A-RAF is a member of the RAF family of serine/threonine protein kinases comprising A-RAF B-RAF and RAF-1. In 1983 and 1986, the viral homologues of RAF were isolated during experiments with the aim to identify novel transforming genes. Viral RAF (v-RAF) homologus were encountered in two different viruses, the avian retrovirus Mill Hill 2 (MH2) and the murine sarcoma virus (MSV) 3611. While MH2 was isolated from a spontaneous chicken ovarian tumor, 3611-MSV was isolated from a mouse with lymphoma and lung adenocarcinoma. The name RAF derives from the observation that 3611-MSV increased the induction of fibrosarcoma in newborn NSF/N mice (rapidly accelerated fibrosarcoma, or RAF). These v-RAF genes originate from the mammalian proto-oncogene RAF-1 (or C-RAF) having incorporated the truncated CRAF kinase domain due to homologous recombination. Shortly afterward, additional C-RAF homologs were found in mammals, referred to as A- and B-RAF.
Several groups identified the ARAF gene as a paralog of RAF-1. Low stringency hybridization of a human fetal liver cDNA library with a v-RAF probe isolated ARAF, which was named PKS (presumably for kinase sequence) at that time. In the same year, Huleihel et al. isolated the A-RAF cDNA from a murine cDNA spleen library. The cDNA showed 85% homology with RAF-1 and had a restricted expression pattern in mouse tissues with highest levels in epididymis. Incorporation of A-RAF into a retrovirus expresses a gag-A-RAF fusion protein that had transforming activity showing ARAF represents a new proto-oncogene (Huleihel et al. 1986).
From an evolutionary point of view, there are no RAF kinases in yeasts, and B-RAF seems to be the phylogenetically oldest isoform already appearing in invertebrates. In comparison, mammals possess three RAF isoforms (RAF-1/C-RAF, B-RAF, and A-RAF), with a shared modular structure. In comparison to RAF-1 and B-RAF, A-RAF is by far the least well-understood member of the RAF family (Matallanas et al. 2011).
A-RAF and MAPK Signaling
Regulation of Activity
The family of RAF serine/threonine protein kinases shares three conserved regions (CR) (Fig. 1b). CR1 contains the RAS-binding domain and a cysteine-rich motif. CR2 features a short cluster of Ser and Thr residues, and CR3 contains the kinase domain. CR1 and CR2 restrain the function of CR3, and activation steps involve the release of this negative interaction as well as posttranslational modifications. The only bona fide substrates identified so far of RAF kinases are MEK1/2.
In general, A-RAF is regulated similar to RAF-1, although important differences have emerged over the years. While binding to active RAS suffices to activate B-RAF, RAF-1 and A-RAF require the presence of both activated RAS and SRC family tyrosine kinases, which are thought to phosphorylate tyrosines 301/302 in the negative-charge regulatory (N-) region upstream of the A-RAF kinase domain (Fig. 1b). In B-RAF, these tyrosines are replaced by aspartates, whose negative charge substitute for N-region phosphorylation normally induced by RAS binding.
A-RAF has a weak, hardly detectable kinase activity toward MEK. The reasons are (i) a substitution of a critical residue (arginine 22 for lysine) in the A-RAF RBD, which weakens the binding to RAS; and (ii) a nonconserved tyrosine 296 in the A-RAF N-region, whose mutation to glycine induced constitutive kinase activity (Baljuls et al. 2007). A-RAF is also positively regulated by phosphorylation (Baljuls et al. 2008). Serine 432 is crucial for the binding of MEK, while phosphorylation of serines 257, 262, and 264 in the Isoform-specific Hinge (IH) segment stimulate A-RAF kinase activity. Full phosphorylation of the IH segment accumulates negative surface charges leading to the electrostatic destabilization of the interaction of A-RAF with the inner part of the plasma membrane and release into the cytosol (Fig. 1b). In addition, A-RAF (like RAF-1) can bind lipids, such as phosphatidic acid and phosphoinositides. Polyphosphorylated phosphoinositides, such as PI(4,5) and PI(3,4,5), suppress kinase activity (Johnson et al. 2005). Thus, while sharing Ras as essential activator and MEK as substrate with other RAF isoforms, A-RAF shows unique mechanisms of regulation.
The regulatory subunit of casein kinase 2 (CK2β) binds and activates A-RAF when coexpressed in insect cells. However, the mechanism and physiological role of CK2β in A-RAF activation in mammalian cells remains to be proven. Other regulatory protein interactions are discussed below.
A-RAF Interacting Proteins
Apart from interacting with components of the ERK pathway, such as RAS, MEK, and other RAF isoforms, A-RAF also interacts with a number of other proteins. We recently summarized these interactions (Rauch et al. 2010), and therefore only discuss selected interactions here.
