Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.

The Met/hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) is deregulated in many cancers and is a recognized target for cancer therapies. Following HGF stimulation, the signaling output of Met is tightly controlled by receptor internalization and sorting for degradation or recycling. Here, we uncover a role for autophagy in selective degradation of Met and regulation of Met-dependent cell migration and invasion. Met engagement with the autophagic pathway is dependent on complex formation with the mammalian ATG8 family member MAP1LC3C. LC3C deletion abrogates Met entry into the autophagy-dependent degradative pathway, allowing identification of LC3C domains required for rescue. Cancer cells with low LC3C levels show enhanced Met stability, signaling, and cell invasion. These findings provide mechanistic insight into RTK recruitment to autophagosomes and establish distinct roles for ATG8 proteins in this process, supporting that differential expression of ATG8 proteins can shape the functional consequences of autophagy in cancer development and progression.

Cell polarization is essential for neuronal development in both the embryonic and postnatal brain. Here, using primary cultures, in vivo postnatal electroporation and conditional genetic ablation, we show that the Ras-like small GTPase RalA and its effector, the exocyst, regulate the morphology and polarized migration of neural progenitors derived from the subventricular zone, a major neurogenic niche in the postnatal brain. Active RalA promotes the direct binding between the exocyst subunit Exo84 and the PDZ domain of Par6 through a non-canonical PDZ-binding motif. Blocking the Exo84-Par6 interaction impairs polarization in postnatal neural progenitors and cultured embryonic neurons. Our results provide the first in vivo characterization of RalA function in the mammalian brain and highlight a novel molecular mechanism for cell polarization. Given that the exocyst and the Par complex are conserved in many tissues, the functional significance of their interaction and its regulation by RalA are likely to be important in a wide range of polarization events.

Background: There are still no effective treatments for superficial bladder cancer (SBC)/non-muscle invasive bladder cancer. Following treatment, 20% of patients still develop metastatic disease. Superficial bladder cancer is often multifocal, has high recurrences after surgical resection and recurs after intravesical live Bacillus Calmette–Guérin. Oncovex GALV/CD , an oncolytic herpes simplex virus-1, has shown enhanced local tumour control by combining oncolysis with the expression of a highly potent pro-drug activating gene and the fusogenic glycoprotein. Methods: In vitro fusion/prodrug/apoptotic cell-based assays. In vivo orthotopic bladder tumour model, visualised by computed microtomography. Results: Treatment of seven human bladder carcinoma cell lines with the virus resulted in tumour cell killing through oncolysis, pro-drug activation and glycoprotein fusion. Oncovex GALV/CD and mitomycin C showed a synergistic effect, whereas the co-administration with cisplatin or gemcitabine showed an antagonistic effect in vitro . Transitional cell cancer (TCC) cells follow an apoptotic cell death pathway after infection with Oncovex GALV/CD with or without 5-FC. In vivo results showed that intravesical treatment with Oncovex GALV/CD + prodrug (5-FC) reduced the average tumour volume by over 95% compared with controls. Discussion: Our in vitro and in vivo results indicate that Oncovex GALV/CD can improve local tumour control within the bladder, and potentially alter its natural history.

Summary Cell chemotaxis, such as migration of fibroblasts towards growth factors during development and wound healing, requires precise spatial coordination of signalling events. Phosphoinositides and signalling enzymes involved in their generation and hydrolysis have been implicated in regulation of chemotaxis; however, the role and importance of specific components remain poorly understood. Here, we demonstrate that phospholipase C epsilon (PLCε) contributes to fibroblast chemotaxis towards platelet-derived growth factor (PDGF-BB). Using PLCe1 null fibroblasts we show that cells deficient in PLCε have greatly reduced directionality towards PDGF-BB without detrimental effect on their basal ability to migrate. Furthermore, we show that in intact fibroblasts, signalling events, such as activation of Rac, are spatially compromised by the absence of PLCε that affects the ability of cells to enlarge their protrusions in the direction of the chemoattractant. By further application of live cell imaging and the use of FRET-based biosensors, we show that generation of Ins(1,4,5)P3 and recruitment of PLCε are most pronounced in protrusions responding to the PDGF-BB gradient. Furthermore, the phospholipase C activity of PLCε is critical for its role in chemotaxis, consistent with the importance of Ins(1,4,5)P3 generation and sustained calcium responses in this process. As PLCε has extensive signalling connectivity, using transgenic fibroblasts we ruled out its activation by direct binding to Ras or Rap GTPases, and suggest instead new unexpected links for PLCε in the context of chemotaxis.

