The Small MrRas-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis inDictyostelium discoideum
| Autor: | Linyi Zhang, Gerald Weeks, James A. Cardelli, Greg Buczynski, David J. Seastone, George B. Spiegelman, Patrick Rebstein |
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| Rok vydání: | 1999 |
| Předmět: |
endocrine system
Genes Protozoan Gene Expression Rap GTP-binding protein Endosomes GTPase Naphthalenes Article Dictyostelium discoideum Diglycerides GTP-binding protein regulators Phagocytosis GTP-Binding Proteins Animals Dictyostelium Enzyme Inhibitors Estrenes Molecular Biology Protein Kinase C Protein kinase C Phospholipase C biology Cell Biology biology.organism_classification Pyrrolidinones Cell biology enzymes and coenzymes (carbohydrates) rap GTP-Binding Proteins Biochemistry Type C Phospholipases Mutation Calcium Rap1 Lysosomes Signal Transduction |
| Zdroj: | Molecular Biology of the Cell. 10:393-406 |
| ISSN: | 1939-4586 1059-1524 |
| Popis: | The function of the small-MrRas-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes inDictyostelium 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. |
| Databáze: | OpenAIRE |
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