Rab23 (Ras-related protein Rab 23) belongs to the Rab family of monomeric small guanosine triphosphatases (GTPases) involved in the regulation of membrane traffic. Rab GTPases are conserved from yeast to humans and coordinate the delivery of cargo to its correct destination within eukaryotic cells. Rab proteins regulate many membrane trafficking steps, including vesicle formation, budding, motility along the cytoskeleton, docking, and membrane fusion (Zerial and McBride 2001; Stenmark 2009). More than 60 members of the Rab family have been identified in humans to date (Zerial and McBride 2001). Rab proteins function as molecular switches cycling from an active GTP (guanosine triphosphate)-bound form to an inactive GDP (guanosine diphosphate)-bound form. The GDP/GTP exchange factors (GEFs) catalyze the conversion from GDP to GTP-bound forms, whereas GTP hydrolysis to GDP is catalyzed by GTPase-activating proteins (GAPs). Once in their active state, GTP-bound Rabs can recruit specific effector molecules to transduce signals in the transport pathway. These effectors include sorting adaptors, tethering factors, kinases, phosphatases, motor proteins, GEFs, and GAPs. Crosstalk between Rab GTPases through their effectors allows the spatiotemporal regulation of vesicle trafficking (Zerial and McBride 2001; Stenmark 2009). Post-translational modification by protein prenylation of C-terminal cysteines is generally required for membrane association and biological function of Rab proteins. (Zerial and McBride 2001; Stenmark 2009).
Rab23 and Hedgehog Signaling
Homozygous mutation of the Rab23 gene is responsible for the mouse open brain (opb) phenotype (Eggenschwiler et al. 2001). There are two independent opb alleles, both of which encode truncated proteins (Eggenschwiler et al. 2001). The opb 1 allele is a natural mutation, while opb 2 was experimentally induced by N-ethyl-N-nitrosurea (ENU). Opb mice are characterized by severe defects in neural tube closure and patterning (Gunther et al. 1994), related to a failure in the correct specification of neurons along the dorsoventral axis of the neural tube. This process is highly dependent on the correct activity of Sonic hedgehog (SHH), a morphogen secreted from the notochord and floorplate (Ericson et al. 1997). The hedgehog (HH) pathway is one of the most pivotal signaling pathways directing embryonic development, and at the cellular level is regulated by trafficking events at the primary cilium (Huangfu et al. 2003). Genetic studies in mice revealed that the opb mutation rescues the Shh mutant phenotype, demonstrating that Rab23 acts as a cell autonomous negative regulator of HH signaling (Eggenschwiler et al. 2001). Since Rab proteins typically regulate vesicle trafficking, Rab23 was initially presumed to have a role in the trafficking of HH pathway components. Localization studies in a range of mammalian cell types showed distribution of exogenously expressed GFP-tagged Rab23 at the plasma membrane and on the endocytic pathway in transferrin-positive endosomes, where it co-localized with the HH receptor patched (PTCH1), but not with the co-receptor smoothened (SMO) (Evans et al. 2003). However, further genetic studies suggested a regulatory role for Rab23 downstream of both PTCH and SMO and upstream of the transcriptional regulators of the HH pathway, the Gli proteins (named after glioblastoma from where they were first isolated) (Eggenschwiler et al. 2006; Yang et al. 2008). Of the three vertebrate Gli proteins, Gli1 and Gli2 generally act as transcriptional activators, while Gli3 is primarily processed to a truncated transcriptional repressor. Studies in the mouse neural tube led to the suggestion that Rab23 negatively regulates HH signaling through trafficking of a molecule that mediates the effects of SMO on the formation of Gli2 activators and Gli3 repressors (Eggenschwiler et al. 2006). To date the identity of such a factor remains elusive, but increasing evidence suggests that the activation and processing of Gli proteins, and hence possibly Rab23 function, is intricately linked to the primary cilium.
Rab23 and Primary Cilia
The discovery that the primary cilium is an essential organelle for mammalian HH signaling (Corbit et al. 2005; Huangfu et al. 2003) is arguably one of the most significant findings in the fields of cell and developmental biology over the past decade. The primary cilium is a microtubule-based organelle that projects from the surface of virtually every vertebrate cell type. The major components of the HH pathway, including the Gli proteins, localize to the ciliary axoneme extension and shuttle in and out in a dynamic fashion (Rohatgi et al. 2007; Kim et al. 2009). The intraflagellar transport (IFT) system is primarily responsible for trafficking of cargo between the cell body and the cilia tip, but a number of other accessory proteins also mediate ciliary trafficking. A screen for Rab GTPases involved in primary cilium formation identified Rab8 as the sole Rab localized on primary cilia. However, in the same study, biochemical analysis of Rab GTPase-activating proteins (Rab GAPs) and their attendant Rabs suggested a role for Rab23 and Rab17 in primary cilia formation (Yoshimura et al. 2007). More recently, exogenously expressed wild-type Rab23 was shown to localize to the primary cilium in Madin-Darby Canine Kidney (MDCK) epithelial cells (Boehlke et al. 2010). In these cells, shRNA-mediated depletion of Rab23 or expression of a GDP-bound form of Rab23 decreased the steady state level of SMO at the cilium, suggesting a role for Rab23 in ciliary turnover. Thus, the precise role of Rab23 at the primary cilium has yet to be determined, but the findings to date hint at a potential role in trafficking cargo to or from the cilium.