A-RAF can associate with the p85 regulatory subunit of the phosphatidylinositide 3-kinase (PI3K) via the p85 SH2 domain. The interaction is constitutive and independent of growth factor stimulation or phosphorylation. A constitutively active A-RAF mutant inhibited p85 associated PI3-kinase activity, but it is unclear whether this interaction is a physiological connection between the ERK and PI3K pathways. The hypothesis that A-RAF coordinates different signaling pathways is supported by the observation that A-RAF associates with the platelet-derived growth factor receptor (PDGFR), and specifically suppresses autophosphorylation and phosphorylation of the phospholipase C-γ (PLCγ) docking site, but not the phosphorylation of binding sites for other signaling molecules. These functional effects were achieved by expression of an activated A-RAF mutant, while the role of endogenous A-RAF was not examined leaving it unclear whether A-RAF can regulate PDGFR signaling under normal conditions.
Using an A-RAF Zebrafish model, Liu and colleagues could demonstrate a novel role for A-RAF in stem cell development and tissue homeostasis (Liu et al. 2013). Here, A-RAF regulates TGF-β signaling by binding and phosphorylating activated Smad2, leading to accelerated degradation and subsequent attenuation of TGF-β signaling (Fig. 2c).
A-RAF Splice Variants
Alternative splicing occurs in more than 90% of human genes, and greatly expands the information content and versatility of the transcriptome in generating tissue, stage, and development specific gene expression patterns.
Recently, A-RAFshort, a third alternative splice form of the A-RAF1 gene, was reported (Rauch et al. 2011). In comparison to DA-RAF1 and DA-RAF2, A-RAFshort incorporates intronic sequences and generates a shortened protein, which lacks the kinase domain. Consequently, A-RAFshort acts as a dominant-negative antagonist by binding and blocking activated Ras and thus is a potent inhibitor of ERK signaling and cellular transformation. The expression of A-RAFshort is reduced in several cancer entities suggesting that A-RAFshort acts as a tumor suppressor protein (Fig. 5b).
Regulation in Cancer and Other Diseases
Initial studies in mice suggested a highly restricted tissue distribution of A-RAF with highest expression levels observed in epididymis, ovary, and intestine. In the meantime, however, A-RAF was found expressed in most normal tissues, but expression levels seem highly regulated and differ dramatically. While neuronal tissues, for example, express A-RAF only at low levels, the urogenital tract shows a high expression. A-RAF mRNA and protein levels are elevated in a number of malignancies. Increased A-RAF mRNA levels were found in peripheral blood mononuclear cells isolated from two patients with angioimmunoblastic lymphadenopathy with dysproteinemia. Elevated levels of A-RAF mRNA were also found in pancreatic ductal carcinoma. In addition, the enhanced A-RAF expression was also found in other tumor types, including astrocytic tumors, where high expression of A-RAF negatively correlated with patients’ prognosis. Elevated A-RAF expression was also found in a number of head and neck squamous cell carcinomas as well as colon carcinomas.
A novel oncogenic driver mutation, A-RAF S214C, was identified in advanced-stage lung cancer and it was demonstrated to transform immortalized human airway epithelial cells in a sorafenib-sensitive manner (Imielinski et al. 2014). Recently, several groups identified a number of A-RAF activating mutations in Langerhans cell histiocytosis, including S214A, A225V, F351 L, and P539H. The mutation Q347_A348del/F351 L was identified in B-RAF wild-type Langerhans cell histiocytosis, leading to MAPK activation and transformation of mouse embryo fibroblasts. Interestingly, the activity of the A-RAF mutant could be inhibited by the B-RAF inhibitor Vemurafenib suggesting possibilities for therapeutic intervention (Nelson et al. 2014). In another study, approximately 11% of intrahepatic cholangiocarcinoma (iCCA), a fatal bile duct cancer, was shown to carry oncogenic A-RAF mutations (N217I and G322S) leading to MAPK activation oncogenic transformation (Sia et al. 2015). In addition, two inactivating mutations were identified in colon carcinoma (G331C, glycine-rich loop) and lymphoblastic leukemia (A451T, activation segment), respectively.
