RalGDS Couples Growth Factor Signaling to Akt Activation

Abstract

The Akt kinase is a key regulator of cell proliferation and survival. It is activated in part by PDK1-induced phosphorylation. Here we show that RalGDS, a Ras effector protein that activates Ral GTPases, has a second function that promotes Akt phosphorylation by PDK1 by bringing these two kinases together. In support of this conclusion is our finding that suppression of RalGDS expression in cells inhibits both epidermal growth factor and insulin-induced phosphorylation of Akt. Moreover, while PDK1 complexes with N-GDS, Akt complexes with the central region of RalGDS through an intermediary, JIP1. The biological significance of this newly discovered RalGDS function is highlighted by the observation that an N-terminally deleted mutant of RalGDS that retains the ability to activate Ral proteins but loses the ability to activate Akt also fails to promote cell proliferation. Thus, RalGDS forms a nexus that transduces growth factor signaling to both Ral GTPase and Akt-mediated signaling cascades.The Akt kinase is a key mediator of the actions of many growth factors and hormones (10). Its effects on cells include regulation of metabolism, gene transcription, cell proliferation, angiogenesis, and apoptosis. Loss or gain of Akt activity contributes to a variety of diseases, including diabetes and cancer. Akt influences these processes by phosphorylating a broad spectrum of substrates, including MDM2, FOXO transcription factors, p27 kip1 , Bad, and GSK3. The three Akt isoforms, Akt1 to -3, belong to the AGC family of kinases, all of which are activated by the PDK1 kinase (for review, see reference 20). The accepted mode of Akt activation involves ligand-induced activation of phosphatidylinositol 3-kinase (PI3-kinase), which leads to the production of PIP2 and PIP3. These phospholipids interact with the PH domain of Akt to target the protein to PDK1 in the plasma membrane, where PDK1 phosphorylates Akt on threonine 308 (2). How specific PDK1 substrates are chosen for activation in response to specific stimuli is poorly understood. A secondary activation event occurs through phosphorylation of Akt at serine 473. Multiple kinases have been reported to be responsible for this event, the most recent of which is the rictor/mTor complex (29).RalGDS is one of a family of guanine nucleotide exchange factors (GEFs) that activate Ral-GTPases in cells by promoting their switch from GDP-bound to GTP-bound states. A subset of these GEFs, including RalGDS, Rlf, Rgl, and Rgl3, are downstream effectors of Ras proteins, as active Ras binds to their C termini and targets them to Ral in cells (25). Recent studies on human cells and in knockout mice have highlighted the importance of this effector pathway in the Ras-mediated oncogenesis. For example, Ral exchange factors can replace Ras in assays designed to test the minimal number of genes that are required to transform primary human cells to a tumorigenic state (14, 26), while a RalGDS knockout suppresses tumor formation in a Ras-mediated tumor model (13).Activation of RalGDS in cells also ...

RalA Functions as an Indispensable Signal Mediator for the Nutrient-sensing System

“…2, C and D), indicating that insulin induces mTORC1 activation in a RalGDS-independent manner. Hao et al (36) recently reported that RalGDS forms a complex with PKB, a critical component in growth factor-regulated mTORC1 activation, and promotes PKB activation in breast cancer cell lines. We are not able to completely explain the conflict between those findings and the results of this study or the discrepancy in the requirement for RalA and RalGDS in insulin-induced mTORC1 activation.…”

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

“…Another explanation for the crucial role of Rgl2 in PDAC transformation is isoform-specific Ral-independent functions, because Rgl2 has been shown to interact with the Ras scaffolding protein CNK (43), whereas RalGDS interacts with PDK1 to promote AKT activation (20,52). In line with this hypothesis is our observations that Rgl2 knockdown and RalA31N expression strongly suppressed anchorage-independent growth in MIA PaCa-2 without suppression of RalA activity and that in MIA PaCa-2 cells, activated RalA was not able to rescue the anchorage-independent growth phenotype of Rgl2 knockdown.…”

“…Thus, depletion of Rgl2 may disrupt the ability of CNK to promote Raf signaling. Another possible mechanism is based on the demonstrated ability of RalGDS to activate AKT in a Ral-independent manner (20). To address these mechanisms, we determined if Rgl2 depletion corresponded with reduced AKT and/or ERK activity levels.…”

“…This sequence divergence may result in important functional differences between the GEFs. For example, a RalGEF-independent function of RalGDS has been described involving activation of AKT (20). Although ectopic overexpression of activated Ras proteins can activate the different RalGEFs in vivo (21)(22)(23)(24), there are currently no data implicating a specific RalGEF in endogenous mutant KRAS-mediated growth transformation.…”

Protein Phosphatase 2A Isoforms Utilizing Aβ Scaffolds Regulate Differentiation through Control of Akt Protein

“…Akt kinases are the most important PI3K effector molecules. Studies of both transformation (11,20,21) and cell death (22) have focused attention on Akt. Translocated to membranes by stimulation of PI3K signaling, Akt is activated by phosphorylation at Thr-308 by PDK1 (PI3K-dependent protein kinase 1) (23) and at Ser-473 by the mTORC2 complex (24).…”