Ras-independent activation of Ral by a Ca2+-dependent pathway

Abstract

The RalA and RalB proteins comprise a distinct family of small GTPases [1]. Ral-specific guanine-nucleotide exchange factors such as RalGDS, Rlf and RGL interact with activated Ras and cooperate with Ras in the transformation of murine fibroblasts [2-5]. Thus, the interaction of RalGDS with Ras and the subsequent activation of Ral are thought to constitute a distinct Ras-dependent signaling pathway. The function of Ral is largely unknown. There is circumstantial evidence that Ral may have a function in regulating the cytoskeleton through its interaction with RIP1 (also known as RLIP or RalBP1), a GTPase-activating protein specific for the small GTPases Cdc42 and Rac [6-8]. Ral also binds to phospholipase D (PLD) and thus may play a role in signaling through phospholipids [9]. We have examined endogenous levels of activated, GTP-bound Ral (Ral-GTP) in Rat-2 fibroblasts stimulated with various mitogens. Lysophosphatidic acid (LPA) and epidermal growth factor (EGF), which activate both Ras-dependent and Ras-independent signaling pathways [10,11], rapidly activated Ral. Inhibition of Ras activation by dominant-negative Ras (RasS17N) or pertussis toxin had little effect on Ral-GTP levels, however. Ral was activated by the Ca2+ ionophore ionomycin, and activation by LPA or EGF could be blocked by a phospholipase C (PLC) inhibitor. The results presented here demonstrate a Ca(2+)-dependent mechanism for the activation of Ral.