Seastone D J, Zhang L, Buczynski G, Rebstein P, Weeks G, Spiegelman G, Cardelli J
Department of Microbiology and Immunology, Shreveport, Louisiana 71130, USA.
Mol Biol Cell. 1999 Feb;10(2):393-406. doi: 10.1091/mbc.10.2.393.
The function of the small-Mr 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(+) 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. In 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.
小分子量Ras样GTP酶Rap1的功能在很大程度上仍然未知，但已证明该蛋白可调节盘基网柄菌中基于皮质肌动蛋白的形态变化以及哺乳动物中性粒细胞中的氧化爆发。为了测试Rap1是否调节吞噬作用，我们对有条件且适度过表达野生型[Rap1 WT(+)]、组成型激活型[Rap1 G12T(+)]和显性负性[Rap1 S17N(+)]形式的盘基网柄菌Rap1的细胞系进行了生化分析。在Rap1 WT(+)和Rap1 G12T(+)细胞中，细菌和乳胶珠的吞噬率显著更高，而在Rap1 S17N(+)细胞中则降低。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激酶的抑制剂不会影响野生型细胞的吞噬率。相反，添加U73122（一种磷脂酶C抑制剂）、钙泊三醇C（一种蛋白激酶C抑制剂）和BAPTA-AM（一种细胞内Ca2+螯合剂）分别使吞噬率降低了90%、50%和65%，这表明磷脂酶C信号通路的两个分支在该过程中均发挥了作用。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，不会降低对照细胞的吞噬率，这表明钙泊三醇C是通过干扰含有二酰基甘油结合结构域的蛋白来影响吞噬作用的。添加钙泊三醇C不会降低Rap1 G12T(+)细胞的吞噬率，这表明假定的二酰基甘油结合蛋白在与Rap1的信号通路中起上游作用。令人惊讶的是，与对照细胞相比，Rap1 WT(+)和Rap1 G12T(+)细胞中的巨胞饮作用显著降低。我们的结果共同表明，Rap1和Ca2+可能共同作用以协调调节吞噬作用的重要早期事件。
