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

• Published in: Molecular biology of the cell Vol. 10; no. 2; pp. 393 - 406
• Main Authors: Seastone, D J, Zhang, L, Buczynski, G, Rebstein, P, Weeks, G, Spiegelman, G, Cardelli, J
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01.02.1999
• Subjects: Animals; Calcium - metabolism; Dictyostelium - drug effects; Dictyostelium - genetics; Dictyostelium - physiology; Diglycerides - metabolism; Endosomes - metabolism; Enzyme Inhibitors - pharmacology; Estrenes - pharmacology; Gene Expression; Genes, Protozoan; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Lysosomes - metabolism; Mutation; Naphthalenes - pharmacology; Phagocytosis - drug effects; Phagocytosis - physiology; Protein Kinase C - antagonists & inhibitors; Pyrrolidinones - pharmacology; rap GTP-Binding Proteins; Signal Transduction; Type C Phospholipases - antagonists & inhibitors; Type C Phospholipases - metabolism
• ISSN: 1059-1524; 1939-4586

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.