗凋亡活性的阻断作用强于KIT-WT。由于自然界的人T细胞表达c-kit，我们通过PCR-SSCP和直接测序检测了这些病例中的c-kit突变。在23例中发现12个单核苷酸替换突变。此外，外显子17的8个突变中有7个（92%）发生在密码子825处，外显子11的4个突变中有3个（75%）发生在密码子56 l处。虽然密码子825突变不是功能获得性突变，但这些突变的发病机制进行了研究。少

The c-kit receptor tyrosine kinase(KIT)plays important roles in embryonhic development, *ematopoiesis and also malignant transformation. We revealed that two point mutations, Val559-->Gly(G559)mutation in the juxtamembrane domain and Asp814-->Val(V814)mutation in the kinase domain, lead to constitutive and oncogenic activation of KIT.Since oncogenic mutants seem to alter their conformation and substrate specificity compared with wild-type(WT)KIT, they may be preferentially inhibited by some compound. Tyrophostines are a class of tyrosine kinase inhibitor either through competition for substrate on through ATP binding. When KIT-WT, KIT-G559 and KIT-V814 expression vector were transfected into an embryonic kidney cell line 293T and treated with increasing dose of tyrophostin AG1296, kinase activity of KIT-G559 protein was more effectively inhibited than that of KIT-WT, whereas that of KIT-V814 was not affected at all. When these KIT genes were introduced into a murine interleukin(IL)-3-d … More ependent cell line Ba/F3, Ba/F3-KIT-WT showed Stem Cell Factor(SCF)-dependent, while Ba/F3-KIT-G559 and Ba/F3-KIT-V814 factor-independent. AG1296 inhibited both factor-independent growth of Ba/F3-KIT-G559 and SCF-dependent growth of Ba/F3-KIT-WT in a dose dependent manner but with different potencies. AG1296 had no effect on factor-independent growth of Ba/F3-KIT-V814 or IL-3-dependent growth of Ba/F3-mock. AG1296 inhibited activation of MAP kinase more effectively in Ba/F3-KIT-G559 than in Ba/F3-KIT-WT.Furthermore, AG1296 blocked antiapoptotic activity of KIT-G559 more potently than that of KIT-WT.These results suggest that a gain-of-function KIT mutant may be used as a molecular target for cancer therapy.Sinonasal natural killer/T-cell lymphoma is one of the major constituents of lethal midline granuloma. Since natural killerT cell expresses c-kit, we examined c-kit mutation in these cases by PCR-SSCP followed by direct sequencing. Twelve single nucleotide substitution mutations were seen in 23 cases. Furthermore, seven of eight mutations(92%)in exon 17 occurred at codon 825 and three of four mutations(75%)in exon 11 occurred at codon 56l. Although the codon 825 mutation was not a gain-of-function mutation, the mechanisms of these mutations in pathogenesis are investigated. Less