ABSTRACT
The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P3), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P2 and PI(3,4,5)P3 attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.
Subject(s)
Adaptor Proteins, Signal Transducing , Breast Neoplasms , Signal Transduction , Trans-Activators , Transcription Factors , YAP-Signaling Proteins , Humans , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Transcription Factors/metabolism , Transcription Factors/genetics , YAP-Signaling Proteins/metabolism , YAP-Signaling Proteins/genetics , Female , Trans-Activators/metabolism , Trans-Activators/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Phosphoproteins/metabolism , Phosphoproteins/genetics , Transcriptional Coactivator with PDZ-Binding Motif Proteins/metabolism , Cell Line, Tumor , Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositols/metabolism , Gene Expression Regulation, Neoplastic , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cell Nucleus/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/geneticsABSTRACT
Phosphatidylserine (PS) is a key lipid that plays important roles in disease-related biological processes, and therefore, the means to track PS in live cells are invaluable. Herein, we describe the metabolic labeling of PS in Saccharomyces cerevisiae cells using analogues of serine, a PS precursor, derivatized with azide moieties at either the amino (N-l-SerN3) or carbonyl (C-l-SerN3) groups. The conservative click tag modification enabled these compounds to infiltrate normal lipid biosynthetic pathways, thereby producing tagged PS molecules as supported by mass spectrometry studies, thin-layer chromatography (TLC) analysis, and further derivatization with fluorescent reporters via click chemistry to enable imaging in yeast cells. This approach shows strong prospects for elucidating the complex biosynthetic and trafficking pathways involving PS.
Subject(s)
Phosphatidylserines , Saccharomyces cerevisiae , Phosphatidylserines/metabolism , Saccharomyces cerevisiae/metabolism , Click ChemistryABSTRACT
Metabolic labeling, in which substrate analogs containing diminutive tags can infiltrate biosynthetic pathways and generate labeled products in cells, has led to dramatic advancements in the means by which complex biomolecules can be detected and biological processes can be elucidated. Within this realm, metabolic labeling of lipid products, particularly in a manner that is headgroup-specific, brings about a number of technical challenges including the complexity of lipid metabolic pathways as well as the simplicity of biosynthetic precursors to headgroup functionality. As such, only a handful of strategies for metabolic labeling of lipids have thus far been reported. However, these approaches provide enticing examples of how strategic modifications to substrate structures, particularly by introducing clickable moieties, can enable the hijacking of lipid biosynthesis. Furthermore, early work in this field has led to an explosion in diverse applications by which these techniques have been exploited to answer key biological questions or detect and track various lipid-containing biological entities. In this article, we review these efforts and emphasize recent advancements in the development and application of lipid metabolic labeling strategies.
Subject(s)
Glycerophospholipids/chemistry , Glycerophospholipids/metabolism , Staining and Labeling/methods , Click Chemistry , HumansABSTRACT
Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.