R-Ras Regulates Exocytosis by Rgl2/Rlf-mediated Activation of RalA on Endosomes

Abstract

R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis. INTRODUCTIONR-Ras is a Ras-family GTPase and its amino acid sequence is 55% identical to those of the classical types of Ras (H-, K-, N-Ras, collectively referred to hereafter as "Ras") (Lowe et al., 1987). As is the case with the other Ras-family GTPases, R-Ras is regulated primarily by two classes of protein, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP). Reflecting the high sequence similarity among Ras-family GTPases, many GEFs and GAPs for R-Ras catalyze other Ras-family GTPases as well (Ohba et al., 2000). Furthermore, R-Ras is known to interact with many effectors of Ras, such as Raf-1, Ral GEFs, and the p110␣ subunit of phosphoinositide 3-kinase (PI3K) (Rey et al., 1994;Marte et al., 1997). Despite this redundancy between R-Ras and Ras, R-Ras exhibits various properties that are distinct from those of Ras. For example, R-Ras preferentially activates Ral GEFs and PI3K, but it does not activate Raf (Huff et al., 1997;Rodriguez-Viciana et al., 2004). The transforming activity of constitutively active R-Ras is substantially less potent than that of the constitutively active Ras (Cox et al., 1994), although it should be noted that a recent report has suggested the involvement of R-Ras in human gastric cancer (Nishigaki et al., 2005). Meanwhile, R-Ras is known to regulate cell adhesion, cell spreading, and phagocytosis through the activation of integrin (Zhang et al., 1996;Keely et al., 1999; Berrier et al., 2000;Self et al., 2001). R-Ras-null mice have recently been shown to exhibit excessive vascular responses, in spite of the fact that they are otherwise normal (Komatsu and Ruoslahti, 2005). This phenotype seems to reflect higher levels of expression of R-Ras in smooth muscle cells, including blood vessel cells. The results obtained with R-Ras-null mice have also demonstrated that an R-Ras defect can be almost entirely compensated for by other ...

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

“…Edge-An additional function of RalGEFs may be their regulation of distinct spatial activation of their GTPase substrates (27). To further investigate the role of Rgl2 in PDAC cells, we examined the subcellular localization of endogenous Rgl2 in CFPAC-I cells.…”

“…All four family members are widely expressed in many tissues, but their relative abilities to activate RalA and RalB have not been investigated. The different RalGEFs may also exhibit distinct subcellular localization, leading to spatially distinct Ral activation and engagement of different effector populations (27).…”

Molecular Machinery Regulating Exocytosis

“…Subsequently, Ral small GTPases were linked to exocytosis in neural, epithelial, endothelial endocrinal, and pancreatic tissues (Hazelett, et al, 2011;Lopez, et al, 2008;Moskalenko, et al, 2003;Polzin, et al, 2002;Rondaij, et al, 2004;Rondaij, et al, 2008;Takaya, et al, 2007). The two mammalian Ral homologues RalA and RalB share 85% protein sequence identity, and are well conserved in evolution (van Dam & Robinson, 2006).…”

How to make FRET biosensors for Rab family GTPases

“…Previous experiments have shown that the YFP/CFP ratio of a Raichu sensor correlates with the GTP/GDP ratio (Mochizuki et al, 2001;Yoshizaki et al, 2003). Raichu sensors for Ras family GTPases (Ras, Rap1, Ral, R-Ras) (Mochizuki et al, 2001;Takaya et al, 2007), Rho family GTPases (RhoA, Rac1, Cdc42, TC10) (Itoh et al, 2002;Yoshizaki et al, 2003), and Rab family GTPase (Rab5) (Kitano et al, 2008) have been published to date.…”