Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Yamazaki 2669, Noda, Chiba, 278-0022, Japan.
Division of Cancer Differentiation, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, 104-0045, Tokyo, Japan.
Cell Commun Signal. 2019 Sep 4;17(1):114. doi: 10.1186/s12964-019-0426-3.
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 KIT 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 KIT in MCL is able to signal on EL.
We used leukemia cell lines, such as Kasumi-1 (KIT, AML), SKNO-1 (KIT, AML), and HMC-1.1 (KIT, 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.
In AML cell lines, KIT 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 KIT 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, KIT in HMC-1.1 migrates and activates downstream in a similar manner to KIT in Kasumi-1.
In AML, KIT mislocalizes to EL. Our findings, however, suggest that the mutant transduces phosphorylation signals on lipid rafts of the Golgi in leukemia cells. Unexpectedly, the KIT signal platform in MCL is similar to that of KIT in AML. These observations provide new insights into the pathogenic role of KIT mutants as well as that of other mutant molecules.
KIT 酪氨酸激酶在肥大细胞、Cajal 间质细胞和造血细胞中表达。永久性激活的 KIT 突变可导致这些宿主细胞发生肿瘤形成，并导致诸如肥大细胞白血病（MCL）、胃肠道间质瘤（GIST）和急性髓系白血病（AML）等疾病。最近，我们报道在 MCL 中，具有突变（D816V，人类；D814Y，小鼠）的 KIT 会运输到内溶酶体（EL），从而启动致癌信号。另一方面，包括 GIST 中的 KIT 突变体在内的 KIT 突变体在内质网中积累，并从那里激活下游。在 30%的核心结合因子-AML（CBF-AML）患者中发现了 KIT 突变，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不清楚。此外，尚不清楚除 MCL 中的 KIT 突变体外，其他 KIT 突变体是否能够在 EL 上发出信号。
我们使用白血病细胞系，如 Kasumi-1（KIT，AML）、SKNO-1（KIT，AML）和 HMC-1.1（KIT，MCL），来探索 KIT 在这些细胞中如何传递信号，并使用免疫荧光显微镜和细胞内运输抑制来检查突变体的信号平台。
在 AML 细胞系中，KIT 异常定位于 EL。在生物合成后，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，这种迁移方式依赖于其激酶活性。然而，磷酸化成像结果表明，KIT 在高尔基体上优先被激活。事实上，用 BFA/M-COPA 阻断 KIT 向高尔基体的迁移可抑制 KIT 下游分子（如 AKT、ERK 和 STAT5）的激活，表明 KIT 信号发生在高尔基体上。此外，高尔基体中的脂筏在 KIT 信号中发挥作用。有趣的是，HMC-1.1 中的 KIT 以类似于 Kasumi-1 中的 KIT 的方式迁移并激活下游。
在 AML 中，KIT 定位异常到 EL。然而，我们的发现表明，突变体在白血病细胞的高尔基体脂筏上传递磷酸化信号。出乎意料的是，MCL 中的 KIT 信号平台与 AML 中的 KIT 信号平台相似。这些观察结果为 KIT 突变体以及其他突变分子的致病作用提供了新的见解。
