We herein describe a PITP/-PI4KIII-MAP4K signaling axis that modulates the Hippo pathway in response to plasma membrane PI4P levels. Our study highlights a novel role for PITP/ in regulating the Hippo kinase cascade through the identification of VT01454, a small-molecule inhibitor and Microcolin B analog that induces YAP phosphorylation. We characterize VT01454 as both a potent and specific YAP inhibitor, use bio-orthogonal methods to identify PITP/ as the unequivocal targets of VT01454, and functionally validate the role of PITP/ in Hippo signaling by genetic deletion in HEK293A cells. Finally, we demonstrate that VT01454 inhibition of PITP/ regulates the Hippo pathway by modulation of a plasma membrane pool of PI4P, and that MAP4K6/7 are potential PI4P-interacting proteins.

The Hippo pathway is a master regulator of tissue growth and homeostasis. As such, the Hippo pathway plays important roles in processes such as cell proliferation, growth, differentiation, and survival. In the past few decades, a slew of studies has characterized a network of proteins that crosstalk with the Hippo pathway, the signals regulating the Hippo pathway, and the biological outputs of the pathway in development, tissue regeneration, and cancer. The transcriptional components of the Hippo pathway consist of transcription co-activators YAP and TAZ as well as transcription factor family TEAD. Past studies of the Hippo pathway largely focus on regulation of the Hippo transcription co-activators YAP and TAZ and studies characterizing mechanisms of TEAD regulation are largely lacking. Thus, studies uncovering new mechanisms of TEAD regulation will provide insight into Hippo signaling and Hippo related pathogeneses.

We demonstrate that upon environmental stresses, such as osmotic stress and high cell density, TEAD localization can be dynamically regulated. Upon osmotic stress, TEAD is translocated to the cytoplasm through direct binding with p38 through its D domain. Importantly, this cytoplasmic translocation of TEAD can override YAP/TAZ activating signals and inhibit Hippo signaling output. Furthermore, in YAP/TAZ driven cancer cell lines, TEAD cytoplasmic translocation can inhibit cell growth in vitro and in vivo.

TEAD cytoplasmic translocation is also seen upon tissue repair and in response to extracellular matrix (ECM) composition. High cell density promoted TEAD cytoplasmic localization can be reversed by inducing a wound in the cell monolayer. This cytoplasmic-nuclear translocation is also seen in an intestinal injury model. Furthermore, TEAD nuclear accumulation occurs in response to composition of ECM proteins, namely laminin and fibronectin. TEAD nuclear localization in response to fibronectin is mediated through FAK/Src signaling. Here we show several Hippo independent mechanisms of TEAD regulation and provide evidence that regulation of TEAD is an alternative mechanism of regulating Hippo signaling output.