Alexey Veraksa

Regulation of Notch signalling by non-visual β-arrestin

Signalling activity of the Notch receptor, which plays a fundamental role in metazoan cell fate determination, is controlled at multiple levels. We uncovered a Notch signal-controlling mechanism that depends on the ability of the non-visual β-arrestin, Kurtz (Krz), to influence the degradation and, consequently, the function of the Notch receptor. We identified Krz as a binding partner of a known Notch-pathway modulator, Deltex (Dx), and demonstrated the existence of a trimeric Notch–Dx–Krz protein complex. This complex mediates the degradation of the Notch receptor through a ubiquitination-dependent pathway. Our results establish a novel mode of regulation of Notch signalling and define a new function for non-visual β-arrestins.

elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8

The formation and function of cilia involves the movement of intraflagellar transport (IFT) particles underneath the ciliary membrane, along axonemal microtubules. Although this process has been studied extensively, its molecular basis remains incompletely understood. For example, it is unknown how the IFT particle interacts with transmembrane proteins. To study the IFT particle further, we examined elipsa, a locus characterized by mutations that cause particularly early ciliogenesis defects in zebrafish. We show here that elipsa encodes a coiled-coil polypeptide that localizes to cilia. Elipsa protein binds to Ift20, a component of IFT particles, and Elipsa homologue in Caenorhabditis elegans, DYF-11, translocates in sensory cilia, similarly to the IFT particle. This indicates that Elipsa is an IFT particle polypeptide. In the context of zebrafish embryogenesis, Elipsa interacts genetically with Rabaptin5, a well-studied regulator of endocytosis, which in turn interacts with Rab8, a small GTPase, known to localize to cilia. We show that Rabaptin5 binds to both Elipsa and Rab8, suggesting that these proteins provide a bridging mechanism between the IFT particle and protein complexes that assemble at the ciliary membrane.

The Drosophila F Box Protein Archipelago Regulates dMyc Protein Levels In Vivo

BACKGROUND The Myc oncoprotein is an important regulator of cellular growth in metazoan organisms. Its levels and activity are tightly controlled in vivo by a variety of mechanisms. In normal cells, Myc protein is rapidly degraded, but the mechanism of its degradation is not well understood. RESULTS Here we present genetic and biochemical evidence that Archipelago (Ago), the F box component of an SCF-ubiquitin ligase and the Drosophila ortholog of a human tumor suppressor, negatively regulates the levels and activity of Drosophila Myc (dMyc) protein in vivo. Mutations in archipelago (ago) result in strongly elevated dMyc protein levels and increased tissue growth. Genetic interactions indicate that ago antagonizes dMyc function during development. Archipelago binds dMyc and regulates its stability, and the ability of Ago to bind dMyc in vitro correlates with its ability to inhibit dMyc accumulation in vivo. CONCLUSIONS Our data indicate that archipelago is an important inhibitor of dMyc in developing tissues. Because archipelago can also regulate Cyclin E levels and Notch activity, these results indicate how a single F box protein can be responsible for the degradation of key components of multiple pathways that control growth and cell cycle progression.

Analyzing protein complexes in Drosophila with tandem affinity purification–mass spectrometry

We describe the application of tandem affinity purification–mass spectrometry (TAP‐MS) to the study of protein complexes in Drosophila. We have constructed vectors for inducible expression of TAP‐tagged fusion proteins in Drosophila cultured cells and in vivo. Using these vectors, we tagged, as a paradigm, several components of the Notch signaling pathway, isolated protein complexes containing the baits and associated proteins from cells and embryos, and identified the subunits by liquid chromatography–tandem mass spectrometry (LC‐MS/MS). Several known interactions involving Notch pathway elements were confirmed, and many novel potential interactions were uncovered. For some of the novel associations, we validated the interaction genetically and biochemically. We conclude that TAP, in combination with MS, can be used as an effective method for the studies of the Drosophila proteome. Developmental Dynamics 232:827–834, 2005.

Tandem affinity purification in Drosophila: the advantages of the GS-TAP system.

Tandem affinity purification (TAP) has been widely used for the analysis of protein complexes. We investigated the parameters of the recently developed TAP method (GS-TAP) and its application in Drosophila. This new tag combination includes two Protein G modules and a streptavidin binding peptide (SBP), separated by one or two TEV protease cleavage sites. We made pMK33-based GS-TAP vectors to allow for generation of stable cell lines using hygromycin selection and inducible expression from a metallothionein promoter, as well as pUAST-based vectors that can be used for inducible expression in flies. Rescue experiments in flies demonstrated that the GS-TAP tag preserves the function of the tagged protein. We have done parallel purifications of proteins tagged with the new GS-TAP tag or with the conventional TAP tag (containing the Protein A and calmodulin binding peptide domains) at the amino terminus, using both cultured cells and embryos. A major difference between the two tags was in the levels of contaminating proteins, which were significantly lower in the GS-TAP purifications. The GS-TAP procedure also resulted in higher yield of the bait protein. Overall, GS-TAP is an improved method of protein complex purification because it provides a superior signal-to-noise ratio of the bait protein relative to contaminants in purified material.

