Mind bomb-2 is an E3 ligase for Notch ligand

The zebrafish gene, mind bomb (mib), encodes a protein that positively regulates of the Delta-mediated Notch signaling. It interacts with the intracellular domain of Delta to promote its ubiquitination and endocytosis. In our search for the mouse homologue of zebrafish mind bomb, we cloned two homologues in the mouse genome: a mouse orthologue (mouse mib1) and a paralogue, named mind bomb-2 (mib2), which is evolutionarily conserved from Drosophila to human. Both Mib1 and Mib2 have an E3 ubiquitin ligase activity in their C-terminal RING domain and interact with Xenopus Delta (XD) via their N-terminal region. Mib2 is also able to ligate ubiquitin to XD and shift the membrane localization of Delta to intracellular vesicles. Importantly, Mib2 rescues both the neuronal and vascular defects in the zebrafish mib(ta52b) mutants. In contrast to the functional similarities between Mib1 and Mib2, mib2 is highly expressed in adult tissues, but almost not at all in embryos, whereas mib1 is abundantly expressed in both embryos and adult tissues. These data suggest that Mib2 has functional similarities to Mib1, but might have distinct roles in Notch signaling as an E3 ubiquitin ligase.     

Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues.     

Dkk3 is required for TGF-beta signaling during Xenopus mesoderm induction

Abstract The Dickkopf (Dkk) family is composed of four main members (Dkk1–4), which typically regulate Wnt/β-catenin signaling. An exception is Dkk3, which does not affect Wnt/β-catenin signaling and whose function is poorly characterized. Here, we describe the Xenopus dkk3 homolog and characterize its expression and function during embryogenesis. Dkk3 is maternally expressed and zygotically in the cement gland, head mesenchyme, and heart. We show that depletion of Dkk3 in Xenopus embryos by Morpholino antisense oligonucleotides induces axial defects as a result of Spemann organizer and mesoderm inhibition. Dkk3 depletion leads to down-regulation of Activin/Nodal signaling by reducing levels of Smad4 protein. Dkk3 overexpression can rescue phenotypic effects resulting from overexpression of the Smad4 ubiquitin ligase Ectodermin. Furthermore, depletion of Dkk3 up-regulates FGF signaling, while Dkk3 overexpression reduces it. These results indicate that Dkk3 modulates FGF and Activin/Nodal signaling to regulate mesoderm induction during early Xenopus development.     

Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis

The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF‐κB signaling pathways. Both Ras/mitogen‐activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high‐throughput electro‐chemiluninescent‐based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self‐ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF‐1α and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah‐KO cells, pointing to its Siah‐dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS‐independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.     

Oral–aboral axis specification in the sea urchin embryo,IV: Hypoxia radializes embryos by preventing the initial spatialization of nodal activity

The oral–aboral axis of the sea urchin embryo is specified conditionally via a regulated feedback circuit involving the signaling gene nodal and its antagonist lefty. In normal development nodal activity becomes localized to the prospective oral side of the blastula stage embryo, a process that requires lefty. In embryos of Strongylocentrotus purpuratus, a redox gradient established by asymmetrically distributed mitochondria provides an initial spatial input that positions the localized domain of nodal expression. This expression is perturbed by hypoxia, leading to development of radialized embryos lacking an oral–aboral axis. Here we show that this radialization is not caused by a failure to express nodal, but rather by a failure to localize nodal activity to one side of the embryo. This occurs even when embryos are removed from hypoxia at late cleavage stage when nodal is first expressed, indicating that the effect involves the initiation phase of nodal activity, rather than its positive feedback-driven amplification and maintenance. Quantitative fluorescence microscopy of MitoTracker Orange-labeled embryos expressing nodal-GFP reporter gene revealed that hypoxia abolishes the spatial correlation between mitochondrial distribution and nodal expression, suggesting that hypoxia eliminates the initial spatial bias in nodal activity normally established by the redox gradient. We propose that absent this bias, the initiation phase of nodal expression is spatially uniform, such that the ensuing Nodal-mediated community effect is not localized, and hence refractory to Lefty-mediated enforcement of localization.     

