The Small M_rRas-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis inDictyostelium discoideum

Abstract

The function of the small-M r 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(ϩ)], constitutively active [Rap1 G12T(ϩ)], and dominant negative [Rap1 S17N(ϩ)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(ϩ) and Rap1 G12T(ϩ) cells and were reduced in Rap1 S17N(ϩ) 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 Ca 2ϩ 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(ϩ) cells, suggesting that the putative diacylglycerolbinding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(ϩ) and Rap1 G12T(ϩ) cells compared with control cells. Together our results suggest that Rap1 and Ca 2ϩ may act together to coordinate important early events regulating phagocytosis.

Shear force-based genetic screen reveals negative regulators of cell adhesion and protrusive activity

“…Because the small G protein Rap1 has been demonstrated to provide a supportive role in Dictyostelium protrusion generation and substrate adhesion, we examined how the expression of the dominant negative Rap1S17N-myc would affect adhesion in the confirmed RAM strains (41)(42)(43)(44)(45). In wild-type cells, Rap1S17N has been shown to increase myosin IIB at the cell cortex which leads to cell retraction and decreased adhesion (44).…”

Molecular Mechanisms of Membrane Trafficking. What do we Learn from Dictyostelium discoideum?

“…Nevertheless, other micropinocytic uptake mechanisms likely exist in this organism, as cells overexpressing Rap1, a member of the Ras-like superfamily of GPTases, show a 60% reduction in fluid-phase uptake but completely lack visible macropinocytic structures (Seastone et al 1999). It is also noteworthy that detergent insoluble lipid rafts, which have been implicated in caveolae-mediated endocytosis in mammalian cells, were found in D. discoideum (Xiao and Devreotes 1997).…”

“…Actin cross-linking proteins and small guanosine triphosphatases (GTPases) of the Rac and Rho family that regulate and organise the actin cytoskeleton are important in the process of ruffle extension in D. discoideum (Seastone et al 1998;Seastone et al 1999). A subset of actin-binding proteins associate transiently with the phagocytic cup of amoebae such as talin (Niewohner et al 1997) and coronin, which is thought to regulate the G-to F-actin equilibrium (Maniak et al 1995).…”

Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

“…Rap proteins have been detected in the endo/phagocytic compartment (59) and have been shown to play a role in phagocytosis in Dictyostelium (44). In mammalian phagocytes, they may contribute to phagocytosis by mediating the activation of integrins, as shown in lymphoid cells (60,61).…”

“…Rap1 regulates phagocytosis in Dictyo- stelium (44) and is required for complement receptor-mediated phagocytosis (45). We previously demonstrated that engagement of Fc␥R cross-activates the complement receptor, CR3, a member of the integrin family, enabling cells to recognize and bind complement-opsonized particles (32).…”