Rho differentially regulates the Hippo pathway by modulating the interaction between Amot and Nf2 in the blastocyst

The Hippo pathway modulates the transcriptional activity of Yap to regulate the differentiation of the inner cell mass (ICM) and the trophectoderm (TE) in blastocysts. Yet, how Hippo signaling is differentially regulated in ICM and TE cells is poorly understood. Through an inhibitor/activator screen, we identified Rho as a negative regulator of Hippo in TE cells, and PKA as a positive regulator of Hippo in ICM cells. We further elucidated a novel mechanism for Rho to suppress Hippo, distinct to the prevailing view that Rho inhibits Hippo signaling through modulating cytoskeleton remodeling and/or cell polarity. Active Rho prevents the phosphorylation of Amot Ser176, thus stabilizing the interaction between Amot and F-actin, and restricting the binding between Amot and Nf2. Moreover, Rho attenuates the interaction between Amot and Nf2 by binding to the coiled-coil domain of Amot. Through blocking the association of Nf2 and Amot, Rho suppresses Hippo in TE cells.