Deficiency of core fucosylation activates cellular signaling dependent on FLT3 expression in a Ba/F3 cell system
Deficiency of core fucosylation activates cellular signaling dependent on FLT3 expression in a Ba/F3 cell system
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DOI：
10.1096/fj.201902313rr
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发表时间：
2020-01-05
期刊：
影响因子：
4.8
通讯作者：
Gu, Jianguo
中科院分区：
文献类型：
--
作者：
Duan, Chengwei;Fukuda, Tomohiko;Gu, Jianguo
FMS样酪氨酸激酶3（FLT 3）是一种糖蛋白，属于III类受体酪氨酸激酶家族。大约三分之一的急性髓性白血病（AML）患者具有该基因的突变，并且FLT 3下游通路的激活在正常和恶性造血中起重要作用。然而，N-糖基化对FLT 3活化的作用仍不清楚。在这项研究中，我们发现野生型（WT），内部串联重复（ITD）和酪氨酸激酶结构域（TKD）突变体的FLT 3的N-聚糖结构是不同的。有趣的是，WT或突变体FLT 3在Ba/F3细胞（一种白细胞介素-3（IL-3）依赖性造血祖细胞）中的表达极大地诱导了核心岩藻糖基化。为了阐明核心岩藻糖基化在FLT 3介导的信号传导中的功能，我们使用CRISPR/Cas9系统来建立α 1，6-岩藻糖基转移酶（Fut 8）敲除（KO）细胞。令人惊讶的是，Fut 8 KO在FLT 3-WT细胞中以IL-3非依赖性方式导致细胞增殖，这在亲本细胞中未观察到，并表明这种增殖依赖于FLT 3表达。Fut 8 KO极大地增加了细胞酪氨酸磷酸化水平，以及STAT 5、AKT和ERK信号传导的激活，这可以通过在KO细胞中用Fut 8恢复而完全中和。因此，酪氨酸激酶抑制剂有效地抑制由Fut 8 KO或特异性岩藻糖基化抑制剂诱导的细胞增殖。此外，用FLT 3进行的免疫染色显示，在KO细胞中蛋白主要表达在细胞表面上，这与FLT 3-WT细胞相似，但与ITD突变体不同。最后，我们发现Fut 8 KO可以在没有配体刺激的情况下诱导FLT 3中的二聚体形成。综上所述，本研究明确了FLT 3核心岩藻糖基化的调控功能，这可能为开发有效治疗AML的药物提供有价值的方向。
Fms-like tyrosine kinase 3 (FLT3) is a glycoprotein, that is a member of the class III receptor tyrosine kinase family. Approximately one-third of acute myeloid leukemia (AML) patients have mutations of this gene, and activation of the FLT3 downstream pathway plays an important role in both normal and malignant hematopoiesis. However, the role of N-glycosylation for FLT3 activation remains unclear. In this study, we showed that the N-glycan structures on wild type (WT), internal tandem duplication (ITD), and tyrosine kinase domain (TKD) mutants of FLT3 were different. Interestingly, expression of either WT or mutant FLT3 in Ba/F3 cells, an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell, greatly induced core fucosylation. To elucidate the function of core fucosylation in FLT3-mediated signaling, we used a CRISPR/Cas9 system to establish alpha 1,6-fucosyltransferase (Fut8) knockout (KO) cells. Surprisingly, the Fut8KO resulted in cell proliferation in an IL-3-independent manner in FLT3-WT cells, which was not observed in the parental cells, and suggested that this proliferation is dependent on FLT3 expression. Fut8KO greatly increased cellular tyrosine phosphorylation levels, together with an activation of STAT5, AKT, and ERK signaling, which could be completely neutralized by restoration with Fut8 in the KO cells. Consistently, a tyrosine kinase inhibitor efficiently inhibited cell proliferation induced by Fut8KO or specific fucosylation inhibitor. Additionally, immunostaining with FLT3 showed that the proteins were mainly expressed on the cell surface in the KO cells, which is similar to FLT3-WT cells, but different from the ITD mutant. Finally, we found that Fut8KO could induce dimer-formation in FLT3 without ligand-stimulation. Taken together, the present study clearly defines the regulatory function of core fucosylation in FLT3, which could provide a valuable direction for development of drugs could be effective in the treatment of AML.