In addition, A-RAF overexpression was observed in several malignancies despite the lack of genetic mutations of the A-RAF gene: As described, the expression of wild-type A-RAF mRNA and protein requires the expression of the splice factor hnRNP H (Rauch et al. 2011) or hnRNP A1/A2 (Shilo et al. 2014), whose levels are enhanced in several tumors including colon, head, and neck cancers, and hepatocellular carcinoma, respectively. High levels of these splice factors ensure the expression of full-length A-RAF protein by suppressing alternative splicing of the A-RAF mRNA, thus allowing the sufficient production of full-length A-RAF protein to counteract MST2-mediated apoptosis. Low levels of hnRNP H and hnRNPA1/A2, as found in non-malignant tissues, cannot suppress alternative splicing of the A-RAF wild-type mRNA, thus favoring the expression of the alternative splice form A-RAFshort (Fig. 5b). As mentioned already above, A-RAFshort acts as a dominant-negative antagonist of the Ras-MAPK cascade, which seems to keep proliferation and transformation in nonmalignant cells in check. In addition, A-RAF’s function to inhibit the MST2/Hippo pathway seems to be instrumental for epithelial to mesenchymal transition (EMT), a crucial process in tumor development. Here, the overexpression of A-RAF and control of the MST2 pathway allows for increased cell migration and scattering thereby promoting HGF-induced EMT (Farrell et al. 2014).
Mouse Models and Phenotypes
In the 1990s, the first mouse model for the A-RAF gene was reported (Pritchard et al. 1996). It was shown that ablation of the A-RAF gene causes neurological defects. Interestingly, in an inbred mouse background, A-RAF ablation resulted in intestinal and neurological abnormalities. A-RAF knockout mice died one to three weeks after birth from megacolon which is reminiscent of Hirschsprung’s disease in humans and was caused by a defect in the migration of visceral neurons controlling bowel contractions. In contrast, in an outbred background, A-RAF knockout animals survived to adulthood. In this genetic background, A-RAF ablation did not lead to intestinal abnormalities, but animals displayed a subset of neurological defects. Interestingly, the regulation of ERK and the oncogene transformation are not impaired in A-RAF knockout cells. These results, together with the low kinase activity toward MEK, suggest that A-RAF does not play a major role in MAPK signaling and that this function is fully compensated by the other two RAF family members. However, A-RAF seems to have a role in the development of the nervous system possibly by regulating neuronal migration. Comparing the knockout phenotypes of all three RAF isoforms in mice indicates that A-RAF and B-RAF may have more specialized functions, while RAF-1 seems to have a more general role in tissue formation.
What did scientists learn about A-RAF since its discovery in the early 1980s? A simple summary is that RAF proteins have distinct functions, and that A-RAF has more functions than initially expected. Over the last few years, scientists made headway in deciphering in which pathways A-RAF might play a role. Science now has entered an era where the mapping of the components of signaling networks is rapidly accelerating producing even longer lists faster than ever before. However, these lists are like a telephone book full of names rather than an ordnance survey type of map that connects the names with pathway topologies. Undoubtedly, much more is known about RAF-1 and B-RAF and 25 years of research mainly focused on these two isoforms, as they play a bigger role in MAPK signaling and oncogenesis and are major targets for drug therapies. In comparison, A-RAF on its own seems to play only a minor role in the canonical MAPK pathway. Multiple studies over the last years have shown A-RAF’s involvement in other processes, such as energy metabolism, the Warburg effect, mitochondrial transport, and antiapoptotic signaling. However, it seems that these interactions with other proteins and processes do not happen at the same time but are rather cell-, tissue-, and time-dependent. Furthermore, A-RAF is found at different localizations of the cell and this spatial regulation is another suitable explanation for the involvement in different pathways. Dissecting these microcomplexes and interacting proteins is a challenging endeavor on the experimental level. To add another level of complexity, three novel alternative splice forms were identified recently with functions that are even antagonistic to the wild-type form.
A-RAF was shown to bind to a plethora of proteins, which suggests A-RAF’s involvement in many other signaling pathways outside the canonical MAPK pathway. However, if there is more than one function in signal transduction, how are these diverse functions coordinated in order to achieve the intended biological outcome and specificity? This question is also relevant for the other members of the RAF family.
Similarly, A-RAF was found at different sub-cellular compartments. How does this localized expression impact on signaling pathways and networks?
What is the impact of dynamic changes in the assembly of A-RAF signaling complexes on biochemical and biological outcome?
A-RAF was shown to play a role in cancer and other diseases. Is this exploitable for the purpose of therapeutic intervention?
In summary, the current state of analysis of the “A-RAF network” offers a glimpse into a new world. New concepts and tools developed hopefully will widen this glimpse into a window overlooking the whole signaling network.
- We apologize to all the colleagues, whose work, for reasons of space, could not be cited in this review.Google Scholar
- Baljuls A, Mueller T, Drexler H, Hekman M, Rapp U. Unique N-region determines low basal activity and limited inducibility of A-RAF kinase: the role of N-region in the evolutionary divergence of RAF kinase function in vertebrates. J Biol Chem. 2007;282:26575–90. doi: 10.1074/jbc.M702429200.CrossRefPubMedGoogle Scholar