A Genome-Wide siRNA Screen Identifies Novel Phospho-enzymes Affecting Wnt/β-Catenin Signaling in Mouse Embryonic Stem Cells

Groenendyk, Jody; Michalak, Marek

doi:10.1007/s12015-011-9265-3

Cited by 6 publications

(6 citation statements)

References 85 publications

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“…Tissue culture media included 10% fetal bovine serum (FBS) in Dulbecco’s Modified Eagle Medium (DMEM) (both from Sigma). The siRNA library (Ambion) was pooled and aliquoted into 96 well full skirt PCR plates (Axygen) (57). Reverse transfection was performed which involves preparation of a mixing plate containing the siRNA, plasmid DNA, Dharmafect Duo (ThermoFisher) and Optimem (Life Technologies), followed by addition of cells and plating into white 96 well plates (Corning) for 48 hours.…”

Section: Methodsmentioning

confidence: 99%

Interplay Between the Oxidoreductase PDIA6 and microRNA-322 Controls the Response to Disrupted Endoplasmic Reticulum Calcium Homeostasis

et al. 2014

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The disruption of the energy or nutrient balance triggers endoplasmic reticulum (ER) stress, a process that mobilizes various strategies, collectively called the unfolded protein response (UPR), which reestablish homeostasis of the ER and cell. Activation of the UPR stress sensor IRE1α (inositol-requiring enzyme 1α) stimulates its endoribonuclease activity, leading to the generation of the mRNA encoding the transcription factor XBP1 (X-box binding protein 1), which regulates the transcription of genes encoding factors involved in controlling the quality and folding of proteins. We found that the activity of IRE1α was regulated by the ER oxidoreductase PDIA6 (protein disulfide isomerase A6) and the microRNA miR-322 in response to disruption of ER Ca2+ homeostasis. PDIA6 interacted with IRE1α and enhanced IRE1α activity as monitored by phosphorylation of IRE1α and XBP1 mRNA splicing, but PDIA6 did not substantially affect the activity of other pathways that mediate responses to ER stress. ER Ca2+ depletion and activation of store operated Ca2+ entry reduced the abundance of the microRNA miR-322, which increased PDIA6 mRNA stability and consequently IRE1α activity during the ER stress response. In vivo experiments with mice and worms showed that the induction of ER stress correlated with decreased miR-322 abundance, increased PDIA6 mRNA abundance, or both. Together these findings demonstrated that ER Ca2+, PDIA6, IRE1α, and miR-322 function in a dynamic feedback loop modulating the UPR under conditions of disrupted ER Ca2+ homeostasis.

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Section: Methodsmentioning

confidence: 99%

Interplay Between the Oxidoreductase PDIA6 and microRNA-322 Controls the Response to Disrupted Endoplasmic Reticulum Calcium Homeostasis

et al. 2014

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“…These high-throughput lossand gain-of-function studies quantitated the response of exogenous Wnt pathway reporters under conditions of elevated signaling (Anton et al, 2011;Caspi and RosinArbesfeld, 2008;DasGupta et al, 2005;Firestein et al, 2008;Groenendyk and Michalak, 2011;Jacob et al, 2011;James et al, 2009;Kategaya et al, 2009;Major et al, 2008;Miller et al, 2009;Port et al, 2011;Tang et al, 2008). In this study, we have performed a comprehensive genome-wide in vivo RNAi screen for kinases and phosphatases in Drosophila to build a phospho-regulatory network of the Wnt pathway.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide identification of phospho-regulators of Wnt signaling inDrosophila

2015

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Evolutionarily conserved intercellular signaling pathways regulate embryonic development and adult tissue homeostasis in metazoans. The precise control of the state and amplitude of signaling pathways is achieved in part through the kinase-and phosphatase-mediated reversible phosphorylation of proteins. In this study, we performed a genome-wide in vivo RNAi screen for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. Our analyses have identified 54 highconfidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway, and numerous phosphoregulators were identified. Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. We identified 29 dual regulators that have the same effect on the Wnt and Hedgehog pathways. As proof of principle, we established that Cdc37 and Gilgamesh/CK1γ inhibit and promote signaling, respectively, by functioning at analogous levels of these pathways in both Drosophila and mammalian cells. The Wnt and Hedgehog pathways function in tandem in multiple developmental contexts, and the identification of several shared phospho-regulators serve as potential nodes of control under conditions of aberrant signaling and disease.

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“…AAK1 can also interact with a membrane-tethered activated form of the Notch receptor and promote its internalization (Gupta-Rossi et al, 2011). AAK1 knock-down upregulated Wnt signalling in a mouse embryonic stem cell-based kinase and phosphatase siRNA screen (Groenendyk and Michalak, 2011). A recent study in human cells confirmed this observation and offered a mechanism.…”

Section: Introductionmentioning

confidence: 86%

Caenorhabditis elegansSEL-5/AAK1 regulates cell migration and cell outgrowth independently of its kinase activity

Knop

Zounarová

Macůrková

2023

Preprint

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During Caenorhabditis elegans development multiple cells migrate long distances or extend processes to reach their final position and/or attain proper shape. Wnt signalling pathway stands out as one of the major coordinators of cell migration or cell outgrowth along the anterior-posterior body axis. Wnt signalling outcome is fine-tuned by various mechanisms including endocytosis. In this study we show that SEL-5, the C. elegans orthologue of mammalian AP2-associated kinase AAK1, acts together with the retromer complex as a positive regulator of EGL-20/Wnt signalling during the migration of QL neuroblast daughter cells. At the same time, SEL-5 in cooperation with the retromer complex is also required during excretory canal cell outgrowth in a Wnt-independent manner. Importantly, SEL-5 kinase activity is not required for its role in either neuronal migration or excretory cell outgrowth and neither of these processes is dependent on DPY-23/AP2M1 phosphorylation. While Wnt proteins do not significantly contribute to the excretory canal guidance, LIN-44/Wnt and LIN-17/Frizzled together generate a stop signal inhibiting further canal outgrowth.

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A Genome-Wide siRNA Screen Identifies Novel Phospho-enzymes Affecting Wnt/β-Catenin Signaling in Mouse Embryonic Stem Cells

Cited by 6 publications

References 85 publications

Interplay Between the Oxidoreductase PDIA6 and microRNA-322 Controls the Response to Disrupted Endoplasmic Reticulum Calcium Homeostasis

Interplay Between the Oxidoreductase PDIA6 and microRNA-322 Controls the Response to Disrupted Endoplasmic Reticulum Calcium Homeostasis

Genome-wide identification of phospho-regulators of Wnt signaling inDrosophila

Caenorhabditis elegansSEL-5/AAK1 regulates cell migration and cell outgrowth independently of its kinase activity

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