In vivo analysis of Yorkie phosphorylation sites.

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.