The Imprinted Gene PEG3 Inhibits Wnt Signaling and Regulates Glioma Growth

The imprinted gene PEG3 confers parenting and sexual behaviors, alters growth and development, and regulates apoptosis. However, a molecular mechanism that can account for the diverse functions of Peg3/Pw1 is not known. To elucidate Peg3-regulated pathways, we performed a functional screen in zebrafish. Enforced overexpression of PEG3 mRNA during zebrafish embryogenesis decreased β-catenin protein expression and inhibited Wnt-dependent tail development. Peg3/Pw1 also inhibited Wnt signaling in human cells by binding to β-catenin and promoting its degradation via a p53/Siah1-dependent, GSK3β-independent proteasomal pathway. The inhibition of the Wnt pathway by Peg3/Pw1 suggested a role in tumor suppression. Hypermethylation of the PEG3 promoter in primary human gliomas led to a loss of imprinting and decreased PEG3 mRNA expression that correlated with tumor grade. The decrease in Peg3/Pw1 protein expression increased β-catenin, promoted proliferation, and inhibited p53-dependent apoptosis in human CD133⁺ glioma stem cells. Thus, mammalian imprinting utilizes Peg3/Pw1 to co-opt the Wnt pathway, thereby regulating development and glioma growth.     

Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.     

Lnx-2b restricts gsc expression to the dorsal mesoderm by limiting Nodal and Bozozok activity

Coordinated Nodal-related signals and Bozozok (Boz) activity are critical for the initial specification of dorsal mesoderm and anterior neuroectoderm during zebrafish embryogenesis. Overexpression of Boz expands gsc expression into the ventro-lateral marginal blastomeres where Nodal signaling is active, but is insufficient to induce ectopic gsc expression in the animal region. We found that overexpression of Boz together with depletion of Lnx-2b (previously named Lnx-like, Lnx-l), but not each manipulation alone, causes robust gsc expression in all blastomeres. Furthermore, nodal-related signals are required for gsc expression in embryos with elevated Boz activity. Through targeted injection into single cells at the 128-cell stage we illustrate the role of maternally deposited Lnx-2b to restrict the expansion of gsc expression into the presumptive ectodermal region. This report provides a novel mechanism for limiting dorsal organizer specification to a defined region of the early zebrafish embryo.     

Siah1/SIP regulates p27kip1 stability and cell migration under metabolic stress

p27^kip1 has been implicated in cell cycle regulation, functioning as an inhibitor of cyclin-dependent kinase activity. In addition, p27 was also shown to affect cell migration, with accumulation of cytoplasmic p27 associated with tumor invasiveness. However, the mechanism underlying p27 regulation as a cytoplasmic protein is poorly understood. Here we show that glucose starvation induces proteasome-dependent degradation of cytoplasmic p27, accompanied by a decrease in cell motility. We also show that the glucose limitation-induced p27 degradation is regulated through an ubiquitin E3 ligase complex involving Siah1 and SIP/CacyBP. SIP^−/− embryonic fibroblasts have increased levels of cytosolic p27 and exhibit increased cell motility compared with wild-type cells. These observations suggest that the Siah1/SIP E3 ligase complex regulates cell motility through degradation of p27.Key words: p27^kip1, Siah1, SIP, glucose starvation, cell migration     

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.     

The effects of Sp7/Osterix gene silencing in the chondroprogenitor cell line, ATDC5

Ubiquitin is a small polypeptide and ubiquitination is the post-translational modification by ubiquitin protein, resulting in degradation of target proteins by the 26S proteasome complex. Here, we found that E3 ubiquitin ligase SINAT5, an Arabidopsis homologue of the Drosophila SINA RING-finger protein, interacts directly with LHY, a component of the circadian oscillator, and DET1, a negative regulator of light-regulated gene expression. We also found that SINAT5 has E3 ubiquitination activity for LHY but not for DET1. Interestingly, LHY ubiquitination by SINAT5 was inhibited by DET1. Late flowering of sinat5 mutants indicates that flowering time can be controlled by DET1 through regulation of LHY stability by SINAT5.