Investigating the Yorkie Interactome and Novel Regulatory Mechanisms in the Hippo Pathway
The Hippo signaling pathway is highly conserved from the fruit fly Drosophila melanogaster to vertebrates. It plays an important role in regulating organ growth by inhibiting the transcription coactivator Yorkie (Yki, YAP/TAZ in mammals). Dysregulation of Hippo signaling leads to abnormal cell proliferation and tissue growth, and is implicated in human cancers. While previous studies have identified a number of proteins that interact with Yki, there is no systematic analysis of Yki protein interactome in vivo. Moreover, there is limited understanding of how the interactome is altered in Yki mutants. Using affinity purification mass spectrometry (AP-MS) in Drosophila S2 cells and embryos, we characterized the Yki protein interactome and identified both previously characterized core Yki interactors and putative novel Yki interacting proteins. To investigate the mechanisms by which these novel interactors regulate Yki function, we performed RNA interference studies and evaluated the effects of knocking down these interactors on adult fly eye overgrowth caused by overactivated Yki. Interestingly, we found that knockdown of RpII215 and CG2186 strongly suppressed the Yki overgrowth phenotype, while knockdown of Spenito (nito) enhanced the overgrowth. Further studies are needed to determine the underlying mechanism. Since these three protein interactors are nuclear proteins, this research may reveal new regulatory mechanisms of Yki nuclear activity. To identify the differences in protein binding profiles of various Yki mutants, we generated gain of function (S168A) and loss of function (P88L and WW domain) Yki mutants, to be used in subsequent experiments. Overall, our data have identified putative novel Yki interactors that may play a role in controlling nuclear Yki activity. We expect that future studies of their physical and genetic interactions will elucidate novel regulatory mechanisms of Yki function in Hippo signaling.