The Hippo-YAP pathway is an evolutionally conserved signaling module that controls tissue growth during development and its dysregulation causes cancer [1]. Core components of the Hippo pathway include a kinase cascade comprising MST1/2 and LATS1/2 kinases, in which MST1/2 phosphorylates and activates LATS1/2 [2]. The major downstream effectors of the Hippo pathway are the transcriptional co-activators YAP and TAZ, which are phosphorylated and inhibited by LATS1/2 [3, 4]. Unphosphorylated YAP/TAZ localizes in the nucleus and promotes target gene expression through binding to the TEAD family transcription factors [5, 6]. Protein kinase C (PKC) controls a broad range of biological processes and can be classified into three sub-groups based on sequence homology and activation mechanisms: the Ca2+-and diacylglycerol (DAG)-dependent conventional PKC (cPKC α, βI, βII, and γ), the DAG-dependent novel PKC (nPKC δ, θ, ε, and η), and the Ca2+-and DAG-independent atypical PKC (aPKC ι and ζ)[7]. Recent studies have established that extracellular diffusible signals act through G-protein coupled receptors (GPCRs) to regulate the Hippo-YAP pathway [8, 9]. PKC represents one of the major effectors downstream of GPCRs (especially Gq/11-coupled receptors). This led us to investigate whether PKC regulates the Hippo-YAP pathway.

We used the DAG analog TPA to activate PKC in HEK293A cells and observed that TPA induced a rapid and robust YAP dephosphorylation as determined by western blotting using the phospho-specific (serine 127) YAP antibody and by differential electrophoretic mobility shift on phos-tag-containing gels. Similarly, TPA also induced TAZ dephosphorylation as indicated by a faster electrophoretic migration (Figure 1A). TPA-induced YAP dephosphorylation was also observed in HeLa and U251MG cells (Supplementary information, Figure S1A and S1B). Phosphorylated YAP localizes in the cytoplasm, whereas dephosphorylated YAP translocates to the nucleus to promote target gene expression. Consistent with this model, TPA induced YAP nuclear localization in HEK293A cells (Figure 1B). Moreover, GO6983, an inhibitor of both