A cell-based assay to screen stimulators of the Hippo pathway reveals the inhibitory effect of dobutamine on the YAP-dependent gene transcription

Abstract

The mammalian Hippo pathway is composed of mammalian Ste20-like (MST) kinases and large tumour suppressor (LATS) kinases. Upon the activation of the pathway, MST kinases phosphorylate and activate LATS kinases, which in turn phosphorylate transcriptional co-activators, yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), recruit them to the cytosol from the nucleus and turn off cell cycle-promoting and anti-apoptotic gene transcriptions. Thus, the pathway restricts cell overgrowth and prevents tumourigenesis. Although a high cell density and stress signallings are known to activate the pathway, no specific stimulators are so far reported. As the dysfunction of the pathway is frequent in human cancers and correlates with poor prognosis, it is important to find out reagents that stimulate the pathway for not only basic research but also clinical medicine. We here developed a cell-based method of screening reagents that induce the recruitment of YAP to the cytosol. Using this method, we found that dobutamine inhibits the YAP-dependent gene transcription. Contrary to our expectations, the effect of dobutamine is independent of the Hippo pathway but our method opens the possibility to discover Hippo pathway stimulators or Hippo-independent YAP inhibitors.

The Hippo pathway: regulators and regulations

“…In the latter scenario, glucagon, epinephrine, and a dopamine receptor agonist induce YAP/TAZ phosphorylation. Interestingly, in a screen for YAP inhibitors, dobutamine has been shown to inhibit YAP (Bao et al 2011). Dobutamine is an agonist for the b1 adrenergic receptor, which likely inhibits YAP by activating Ga s .…”

Screening with a Novel Cell-Based Assay for TAZ Activators Identifies a Compound That Enhances Myogenesis in C2C12 Cells and Facilitates Muscle Repair in a Muscle Injury Model

“…The rat anti-YAP monoclonal antibody used was described previously (43). The following antibodies and reagents were obtained from commercial sources.…”

“…The TEAD reporter assay was performed with HEK293 cells as described previously (43). C2C12 cells were plated at 1 ϫ 10 5 /well in 12-well plates and cultured overnight.…”

Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ)

“…However, the role of the classical Hippo cascade and its downstream effectors YAP and TAZ remained unclear in this study. H 2 O 2 -induced activation of the canonical Hippo cascade could promote phosphorylation and nuclear localization of the downstream effectors, which has been demonstrated recently for YAP (37). This inactivation of YAP would promote cell death, and consistently it has been shown that both decreased Lats1 activity (31) as well as increased YAP activity could protect cardiomyocytes against H 2 O 2 -induced cell death (38).…”