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Updated: Jun 24, 2026
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
In vivo analysis of Yorkie phosphorylation sites
1Department of Molecular Biology and Biochemistry, Waksman Institute, Howard Hughes Medical Institute, Rutgers The State University of New Jersey, Piscataway, NJ 8854, USA.
Abstract:
The co-activator Yorkie (Yki) mediates transcriptional regulation effected by the Drosophila Fat-Warts (Wts)-Hippo (Hpo) pathways. Yki is inhibited by Wts-mediated phosphorylation, and a Wts phosphorylation site at Ser168 has been identified. Here we identify two additional Wts phosphorylation sites on Yki, and examine the respective contribution of all three sites to Yki nuclear localization and activity. Our results show that although Ser168 is the most critical site, all three phosphorylation sites influence Yki localization and activity in vivo, and can be sites of regulation by Wts. Thus, investigations of the role of Yki and its mammalian homolog Yes-associated protein (YAP) in development and oncogenesis should include evaluations of additional sites. The WW domains of Yki are not required for its phosphorylation, but instead are positively required for its activity. We also identify two potential sites of phosphorylation by an unknown kinase, which could influence phosphorylation of Ser168 by Wts, suggesting that there are additional mechanisms for regulating Yki/YAP activity.
Insights
The Yorkie (Yki) protein
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The Hippo-Warts-Yorkie pathway regulates organ size and tissue homeostasis.
- Yorkie (Yki) is a transcriptional co-activator and key downstream effector of this pathway.
- Warts (Wts) kinase phosphorylates and inhibits Yki activity, with Ser168 identified as a key phosphorylation site.
Purpose of the Study:
- To identify and characterize additional Warts (Wts) phosphorylation sites on Yorkie (Yki).
- To investigate the functional contribution of all identified Wts phosphorylation sites to Yki nuclear localization and transcriptional activity.
- To explore the role of Yki's WW domains and potential regulation by other kinases.
Main Methods:
- Site-directed mutagenesis to alter identified phosphorylation sites.
- In vivo assays to assess Yki nuclear localization and transcriptional activity.
- Analysis of Yki WW domains and potential upstream regulatory kinases.
Main Results:
- Two novel Warts (Wts) phosphorylation sites on Yorkie (Yki) were identified, in addition to Ser168.
- All three Wts phosphorylation sites collectively influence Yki nuclear localization and activity in vivo.
- Yki's WW domains are essential for its activity but not for Wts-mediated phosphorylation; two potential novel kinase phosphorylation sites were found.
Conclusions:
- Multiple Warts (Wts) phosphorylation sites regulate Yorkie (Yki) activity and localization.
- Future studies on Yki and its mammalian homolog YAP in development and cancer should consider these additional regulatory sites.
- Novel regulatory mechanisms involving other kinases may further control Yki/YAP activity.
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