RAL small GTPases, encoded by the Rala and Ralb genes, are members of the RAS superfamily of small GTPases and can act as downstream effectors of RAS [1]. Although highly similar, distinct functions have been identified for RALA and RALB: RALA has been implicated in epithelial cell polarity [2], insulin secretion [3], GLUT4 translocation [4, 5], neurite branching, and neuronal polarity [6, 7], and RALB in tumor cell survival [8], migration/invasion [9-12], TBK1 activation [13], and autophagy [14]. To investigate RAL GTPases in vivo, we generated null and conditional knockout mice. Ralb null mice are viable with no overt phenotype; the Rala null leads to exencephaly and embryonic lethality. The exencephaly phenotype is exacerbated in Rala(-/-);Ralb(+/-) embryos; embryos null for Rala and Ralb do not live past gastrulation. Using a Kras-driven non-small cell lung carcinoma mouse model, we found that either RALA or RALB is sufficient for tumor growth. However, deletion of both Ral genes blocks tumor formation. Either RALA or RALB is sufficient for cell proliferation, but cells lacking both fail to proliferate. These studies demonstrate functions of RAL proteins in development, tumorigenesis, and cell proliferation and show that RALA and RALB act in a redundant fashion.

Cbl proteins are E3 ubiquitin ligases that are negative regulators of many receptor tyrosine kinases. Cbl-b and c-Cbl contain a ubiquitinassociated (UBA) domain, which is present in a variety of proteins involved in ubiquitinmediated processes. Despite high sequence identity, Cbl UBA domains display remarkably different ubiquitin-binding properties. Here, we report the crystal structure of the UBA domain of Cbl-b in complex with ubiquitin at 1.9 angstrom resolution. The structure reveals an atypical mechanism of ubiquitin recognition by the first helix of the UBA. Helices 2 and 3 of the UBA domain form a second binding surface, which mediates IJIBA dimerization in the crystal and in solution. Site-directed mutagenesis demonstrates that Cbl-b dimerization is regulated by ubiquitin binding and required for tyrosine phosphorylation of Cbl-b and ubiquitination of Cbl-b substrates. These studies demonstrate a role for ubiquitin in regulating biological activity by promoting protein dimerization.

Ligand-induced down-regulation by the ubiquitin-protein ligases, c-Cbl and Cbl-b, controls signaling downstream from many receptor-tyrosine kinases (RTK). Cbl proteins bind to phosphotyrosine residues on activated RTKs to affect ligand-dependent ubiquitylation of these receptors targeting them for degradation in the lysosome. Both c-Cbl and Cbl-b contain a ubiquitin-associated (UBA) domain, which is important for Cbl dimerization and tyrosine phosphorylation; however, the mechanism of UBA-mediated dimerization and its requirement for Cbl biological activity is unclear. Here, we report the crystal structure of the UBA domain of c-Cbl refined to 2.1-angstrom resolution. The structure reveals the protein is a symmetric dimer tightly packed along a large hydrophobic surface formed by helices 2 and 3. NMR chemical shift mapping reveals heterodimerization can occur with the related Cbl-b UBA domain via the same surface employed for homodimerization. Disruption of c-Cbl dimerization by site-directed mutagenesis impairs c-Cbl phosphorylation following activation of the Met/hepatocyte growth factor RTK and c-Cbl-dependent ubiquitination of Met. This provides direct evidence for a role of Cbl dimerization in terminating signaling following activation of RTKs.

Deregulation of growth factor receptor tyrosine kinases (RTKs) is linked to a large number of malignancies. This occurs through a variety of mechanisms that result in enhanced activity of the receptor. Considerable evidence now supports the idea that loss of negative regulation plays an important role in receptor deregulation. RTKs are removed from the cell surface via endocytosis and many are subsequently degraded in the lysosome. Lysosomal targeting has recently been linked with receptor ubiquitination. We review here molecular alterations that uncouple RTKs from ubiquitination and implicate loss of ubiquitination as a process that plays a significant role in the pathogenesis of cancer.

Sonic hedgehog (Shh) signaling is essential for many normal developmental processes. The Shh signal is interpreted by the Gli transcription factors. ElevatedGli-1 expression has been associated with several neoplasms, including basal cell carcinoma. All-transretinoic acid (RA) has strong effects on epidermal growth and differentiation and has been used for the treatment of various epithelial disorders. In this report, we show that RA can inhibit Gli activity in immortalized murine keratinocytes in a RA receptor-specific manner. This inhibition may occur, at least in part, through sequestration of the transcriptional coactivator cyclic AMPresponsive element-binding protein-binding protein and suggests a novel effect of retinoid excess on Shh signaling.