Role of Src-family kinases in formation and trafficking of macropinosomes
Role of Src-family kinases in formation and trafficking of macropinosomes
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DOI：
10.1002/jcp.20931
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发表时间：
2007-04-01
影响因子：
5.6
通讯作者：
Yamaguchi, Naoto
中科院分区：
文献类型：
--
作者：
Kasahara, Kousuke;Nakayama, Yuji;Yamaguchi, Naoto
src家族激酶通过脂质修饰定位于细胞膜的细胞质侧，在包括膜运输在内的信号事件中发挥作用。巨饮作用是一种内吞过程，由称为巨饮小体的大囊泡摄取溶质。虽然在摄取荧光大分子后可以看到巨量酶体，但对活细胞中巨量酶体的动力学知之甚少。在这里，我们发现构成型c-Src表达以激酶依赖的方式产生大蛋白酶体。gfp标记的c-Src （Src-GFP）的活细胞成像显示，c-Src从膜皱处生成开始，通过其n端持续与大蛋白酶体结合，通过向心运输，与晚期核内体和溶酶体融合。Src-GFP的光漂白后荧光恢复（FRAP）表明，即使存在蛋白质合成抑制剂，Src-GFP也能通过囊泡运输从质膜和胞内细胞器迅速招募到大蛋白酶体膜上。此外，通过使用过表达诱导型c-Src的HeLa细胞系，我们发现在表皮生长因子（EGF）刺激后，高水平的c-Src激酶活性促进了与溶酶体室相关的大蛋白酶体的形成。与c-Src不同的是，Lyn和Fyn是棕榈酰化的Src激酶，它们只能最低限度地诱导巨嗜小体，尽管Lyn突变体中棕榈酰化位点的突变有效地诱导巨嗜红细胞增生。我们得出结论，包括c-Src在内的非棕榈酰化Src激酶的激酶活性可能在巨肽小体的生物发生和运输中发挥重要作用。
Src-family kinases that localize to the cytoplasmic side of cellular membranes through lipid modification play a role in signaling events including membrane trafficking. Macropinocytosis is an endocytic process for solute uptake by large vesicles called macropinosomes. Although macropinosomes can be visualized following uptake of fluorescent macromolecules, little is known about the dynamics of macropinosomes in living cells. Here, we show that constitutive c-Src expression generates macropinosomes in a kinase-dependent manner. Live-cell imaging of GFP-tagged c-Src (Src-GFP) reveals that c-Src associates with macropinosomes via its N-terminus continuously from their generation at membrane ruffles, through their centripetal trafficking, to fusion with late endosomes and lysosomes. Fluorescence recovery after photobleaching (FRAP) of Src-GFP shows that Src-GFP is rapidly recruited to macropinosomal membranes from the plasma membrane and intracellular organelles through vesicle transport even in the presence of a protein synthesis inhibitor. Furthermore, using a HeLa cell line overexpressing inducible c-Src, we show that following stimulation with epidermal growth factor (EGF), high levels of c-Src kinase activity promote formation of macropinosomes associated with the lysosomal compartment. Unlike c-Src, Lyn and Fyn, which are palmitoylated Src kinases, only minimally induce macropinosomes, although a Lyn mutant in which the palmitoylation site is mutated efficiently induces macropinocytosis. We conclude that kinase activity of nonpalmitoylated Src kinases including c-Src may play an important role in the biogenesis and trafficking of macropinosomes.