RLIP76 (RalBP1) is an R-Ras effector that mediates adhesion-dependent Rac activation and cell migration

Abstract

The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…Conversely, activation of endogenous Arf6 by expression of the Arf GEF ARNO (Arf nucleotide-binding site opener) was found to trigger the activation of Rac, extension of lamellipodia and the onset of migration in normally stationary epithelial cells (Figure 2) [31]. Subsequent studies have confirmed that Arf6 activation is coupled to Rac activation in several settings, including integrin ligation [32,33] or stimulation of cells with vascular endothelial growth factor (VEGF) [34], platelet-derived growth factor (PDGF) [35] or angiotensin II [36].…”

BRAG2/GEP100/IQSec1 Interacts with Clathrin and Regulates α5β1 Integrin Endocytosis through Activation of ADP Ribosylation Factor 5 (Arf5)

“…4A), Arf6-depleted cells did not spread more rapidly than controls on fibronectin-coated substrates. One explanation for this is that Arf6 is involved in membrane targeting and activation of Rac1 that occurs upon integrin ligation (18,21,34) and that impaired Rac1 activation prevents the formation of lamellipodia that promote spreading. Here, we show that Rac1 activation is significantly enhanced in cells depleted of BRAG2 or Arf5 and that this correlates with more rapid spreading.…”

“…Arf6 has been shown to regulate delivery of Rac1 to the plasma membrane and its activation in response to integrin ligation (18,21). We considered it likely that although Arf6-depleted cells express more ␤1 integrin on their surface, their spreading is attenuated because Arf6 deple- Fig.…”

Regression of Lung and Colon Cancer Xenografts by Depleting or Inhibiting RLIP76 (Ral-Binding Protein 1)

“…Functions attributed to the intracellular RALBP1 (e.g., its signaling role as a Ral or Ras-R GTPase-activating protein; refs. [22][23][24][25][26] are least likely to be affected by anti-RALBP1 antibodies that cannot enter the cells and specifically interact with epitopes on membrane-anchored RALBP1 to inhibit its transport function. In case the lack of presence of RALBP1 within cells also contributes to the observed suppression of tumors due to the cessation of some or all of its known cellular functions (e.g., involvement in Ral or Ras-R signaling, endocytosis and mitosis, spindle motor functions, and the regulation of the expression of heat shock proteins), one would expect a more tumor-suppressive activity associated with antisense as compared with anti-RALBP1 IgG.…”