Kasahara Kousuke, Nakayama Yuji, Sato Izumi, Ikeda Kikuko, Hoshino Masaki, Endo Takeshi, Yamaguchi Naoto
Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
J Cell Physiol. 2007 Apr;211(1):220-32. doi: 10.1002/jcp.20931.
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.
通过脂质修饰定位于细胞膜胞质侧的Src家族激酶在包括膜运输在内的信号转导事件中发挥作用。巨胞饮作用是一种通过称为巨胞饮体的大囊泡摄取溶质的内吞过程。尽管在摄取荧光大分子后可以观察到巨胞饮体，但对于活细胞中巨胞饮体的动态变化知之甚少。在这里，我们表明组成型c-Src表达以激酶依赖性方式产生巨胞饮体。对绿色荧光蛋白标记的c-Src（Src-GFP）进行活细胞成像显示，c-Src从其在膜皱褶处产生开始，通过向心运输，直至与晚期内体和溶酶体融合，持续通过其N端与巨胞饮体结合。Src-GFP的光漂白后荧光恢复（FRAP）显示，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细胞内细胞器迅速募集到巨胞饮体膜上。此外，使用过表达可诱导c-Src的HeLa细胞系，我们发现用表皮生长因子（EGF）刺激后，高水平的c-Src激酶活性促进了与溶酶体区室相关的巨胞饮体的形成。与c-Src不同，Lyn和Fyn是棕榈酰化的Src激酶，它们仅能轻微诱导巨胞饮体形成，尽管一个棕榈酰化位点发生突变的Lyn突变体能够有效地诱导巨胞饮作用。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胞饮体的生物发生和运输中起重要作用。
