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Vol. 10, Issue 2, 393-406, February 1999
and
§
*Department of Microbiology and Immunology and
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.
Feist-Weiller Cancer Center; Louisiana State University
Medical Center, Shreveport, Louisiana 71130; and
Department of Microbiology, University of British
Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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