PIP2 Lipids Research Articles

Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P2 with positive feedback

The ability for cells to localize and activate peripheral membrane-binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes are phosphatidylinositol phosphate (PIP) lipids, which are dynamically phosphorylated by PIP lipid kinases on intracellular membranes. Functioning primarily at the plasma membrane, phosphatidylinositol-4-phosphate 5-kinases (PIP5K) catalyzes the phosphorylation of PI(4)P to generate most of the PI(4,5)P2 lipids found in eukaryotic plasma membranes. Recently, we determined that PIP5K displays a positive feedback loop based on membrane-mediated dimerization and cooperative binding to its product, PI(4,5)P2. Here, we examine how two motifs contribute to PI(4,5)P2 recognition to control membrane association and catalysis of PIP5K. Using a combination of single molecule TIRF microscopy and kinetic analysis of PI(4)P lipid phosphorylation, we map the sequence of steps that allow PIP5K to cooperatively engage PI(4,5)P2. We find that the specificity loop regulates the rate of PIP5K membrane association and helps orient the kinase to more effectively bind PI(4,5)P2 lipids. After correctly orienting on the membrane, PIP5K transitions to binding PI(4,5)P2 lipids near the active site through a motif previously referred to as the substrate or PIP-binding motif (PIPBM). The PIPBM has broad specificity for anionic lipids and serves a role in regulating membrane association invitro and invivo. Overall, our data supports a two-step membrane-binding model where the specificity loop and PIPBM act in concert to help PIP5K orient and productively engage anionic lipids to drive the positive feedback during PI(4,5)P2 production.

Plasticity of the selectivity filter is essential for permeation in lysosomal TPC2 channels

Two-pore channels are pathophysiologically important Na+- and Ca2+-permeable channels expressed in lysosomes and other acidic organelles. Unlike most other ion channels, their permeability is malleable and ligand-tuned such that when gated by the signaling lipid PI(3,5)P2, they are more Na+-selective than when gated by the Ca2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate. However, the structural basis that underlies such plasticity and single-channel behavior more generally remains poorly understood. A recent Cryo-electron microscopy (cryo-EM) structure of TPC2 bound to PI(3,5)P2 in a proposed open-channel conformation provided an opportunity to address this via molecular dynamics (MD) simulation. To our surprise, simulations designed to compute conductance through this structure revealed almost no Na+ permeation events even at very high transmembrane voltages. However further MD simulations identified a spontaneous transition to a dramatically different conformation of the selectivity filter that involved expansion and a flip in the orientation of two core asparagine residues. This alternative filter conformation was remarkably stable and allowed Na+ to flow through the channel leading to a conductance estimate that was in very good agreement with direct single-channel measurements. Furthermore, this conformation was more permeable for Na+ over Ca2+. Our results have important ramifications not just for understanding the control of ion selectivity in TPC2 channels but also more broadly in terms of how ion channels discriminate ions.

Abstract 1385 Crosstalk between V-ATPase and PI(3,5)P2 lipid in Osmotic Stress. How, When, and Where? Kalaivani Saravanan and Patricia Kane Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, United States

Abstract 1385 Crosstalk between V-ATPase and PI(3,5)P2 lipid in Osmotic Stress. How, When, and Where? Kalaivani Saravanan and Patricia Kane Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, United States

Molecular dynamics simulations of HIV-1 matrix-membrane interactions at different stages of viral maturation

Although the structural rearrangement of the membrane-bound matrix (MA) protein trimers upon HIV-1 maturation has been reported, the consequences of MA maturation on the MA-lipid interactions are not well understood. Long-timescale molecular dynamics simulations of the MA multimeric assemblies of immature and mature virus particles with our realistic asymmetric membrane model have explored MA-lipid interactions and lateral organization of lipids around MA complexes. The number of stable MA-phosphatidylserine and MA-phosphatidylinositol 4,5-bisphosphate (PIP2) interactions at the trimeric interface of the mature MA complex is observed to be greater compared to that of the immature MA complex. Our simulations identified an alternative PIP2-binding site in the immature MA complex where the multivalent headgroup of a PIP2 lipid with a greater negative charge binds to multiple basic amino acid residues such as ARG3 residues of both the MA monomers at the trimeric interface and highly basic region (HBR) residues (LYS29, LYS31) of one of the MA monomers. Our enhanced sampling simulations have explored the conformational space of phospholipids at different binding sites of the trimer-trimer interface of MA complexes that are not accessible by conventional unbiased molecular dynamics. Unlike the immature MA complex, the 2′ acyl tail of two PIP2 lipids at the trimeric interface of the mature MA complex is observed to sample stable binding pockets of MA consisting of helix-4 residues. Together, our results provide molecular-level insights into the interactions of MA trimeric complexes with membrane and different lipid conformations at the specific binding sites of MA protein before and after viral maturation.

SF-1 Induces Nuclear PIP2.

