Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides.

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.

Rab GTPases and phosphoinositides fine-tune SNAREs dependent targeting specificity of intracellular vesicle traffic.

In the secretory pathway the destination of trafficking vesicles is determined by specific proteins that, with the notable exception of SNAREs, are recruited from soluble pools. Previously we have shown that microinjected proteoliposomes containing early or late endosomal SNAREs, respectively, are targeted to the corresponding endogenous compartments, with targeting specificity being dependent on the recruitment of tethering factors by some of the SNAREs. Here, we show that targeting of SNARE-containing liposomes is refined upon inclusion of polyphosphoinositides and Rab5. Intriguingly, targeting specificity is dependent on the concentration of PtdIns(3)P, and on the recruitment of PtdIns(3)P binding proteins such as rabenosyn-5 and PIKfyve, with conversion of PtdIns(3)P into PtdIns(3,5)P2 re-routing the liposomes towards late endosomes despite the presence of GTP-Rab5 and early endosomal SNAREs. Our data reveal a complex interplay between permissive and inhibitory targeting signals that sharpen a basic targeting and fusion machinery for conveying selectivity in intracellular membrane traffic.

Inositol-Exchange Activity in Human Primordial Placenta.

Human placenta is an intensively growing tissue. Phosphatidylinositol (PI) and its derivatives are part of the signaling pathway in the regulation of trophoblast cell differentiation. There are two different enzymes that take part in the direct PI synthesis: phosphatidylinositol synthase (PIS) and inositol exchange enzyme (IE). The presence of PIS is known in the human placenta, but IE activity has not been documented before. In our study, we describe the physiological properties of the two enzymes in vitro. PIS and IE were studied in different Mn2+ and Mg2+ concentrations that enabled us to separate the individual enzyme activities. Enzyme activity was measured by incorporation of 3[H]inositol in human primordial placenta tissue or microsomes. Optimal PIS activity was achieved between 0.5 and 2.0 mM Mn2+ concentration, but higher concentrations inhibit enzyme activity. In the presence of Mg2+, the enzyme activity increases continuously up to a concentration of 100 mM. PIS was inhibited by nucleoside di- and tri-phosphates. PI production increases between 0.1 and 10 mM Mn2+ concentration. The incorporation of [3H]inositol into PI increased by 57% when adding stabile GTP analog. The described novel pathway of inositol synthesis may provide an additional therapeutic approach of inositol supplementation before and during pregnancy.

Extracellular Kir2.1C122Y Mutant Upsets Kir2.1-PIP2 Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome.

BACKGROUND: Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys (cysteine)122-to-Cys154 disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS: We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments. RESULTS: Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate-binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins. CONCLUSIONS: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate-dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.

Phosphoinositide switches in cell physiology - From molecular mechanisms to disease.

Phosphoinositides are amphipathic lipid molecules derived from phosphatidylinositol that represent low abundance components of biological membranes. Rather than serving as mere structural elements of lipid bilayers, they represent molecular switches for a broad range of biological processes, including cell signaling, membrane dynamics and remodeling, and many other functions. Here, we focus on the molecular mechanisms that turn phosphoinositides into molecular switches and how the dysregulation of these processes can lead to disease.

The phosphatidylinositol-5' phosphatase synaptojanin1 limits integrin-mediated invasion of Staphylococcus aureus.

