Identification and Characterization of Novel Upstream Regulators of the Hippo Signalling Pathway

The evolutionarily conserved Hippo signalling pathway is a major determinant of growth control and organ size. The co-transcription factors YAP/TAZ are downstream effectors of the Hippo pathway that regulate vital processes like cell proliferation, cell migration, apoptosis, stem cell self-renewal and differentiation. YAP/TAZ are regulated by the core Ser/Thr kinases of the Hippo pathway MST1/2 and LATS1/2. Upon activation, MST1/2 in association with the adaptor protein SAV, phosphorylate and activate the downstream kinases LATS1/2. In turn, activated LATS1/2 in association with the adaptor protein MOB, phosphorylate YAP/TAZ leading to cytoplasmic accumulation, degradation and transcriptional inactivation of YAP/TAZ. The Hippo pathway can be regulated by various intrinsic and extrinsic stimuli such as mechanotransduction, actin cytoskeleton dynamics, cell-cell contact and cell polarity, G-protein coupled receptor signalling and metabolic pathways. However, the mechanistic details and molecular mediators that connect the upstream stimuli to the core of the pathway are not fully understood. With the aim of identifying novel upstream regulators of the Hippo pathway we undertook a high-throughput LUMIER-based protein interaction screen complemented with a TEAD-luciferase reporter screen that led to the identification of betaPIX and MARKs as positive and negative regulators of the Hippo pathway, respectively. Mechanistically, betaPIX mediates the interaction between YAP/TAZ and the Hippo core kinase LATS to promote YAP/TAZ phosphorylation and transcriptional inhibition. Conversely, MARK4 enhances YAP/TAZ transcriptional activity in a kinase-dependent manner by phosphorylating MST and SAV, and disrupting complex formation with the downstream LATS kinase. Furthermore, abrogation of MARK4 or betaPIX overexpression in breast cancer cells attenuates tumorigenic features such as proliferation and migration by activating the Hippo kinase cascade. Finally, betaPIX and MARK binding partners GIT1 and DLG5 are respectively characterized as positive and negative regulators of YAP/TAZ transcriptional activity.