Ral small GTPase signaling and oncogenesis: More than just 15minutes of fame

Abstract

Since their discovery in 1986, Ral (Ras-like) GTPases have emerged as critical regulators of diverse cellular functions. Ral-selective guanine nucleotide exchange factors (RalGEFs) function as downstream effectors of the Ras oncoprotein, and the RalGEF-Ral signaling network comprises the third best characterized effector of Ras-dependent human oncogenesis. Because of this, Ral GTPases as well as their effectors are being explored as possible therapeutic targets in the treatment of RAS mutant cancer. The two Ral isoforms, RalA and RalB, interact with a variety of downstream effectors and have been found to play key and distinct roles in both normal and neoplastic cell physiology including regulation of vesicular trafficking, migration and invasion, tumor formation, metastasis, and gene expression. In this review we provide an overview of Ral biochemistry and biology, and we highlight recent discoveries.

Keywords: Exocyst; GTPase activating protein; Guanine nucleotide exchange factor; Ras; Rho; Small GTPase.

Fig. 1

Evolutionary conservation of Ral small GTPases. A. Human and invertebrate Ral orthologs exhibit strong sequence identity. The RalA and RalB isoforms are found in all vertebrate species [17]. There is one Ral ortholog in C. elegans (Ce) and D. melanogaster (Dm). Overall sequence identity was determined by CLUSTALW multiple sequence alignment. B. Ral GTPases are members of the Ras branch of the Ras superfamily. Shown here is a comparison with the four Ras proteins and representative members of the Ras family. The dendrogram was generated by CLUSTALW multiple sequence alignment. C. Dendrogram showing sequence relationship of human and invertebrate Ral proteins. D. Ral domain structure. Human RalA and RalB G domains (12-176) shares 88% sequence identity and contain the SI and SII domains that change in conformation during GDP-GTP cycling and are involved in interaction with regulators and effectors. The switch regions are conserved between human Ral proteins and Drosophila Ral and differ by a single residue in each switch in C. elegans Ral (identical residues indicated in blue text). The hypervariable (HV) C-terminus (50% identity) consists of the membrane targeting region and contains key post-translational phosphorylation sites that regulate Ral subcellular localization and effector interaction. Multiple sequence alignment was done by ClustalW analyses and domain topology by SMART analyses. Numbers correspond to the human Ral amino acid sequences.

Fig. 2

Regulators of the Ral GDP-GTP cycle. A. Regulation of Ral GDP-GTP cycling. Ral-selective GEFs and GAPs accelerate the low intrinsic exchange and GTP hydrolysis activities to promote formation of active GTP-bound and inactive GDP-bound Ral. B. The RalGEFs are highly conserved across species. All RalGEFs contain a CDC25 homology domain, which is responsible for catalytic activity. There are four human isoforms of RalGEF that contain Ras association (RA) domain. These isoforms also contain a Ras exchanger motif (REM) that likely stabilizes the CDC25 homology domain and is essential for RalGEF catalytic activity. There is one homolog in C. elegans and two in Drosophila. The RalGEF homolog in C. elegans is most similar to RalGDS. The RalGPS RalGEFs lack a REM domain and do not associate with Ras, but instead contain a pleckstrin homology (PH) domain. RGL4 contains a CDC25 homology domain, but lacks a REM, RA or PH domain. C. The RalGAPs are heterodimeric complexes formed by either a RalGAPα1 or RalGAPα2 catalytic subunit with the regulatory RalGAPβ subunit. The RalGAPβ subunit serves to regulate the catalytic activity of the RalGAPα subunits, similar to TSC1 regulation of TSC2. Percentages indicate sequence identity with the RalGAPα1 catalytic domain. Orthologs of the human RalGAPα and RalGAPβ subunits are present in C. elegans and Drosophila. Multiple sequence alignment and sequence identity was determined by ClustalW analyses and domain topology by SMART analyses.

