Regulation of hedgehog Ligand Expression by the N-End Rule Ubiquitin-Protein Ligase Hyperplastic Discs and the Drosophila GSK3ß Homologue, Shaggy.

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3ß, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals.

EDD, a ubiquitin-protein ligase of the N-end rule pathway, associates with spindle assembly checkpoint components and regulates the mitotic response to nocodazole.

In this work, we identify physical and genetic interactions that implicate E3 identified by differential display (EDD) in promoting spindle assembly checkpoint (SAC) function. During mitosis, the SAC initiates a mitotic checkpoint in response to chromosomes with kinetochores unattached to spindle pole microtubules. Similar to Budding uninhibited by benzimidazoles-related 1 (BUBR1) siRNA, a bona fide SAC component, EDD siRNA abrogated G2/M accumulation in response to the mitotic destabilizing agent nocodazole. Furthermore, EDD siRNA reduced mitotic cell viability and, in nocodazole-treated cells, increased expression of the promitotic progression protein cell division cycle 20 (CDC20). Copurification studies also identified physical interactions with CDC20, BUBR1, and other components of the SAC. Taken together, these observations highlight the potential role of EDD in regulating mitotic progression and the cellular response to perturbed mitosis.

The E3 ubiquitin ligase WWP1 regulates ΔNp63-dependent transcription through Lys63 linkages.

The transcription factor p63, a member of the p53 family, plays a crucial role in epithelial development and tumorigenesis through the regulation of epithelial progenitor cell proliferation, differentiation and apoptosis. Similarly to p53, p63 activity is regulated by post-translational modifications, including ubiquitylation. Here, we report that the WWP1 E3 ubiquitin ligase binds specifically to ΔNp63 isoform but it does not trigger ΔNp63 proteasome-dependent degradation. Accordingly, we found that WWP1-dependent ubiquitylation of ΔNp63 occurs through the formation of Lys63-linked poly-ubiquitin chains. Importantly, we found that WWP1 is able to increase ΔNp63-dependent transcription and depletion of WWP1 in human primary keratinocytes induces cell cycle arrest. All together these results indicate that WWP1 regulates ΔNp63 transcriptional activity, acting thus as a potential regulator of the proliferation and survival of epithelial-derived cells.

Itch self-polyubiquitylation occurs through lysine-63 linkages.

Itch, an E3 protein ubiquitin ligase (E3), which belongs to the homologous to E6-AP carboxy terminus (HECT)-type subfamily, catalyzes its own ubiquitylation. The precise nature of Itch-mediated self-modification and its biological outcome are not completely understood. Here, we show that Itch auto-ubiquitylation is an intermolecular reaction generating Lys63-linkages, rather than the Lys48-linked polyubiquitin chains that target proteins for proteasomal degradation. As a result, Itch is a relatively high stable protein, whose levels are not significantly affected by treatment by either proteasome or lysosome inhibitors. Furthermore, we demonstrate that the decay rate of a catalytic inactive Itch mutant, which is devoided of self-ubiquitylating activity, is barely indistinguishable from the one of the wild-type protein. These data definitely establish a nondegradative role for Lys63-linked Itch self-ubiquitylation.

Biochemical characterization of MLC1 protein in astrocytes and its association with the dystrophin-glycoprotein complex.

MLC1 gene mutations have been associated with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare neurologic disorder in children. The MLC1 gene encodes a membrane protein (MLC1) with unknown function which is mainly expressed in astrocytes. Using a newly developed anti-human MLC1 polyclonal antibody, we have investigated the biochemical properties and localization of MLC1 in cultured astrocytes and brain tissue and searched for evidence of a relationship between MLC1 and proteins of the dystrophin-glycoprotein complex (DGC). Cultured astrocytes express two MLC1 components showing different solubilisation properties and subcellular distribution. Most importantly, we show that the membrane-associated component of MLC1 (60-64 kDa) localizes in astrocytic lipid rafts together with dystroglycan, syntrophin and caveolin-1, and co-fractionates with the DGC in whole rat brain tissue. In the human brain, MLC1 protein is expressed in astrocyte processes and ependymal cells, where it colocalizes with dystroglycan and syntrophin. These data indicate that the DGC may be involved in the organization and function of the MLC1 protein in astrocyte membranes.