Kitayama H, Kanakura Y, Furitsu T, Tsujimura T, Oritani K, Ikeda H, Sugahara H, Mitsui H, Kanayama Y, Kitamura Y
Second Department of Internal Medicine, Osaka University Medical School, Japan.
Blood. 1995 Feb 1;85(3):790-8.
The c-kit receptor tyrosine kinase (KIT) is activated upon ligand binding, thereby leading to a variety of signaling events that play a fundamental role in hematopoiesis. In addition to ligand-dependent activation, we have previously shown that KIT is constitutively activated in a ligand-independent manner by two point mutations, Val-559-->Gly (G559) mutation in the juxtamembrane domain and Asp-814-->Val (V814) mutation in the phosphotransferase domain. To investigate the biochemical consequence and biologic significance of these mutations, retroviral vectors encoding KITG559 or KITV814 were introduced into murine pro-B-type Ba/F3 cells and myeloid FDC-P1 cells, both of which require interleukin-3 (IL-3) for their growth and survival. In the cells, KITG559 or KITV814 were found to be constitutively phophorylated on tyrosine in the absence of stem cell factor (SCF) that is a ligand for KIT. Chemical cross-linking analysis showed that a substantial fraction of the phosphorylated KITG559 underwent dimerization even in the absence of SCF, whereas the phosphorylated KITV814 did not, suggesting the distinct mechanisms underlying constitutive activation of KIT by G559 and V814 mutations. Furthermore, the cells expressing either KITG559 or KITV814 were found to show a factor-independent growth, whereas the cells expressing wild-type KIT (KITWT) proliferated in response to SCF as well as IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing KITG559 or KITV814 into nude mice resulted in production of large tumors at all sites of the injection within 2 weeks, and all nude mice quickly succumbed to leukemia and died. These results suggest that, although the mechanisms underlying constitutive activation of KITG559 or KITV814 may be different, both of the activating mutations have a function to induce a factor-independent and tumorigenic phenotype. Also, the data of this study raise the possibility that the constitutively activating mutations of c-kit may play a causal role in development of hematologic malignancies.
c-kit受体酪氨酸激酶（KIT）在配体结合后被激活，从而引发多种信号转导事件，这些事件在造血过程中发挥着重要作用。除了依赖配体的激活外，我们之前还表明，KIT通过两个点突变以非配体依赖的方式被组成性激活，即近膜结构域中的Val-559→Gly（G559）突变和磷酸转移酶结构域中的Asp-814→Val（V814）突变。为了研究这些突变的生化后果和生物学意义，将编码KITG559或KITV814的逆转录病毒载体导入小鼠前B型Ba/F3细胞和髓系FDC-P1细胞，这两种细胞的生长和存活都需要白细胞介素-3（IL-3）。在这些细胞中，发现KITG559或KITV814在没有作为KIT配体的干细胞因子（SCF）的情况下，酪氨酸会被组成性磷酸化。化学交联分析表明，即使在没有SCF的情况下，相当一部分磷酸化的KITG559也会发生二聚化，而磷酸化的KITV814则不会，这表明G559和V814突变对KIT组成性激活的潜在机制不同。此外，发现表达KITG559或KITV814的细胞表现出因子非依赖性生长，而表达野生型KIT（KITWT）的细胞则对SCF和IL-3都有增殖反应。此外，将表达KITG559或KITV814的Ba/F3细胞皮下注射到裸鼠体内，在2周内所有注射部位都产生了大肿瘤，所有裸鼠很快患上白血病并死亡。这些结果表明尽管KITG559或KITV814组成性激活的潜在机制可能不同，但这两种激活突变都具有诱导因子非依赖性和致瘤表型的功能。此外，本研究的数据还提出了c-kit的组成性激活突变可能在血液系统恶性肿瘤发生中起因果作用的可能性。
