The small M-r Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum

The function of the small-M, Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based mor phologic changes in Dictyostelium and the oxidative burst in mammalian neut rophils. To test whether Rap1 regulates phagocytosis, we biochemically anal yzed cell lines that conditionally and modestly overexpressed wild-type [Ra p1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap 1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacter ia 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 o f protein kinase A, protein kinase G, protein tyrosine kinase, or phosphati dylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. Ln contrast, the addition of U73122 (a phospholipase C inhibitor), calphos tin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ c helator) reduced phagocytosis rates by 90, 50, and 65%, respectively, sugge sting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C-specific inhibitors, such as cheleryth rine and bisindolylmaleimide I, did not reduce phagocytosis rates in contro l 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, sugge sting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significant ly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells . Together our results suggest that Rap1 and Ca2+ may act together to coord inate important early events regulating phagocytosis.