Characterization of the GbpD-activated Rap1 Pathway Regulating Adhesion and Cell Polarity in Dictyostelium discoideum

Abstract

The regulation of cell polarity plays an important role in chemotaxis. GbpD, a putative nucleotide exchange factor for small G-proteins of the Ras family, has been implicated in adhesion, cell polarity, and chemotaxis in Dictyostelium. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. These cells overexpressing GbpD are severely impaired in chemotaxis, most likely due to the induction of many protrusions rather than an enhanced adhesion. The GbpD-overexpression phenotype is similar to that of cells overexpressing Rap1. Here we demonstrate that GbpD activates Rap1 both in vivo and in vitro but not any of the five other characterized Ras proteins. In a screen for Rap1 effectors, we overexpressed GbpD in several mutants defective in adhesion or cell polarity and identified Phg2 as Rap1 effector necessary for adhesion, but not cell polarity. Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain.

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

“…Moreover, almost all of the phenotypes can be suppressed by overexpression of dominant negative Rap1 which is thought to be a regulator in multiple pathways involved in chemotaxis. In Dictyostelium, Rap1 has been reported to either activate Ras or negatively regulate myosin leading to increased PKB signaling and decreased contraction (41,42,44). These mutants show increased patches of actin polymerization suggesting that directly or indirectly, the functions of the RAM proteins feed into negatively regulating cytoskeletal 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).…”

Big roles for small GTPases in the control of directed cell movement

“…However, Rap1 is likely to be regulated by more than one GEF. Phg2 was reported to promote the Rap1-regulated cell adhesion [162], but we have evidence suggesting that Phg2 also regulates the chemoattractant-induced phosphorylation of myosin II (T. Mammalian Rap1 has attracted much attention because of its involvement in several aspects of cell adhesion [161]. However, recent data suggest that chemoattractant-induced Rap1 signalling at the leading edge is critical for lymphocyte polarity and migration [166,167].…”

“…Lee, S. Merlot, K. Takeda, G. Weeks and R. A. Firtel, unpublished work). Rap1 is well known for its control of cell adhesion in mammalian cells and appears to play a similar role in Dictyostelium [161,162], but the results from several studies suggest that Rap1 also regulates cell polarity and motility by controlling myosin II function. Rap1 is rapidly activated in response to chemoattractant stimulation.…”

“…Lee, S. Merlot, K. Takeda, G. Weeks and R. A. Firtel, unpublished work), which is similar to the reported localization and activation of mammalian Rap1 in fibroblasts and leucocytes [163,164]. Evidence hinting that Rap1 controls myosin II includes the observation that the azide-promoted myosin II-dependent cell contraction is inhibited in cells that overexpress Rap1 or a constitutively active Rap1 mutant (Rap1G12V) [165] Rap1's function in Dictytostelium chemotaxis most likely involves the recently identified Rap1 effector kinase Phg2 as well as the putative cGMP-binding protein GbpD, which was found to be a specific Rap1 GEF [162]. The latter finding supports the possibility that cGMP controls myosin II function at the leading edge by preventing its assembly at this site, in addition to promoting myosin II assembly and contraction at the rear of chemotactic cells.…”