Abstract
Yorkie (Yki), a transcription factor of the Fat and Hippo signaling pathways, is negatively regulated by the Warts kinase. Here, we use Phos-tag gels to characterize Warts-dependent phosphorylation of Yki in vivo, and show that Warts promotes phosphorylation of Yki at multiple sites. We also show that Warts inhibits Yki nuclear localization in vivo, and can promote binding of Yki to 14-3-3 proteins in cultured cells. In vivo assessment of the influence of individual upstream regulators of Warts reveals that some mutants (e.g. fat) have only partial effects on Yki phosphorylation, and weak effects on Yki localization, whereas other genotypes (e.g. ex fat double mutants) have stronger effects on both Yki phosphorylation and localization. We also identify serine 168 as a critical site through which negative regulation of Yki by Warts-mediated phosphorylation occurs, but find that this site is not sufficient to explain effects of Hippo signaling on Yki in vivo. These results identify modulation of subcellular localization as a mechanism of Yki regulation, and establish that this regulation occurs in vivo through multiple sites of Warts-dependent phosphorylation on Yki.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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14-3-3 Proteins / metabolism
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Amino Acid Substitution
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Animals
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Animals, Genetically Modified
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Binding Sites
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Cell Adhesion Molecules / genetics
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Cell Adhesion Molecules / metabolism
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Cell Line
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Cell Nucleus / metabolism
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Drosophila / genetics
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Drosophila / metabolism*
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Drosophila Proteins / chemistry
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Drosophila Proteins / genetics
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Drosophila Proteins / metabolism*
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Female
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Genes, Insect
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism
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Male
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Mutagenesis, Site-Directed
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Mutation
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Phosphorylation
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Protein Binding
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Protein Kinases / genetics
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Protein Kinases / metabolism
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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Serine / chemistry
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Signal Transduction
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Trans-Activators / chemistry
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Trans-Activators / genetics
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Trans-Activators / metabolism*
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YAP-Signaling Proteins
Substances
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14-3-3 Proteins
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Cell Adhesion Molecules
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Drosophila Proteins
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Intracellular Signaling Peptides and Proteins
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Nuclear Proteins
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Trans-Activators
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YAP-Signaling Proteins
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Yki protein, Drosophila
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ft protein, Drosophila
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Serine
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Protein Kinases
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wts protein, Drosophila
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Protein Serine-Threonine Kinases
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hpo protein, Drosophila