RET Functions as a Dual-Specificity Kinase that Requires Allosteric Inputs from Juxtamembrane Elements.

Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM) segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.

The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles.

Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-ß to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.

Genetic characterization of ebi reveals its critical role in Drosophila wing growth.

The ebi gene of Drosophila melanogaster has been implicated in diverse signalling pathways, cellular functions and developmental processes. However, a thorough genetic analysis of this gene has been lacking and the true extent of its biological roles is unclear. Here, we characterize eleven ebi mutations and find that ebi has a novel role in promoting growth of the wing imaginal disc: viable combinations of mutant alleles give rise to adults with small wings. Wing discs with reduced EBI levels are correspondingly small and exhibit down-regulation of Notch target genes. Furthermore, we show that EBI colocalizes on polytene chromosomes with Smrter (SMR), a transcriptional corepressor, and Suppressor of Hairless (SU(H)), the primary transcription factor involved in Notch signalling. Interestingly, the mammalian orthologs of ebi, transducin ß-like 1 (TBL1) and TBL-related 1 (TBLR1), function as corepressor/coactivator exchange factors and are required for transcriptional activation of Notch target genes. We hypothesize that EBI acts to activate (de-repress) transcription of Notch target genes important for Drosophila wing growth by functioning as a corepressor/coactivator exchange factor for SU(H).

Inhibitor of apoptosis proteins in Drosophila: gatekeepers of death.

Regulation of apoptosis is crucial to ensure cellular viability, and failure to do so is linked to several human pathologies. The apoptotic cell death programme culminates in the activation of caspases, a family of highly specific cysteine proteases essential for the destruction of the cell. Although best known for their role in executing apoptosis, caspases also play important signalling roles in non-apoptotic processes, such as regulation of actin dynamics, innate immunity, cell proliferation, differentiation and survival. Under such conditions, caspases are activated without killing the cell. Caspase activation and activity is subject to complex regulation, and various cellular and viral inhibitors have been identified that control the activity of caspases in their apoptotic and non-apoptotic roles. Members of the Inhibitor of APoptosis (IAP) protein family ensure cell viability in Drosophila by directly binding to caspases and regulating their activities in a ubiquitin-dependent manner. The observation that IAPs are essential for cell survival in Drosophila, and are frequently deregulated in human cancer, contributing to tumourigenesis, chemoresistance, disease progression and poor patient survival, highlights the importance of this family of caspase regulators in health and disease. Here we summarise recent advances from Drosophila that start to elucidate how the cellular response to caspase activation is modulated by IAPs and their regulators.

Input from Ras is required for maximal PI(3)K signalling in Drosophila.

Class I phosphoinositide 3-kinases (PI(3)Ks) are activated through associated adaptor molecules in response to G protein-coupled and tyrosine kinase receptor signalling. They contain Ras-binding domains (RBDs) and can also be activated through direct association with active GTP-bound Ras. The ability of Ras to activate PI(3)K has been established in vitro and by overexpression analysis, but its relevance for normal PI(3)K function in vivo is unknown. The Drosophila class I PI(3)K, Dp110, is activated by nutrient-responsive insulin signalling and modulates growth, oogenesis and metabolism. To investigate the importance of Ras-mediated PI(3)K activation for normal PI(3)K function, we replaced Dp110 with Dp110(RBD), which is unable to bind to Ras but otherwise biochemically normal. We found that Ras-mediated Dp110 regulation is dispensable for viability. However, egg production, which requires large amounts of growth, is dramatically lowered in Dp110(RBD) flies. Furthermore, insulin cannot maximally activate PI(3)K signalling in Dp110(RBD) imaginal discs and Dp110(RBD) flies are small. Thus, Dp110 integrates inputs from its phosphotyrosine-binding adaptor and Ras to achieve maximal PI(3)K signalling in specific biological situations.

Flies on steroids: the interplay between ecdysone and insulin signaling.

In the fruit fly Drosophila melanogaster, the insulin and ecdysone signaling pathways have long been known to regulate growth and developmental timing, respectively. Recent findings reveal that crosstalk between these pathways allows coordination of growth and developmental timing and thus determines final body size.

Nuclear export of alpha-catenin: overlap between nuclear export signal sequences and the beta-catenin binding site.

Mutations in APC or in beta-catenin, which are common in colon cancer, lead to constitutive activation of beta-catenin/Tcf-dependent signaling. alpha-Catenin is also found in some colon cancer cell nuclei, and loss of its expression correlates with increased beta-catenin/Tcf transcriptional activity. Moreover, targeted expression of alpha-catenin in the nucleus inhibits beta-catenin/Tcf-dependent transcription. Thus, an understanding of the regulation of alpha-catenin localization could provide insight into the control of beta-catenin signaling. While the beta-catenin/Tcf complex can promote nuclear import of alpha-catenin, the mechanism for its nuclear export is not known. We found that leptomycin B (LMB) inhibited nuclear export of GFP-alpha-catenin in HCT116 colon cancer cells, suggesting that alpha-catenin localization is regulated by CRM-1-dependent nuclear export. We identified two putative nuclear export signals in a domain of alpha-catenin that overlaps with the beta-catenin binding domain. Using a nuclear export assay, we determined that one of these (NES1) is a weak LMB-insensitive NES, whereas the other (NES2) is strong and LMB-sensitive. Mutations in either NES reduced nuclear export of alpha-catenin in HCT116 cells. In addition, mutations in NES1, but not NES2, reduced binding of alpha-catenin to beta-catenin and impaired the ability of alpha-catenin to repress beta-catenin/Tcf-dependent transcription. Therefore, NES1 is required both for repression of beta-catenin signaling and for nuclear export, while NES2 is required only for nuclear export.