The gram-positive bacterium Staphylococcus aureus can invade non-professional phagocytic cells by associating with the plasma protein fibronectin to exploit host cell integrins. Integrin-mediated internalization of these pathogens is facilitated by the local production of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) via an integrin-associated isoform of phosphatidylinositol-5' kinase. In this study, we addressed the role of PI-4,5-P2-directed phosphatases on internalization of S. aureus. ShRNA-mediated knockdown of individual phosphoinositide 5-phosphatases revealed that synaptojanin1 (SYNJ1) is counteracting invasion of S. aureus into mammalian cells. Indeed, shRNA-mediated depletion as well as genetic deletion of synaptojanin1 via CRISPR/Cas9 resulted in a gain-of-function phenotype with regard to integrin-mediated uptake. Surprisingly, the surface level of integrins was slightly downregulated in Synj1-KO cells. Nevertheless, these cells showed enhanced local accumulation of PI-4,5-P2 and exhibited increased internalization of S. aureus. While the phosphorylation level of the integrin-associated protein tyrosine kinase FAK was unaltered, the integrin-binding and -activating protein talin was enriched in the vicinity of S. aureus in synaptojanin1 knockout cells. Scanning electron microscopy revealed enlarged membrane invaginations in the absence of synaptojanin1 explaining the increased capability of these cells to internalize integrin-bound microorganisms. Importantly, the enhanced uptake by Synj1-KO cells and the exaggerated morphological features were rescued by the re-expression of the wild-type enzyme but not phosphatase inactive mutants. Accordingly, synaptojanin1 activity limits integrin-mediated invasion of S. aureus, corroborating the important role of PI-4,5-P2 during this process.IMPORTANCEStaphylococcus aureus, an important bacterial pathogen, can invade non-professional phagocytes by capturing host fibronectin and engaging integrin α5ß1. Understanding how S. aureus exploits this cell adhesion receptor for efficient cell entry can also shed light on the physiological regulation of integrins by endocytosis. Previous studies have found that a specific membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), supports the internalization process. Here, we extend these findings and report that the local levels of PIP2 are controlled by the activity of the PIP2-directed lipid phosphatase Synaptojanin1. By dephosphorylating PIP2 at bacteria-host cell attachment sites, Synaptojanin1 counteracts the integrin-mediated uptake of the microorganisms. Therefore, our study not only generates new insight into subversion of cellular receptors by pathogenic bacteria but also highlights the role of host cell proteins acting as restriction factors for bacterial invasion at the plasma membrane.

PI4KIIIß-Mediated Phosphoinositides Metabolism Regulates Function of the VTA Dopaminergic Neurons and Depression-Like Behavior.

Phosphoinositides, including phosphatidylinositol-4,5-bisphosphate (PIP2), play a crucial role in controlling key cellular functions such as membrane and vesicle trafficking, ion channel, and transporter activity. Phosphatidylinositol 4-kinases (PI4K) are essential enzymes in regulating the turnover of phosphoinositides. However, the functional role of PI4Ks and mediated phosphoinositide metabolism in the central nervous system has not been fully revealed. In this study, we demonstrated that PI4KIIIß, one of the four members of PI4Ks, is an important regulator of VTA dopaminergic neuronal activity and related depression-like behavior of mice by controlling phosphoinositide turnover. Our findings provide new insights into possible mechanisms and potential drug targets for neuropsychiatric diseases, including depression. Both sexes were studied in basic behavior tests, but only male mice could be used in the social defeat depression model.

Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the ß2 adrenergic receptor (ß2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on ß2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of ß2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, ß2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of ß2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease ß2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.

Bidirectional Allosteric Coupling between PIP2 Binding and the Pore of the Oncochannel TRPV6.

The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP2 in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP2 depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.

The expanding roles of PI4P and PI(4,5)P2 at the plasma membrane: Role of phosphatidylinositol transfer proteins.

Phosphoinositides are phosphorylated derivatives of phosphatidylinositol, a phospholipid that is synthesised at the endoplasmic reticulum. The plasma membrane contains the enzymes to phosphorylate phosphatidylinositol and is therefore rich in the phosphorylated derivatives, PI4P and PI(4,5)P2. PI(4,5)P2 is a substrate for phospholipase C and during cell signaling, PI(4,5)P2 levels are reduced. Here I discuss a family of proteins, phosphatidylinositol transfer proteins (PITPs) that can restore PI(4,5)P2 levels.

Synthesis of azide-modified glycerophospholipid precursor analogs for detection of enzymatic reactions.

Glycerophospholipids (GPLs) are major cell membrane components. Although various phosphorylated molecules are attached to lipid moieties as their headgroups, GPLs are biosynthesized from phosphatidic acid (PA) via its derivatives, diacylglycerol (DAG) or cytidine diphosphate diacylglycerol (CDP-DAG). A variety of molecular probes capable of introducing detection tags have been developed to investigate biological events involved in GPLs. In this study, we report the design, synthesis, and evaluation of novel analytical tools suitable to monitor the activity of GPL biosynthetic enzymes in vitro. Our synthetic targets, namely, azide-modified PA, azide-modified DAG, and azide-modified CDP-DAG, were successfully obtained from solketal as their common starting material. Moreover, using CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT), an enzyme that catalyzed the final reaction step in synthesizing phosphatidylinositol, we demonstrated that azide-modified CDP-DAG worked as a substrate for CDIPT.

