Abstract 3108: Functional delineation of structural domains in Yorkie/Yes-associated protein

Abstract

Abstract The Salvador/Warts/Hippo (SWH) pathway plays an important role in organ size control, and deregulation of the pathway can lead to organ overgrowth and human cancer as well. The SWH pathway regulates tissue growth through inhibiting the activity of the downstream regulator, Yorkie (Yki) in D. melanogaster and Yes-associated protein (YAP) in mammals, which act as transcriptional co-activators. Yki and YAP have a conserved N-terminal domain, through which they associate and cooperate with the transcription factors, Scalloped (Sd)/TEAD, to promote tissue growth. In addition, Yki and YAP possess two conserved WW domains that mediate interaction between several proteins that either promote or inhibit YAP's ability to drive transcription. Furthermore, the C-terminus of YAP includes three further motifs: a SH3 domain-binding motif, transcriptional activation domain and a PDZ domain-binding motif, none of which are conserved in Yki. To study the role of the Yki/YAP WW domains and the YAP C-terminus, several Yki/YAP mutants were constructed and their function was analyzed with assays for tissue growth in Drosophila, or with assays for colony growth, cell transformation, migration and invasion in MCF10A or NIH-3T3 cells. Results showed that: 1. WW domain mutant YAP promoted transformation, migration and invasive ability of MCF10A cells, suggesting that WW domains impart inhibitory regulation of YAP in these cells. In contrast, the YAP WW domains were essential to promote NIH-3T3 cell growth and transformation, and for the capability of Yki to drive tissue growth in D. melanogaster. These results also suggested that YAP WW domains are required for tissue growth but inhibit invasion. 2. Deletion of YAP's transcriptional activation domain abolished its ability to enhance transcriptional activity of TEAD2, as well as YAP's ability to stimulate invasion of MCF10A cells. However, this mutant promoted growth and transformation of both MCF10A and NIH-3T3 cells. These results suggest that the transcriptional activation domain is required for YAP-mediated invasion but inhibits its ability to stimulate colony growth and cell transformation. 3. Mutation of YAP's PDZ domain-binding motif lowered its ability to activate TEAD2 and promote invasive behavior of MCF10A cells. In contrast, PDZ binding motif-deleted YAP promoted 3D growth of MCF10A cells and transformation in both MCF10A and NIH-3T3 cells. These results suggest that YAP's PDZ domain-binding motif is required for YAP-mediated invasion but has an inhibitory effect on growth and transformation. Taken together, these results suggest that YAP is able to mediate tissue growth, transformation and invasion through different domains and the total effect of YAP on growth and invasion is dependent on the coordination of protein interactions via its WW and transcriptional activation domains, as well as its PDZ domain-binding motif. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3108.