The Hippo pathway plays a central role in tissue homoeostasis, and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include a kinase cascade of MST1/2 and LATS1/2 and the transcription co-activators YAP/TAZ. In response to stimulation, LATS1/2 phosphorylate and inhibit YAP/TAZ, the main effectors of the Hippo pathway. Accumulating evidence suggests that MST1/2 are not required for the regulation of YAP/TAZ. Here we show that deletion of LATS1/2 but not MST1/2 abolishes YAP/TAZ phosphorylation. We have identified MAP4K family members—Drosophila Happyhour homologues MAP4K1/2/3 and Misshapen homologues MAP4K4/6/7—as direct LATS1/2-activating kinases. Combined deletion of MAP4Ks and MST1/2, but neither alone, suppresses phosphorylation of LATS1/2 and YAP/TAZ in response to a wide range of signals. Our results demonstrate that MAP4Ks act in parallel to and are partially redundant with MST1/2 in the regulation of LATS1/2 and YAP/TAZ, and establish MAP4Ks as components of the expanded Hippo pathway.

Mammalian cells are surrounded by neighbouring cells and extracellular matrix (ECM), which provide cells with structural support and mechanical cues that influence diverse biological processes. The Hippo pathway effectors YAP (also known as YAP1) and TAZ (also known as WWTR1) are regulated by mechanical cues and mediate cellular responses to ECM stiffness. Here we identified the Ras-related GTPase RAP2 as a key intracellular signal transducer that relays ECM rigidity signals to control mechanosensitive cellular activities through YAP and TAZ. RAP2 is activated by low ECM stiffness, and deletion of RAP2 blocks the regulation of YAP and TAZ by stiffness signals and promotes aberrant cell growth. Mechanistically, matrix stiffness acts through phospholipase Cγ1 (PLCγ1) to influence levels of phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, which activates RAP2 through PDZGEF1 and PDZGEF2 (also known as RAPGEF2 and RAPGEF6). At low stiffness, active RAP2 binds to and stimulates MAP4K4, MAP4K6, MAP4K7 and ARHGAP29, resulting in activation of LATS1 and LATS2 and inhibition of YAP and TAZ. RAP2, YAP and TAZ have pivotal roles in mechanoregulated transcription, as deletion of YAP and TAZ abolishes the ECM stiffness-responsive transcriptome. Our findings show that RAP2 is a molecular switch in mechanotransduction, thereby defining a mechanosignalling pathway from ECM stiffness to the nucleus.

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