Highlights of the 2nd International Symposium on Tribbles and Diseases: Tribbles tremble in therapeutics for immunity, metabolism, fundamental cell biology and cancer

The Tribbles (TRIB) family of pseudokinase proteins has been shown to play key roles in cell cycle, metabolic diseases, chronic inflammatory disease, and cancer development. A better understanding of the mechanisms of TRIB pseudokinases could provide new insights for disease development and help promote TRIB proteins as novel therapeutic targets for drug discovery. At the 2nd International Symposium on Tribbles and Diseases held on May 7‒9, 2018 in Beijing, China, a group of leading Tribbles scientists reported their findings and ongoing studies about the effects of the different TRIB proteins in the areas of immunity, metabolism, fundamental cell biology and cancer. Here, we summarize important and insightful overviews from 4 keynote lectures, 13 plenary lectures and 8 short talks that took place during this meeting. These findings may offer new insights for the understanding of the roles of TRIB pseudokinases in the development of various diseases

Ecdysterone regulatory elements function as both transcriptional activators and repressors.

A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene also led to high levels of ecdysterone-induced CAT expression, but the repression of high constitutive levels of CAT activity failed to occur. The EcRE-CAT construct was cotransfected with plasmids containing tandem 10-mers or 40-mers of the EcRE but lacking a reporter gene. These additional EcREs led to a reduced level of ecdysterone-induced CAT activity and to an elevation of basal CAT activity in the absence of hormone. The data suggest that the receptor binds to the EcRE in the absence of hormone, blocking basal transcription from a constitutive promoter. In the presence of ecdysterone, receptor-hormone binding to the EcRE leads to greatly enhanced transcription

Control of Cell Growth and Proliferation by the Tribbles Pseudokinase: Lessons from Drosophila

The Tribbles (Trib) family of pseudokinase proteins regulate cell growth, proliferation, and differentiation during normal development and in response to environmental stress. Mutations in human Trib isoforms (Trib1, 2, and 3) have been associated with metabolic disease and linked to leukemia and the formation of solid tumors, including melanomas, hepatomas, and lung cancers. Drosophila Tribbles (Trbl) was the first identified member of this sub-family of pseudokinases and shares a conserved structure and similar functions to bind and direct the degradation of key mediators of cell growth and proliferation. Common Trib targets include Akt kinase (also known as protein kinase B), C/EBP (CAAT/enhancer binding protein) transcription factors, and Cdc25 phosphatases, leading to the notion that Trib family members stand athwart multiple pathways modulating their growth-promoting activities. Recent work using the Drosophila model has provided important insights into novel facets of conserved Tribbles functions in stem cell quiescence, tissue regeneration, metabolism connected to insulin signaling, and tumor formation linked to the Hippo signaling pathway. Here we highlight some of these recent studies and discuss their implications for understanding the complex roles Tribs play in cancers and disease pathologies

FijiWingsPolarity: An open source toolkit for semi-automated detection of cell polarity

<p>Epithelial cells are defined by apical-basal and planar cell polarity (PCP) signaling, the latter of which establishes an orthogonal plane of polarity in the epithelial sheet. PCP signaling is required for normal cell migration, differentiation, stem cell generation and tissue repair, and defects in PCP have been associated with developmental abnormalities, neuropathologies and cancers. While the molecular mechanism of PCP is incompletely understood, the deepest insights have come from <i>Drosophila</i>, where PCP is manifest in hairs and bristles across the adult cuticle and organization of the ommatidia in the eye. Fly wing cells are marked by actin-rich trichome structures produced at the distal edge of each cell in the developing wing epithelium and in a mature wing the trichomes orient collectively in the distal direction. Genetic screens have identified key PCP signaling pathway components that disrupt trichome orientation, which has been measured manually in a tedious and error prone process. Here we describe a set of image processing and pattern-recognition macros that can quantify trichome arrangements in micrographs and mark these directly by color, arrow or colored arrow to indicate trichome location, length and orientation. Nearest neighbor calculations are made to exploit local differences in orientation to better and more reliably detect and highlight local defects in trichome polarity. We demonstrate the use of these tools on trichomes in adult wing preps and on actin-rich developing trichomes in pupal wing epithelia stained with phalloidin. FijiWingsPolarity is freely available and will be of interest to a broad community of fly geneticists studying the effect of gene function on PCP.</p