Metazoan cell nuclei contain non-membrane pools of the phosphoinositide lipid PI(4,5)P2 (PIP2), but how this hydrophobic lipid exists within the aqueous nucleoplasm remains unclear. Steroidogenic Factor-1 (NR5A1, SF-1) is a nuclear receptor that binds PIP2 in vitro, and a co-crystal structure of the complex suggests the acyl chains of PIP2 are hidden in the hydrophobic core of the SF-1 protein while the PIP2 headgroup is solvent-exposed. This binding mode explains how SF-1 can solubilize nuclear PIP2; however, cellular evidence that SF-1 expression associates with nuclear PIP2 has been lacking. Here, we examined if tetracycline induction of SF-1 expression would associate with nuclear accumulation of PIP2, using antibodies directed against the PIP2 headgroup. Indeed, tetracycline induction of wild-type SF-1 induced a signal in the nucleus of HEK cells that cross-reacts with PIP2 antibodies, but did not cross-react with antibodies against the lower abundance phosphoinositide PI(3,4,5)P3 (PIP3). The nuclear PIP2 signal co-localized with FLAG-tagged SF-1 in the nuclear compartment. To determine if the nuclear PIP2 signal was dependent on the ability of SF-1 to bind PIP2, we examined a "pocket mutant" of SF-1 (A270W, L345F) shown to be deficient in phospholipid binding by mass spectrometry. Tetracycline induction of this pocket mutant SF-1 in HEK cells failed to induce a detectable PIP2 antibody cross-reactive signal, despite similar Tet-induced expression levels of the wild-type and pocket mutant SF-1 proteins in these cells. Together, these data are the first to suggest that expression of SF-1 induces a PIP2 antibody cross-reactive signal in the nucleus, consistent with X-ray crystallographic and biochemical evidence suggesting SF-1 binds PIP2 in human cells.

Intrinsic Disorder in α-Synuclein Regulates the Exocytotic Fusion Pore Transition.

Today, it is widely accepted that intrinsic disorder is strongly related to the cell cycle, during mitosis, differentiation, and apoptosis. Of particular interest are hybrid proteins possessing both structured and unstructured domains that are critical in human health and disease, such as α-synuclein. In this work, we describe how α-synuclein interacts with the nascent fusion pore as it evolves toward expansion. We unveil the key role played by its intrinsically disordered region as a thermodynamic regulator of the nucleation-expansion energy barrier. By analyzing a truncated variant of α-synuclein that lacks the disordered region, we find that the landscape of protein interactions with PIP2 and POPS lipids is highly altered, ultimately increasing the energy cost for the fusion pore to transit from nucleation to expansion. We conclude that the intrinsically disordered region in full-length α-synuclein recognizes and allocates pivotal protein:lipid interactions during membrane remodeling in the first stages of the fusion pore.

The role of high cholesterol in SARS-CoV-2 infectivity

Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2. The virus binds to angiotensinogen converting enzyme 2 (ACE2), which mediates viral entry into mammalian cells. COVID-19 is notably severe in the elderly and in those with underlying chronic conditions. The cause of selective severity is not well understood. Here we show cholesterol and the signaling lipid phosphatidyl-inositol 4,5 bisphosphate (PIP2) regulate viral infectivity through the localization of ACE2's into nanoscopic (<200nm) lipid clusters. Uptake of cholesterol into cell membranes (a condition common to chronic disease) causes ACE2 to move from PIP2 lipids to endocytic ganglioside (GM1) lipids, where the virus is optimally located for viral entry. In mice, age and high-fat diet increase lung tissue cholesterol by up to 40%. And in smokers with chronic disease, cholesterol is elevated 2-fold, a magnitude of change that dramatically increases infectivity of virus in cell culture. We conclude increasing the ACE2 location near endocytic lipids increases viral infectivity and may help explain the selective severity of COVID-19 in aged and diseased populations.

Why Does Synergistic Activation of WASP, but Not N-WASP, by Cdc42 and PIP2 Require Cdc42 Prenylation?

Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the N-terminal WH1 domain. The basic region also participates in Cdc42 binding, especially for WASP; consequently Cdc42 binding significantly compromises the ability of the basic region in WASP, but not N-WASP, to bind PIP2. PIP2 binding to the WASP basic region is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the activation of WASP and N-WASP may contribute to their different functional roles.

Molecular dynamics characterization of the mechanism of inhibition of ClC-7 activity by PI(3)P.

