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。我们发现了两个点突变，即膜旁区域的Val559-&Gt；Gly(G559)突变和激活区的Asp814-&Gt；Val(V814)突变，导致KIT的结构性和致癌活性。由于癌基因突变似乎改变了它们的构象和底物特异性，与野生型(WT)试剂盒相比，它们可能被某些化合物优先抑制。酪氨酸氨基转移酶是一类酪氨酸激酶的抑制剂，它通过与ATP结合来竞争底物。将KIT-WT、KIT-G559和KIT-V814表达载体分别导入293T胚胎肾细胞系293T，再用剂量递增的Tyrophostin AG1296处理后，KIT-G559蛋白的活性被抑制得比KIT-WT更有效，而KIT-V814蛋白的活性则完全不受影响。将这些KIT基因导入小鼠白细胞介素3-d…BA/F3、BA/F3-KIT-WT依赖干细胞因子(SCF)，而BA/F3-KIT-G559和BA/F3-KIT-V814不依赖干细胞因子。AG1296对BA/F3-KIT-G559细胞生长的抑制作用呈剂量依赖性，但抑制作用强度不同。AG1296对BA/F3-KIT-V814细胞的生长无影响，对依赖IL-3的细胞生长也无影响。AG1296比BA/F3-KIT-G559更有效地抑制MAPK的激活，并且比KIT-WT更有效地阻断KIT-G559的抗凋亡活性。这些结果表明，功能获得型KIT突变体可能成为癌症治疗的分子靶点。鼻腔自然杀伤T细胞淋巴瘤是致死性中线肉芽肿的主要成分之一。由于自然杀伤T细胞表达c-kit，我们用聚合酶链式反应-单链构象多态性直接测序的方法检测了这些病例中的c-kit突变。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个突变(92%)发生在密码子825，而外显子11的4个突变中有3个(75%)发生在密码子561。虽然密码子825突变不是功能获得突变，但这些突变在发病机制上是被研究的。较少

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