BACKGROUND KIT tyrosine kinase is expressed in mast cells, interstitial cells of Cajal, and hematopoietic cells. Permanently active KIT mutations lead these host cells to tumorigenesis, and to such diseases as mast cell leukemia (MCL), gastrointestinal stromal tumor (GIST), and acute myeloid leukemia (AML). Recently, we reported that in MCL, KIT with mutations (D816V, human; D814Y, mouse) traffics to endolysosomes (EL), where it can then initiate oncogenic signaling. On the other hand, KIT mutants including KITD814Y in GIST accumulate on the Golgi, and from there, activate downstream. KIT mutations, such as N822K, have been found in 30% of core binding factor-AML (CBF-AML) patients. However, how the mutants are tyrosine-phosphorylated and where they activate downstream molecules remain unknown. Moreover, it is unclear whether a KIT mutant other than KITD816V in MCL is able to signal on EL. METHODS We used leukemia cell lines, such as Kasumi-1 (KITN822K, AML), SKNO-1 (KITN822K, AML), and HMC-1.1 (KITV560G, MCL), to explore how KIT transduces signals in these cells and to examine the signal platform for the mutants using immunofluorescence microscopy and inhibition of intracellular trafficking. RESULTS In AML cell lines, KITN822K aberrantly localizes to EL. After biosynthesis, KIT traffics to the cell surface via the Golgi and immediately migrates to EL through endocytosis in a manner dependent on its kinase activity. However, results of phosphorylation imaging show that KIT is preferentially activated on the Golgi. Indeed, blockade of KITN822K migration to the Golgi with BFA/M-COPA inhibits the activation of KIT downstream molecules, such as AKT, ERK, and STAT5, indicating that KIT signaling occurs on the Golgi. Moreover, lipid rafts in the Golgi play a role in KIT signaling. Interestingly, KITV560G in HMC-1.1 migrates and activates downstream in a similar manner to KITN822K in Kasumi-1. CONCLUSIONS In AML, KITN822K mislocalizes to EL. Our findings, however, suggest that the mutant transduces phosphorylation signals on lipid rafts of the Golgi in leukemia cells. Unexpectedly, the KITV560G signal platform in MCL is similar to that of KITN822K in AML. These observations provide new insights into the pathogenic role of KIT mutants as well as that of other mutant molecules.

中文翻译：

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N822K或V560G突变的KIT激活优先发生在白血病细胞中高尔基体的脂质筏中。

背景技术KIT酪氨酸激酶在肥大细胞，Cajal的间质细胞和造血细胞中表达。永久活跃的KIT突变导致这些宿主细胞发生肿瘤，并导致诸如肥大细胞白血病（MCL），胃肠道间质瘤（GIST）和急性髓细胞性白血病（AML）等疾病。最近，我们报道了在MCL中，具有突变的KIT（D816V，人； D814Y，小鼠）运输到了溶酶体（EL），然后它可以启动致癌信号。另一方面，包括GIST中的KITD814Y在内的KIT突变体在高尔基体上积累，并从那里向下游激活。在30％的核心结合因子AML（CBF-AML）患者中发现了KIT突变，例如N822K。然而，突变体如何被酪氨酸磷酸化以及它们在何处激活下游分子仍是未知的。而且，目前尚不清楚MCL中除KITD816V以外的KIT突变体是否能够在EL上发出信号。方法我们使用Kasumi-1（KITN822K，AML），SKNO-1（KITN822K，AML）和HMC-1.1（KITV560G，MCL）等白血病细胞系来研究KIT如何在这些细胞中转导信号并检查使用免疫荧光显微镜和抑制细胞内运输的突变体的信号平台。结果在AML细胞系中，KITN822K异常定位于EL。生物合成后，KIT通过高尔基体运输到细胞表面，并通过内吞作用立即迁移到EL，其方式取决于其激酶活性。然而，磷酸化成像的结果表明，KIT在高尔基体上被优先激活。实际上，用BFA / M-COPA阻止KITN822K迁移到高尔基体会抑制KIT下游分子（例如AKT）的激活，ERK和STAT5，表示在高尔基体上发生KIT信号。此外，高尔基体中的脂筏在KIT信号传导中起作用。有趣的是，HMC-1.1中的KITV560G以与Kasumi-1中的KITN822K相似的方式向下游迁移并激活。结论在AML中，KITN822K错误地定位到EL。然而，我们的发现表明，该突变体在白血病细胞中的高尔基体脂筏上转导了磷酸化信号。出乎意料的是，MCL中的KITV560G信号平台类似于AML中的KITN822K。这些观察结果提供了对KIT突变体以及其他突变分子的致病作用的新见解。1以与Kasumi-1中的KITN822K相似的方式向下游迁移并激活。结论在AML中，KITN822K错误地定位到EL。然而，我们的发现表明，该突变体在白血病细胞中的高尔基体脂筏上转导了磷酸化信号。出乎意料的是，MCL中的KITV560G信号平台类似于AML中的KITN822K。这些观察结果提供了对KIT突变体以及其他突变分子的致病作用的新见解。1以与Kasumi-1中的KITN822K相似的方式向下游迁移并激活。结论在AML中，KITN822K错误地定位到EL。然而，我们的发现表明，该突变体在白血病细胞中的高尔基体脂筏上转导了磷酸化信号。出乎意料的是，MCL中的KITV560G信号平台类似于AML中的KITN822K。这些观察结果提供了对KIT突变体以及其他突变分子的致病作用的新见解。