Ueda Shuji, Mizuki Masao, Ikeda Hirokazu, Tsujimura Tohru, Matsumura Itaru, Nakano Kazushi, Daino Hanako, Honda Zi Zen-ichiro, Sonoyama Junko, Shibayama Hirohiko, Sugahara Hiroyuki, Machii Takashi, Kanakura Yuzuru
Department of Hematology and Oncology, and the Department of Microbiology, Osaka University Graduate School of Medicine, Osaka, Japan.
Blood. 2002 May 1;99(9):3342-9. doi: 10.1182/blood.v99.9.3342.
Stem cell factor (SCF) has crucial roles in proliferation, survival, and differentiation of hematopoietic stem cells and mast cells through binding to c-Kit receptor (KIT). Chemotaxis is another unique function of SCF. However, little is known about the intracellular signaling pathway of SCF/KIT-mediated cell migration. To investigate the signaling cascade, we made a series of 22 KIT mutants, in which tyrosine (Y) residue was substituted for phenylalanine (F) in the cytoplasmic domain, and introduced into BAF3 cells or 293T cells. On stimulation with SCF, BAF3 expressing KIT(WT)(WT) showed cell migration and Ca(2+) mobilization. Among 22 YF mutants, Y567F, Y569F, and Y719F showed significantly reduced cell migration and Ca(2+) mobilization compared to WT. In Y567F, Lyn activation on SCF stimulation decreased and C-terminal Src kinase (Csk) suppressed KIT-mediated Ca(2+) influx and cell migration, suggesting that Y567-mediated Src family kinase (SFK) activation leads to Ca(2+) influx and migration. Furthermore, we found that p38 mitogen-activated protein kinase (p38 MAPK) and Erk1/2 were also regulated by Y567/SFK and involved in cell migration, and that p38 MAPK induced Ca(2+) influx, thereby leading to Erk1/2 activation. In Y719F, the binding of phosphatidylinositol 3'-kinase (PI3K) to KIT was lost and KIT-mediated cell migration and Ca(2+) mobilization were suppressed by PI3K chemical inhibitors or dominant-negative PI3K, suggesting the involvement of Y719-mediated PI3K pathway in cell migration. Combination of Csk and the PI3K inhibitor synergistically reduced cell migration, suggesting the cooperation of SFK and PI3K. Taken together, these results indicate that 2 major KIT signaling pathways lead to cell migration, one is Y567-SFK-p38 MAPK-Ca(2+) influx-Erk and the other is Y719-PI3K-Ca(2+) influx.
干细胞因子（SCF）通过与c-Kit受体（KIT）结合，在造血干细胞和肥大细胞的增殖、存活及分化过程中发挥关键作用。趋化性是SCF的另一独特功能。然而，关于SCF/KIT介导的细胞迁移的细胞内信号通路却知之甚少。为了研究信号级联反应，我们构建了一系列22个KIT突变体，其中细胞质结构域中的酪氨酸（Y）残基被苯丙氨酸（F）取代，并将其导入BAF3细胞或293T细胞。在用SCF刺激时，表达KIT(WT)(WT)的BAF3细胞表现出细胞迁移和Ca(2+)动员。在22个YF突变体中，与野生型相比，Y567F、Y569F和Y719F的细胞迁移和Ca(2+)动员显著减少。在Y567F中，SCF刺激时Lyn激活减少，C末端Src激酶（Csk）抑制KIT介导的Ca(2+)内流和细胞迁移，这表明Y567介导的Src家族激酶（SFK）激活导致Ca(2+)内流和迁移。此外，我们发现p38丝裂原活化蛋白激酶（p38 MAPK）和Erk1/2也受Y567/SFK调节并参与细胞迁移，并且p38 MAPK诱导Ca(2+)内流，从而导致Erk1/2激活。在Y719F中，磷脂酰肌醇3'-激酶（PI3K）与KIT的结合丧失，KIT介导的细胞迁移和Ca(2+)动员被PI3K化学抑制剂或显性负性PI3K抑制，这表明Y719介导的PI3K途径参与细胞迁移。Csk和PI3K抑制剂联合使用可协同减少细胞迁移，表明SFK和PI3K存在协同作用。综上所述，这些结果表明两条主要的KIT信号通路导致细胞迁移，一条是Y567-SFK-p38 MAPK-Ca(2+)内流-Erk，另一条是Y719-PI3K-Ca(2+)内流。
