Thursday, April 3, 2025
4/3/2025
Regulation of Innate Immunity by the Hippo Kinases - Project Summary The Hippo pathway is conserved from yeasts to humans. In mammals, this signaling pathway controls tissue development by balancing cell proliferation and death. The Hippo kinases, MST1 and MST2, transduce signals via phosphorylation of the scaffold protein MOB1 and the downstream kinase LAST1. Animal models show that tissue-specific Mst1/2 gene knockouts lead to abnormal organ development and tumor formation. In addition to its well-known functions in development and cancer, recent studies found the Hippo pathway is targeted and manipulated by bacterial pathogens, suggesting a role of this conserved host pathway in bacterial pathogenesis. Notably, individuals with loss-of-function mutations in the MST1 gene are immunocompromised. Consistent with this observation in humans, our lab knocked out both Mst1 and Mst2 genes in macrophage and found that these macrophages are highly susceptible to infection by the human pathogen Legionella pneumophila, highlighting an emerging role of the Hippo kinases in immunity against infection. Using the Mst1/2 double knockout cell model, we further discovered that MST1/2 are the key regulators for immune gene expression and programmed cell death during infection. Importantly, upon challenge of virulent L. pneumophila, MST1/2 full-length proteins are proteolytically cleaved, resulting in MST1/2 fragments (MST1/2-NT) that contain the kinase domains but are separated from the regulatory domains. In biochemical assays, MST1/2-NT, surprisingly, no longer phosphorylate their cognate substrate, MOB1, but exhibit altered substrate specificity for protein phosphorylation. In cell-based assays, MST1/2-NT production contributes to activation of apoptotic cell death that is critical for restricting virulent L. pneumophila. Based on the preliminary data collected in our lab, we propose a model that, upon infection, macrophages trigger cleavage of MST1/2 to activate an inflammatory Hippo signaling mediated by the proapoptotic MST1/2-NT as a defense mechanism. The objective of the proposed study is to dissect the importance of this novel inflammatory Hippo signaling. In Aim 1, we will use an innovative technique, Thiophosphorylation Proteome Screen (TPS), that allows us to screen more than 21,000 human proteins for direct substrates of full-length MST1/2 and MST1/2-NT. We will use the established substrate validation methods which include in vitro and cell-based assays to validate genuine substrates of these kinases. This aim will yield a comprehensive phosphoproteome controlled by the Hippo kinases that is fundamental for understanding both the canonical and inflammatory Hippo signaling. In Aim 2, we will focus on how MST1/2 influence gene expression and communication in immune cells. Specifically, we discovered that expression of the complement protein C1q is depleted in Mst1/2 double knockout macrophages. We will use genetic and cell-based assays to elucidate the molecular mechanism by which MST1/2 regulate C1q, the complement protein with a crucial role in bacteria killing. This study will provide insights for boosting innate immunity against infection through modulating the activities of the Hippo kinases.
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