Rab23 and Planar Cell Polarity
The planar cell polarity (PCP) pathway coordinates cell polarization in a given plane across a cell layer, a process essential for the correct formation of certain highly ordered differentiated tissues during development (Fanto and McNeill 2004). A recent study in Drosophila provides new evidence for Rab23 as a PCP regulator (Pataki et al. 2010). Mutations in the Drosophila Rab23 gene resulted in abnormal trichome orientation and the formation of multiple hairs on the wing, leg, and abdomen. This work also showed that Rab23 associates with the PCP protein Prickle, likely contributing to its asymmetric cellular accumulation to regulate the hexagonal packing of Drosophila wing cells and the orientation of cuticular hairs. Since components of the PCP pathway are important for the correct formation and positioning of cilia in vertebrate cells (Park et al. 2006), these data potentially provide a further link between Rab23 and cilia. However, cilia do not appear to be important for PCP or HH signaling in Drosophila, and a role for Rab23 in vertebrate PCP signaling has not yet been elucidated.
Rab23 in Human Disease
The physiological relevance of Rab23 has been highlighted by its involvement in a number of human disorders. Homozygous loss-of-function mutations in RAB23 are responsible for Carpenter syndrome, a pleiotropic disorder with autosomal recessive inheritance, characterized by premature closure of the cranial sutures, polysyndactyly, obesity, and cardiac defects (Jenkins et al. 2007). Six independent mutations in RAB23 have been identified in Carpenter syndrome patients (Alessandri et al. 2010; Jenkins et al. 2007). Five of the six mutations are predicted to result in premature protein truncation, and one is a missense mutation thought to interfere with protein folding (Fig. 1). They show no apparent clustering to specific domains within the RAB23 protein and all are likely to represent loss-of-function alleles. While craniosynostosis and obesity are not classic features of perturbed HH signaling, obesity in particular has been associated with disrupted cilia in humans and mice (Sheffield 2010), again reinforcing a role for Rab23 in ciliogenesis.
Rab23 appears to have functions beyond mammalian embryonic development, as overexpression of Rab23 has been associated with human cancers. In the gastric cancer cell line Hs746T, siRNA-mediated silencing of Rab23 significantly reduced cellular migration and invasion, whereas overexpression of Rab23 enhanced invasion in gastric epithelial (AGS) cells (Hou et al. 2008). Rab23 expression is also upregulated in hepatocellular carcinoma (Liu et al. 2007). The finding that Rab23 is upregulated in a number of human cancers seems contradictory given that Rab23 antagonizes HH signaling, and enhanced expression would be expected to result in pathway inhibition. In a wide range of tumor types, activation of HH signaling, rather than inhibition, is generally associated with tumorigenesis. It is possible that the role of Rab23 in cancer is unrelated to its regulation of HH signaling, or alternatively that a fine balance of Rab23 is required for correct functioning in the tumor environment. Future elucidation of the precise role of Rab23 in regulating HH signaling, ciliogenesis, and other cellular events will shed light on its involvement in cancer and other disease states.
Summary and Perspectives
Genetic and biochemical studies have implicated Rab23 in embryonic development, thus highlighting the role of vesicular trafficking in regulating embryogenesis at the cellular level. However, no Rab23 effectors or interacting partners have been identified to date in vertebrates, and as a result no molecular mechanism for Rab23 action has yet been elucidated. It may be that Rab23 acts indirectly to inhibit HH signaling. The key to Rab23 function might be found at the primary cilium, as the two signaling pathways in which Rab23 is involved (PCP and HH), converge at this organelle (Veland et al. 2009). Rab23 is not essential for Drosophila development (Pataki et al. 2010) but it is possible that, unlike Rab23 involvement in HH signaling, the Rab23-PCP link is evolutionarily conserved. PCP genes are required for neural tube closure, a characteristic phenotype of Rab23 and ciliogenesis mouse mutants (Doudney and Stanier 2005). Taken together, these observations suggest a key role for Rab23 at the intersection of the cilia-related HH and PCP pathways. Elucidation of such a link, along with the more precise definition of Rab23 function, is likely to come from future detailed studies at both the whole organism and cellular levels.