The RalGEF-Ral Effector Signaling Network: The Road Less Traveled for Anti-Ras Drug Discovery

Abstract

The high frequency of RAS mutations in human cancers (33%) has stimulated intense interest in the development of anti-Ras inhibitors for cancer therapy. Currently, the major focus of these efforts is centered on inhibitors of components involved in Ras downstream effector signaling. In particular, more than 40 inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase cascade and phosphoinositide 3-kinase-AKT-mTOR effector signaling networks are currently under clinical evaluation. However, these efforts are complicated by the fact that Ras can utilize at least 9 additional functionally distinct effectors, with at least 3 additional effectors with validated roles in Ras-mediated oncogenesis. Of these, the guanine nucleotide exchange factors of the Ras-like (Ral) small GTPases (RalGEFs) have emerged as important effectors of mutant Ras in pancreatic, colon, and other cancers. In this review, we summarize the evidence for the importance of this effector pathway in cancer and discuss possible directions for therapeutic inhibition of aberrant Ral activation and signaling.

Keywords: Aurora A; RalBP1/RLIP76; exocyst complex; geranylgeranyltransferase-I inhibitor.

Figure 1.

Effectors implicated in Ras-mediated oncogenesis. Missense mutations at Ras residues, primarily at Ras residues G12, G13, and Q61, impair GAP stimulation of intrinsic Ras GTP hydrolysis activity. Thus, mutant Ras is persistently GTP bound and active. In addition to the Raf, PI3K, and RalGEF effector families, the Tiam1 Rho family GEF and phospholipase Cϵ have also been identified as effectors important in Ras-mediated tumorigenesis. RAS mutation frequencies are compiled from COSMIC (http://www.sanger.ac.uk/genetics/CGP/cosmic/). PIP3 = phosphatidylinositol 3,4,5-bisphosphate; PIP2 = phosphatidylinositol 4,5-bisphosphate; IP3 = inositol 1,4,5-trisphosphate; DAG = diacyglycerol.

Figure 2.

Ral small GTPases. (A) Sequence alignment of human (Hs) RalA and RalB. There is 100% identity in the switch I (SI: 41-51) and switch II (SII: 69-81) sequences that change conformation in the GDP- and GTP-bound states and are involved in effector binding. The greatest sequence divergence is in the C-terminal membrane-targeting sequences, with polybasic (green), phosphorylation site (red), and CAAX motif (blue) residues indicated. G domain = residues 15-178/9 (corresponding to Ras residues 4-166). Asterisks indicate sequence identity. (B) Conservation of Ral GTPases in evolution. Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) possess a single Ral GTPase ortholog with strong sequence identity to human Ral. The dendrogram was generated by Clustal/W multiple sequence alignment of the indicated Ral proteins, human K-Ras4B and Rac1 (Rho family).

Figure 3.

Regulators and effectors of Ral. (A) Regulators of the Ral GDP-GTP cycle. The 2 classes of RalGEFs are defined by the presence of an RA or PH domain. (B) RalGEF domain structure and evolutionary conservation. REM = Ras exchange motif; CDC25 homology domain = RalGEF catalytic domain; RA = Ras association domain.

Figure 4.

Ral effectors. RalA and/or RalB have been determined to interact with a diverse spectrum of downstream effectors. Most bind preferentially to the GTP-bound protein, whereas some are nucleotide independent. Ral effector networks can regulate endocytosis, exocytosis, actin organization, phospholipid metabolism, and generation of second messengers. Ral has been found to activate various transcription factors or promote elements, to regulate gene expression, through pathways that may or may not be distinct from known effectors. PC = phosphatidylcholine; PA = phosphatidic acid.

Figure 5.

Ral posttranslational processing. Ral is synthesized initially as an inactive cytosolic protein. The cytoplasmic (GGTase-I)– or endoplasmic reticulum (Rce1 and ICMT)–associated enzymes recognize the Ral C-terminal CAAX motif, leading to covalent addition of a geranylgeranyl isoprenoid to the cysteine residue of the CAAX motif, followed by proteolytic cleavage of the AAX residues and carboxylmethylation of the now terminal prenylated cysteine. Sequences upstream of the CAAX motif are rich in K or R basic amino acids. These polybasic stretches comprise a second membrane-targeting signal. Reversible phosphorylation of S194 in RalA modulates the targeting activity of the C-terminus, altering subcellular location. CAAL = cysteine–aliphatic amino acid–aliphatic amino acid–leucine; the terminal amino acid of the CAAX tetrapeptide motif dictates prenyltransferase specificity, with leucine recognized by geranylgeranyltransferase-I (GGTase-I); K/R = basic amino acid–rich sequences; Aur-A = Aurora A; GGTI = GGTase-I inhibitor; PKI = protein kinase inhibitor; Rce1 = Ras-converting enzyme 1; Icmt = isoprenylcysteine carboxyl methyltransferase.