The small Mr Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum
The small Mr Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum
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DOI：
10.1091/mbc.10.2.393
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发表时间：
1999-02-01
影响因子：
3.3
通讯作者：
Cardelli, J
中科院分区：
文献类型：
--
作者：
Seastone, DJ;Zhang, LY;Cardelli, J
小-M，Ras样GTdR Rap 1的功能在很大程度上仍然未知，但这种蛋白质已被证明可以调节皮质肌动蛋白为基础的形态学变化，在Dictyosteopathy和哺乳动物中性粒细胞的氧化爆发。为了测试Rap 1是否调节吞噬作用，我们生化分析了条件性和适度过表达野生型[Rap 1 WT（+）]、组成型活性[Rap 1 G12 T（+）]和显性阴性[Rap 1 S17 N（+）]形式的D.盘状叶Rap 1.细菌和乳胶珠的吞噬率在Rap 1 WT（+）和Rap 1 G12 T（+）细胞中显著更高，而在Rap 1 S17 N（S）细胞中降低。添加蛋白激酶A、蛋白激酶G、蛋白酪氨酸激酶或磷脂酰肌醇3-激酶抑制剂不影响野生型细胞的吞噬率。相反，添加U 73122（磷脂酶C抑制剂）、calphostin C（蛋白激酶C抑制剂）和BAPTA-AM（细胞内Ca 2+螯合剂）分别使吞噬率降低90%、50%和65%，表明磷脂酶C信号通路的两个分支在此过程中发挥了作用。其他蛋白激酶C特异性抑制剂，如白屈菜红碱和双吲哚马来酰亚胺I，没有降低对照细胞的吞噬率，表明钙磷蛋白C通过干扰含有二酰基甘油结合结构域的蛋白质来影响吞噬作用。添加钙磷蛋白C并没有降低Rap 1 G12 T（+）细胞的吞噬率，这表明假定的二酰基甘油结合蛋白在Rap 1的信号通路上游起作用。令人惊讶的是，与对照细胞相比，Rap 1 WT（+）和Rap 1 G12 T（+）细胞中的巨胞饮显著减少。总之，我们的研究结果表明，Rap 1和Ca 2+可能共同作用，以协调重要的早期事件调节吞噬作用。
The function of the small-M, Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(S) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. Ln contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C-specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.