A relatively constant organ size is one of the most striking yet mysterious features of multicellular organisms, which was recently found to be regulated by the Hippo pathway in an evolutionarily conserved manner. Inactivation of this pathway leads to drastic enlargement of organ size due to a marked increase of cell number1. Furthermore, deregulation of the Hippo pathway plays broad roles in human cancer development from tumorigenesis to cancer relapse2. Mechanistically, Yes-associated protein (YAP), a transcription co-activator and a potent growth promoter, is directly phosphorylated by the Hippo pathway kinases Lats1 and Lats2, and then inhibited through cytoplasmic retention and degradation. Chemical and physical signals such as GPCR ligands and matrix stiffness dampen the Hippo pathway, thus leading to YAP activation, which facilitates cell proliferation, evasion from apoptosis, and stem cell self-renewal3. Therefore, the amplitude and duration of YAP activation in response to physiological stimulations should be precisely controlled to achieve proper development and tissue homeostasis. Here we report that YAP activation strongly activates its upstream inhibitory kinases Lats1/2 partially via directly inducing transcription of Lats2 and AMOTL2 and possibly further via increasing the protein level of NF2, the Hippo pathway upstream component. Our data suggest that YAP activity is limited by a negative feedback mechanism, which serves to cancel signal noise during development and thus ensure tissue homeostasis.

By examining the expression of Hippo pathway proteins we found that Lats2 protein level was consistently elevated by YAP activation across cell lines while Lats1 and NF2 protein levels were elevated in a cell type-dependent manner (Figure 1A, 1C and Supplementary information, Figure S1A). In contrast, Mst1/2 and Sav protein levels remain similar between control and YAP-5SA-expression cells (Figure 1A). We further examined whether the elevation of Lats1/2 phosphorylation and Lats2 protein levels depends on YAP transcriptional activity. YAP serine 94 (S94) is essential for its interaction with TEADs, major transcription factors mediating YAP functions7. S94A mutation largely abolished YAP-5SA-induced increase in Lats1/2 phosphorylation and Lats2 protein levels (Figure 1C). Furthermore, deletion of the C-terminal YAP transcription activation domain also abolished YAP-5SA-induced Lats1/2 activation (Figure 1C). Therefore, YAP-induced negative feedback is dependent on YAP-TEAD transcriptional activity. We next asked whether the elevation of Lats2 and sometimes Lats1 and NF2 protein levels induced by YAP activation is due to their enhanced transcription. We found that Lats2 but not Lats1 or NF2 transcription was induced by YAP activation to different extents across cell lines (Figure 1D and Supplementary information, Figure S1B). In contrast, expression of active YAP could not block Lats1/2 degradation upon inhibition of protein synthesis, although at an intermediate time point Lats2 degradation seems to be slower possibly due to a much higher basal Lats2 protein level (Supplementary information, Figure S1C). Thus alteration of Lats2 stability is unlikely to be the major reason for elevated Lats2 protein level induced by YAP activation. Furthermore, chromatin immunoprecipitation (ChIP) experiments showed that TEAD1 and YAP directly bind to the proximal region of the Lats2 promoter, where potential TEAD-binding elements could be spotted, but not to other regions of the Lats2 promoter or Lats1 and NF2 promoters (Figure 1E). These findings demonstrate that YAP directly induces Lats2 transcription through TEADs, which partially contributes to a feedback activation of the Hippo pathway. The observed alteration of Lats1 protein levels (Figure 1A) could be partially due to cross-recognition of Lats2 by Lats1 antibody as could be seen in Figure 1B.

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