¹Department of Molecular Biology and Biochemistry, Waksman Institute, Howard Hughes Medical Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA

Correspondence: Dr KD Irvine, Department of Molecular Biology and Biochemistry, Waksman Institute, Rutgers The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 8854, USA. E-mail: irvine@waksman.rutgers.edu

Received 4 November 2008; Revised 20 January 2009; Accepted 24 February 2009; Published online 30 March 2009.

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.