Opposing roles of conventional and novel PKC isoforms in Hippo-YAP pathway regulation

Abstract

LETTER TO THE EDITOR Cell Research (2015) 25:985-988. © 2015 IBCB, SIBS, CAS All rights reserved 1001-0602/15 $ 32.00 www.nature.com/cr npg Opposing roles of conventional and novel PKC isoforms in Hippo-YAP pathway regulation Cell Research (2015) 25:985-988. doi:10.1038/cr.2015.88; published online 24 July 2015 Dear Editor, The Hippo-YAP pathway is an evolutionally conserved signaling module that controls tissue growth during de- velopment and its dysregulation causes cancer [1]. Core components of the Hippo pathway include a kinase cas- cade 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]. Un- phosphorylated 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 Ca 2+ - and dia- cylglycerol (DAG)-dependent conventional PKC (cPKC α, βI, βII, and γ), the DAG-dependent novel PKC (nPKC δ, θ, e, and η), and the Ca 2+ - 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 recep- tors). This led us to investigate whether PKC regulates the Hippo-YAP pathway. We used the DAG analog TPA to activate PKC in HE- K293A cells and observed that TPA induced a rapid and robust YAP dephosphorylation as determined by western blotting using the phospho-specific (serine 127) YAP an- tibody and by differential electrophoretic mobility shift on phos-tag-containing gels. Similarly, TPA also induced TAZ dephosphorylation as indicated by a faster electro- phoretic migration (Figure 1A). TPA-induced YAP de- phosphorylation 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 pro- mote target gene expression. Consistent with this model, TPA induced YAP nuclear localization in HEK293A cells (Figure 1B). Moreover, GO6983, an inhibitor of both cPKC and nPKC, effectively blocked TPA-induced YAP/ TAZ dephosphorylation (Figure 1A). These observations suggest a role of PKC in YAP/TAZ activation. Next, we examined whether activation of PKC by physiological stimuli would activate YAP/TAZ. We found that addition of acetylcholine to U251MG cells, which express the Gq/11-coupled muscarinic acetylcholine re- ceptor M3, resulted in significant YAP dephosphorylation (Figure 1C). Inhibition of either M3 receptor by 4-DAMP or PKC by GO6983 suppressed acetylcholine-induced YAP dephosphorylation (Figure 1C), suggesting that YAP activation by acetylcholine is mediated by the M3 recep- tor and PKC. Moreover, Gq/11 knockdown in U251MG cells suppressed YAP dephosphorylation by acetylcho- line (Supplementary information, Figure S1C). Collec- tively, these data indicate that PKC acts downstream of Gq/11-coupled receptors to activate YAP. In the efforts to examine the effect of TPA on YAP/ TAZ phosphorylation in different cell types, we were surprised that TPA induced a dramatic YAP/TAZ phos- phorylation in Swiss3T3 cells (Figure 1D), a response completely opposite to what was observed in HEK293A and several other cell lines (Figure 1A and Supplementa- ry information, Figure S1A and S1B). Notably, TPA-in- duced YAP/TAZ phosphorylation could be blocked by GO6983 (Figure 1D), indicating that PKC activation was also responsible for the increase of YAP/TAZ phosphory- lation in Swiss3T3 cells. Similar effects were observed in MEF cells and the lung cancer A549 cells (Supplementary information, Figure S1D and S1E). Consistent with the increased YAP phosphorylation, TPA treatment promoted a YAP cytoplasmic localization in MEF cells (Figure 1E). Although seemingly paradoxical, the above results are very interesting and show that PKC can either positively or negatively regulate YAP activity in a cell-type-depen- dent manner. The opposing effects of TPA on YAP phosphorylation observed in different cell types prompted us to speculate that different PKC isoforms may exert opposite effects on YAP regulation. To test this, individual cPKC or nPKC isoform was co-transfected with YAP into HEK293A cells and TPA-induced YAP phosphorylation/dephosphoryla-