Fig. 2

Regulators of the Ral GDP-GTP cycle. A. Regulation of Ral GDP-GTP cycling. Ral-selective GEFs and GAPs accelerate the low intrinsic exchange and GTP hydrolysis activities to promote formation of active GTP-bound and inactive GDP-bound Ral. B. The RalGEFs are highly conserved across species. All RalGEFs contain a CDC25 homology domain, which is responsible for catalytic activity. There are four human isoforms of RalGEF that contain Ras association (RA) domain. These isoforms also contain a Ras exchanger motif (REM) that likely stabilizes the CDC25 homology domain and is essential for RalGEF catalytic activity. There is one homolog in C. elegans and two in Drosophila. The RalGEF homolog in C. elegans is most similar to RalGDS. The RalGPS RalGEFs lack a REM domain and do not associate with Ras, but instead contain a pleckstrin homology (PH) domain. RGL4 contains a CDC25 homology domain, but lacks a REM, RA or PH domain. C. The RalGAPs are heterodimeric complexes formed by either a RalGAPα1 or RalGAPα2 catalytic subunit with the regulatory RalGAPβ subunit. The RalGAPβ subunit serves to regulate the catalytic activity of the RalGAPα subunits, similar to TSC1 regulation of TSC2. Percentages indicate sequence identity with the RalGAPα1 catalytic domain. Orthologs of the human RalGAPα and RalGAPβ subunits are present in C. elegans and Drosophila. Multiple sequence alignment and sequence identity was determined by ClustalW analyses and domain topology by SMART analyses.

Fig. 3

Ral effectors and effector functions. Active Ral can bind to a variety of downstream effectors and modulate numerous cellular activities. RalBP1 acts as a RhoGAP as well as a scaffold for other proteins that regulate endocytosis and other cellular processes. Ral association with Sec5 or Exo84 can regulate exocyst-dependent and –independent processes. Other effector processes include regulation of cell cycle progression through PLD1-dependent cytokinesis and cytoskeletal changes through filamin A, IP3 signaling through PLCδ1, and gene transcription through ZONAB. Ral activation also stimulates signaling pathways that lead to the activation of various transcription factors (TF), stimulating gene expression.

Fig. 4

Regulation of Ral subcellular localization and membrane association. CAAX motif-signaled posttranslational modifications. Ral is geranylgeranylated by GGTase-1 on the first cysteine residue of the CAAX (RalA: CCIL; RalB: CCLL). By the canonical CAAX processing pathway, Ral is then modified at the endoplasmic reticulum (ER) by Ras converting enzyme (Rce1) which cleaves between the two cysteine residues. Isoprenyl cysteine carboxyl methyltransferase (ICMT) then catalyzes methylation of the isoprenylated free cysteine residue, facilitating recruitment to the plasma membrane. A second non-canonical pathway has been described by which Ral is palmitoylated at the cysteine residue at the A1 position by Golgi-associated protein acetyltransferase (PAT) after geranylgeranylation. Palmitate addition is reversible and depalmitoylation is catalyzed by acylprotein thioesterase (APT). Whether this double lipid modified form is associated with a different membrane compartment has not been determined. Ral subcellular localization is also regulated by a dynamic and reversible protein kinase (PK)-mediated phosphorylation and protein phosphatase (PP)-mediated dephosphorylation cycle. RalA and RalB possess distinct C-terminal phosphorylation sites for different protein kinases (PK). Phosphorylation causes dissociation from the plasma membrane and translocation to specific endomembrane compartments, resulting in a switch in effector (E) interaction.

Fig. 5

EGFR signaling toggles C. elegans developmental output by effector switching. The C. elegans Ras ortholog (LET-60) can interact with orthologs of human Raf (LIN-45) and RalGEF (RGL-1). The nearby anchor cell (AC) secretes EGF/LIN-3, creating a concentration gradient, inducing vulval precursor cell (VPC) development. This concentration gradient, in combination with sequential induction, patterns vulval cell fates. Active pro-1° signaling is shown in blue, with active pro-2° signaling shown in red, and quenched signaling is in gray. In presumptive 1° cells, typically P6.p, EGF activates Ras to utilize Raf to promote 1° cell fate. Pro-2° signaling through Notch is quenched. RGL-1→RAL-1 pro-2° quenching is based on RAL-1 exclusion from presumptive 1° cells. Additionally, presumptive 1° cells express and secrete notch ligand (DSL; Delta/Serrate/LAG-2) that then induces neighboring vulval precursor cells via the Notch/LIN-12 receptor to assume a 2° fate. In presumptive 2° cells, Notch induces expression of an ERK MAPK phosphatase, and other 2°-specific proteins to quench the Raf pro-1° signal. EGF activates Ras to utilize the RGL-1 RalGEF effector to promote 2° fate.