A new role for phosphoinositides in regulating mitochondrial dynamics.

Phosphoinositides are a minor group of membrane-associated phospholipids that are transiently generated on the cytoplasmic leaflet of many organelle membranes and the plasma membrane. There are seven functionally distinct phosphoinositides, each derived via the reversible phosphorylation of phosphatidylinositol in various combinations on the inositol ring. Their generation and termination is tightly regulated by phosphatidylinositol-kinases and -phosphatases. These enzymes can function together in an integrated and coordinated manner, whereby the phosphoinositide product of one enzyme may subsequently serve as a substrate for another to generate a different phosphoinositide species. This regulatory mechanism not only enables the transient generation of phosphoinositides on membranes, but also more complex sequential or bidirectional conversion pathways, and phosphoinositides can also be transferred between organelles via membrane contacts. It is this capacity to fine-tune phosphoinositide signals that makes them ideal regulators of membrane organization and dynamics, through their recruitment of signalling, membrane altering and lipid transfer proteins. Research spanning several decades has provided extensive evidence that phosphoinositides are major gatekeepers of membrane organization, with roles in endocytosis, exocytosis, autophagy, lysosome dynamics, vesicular transport and secretion, cilia, inter-organelle membrane contact, endosome maturation and nuclear function. By contrast, there has been remarkably little known about the role of phosphoinositides at mitochondria - an enigmatic and major knowledge gap, with challenges in reliably detecting phosphoinositides at this site. Here we review recent significant breakthroughs in understanding the role of phosphoinositides in regulating mitochondrial dynamics and metabolic function.

Phosphoinositide Regulation of TRP Channels: A Functional Overview in the Structural Era.

Transient receptor potential (TRP) ion channels have diverse activation mechanisms including physical stimuli, such as high or low temperatures, and a variety of intracellular signaling molecules. Regulation by phosphoinositides and their derivatives is their only known common regulatory feature. For most TRP channels, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] serves as a cofactor required for activity. Such dependence on PI(4,5)P2 has been demonstrated for members of the TRPM subfamily and for the epithelial TRPV5 and TRPV6 channels. Intracellular TRPML channels show specific activation by PI(3,5)P2. Structural studies uncovered the PI(4,5)P2 and PI(3,5)P2 binding sites for these channels and shed light on the mechanism of channel opening. PI(4,5)P2 regulation of TRPV1-4 as well as some TRPC channels is more complex, involving both positive and negative effects. This review discusses the functional roles of phosphoinositides in TRP channel regulation and molecular insights gained from recent cryo-electron microscopy structures.

Peroxisome population control by phosphoinositide signaling at the endoplasmic reticulum-plasma membrane interface.

Phosphoinositides are lipid signaling molecules acting at the interface of membranes and the cytosol to regulate membrane trafficking, lipid transport and responses to extracellular stimuli. Peroxisomes are multicopy organelles that are highly responsive to changes in metabolic and environmental conditions. In yeast, peroxisomes are tethered to the cell cortex at defined focal structures containing the peroxisome inheritance protein, Inp1p. We investigated the potential impact of changes in cortical phosphoinositide levels on the peroxisome compartment of the yeast cell. Here we show that the phosphoinositide, phosphatidylinositol-4-phosphate (PI4P), found at the junction of the cortical endoplasmic reticulum and plasma membrane (cER-PM) acts to regulate the cell's peroxisome population. In cells lacking a cER-PM tether or the enzymatic activity of the lipid phosphatase Sac1p, cortical PI4P is elevated, peroxisome numbers and motility are increased, and peroxisomes are no longer firmly tethered to Inp1p-containing foci. Reattachment of the cER to the PM through an artificial ER-PM "staple" in cells lacking the cER-PM tether does not restore peroxisome populations to the wild-type condition, demonstrating that integrity of PI4P signaling at the cell cortex is required for peroxisome homeostasis.

Phosphoinositides take a central stage in regulating blood platelet production and function.

