In vivo regulation of Yorkie phosphorylation and localization.
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.
Oh
Hyangyee
H
Howard Hughes Medical Institute, Waksman Institute, Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA.
Irvine
Kenneth D
KD
D048948:14-3-3 Proteins / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D017344:Genes, Insect; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000091102:YAP-Signaling Proteins; D000099317:Hippo Kinases
D048948:14-3-3 Proteins; D015815:Cell Adhesion Molecules; D029721:Drosophila Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D015534:Trans-Activators; D000091102:YAP-Signaling Proteins; C503814:Yki protein, Drosophila; C472307:ft protein, Drosophila; C093503:wts protein, Drosophila; C499474:hpo protein, Drosophila; D000099317:Hippo Kinases