RalA promotes a direct exocyst-Par6 interaction to regulate polarity in neuronal development.

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.

Activity of PLCε contributes to chemotaxis of fibroblasts towards PDGF.

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)P(3) 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)P(3) 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.

Escape from Cbl-mediated downregulation: a recurrent theme for oncogenic deregulation of receptor tyrosine kinases.

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.

RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes.

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.

LC3C-Mediated Autophagy Selectively Regulates the Met RTK and HGF-Stimulated Migration and Invasion.

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.

Met/Hepatocyte growth factor receptor ubiquitination suppresses transformation and is required for Hrs phosphorylation.

The Met receptor tyrosine kinase (RTK) regulates epithelial remodeling, dispersal, and invasion and is deregulated in many human cancers. It is now accepted that impaired down-regulation, as well as sustained activation, of RTKs could contribute to their deregulation. Down-regulation of the Met receptor involves ligand-induced internalization, ubiquitination by Cbl ubiquitin ligases, and lysosomal degradation. Here we report that a ubiquitination-deficient Met receptor mutant (Y1003F) is tumorigenic in vivo. The Met Y1003F mutant is internalized, and undergoes endosomal trafficking with kinetics similar to the wild-type Met receptor, yet is inefficiently targeted for degradation. This results in sustained activation of Met Y1003F and downstream signals involving the Ras-mitogen-activated protein kinase pathway, cell transformation, and tumorigenesis. Although Met Y1003F undergoes endosomal trafficking and localizes with the cargo-sorting protein Hrs, it is unable to induce phosphorylation of Hrs. Fusion of monoubiquitin to Met Y1003F is sufficient to decrease Met receptor stability and prevent sustained MEK1/2 activation. In addition, this rescues Hrs tyrosine phosphorylation and decreases transformation in a focus-forming assay. These results demonstrate that Cbl-dependent ubiquitination is dispensable for Met internalization but is critical to target the Met receptor to components of the lysosomal sorting machinery and to suppress its inherent transforming activity.

Genetic deletion of RALA and RALB small GTPases reveals redundant functions in development and tumorigenesis.

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.

Structural basis for UBA-mediated dimerization of c-Cbl ubiquitin ligase.

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-A 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.

Structural basis for ubiquitin-mediated dimerization and activation of the ubiquitin protein ligase Cbl-b.

Cbl proteins are E3 ubiquitin ligases that are negative regulators of many receptor tyrosine kinases. Cbl-b and c-Cbl contain a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated 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 A 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 UBA 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.

A conserved DpYR motif in the juxtamembrane domain of the Met receptor family forms an atypical c-Cbl/Cbl-b tyrosine kinase binding domain binding site required for suppression of oncogenic activation.

The activation and phosphorylation of Met, the receptor tyrosine kinase (RTK) for hepatocyte growth factor, initiates the recruitment of multiple signaling proteins, one of which is c-Cbl, a ubiquitin-protein ligase. c-Cbl promotes ubiquitination and enhances the down-modulation of the Met receptor and other RTKs, targeting them for lysosomal sorting and subsequent degradation. The ubiquitination of Met by c-Cbl requires the direct interaction of the c-Cbl tyrosine kinase binding (TKB) domain with tyrosine 1003 in the Met juxtamembrane domain. Although a consensus for c-Cbl TKB domain binding has been established ((D/N)XpYXX(D/E0phi), this motif is not present in Met, suggesting that other c-Cbl TKB domain binding motifs may exist. By alanine-scanning mutagenesis, we have identified a DpYR motif including Tyr(1003) as being important for the direct recruitment of the c-Cbl TKB domain and for ubiquitination of the Met receptor. The substitution of Tyr(1003) with phenylalanine or substitution of either aspartate or arginine residues with alanine impairs c-Cbl-recruitment and ubiquitination of Met and results in the oncogenic activation of the Met receptor. We demonstrate that the TKB domain of Cbl-b, but not Cbl-3, binds to the Met receptor and requires an intact DpYR motif. Modeling studies suggest the presence of a salt bridge between the aspartate and arginine residues that would position pTyr(1003) for binding to the c-Cbl TKB domain. The DpYR motif is conserved in other members of the Met RTK family but is not present in previously identified c-Cbl-binding proteins, identifying DpYR as a new binding motif for c-Cbl and Cbl-b.

The Shc adaptor protein is critical for VEGF induction by Met/HGF and ErbB2 receptors and for early onset of tumor angiogenesis.

The etiology and progression of a variety of human malignancies are linked to the deregulation of receptor tyrosine kinases (RTKs). To define the role of RTK-dependent signals in various oncogenic processes, we have previously engineered RTK oncoproteins that recruit either the Shc or Grb2 adaptor proteins. Although these RTK oncoproteins transform cells with similar efficiencies, fibroblasts expressing the Shc-binding RTK oncoproteins induced tumors with short latency (approximately 7 days), whereas cells expressing the Grb2-binding RTK oncoproteins induced tumors with delayed latency (approximately 24 days). The early onset of tumor formation correlated with the ability of cells expressing the Shc-binding RTK oncoproteins to produce vascular endothelial growth factor (VEGF) in culture and an angiogenic response in vivo. Consistent with this, treatment with a VEGF inhibitor, VEGF-Trap, blocked the in vivo angiogenic and tumorigenic properties of these cells. The importance of Shc recruitment to RTKs for the induction of VEGF was further demonstrated by using mutants of the Neu/ErbB2 RTK, where the Shc, but not Grb2, binding mutant induced VEGF. Moreover, the use of fibroblasts derived from ShcA-deficient mouse embryos, demonstrated that Shc was essential for the induction of VEGF by the Met/hepatocyte growth factor RTK oncoprotein and by serum-derived growth factors. Together, our findings identify Shc as a critical angiogenic switch for VEGF production downstream from the Met and ErbB2 RTKs.