<i>Drosophila</i> Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

<div><p><i>Drosophila</i> Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that <i>Drosophila</i> Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation.</p></div

Novel functions of Ecdysone receptor B1 isoform during Drosophila oogenesis

In Drosophila, ecdysone signaling is mediated by a heteromeric receptor composed of the Ecdysone Receptor protein (EcR) and the RXR homolog transcription factor Ultraspiracle (USP). There are three different EcR isoforms: EcR-A, EcR-B1 and EcR-B2 that differ in their N termini and have different spatial\u2013temporal functions. Here we analized the effects of EcR-B1 or USP loss of function in the follicular epithelium during oogenesis. We find that targeting RNAi of EcR-B1 by using the ubiquitous Tubulin-Gal4 driver causes severe alteration in egg chamber development. Silencing of EcR-B1 isoform in flp-out clones causes apoptotic follicle cell death and affects follicular epithelium monolayer structure. Multilayered follicle cells are also detected knocking down EcR-B1 isoform by using enhancer trap lines that drive Gal4 expression at mid-oogenesis. We show that multilayered follicle cells lack proper cell polarity with altered distribution of apical and baso-lateral cell polarity markers (atypical-PKC, Armadillo, Discs-large and Scribble). Furthermore, these delaminating follicle cells show accumulation of adherens junctions (DE-Cadherin) and their F-actin cytoskeleton is strongly affected. Interestingly, we found that mosaic follicle cells homozigous for the usp3 mutation that interferes with USP repressor activity die by apoptosis at early stages of oogenesis and show only slightly increased Discs-large expression levels but not alteration in DE-cadherin expression levels. These data indicate that follicular epithelium morphogenesis and follicle cell survival require EcR-B1 and USP activities

Trbl binds Akt and suppresses Akt-mediated cuticle growth phenotypes.

<p>(A). The ability of yeast cells to grow on increasing concentrations of 3AT growth inhibitor depends on the strength of the protein-protein interaction. Yeast cells co-expressing Akt prey and WT Trbl bait are able to grow in presence of up to 50 mM 3AT whereas yeast cells co-expressing Akt and Trbl D/NLK bait are unable to grow in presence of 10 mM 3AT, similar to negative controls. (B–D). Akt misexpression in head capsule by the GMRGAL4 driver results in increased head size (C), which is effectively suppressed by Trbl co-misexpression (D). Genotypes: (B) GMRGAL4, (C) GMRGAL4>UAS-Akt1, (D) GMRGAL4>UAS-Akt1, UAS-Trbl. (E–G). Akt misexpression in the larval muscle using the MefGAL4 driver results in an increase in muscle size (cf. E,F) while Trbl effectively suppresses this (G). Genotypes: (E) Mef2GAL4, (F) Mef2GAL4>UAS-Akt1, (G) Mef2GAL4>UAS-Akt1, UAS-Trbl. (H–J). Age matched mid 3rd instar larval fat body cells from PplGAL4>UAS-Akt (I) are detectably larger that WT (H). This Akt-mediated increase in cell size is noticeably suppressed in fat body cells co-expressing Trbl and Akt (J). Tissues were stained with Phalloidin (red) to reveal cell boundary and DAPI (green) to reveal nucleus. Note that Fig 4B-D, E-G, H-J are taken under the same magnification, respectively. (K). Quantification of fat body cell size in pixels (n≈100 cells/genotype; for K and L, P values from two-tailed T test data are indicated (n.s. =  not significant; *P<0.05; **P<0.01; ***P<0.001) and are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109530#pone.0109530.s001" target="_blank">Table S1</a> and all error bars are ± S.D. (L). Quantification of fat body nuclear size in pixels (n≈100 nuclei/genotype).</p