Functions And Mechanisms Of AMOT Family Proteins In The Hippo Pathway

A relatively constant organ size is one of the most striking yet mysterious features of multicellular organisms,which was recently found regulated by the Hippo pathway.Mechanistically,the Hippo pathway kinases large tumor suppressor kinase 1/2(LATS1/2)can phosphorylate transcription co-activators Yes-associated protein(YAP)/transcriptional coactivator with a PDZ-binding domain(TAZ)and then promote YAP/TAZ cytoplasmic retention and degradation.However,YAP/TAZ-independent functions of the Hippo pathway were poorly studied.By screening potential LATS 1/2 substrates with HXRXXS motifs,we found angiomotin(AMOT)family proteins are new substrates of LATS 1/2 kinases.AMOTp130 is phosphorylated in a conserved HXRXXS motif by LATS 1/2 downstream of GPCR signaling.Phosphorylation disrupts AMOTp130 interaction with F-actin and correlates with reduced stress fibers and focal adhesions.Furthermore,phosphorylation of AMOT by LATS 1/2 inhibits endothelial cell migration in vitro and angiogenesis in zebrafish embryos in vivo.Thus AMOTp130 is a direct substrate of LATS 1/2 mediating functions of the Hippo pathway in endothelial cell migration and angiogenesis.On the other hand,AMOT family proteins also play a regulatory role in the Hippo signaling pathway.YAP as a key effector of the Hippo pathway can facilitate cell proliferation and evasion from apoptosis.However,dysregulation of the activity of YAP plays broad roles in human cancer development from tumorigenesis to cancer relapse.Therefore,the amplitude and duration of YAP activation should be precisely controlled to achieve proper development and tissue homeostasis.We found that YAP activation strongly activates its upstream inhibitory kinases LATS 1/2 via directly inducing transcription of LATS2 and angiomotin-like 2(AMOTL2)and possibly further via increasing the protein level of neurofibromin 2(NF2),a Hippo pathway upstream component.This negative feedback regulation exerts important function in the normalization of Hippo pathway activity upon loss of cell-cell junctions or serum stimulation.In YAP induced tumors and Mst1/2 knockout mice livers,YAP also activates the negative feedback regulation.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.In summary,our study demonstrates that AMOT family proteins involved in the Hippo pathway at different levels.On one hand,AMOTp130 is a direct substrate of LATS1/2 kinases in the Hippo pathway mediating regulation of angiogenesis.On the other hand,AMOTL2 is a target gene of YAP which limits YAP activity by a negative feedback mechanism.Our study expands functional and regulatory roles of AMOT family proteins in the Hippo pathway in physiological and pathological contexts.