Riquiqui and Minibrain are regulators of the Hippo pathway downstream of Dachsous

The atypical cadherins Fat (Ft) and Dachsous (Ds) control tissue growth through the Salvador–Warts–Hippo (SWH) pathway, and also regulate planar cell polarity and morphogenesis. Ft and Ds engage in reciprocal signalling as both proteins can serve as receptor and ligand for each other. The intracellular domains (ICDs) of Ft and Ds regulate the activity of the key SWH pathway transcriptional co-activator protein Yorkie (Yki). Signalling from the FtICD is well characterized and controls tissue growth by regulating the abundance of the Yki-repressive kinase Warts (Wts). Here we identify two regulators of the Drosophila melanogaster SWH pathway that function downstream of the DsICD: the WD40 repeat protein Riquiqui (Riq) and the DYRK-family kinase Minibrain (Mnb). Ds physically interacts with Riq, which binds to both Mnb and Wts. Riq and Mnb promote Yki-dependent tissue growth by stimulating phosphorylation-dependent inhibition of Wts. Thus, we describe a previously unknown branch of the SWH pathway that controls tissue growth downstream of Ds.

In Vivo Analysis of the Notch Receptor S1 Cleavage

A ligand-independent cleavage (S1) in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.

The Ecdysone Receptor Coactivator Taiman Links Yorkie to Transcriptional Control of Germline Stem Cell Factors in Somatic Tissue

The Hippo pathway is a conserved signaling cascade that modulates tissue growth. Although its core elements are well defined, factors modulating Hippo transcriptional outputs remain elusive. Here we show that components of the steroid-responsive ecdysone (Ec) pathway modulate Hippo transcriptional effects in imaginal disc cells. The Ec receptor coactivator Taiman (Tai) interacts with the Hippo transcriptional coactivator Yorkie (Yki) and promotes expression of canonical Yki-responsive genes. Tai enhances Yki-driven growth, while Tai loss, or a form of Tai unable to bind Yki, suppresses Yki-driven tissue growth. This growth suppression is not correlated with impaired induction of canonical Hippo-responsive genes but with suppression of a distinct pro-growth program of Yki-induced/Tai-dependent genes, including the germline stem cell factors nanos and piwi. These data reveal Hippo/Ec pathway crosstalk in the form a Yki-Tai complex that collaboratively induces germline genes as part of a transcriptional program that is normally repressed in developing somatic epithelia.

β-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development.

β‐Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila β‐arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF‐κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of β‐arrestins and present a new mechanism of ERK inhibition by the Drosophila β‐arrestin Krz.

The GTPase Regulatory Proteins Pix and Git Control Tissue Growth via the Hippo Pathway

The Salvador-Warts-Hippo (Hippo) pathway is a conserved regulator of organ size and is deregulated in human cancers. In epithelial tissues, the Hippo pathway is regulated by fundamental cell biological properties, such as polarity and adhesion, and coordinates these with tissue growth. Despite its importance in disease, development, and regeneration, the complete set of proteins that regulate Hippo signaling remain undefined. To address this, we used proteomics to identify proteins that bind to the Hippo (Hpo) kinase. Prominent among these were PAK-interacting exchange factor (known as Pix or RtGEF) and G-protein-coupled receptor kinase-interacting protein (Git). Pix is a conserved Rho-type guanine nucleotide exchange factor (Rho-GEF) homologous to Beta-PIX and Alpha-PIX in mammals. Git is the single Drosophila melanogaster homolog of the mammalian GIT1 and GIT2 proteins, which were originally identified in the search for molecules that interact with G-protein-coupled receptor kinases. Pix and Git form an oligomeric scaffold to facilitate sterile 20-like kinase activation and have also been linked to GTPase regulation. We show that Pix and Git regulate Hippo-pathway-dependent tissue growth in D. melanogaster and that they do this in parallel to the known upstream regulator Fat cadherin. Pix and Git influence activity of the Hpo kinase by acting as a scaffold complex, rather than enzymes, and promote Hpo dimerization and autophosphorylation of Hpos activation loop. Therefore, we provide important new insights into an ancient signaling network that controls the growth of metazoan tissues.