Blood platelets are produced by megakaryocytes through a complex program of differentiation and play a critical role in hemostasis and thrombosis. These anucleate cells are the target of antithrombotic drugs that prevent them from clumping in cardiovascular disease conditions. Platelets also significantly contribute to various aspects of physiopathology, including interorgan communications, healing, inflammation, and thromboinflammation. Their production and activation are strictly regulated by highly elaborated mechanisms. Among them, those involving inositol lipids have drawn the attention of researchers. Phosphoinositides represent the seven combinatorially phosphorylated forms of the inositol head group of inositol lipids. They play a crucial role in regulating intracellular mechanisms, such as signal transduction, actin cytoskeleton rearrangements, and membrane trafficking, either by generating second messengers or by directly binding to specific domains of effector proteins. In this review, we will explore how phosphoinositides are implicated in controlling platelet production by megakaryocytes and in platelet activation processes. We will also discuss the diversity of phosphoinositides in platelets, their role in granule biogenesis and maintenance, as well as in integrin signaling. Finally, we will address the discovery of a novel pool of phosphatidylinositol 3-monophosphate in the outerleaflet of the plasma membrane of human and mouse platelets.

Lipid exchange at ER-trans-Golgi contact sites governs polarized cargo sorting.

Oxysterol binding protein (OSBP) extracts cholesterol from the ER to deliver it to the TGN via counter exchange and subsequent hydrolysis of the phosphoinositide PI(4)P. Here, we show that this pathway is essential in polarized epithelial cells where it contributes not only to the proper subcellular distribution of cholesterol but also to the trans-Golgi sorting and trafficking of numerous plasma membrane cargo proteins with apical or basolateral localization. Reducing the expression of OSBP, blocking its activity, or inhibiting a PI4Kinase that fuels OSBP with PI(4)P abolishes the epithelial phenotype. Waves of cargo enrichment in the TGN in phase with OSBP and PI(4)P dynamics suggest that OSBP promotes the formation of lipid gradients along the TGN, which helps cargo sorting. During their transient passage through the trans-Golgi, polarized plasma membrane proteins get close to OSBP but fail to be sorted when OSBP is silenced. Thus, OSBP lipid exchange activity is decisive for polarized cargo sorting and distribution in epithelial cells.

Research progress on phosphatidylinositol 4-kinase inhibitors.

Phosphatidylinositol 4-kinases (PI4Ks) could phosphorylate phosphatidylinositol (PI) to produce phosphatidylinositol 4-phosphate (PI4P) and maintain its metabolic balance and location. PI4P, the most abundant monophosphate inositol in eukaryotic cells, is a precursor of higher phosphoinositols and an essential substrate for the PLC/PKC and PI3K/Akt signaling pathways. PI4Ks regulate vesicle transport, signal transduction, cytokinesis, and cell unity, and are involved in various physiological and pathological processes, including infection and growth of parasites such as Plasmodium and Cryptosporidium, replication and survival of RNA viruses, and the development of tumors and nervous system diseases. The development of novel drugs targeting PI4Ks and PI4P has been the focus of the research and clinical application of drugs, especially in recent years. In particular, PI4K inhibitors have made great progress in the treatment of malaria and cryptosporidiosis. We describe the biological characteristics of PI4Ks; summarize the physiological functions and effector proteins of PI4P; and analyze the structural basis of selective PI4K inhibitors for the treatment of human diseases in this review. Herein, this review mainly summarizes the developments in the structure and enzyme activity of PI4K inhibitors.

Toxoplasma membrane inositol phospholipid binding protein TgREMIND is essential for secretory organelle function and host infection.

Apicomplexan parasites possess specialized secretory organelles called rhoptries, micronemes, and dense granules that play a vital role in host infection. In this study, we demonstrate that TgREMIND, a protein found in Toxoplasma gondii, is necessary for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes-CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, which binds to membrane phospholipids, as well as a novel uncharacterized domain that we have named REMIND (regulator of membrane-interacting domain). Both the F-BAR domain and the REMIND are crucial for TgREMIND functions. When TgREMIND is depleted, there is a significant decrease in the abundance of dense granules and abnormal transparency of rhoptries, leading to a reduction in protein secretion from these organelles. The absence of TgREMIND inhibits host invasion and parasite dissemination, demonstrating that TgREMIND is essential for the proper function of critical secretory organelles required for successful infection by Toxoplasma.

Actin-binding protein profilin1 is an important determinant of cellular phosphoinositide control.

Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.

Epinephrine inhibits PI3Kα via the Hippo kinases.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.