Identification and Characterization of Potential Effector Molecules of the Ras-related GTPase Rap2

Abstract

In search for effectors of the Ras-related GTPase Rap2, we used the yeast two-hybrid method and identified the C-terminal Ras/Rap interaction domain of the Ral exchange factors (RalGEFs) Ral GDP dissociation stimulator (RalGDS), RalGDS-like (RGL), and RalGDSlike factor (Rlf). These proteins, which also interact with activated Ras and Rap1, are effectors of Ras and mediate the activation of Ral in response to the activation of Ras. Here we show that the full-length RalGEFs interact with the GTP-bound form of Rap2 in the two-hybrid system as well as in vitro. When co-transfected in HeLa cells, an activated Rap2 mutant (Rap2Val-12) but not an inactive protein (Rap2Ala-35) co-immunoprecipitates with RalGDS and Rlf; moreover, Rap2-RalGEF complexes can be isolated from the particulate fraction of transfected cells and were localized by confocal microscopy to the resident compartment of Rap2, i.e. the endoplasmic reticulum. However, the overexpression of activated Rap2 neither leads to the activation of the Ral GTPase via RalGEFs nor inhibits Ras-dependent Ral activation in vivo. Several hypotheses that could explain these results, including compartmentalization of proteins involved in signal transduction, are discussed. Our results suggest that in cells, the interaction of Rap2 with RalGEFs might trigger other cellular responses than activation of the Ral GTPase.Ras proteins are monomeric GTPases that play a pivotal role in the control of cell proliferation; they function as binary switches by cycling between an inactive form bound to GDP and the active GTP-bound state (1). Activation, through the dissociation of bound GDP and subsequent binding of GTP, is catalyzed by GEFs, 1 such as CDC25/Ras-GRF and Sos (2-4). Return to the inactive state is ensured via stimulation of the low intrinsic GTPase activity of Ras by GTPase-activating proteins, such as p120-GTPase-activating protein, neurofibromin, and Gap IP4BP (2, 5). In the active GTP-bound state, Ras exerts its biological effects by turning on several effectors that activate downstream pathways. Soluble serine/threonine kinases B-Raf and c-Raf, once activated by Ras-GTP through a mechanism that is not fully understood, trigger a cascade of protein kinases that results in the activation of mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (6). Another target of Ras is the catalytic subunit of phosphatidylinositol 3-OH kinase (PI3K) (7); this pathway leads to the activation of the protein kinase Akt (8, 9), as well as the activation of Ras-related proteins of the Rho/Rac/Cdc42 family involved in controlling the polymerization state of the actin cytoskeleton, cell adhesion, and gene transcription (10, 11). Ras is also able to activate the related GTPase Ral through RalGEFs that constitute direct effectors of Ras (12-15); three such proteins, namely RalGDS (16), RGL (17), and Rlf (18) have been extensively characterized, and the isolation of another member of this family has been recently reported (19). Elegant genetic experiments have show...

Control of embryonicXenopusmorphogenesis by a Ral-GDS/Xral branch of the Ras signalling pathway

“…The potential interaction of Ral-GDS with either activated Ras or Rap1 suggests that Ral can be activated by at least two pathways. However, although Rap1/Ral-GDS interaction has been seen in the two-hybrid system and in vitro, Rap1 fails to co-immunoprecipitate with Ral-GDS in co-transfected cells, so compartmentalisation of the proteins was suggested (Nancy et al, 1999). Here we have demonstrated that Ras activates Ral through the Ral-GDS effector in Xenopus embryos.…”

Positive regulation of skeletal myogenesis by R-Ras

“…When the activating abilities of these three eectors among Ras-family proteins are compared, Ras can activate all of the eectors (Campbell et al, 1998), Rap1 activates B-Raf and Ral-GEFs (Ohtsuka et al, 1996;Kishida et al, 1997;Zwartkruis et al, 1998), and TC21 activates B-Raf and Raf-1 (Rosa rio et al, 1999). In contrast, although R-Ras has been reported to bind Raf and Ral-GEF in a GTP-dependent fashion, the binding anities to these molecules are much lower than those of Ras (Herrmann et al, 1996;Nancy et al, 1999). Corresponding with this, R-Ras causes only a marginal increase in the activity of ERK, a down- Figure 4 Eect of R-Ras Q87L on myoblast migration.…”

The Rap GTPases Regulate B Cell Migration Toward the Chemokine Stromal Cell-Derived Factor-1 (CXCL12): Potential Role for Rap2 in Promoting B Cell Migration

“…However, we cannot rule out a role for Rap1. Because activated Rap1 and Rap2 bind many of the same effector proteins (43), it is possible that activation of either Rap1 or Rap2 is sufficient to allow B cells to migrate toward SDF-1. Unfortunately, expressing dominant-negative forms of Rap1 and Rap2 would not allow us to determine the relative roles of Rap1 and Rap2 in B cell migration because dominant-negative GTPases act by sequestering upstream activators and Rap1 and Rap2 share the same upstream activators.…”