PROJECT SUMMARY Over the past decades, the Hippo pathway has been recognized as a crucial signaling pathway that controls organ and tissue size, by restricting cell proliferation and anti-apoptosis. The Hippo pathway can be regulated by a wide range of extracellular signaling, including perceived physical signals from cell microenvironment (i.e. contact inhibition, cell polarity, cytoskeleton-based mechanical cues), growth factors/mitogenic hormones (i.e. LPA and S1P regulated GPCR signaling), and recently discovered metabolic status (i.e. energy stress, hypoxia). Mechanistically, almost all these stimuli or conditions operate YAP activity through LATS1/2 kinases. The major knowledge gap for current Hippo signaling comes from the uncharacterized mechanism for LATS1/2 regulation. The overall objective of this proposal is to elucidate the regulatory mechanism for LATS1/2 kinases in response to Hippo upstream signaling events. The detailed regulatory mechanism for LATS1/2 kinases could be complex, since multiple upstream kinases including MST1/2, MAP4Ks, TAO1-3, are able to phosphorylate LATS1/2 and required for LATS1/2 activation. It is still unclear how these identified kinases are coordinated to transduce upstream signaling to activate LATS1/2. Besides, small Rho GTPase together with F-actin can sense upstream signaling to modulate LATS1/2 activities, however, the underlying mechanism is also largely unknown. Therefore, over the past few years, we have conducted a proteomic analysis of the major components and regulators in the Hippo pathway to pursue the answer to these questions. Unfortunately, our findings and findings from other labs failed identifying such a putative “mediator” to fill the current knowledge gap in the Hippo pathway. Unexpectedly and excitingly, our preliminary studies have discovered phosphatidic acid (PA) and it related lipid signaling as a critical signaling axis involved in the Hippo pathway regulation. PA could function as a second messenger to directly associate with LATS1/2 kinases and regulate LATS1/2 activities. Remarkably, PA’s level as well as the activity of PLD1, a key enzyme that catalyzes PA production, are both decreased in response to the Hippo- activating stimuli or conditions. On the basis of these observations, we hypothesized that PLD1-PA axis could play a crucial role in regulation of LATS1/2 activities. Specifically, we propose to 1) determine the role of PA in YAP regulation, particularly focusing on the independent role of PA from LPA in YAP activation;; 2) elucidate the mechanism by which PA activates YAP. We will dissect the role of PA in LATS1/2 suppression through its association with LATS1/2 and NF2;; and 3) investigate the role of PLD1-PA axis in LATS1/2 regulation in response to Hippo upstream signaling. Collectively, our proposed study will reveal a crucial regulatory mechanism to control LATS1/2 activation in response to the Hippo upstream signaling and conc...