The Ste20 kinases SPAK and OSR1 travel between cells through exosomes.
The Ste20 kinases SPAK and OSR1 travel between cells through exosomes.
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Ste20 激酶 SPAK 和 OSR1 通过外泌体在细胞之间传播。
DOI：
10.1152/ajpcell.00080.2016
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Delpire,Eric
中科院分区：
文献类型：
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作者：
Koumangoye,Rainelli;Delpire,Eric
蛋白质组学研究已经在血液、唾液和尿液等体液中分离出与Ste20相关的富含脯氨酸/丙氨酸的激酶(SPAK)和氧化应激反应1(OSR1)。由于蛋白质组学研究可能高估了外切体蛋白的数量，我们试图使用传统的生化和细胞生物学方法来证实和扩大这一观察结果。我们利用培养中的HEK293细胞来验证这些Ste20激酶在外切体中的包装。利用从HEK293细胞分离的条件培养液进行一系列的离心和过滤步骤，我们分离出了40-100 nm范围的纳米囊泡。我们发现这些小泡表达Tetraspanin蛋白CD63，缺乏内质网和高尔基标记，这与这些外体是一致的。免疫印迹和免疫胶体分析表明，这些外体表达SPAK、OSR1和Na-K-Cl协转运蛋白1(NKCC1)。我们发现，外切体不仅由细胞分泌，还可由邻近细胞积累。事实上，将培养的细胞暴露于其他表达荧光标记的激酶的细胞产生的外切体，会导致该激酶进入这些细胞的细胞质，这与外切体充当细胞间通信血管的想法是一致的。同样，共培养表达不同荧光标记蛋白的细胞会导致细胞之间的蛋白质交换。此外，我们还发现通过外体进入细胞的Spak和OSR1蛋白都优先在质膜上表达，并且外体中的蛋白具有功能，并维持NKCC1的磷酸化状态。
Proteomics studies have identified Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) in exosomes isolated from body fluids such as blood, saliva, and urine. Because proteomics studies likely overestimate the number of exosome proteins, we sought to confirm and extend this observation using traditional biochemical and cell biology methods. We utilized HEK293 cells in culture to verify the packaging of these Ste20 kinases in exosomes. Using a series of centrifugation and filtration steps of conditioned culture medium isolated from HEK293 cells, we isolated nanovesicles in the range of 40–100 nm. We show that these small vesicles express the tetraspanin protein CD63 and lack endoplasmic reticulum and Golgi markers, consistent with these being exosomes. We show by Western blot and immunogold analyses that these exosomes express SPAK, OSR1, and Na-K-Cl cotransporter 1 (NKCC1). We show that exosomes are not only secreted by cells, but also accumulated by adjacent cells. Indeed, exposing cultured cells to exosomes produced by other cells expressing a fluorescently labeled kinase resulted in the kinase finding its way into the cytoplasm of these cells, consistent with the idea of exosomes serving as cell-to-cell communication vessels. Similarly, coculturing cells expressing different fluorescently tagged proteins resulted in the exchange of proteins between cells. In addition, we show that both SPAK and OSR1 kinases entering cells through exosomes are preferentially expressed at the plasma membrane and that the kinases in exosomes are functional and maintain NKCC1 in a phosphorylated state.