ClC-7 is a Cl-/H+ antiporter expressed in the lysosome and has been identified as a player in the control of lysosomal pH and ion homeostasis. Known pathologies associated with CLC-7 mutations include osteopetrosis and lysosomal storage diseases. Both pH and current measurements have shown that the signaling lipid PI(3,5)P2 is critical in tuning ClC-7 activity. However, the mechanism by which PI(3,5)P2 achieves this is still unknown. We hypothesized that PI(3,5)P2 stabilizes interactions at the interface between ClC-7's transmembrane domain (TMD) and the cytoplasmic cystathionine beta synthase (CBS) domain, resulting in a TMD-CBS docked conformation that inhibits ClC-7 currents. To test this hypothesis, we have used atomistic molecular dynamics simulations of the transporter in the presence and absence of PI(3)P, a phosphoinositide bound in a recent Cryo-EM structure of CLC-7. Our simulations show that coordination of residues on the CBS with PI(3)P, as well as interactions between residues on the CBS with residues on the TMD play a role in stabilizing the TMD-CBS docked conformation. furthermore, the absence of PI(3)P destabilizes the TMD-CBS docked conformation, increasing the dynamics of the CBS domain. Interestingly, a known disease-causing gain-of-function mutation has similar effects at the TMD-CBS interface. Together our results hint at a mechanism of lipid modulation of ClC-7 activity and pave a way for the development of targeted therapeutics for ClC-7 related pathologies.

Role of lipid composition on human plasma membrane interactions with the Ebola virus matrix protein VP40.

The Ebola virus is a very dangerous zoonotic virus and its infection can cause hemorrhagic fever with high fatality rates. The Ebola virus matrix protein VP40 is the major protein responsible for the formation of the viral matrix by localizing and assembling it at the inner leaflet of the human plasma membrane (PM). This assembly leads to the formation of virus-like-particles (VLP) and eventual budding. Anionic lipids PI(4,5)P2 (PIP2) and Phosphatidylserine (PS) have been shown to facilitate VP40-membrane binding through electrostatic interactions with cationic VP40 residues. It has been determined that Phosphatidic Acid (PA), resulting from the enzyme Phospholipase-D (PLD), may also play an active role in this process. We performed coarse-grained molecular dynamics (CGMD) simulations to investigate the effects various lipids have on VP40-lipid interactions. We used a triple-dimer structure of VP40 and a PM composed of various lipid types. Contact analyses show interactions with specific cationic VP40 residues that facilitate most of the lipid interactions. Interaction strengths between VP40 residues and lipid headgroups are found to vary when PA was included in the PM compared to when it was not. The radial distribution functions also show differences in local lipid clustering between the two systems. These simulations provide new insights on VP40 interactions with the human PM and how different lipids may influence the overall membrane association and assembly of the VP40 dimers.

PIP2 sterically inhibits pore opening of a CNG channel.

Cyclic nucleotide-gated (CNG), and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels are key players in signaling cascades related to vision and olfaction, as well as pacemaking and neuropathic pain, respectively. Despite their structural homology, their functional regulation is quite different. For instance, the signaling lipid PIP2 inhibits CNG channels, but activates HCN channels. In both cases, the underlying mechanism is unknown.

Full activation of WASP, not N-WASP, by Cdc42 and PIP2 requires Cdc42 prenylation.

WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GPTase Cdc42, to relieve auto-inhibition before they can stimulate the initiation of actin polymerization. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the upstream WH1 domain. The basic region also participates in Cd42 binding, especially for WASP; consequently, Cdc42 binding significantly compromises the ability of WASP, but not N-WASP, to bind PIP2. PIP2 binding of WASP is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the full activation of WASP and N-WASP may contribute to their different function roles.

Predicting protein-lipid interactions through machine learning methods employing new tokenization techniques.

Machine learning (ML) methods have had a broad and tremendous impact on structural and dynamical studies of different classes of proteins. Nonetheless, the application of ML models to protein-membrane interactions have received less attention. Given their crucial importance to several aspects of cell communication, we focus our attention to peripheral proteins. In this work, we develop a novel tokenization algorithm for protein-membrane complexes. This method accounts for spatial relationships between the membrane-bound phase of a protein and the lipid bilayer. With this tokenization method, we prepare an autoencoder-based ML workflow for predicting contact maps between residues in a peripheral membrane-binding protein and select key lipids in a lipid bilayer. Our ML model is trained with a series of molecular dynamics (MD) simulations including different PIP-binding proteins and lipid bilayers including PIP lipids. Then, we seek to predict the contact map of PIP-binding proteins, not present in the training set, and verify the predictions of the ML model by MD results. A key element of our tokenization scheme is the embedding of distances, between the protein's membrane-bound phase residues and lipids in the bilayer; we believe this provides a productive feature space for our ML workflow. Details of the ML workflow and results will be discussed. The tokenization method we employ for protein-membrane interactions may facilitate developing applications of previously un-tested ML models in problems regarding protein-membrane interactions. Furthermore, our results for contact map prediction serve as a motivation to further explore ML applications for systems containing a lipid bilayer.

Altered phenotypes due to genetic interaction between the mouse phosphoinositide biosynthesis genes Fig4 and Pip4k2c.

Loss-of-function mutations of FIG4 are responsible for neurological disorders in human and mouse that result from reduced abundance of the signaling lipid PI(3,5)P2. In contrast, loss-of-function mutations of the phosphoinositide kinase PIP4K2C result in elevated abundance of PI(3,5)P2. These opposing effects on PI(3,5)P2 suggested that we might be able to compensate for deficiency of FIG4 by reducing expression of PIP4K2C. To test this hypothesis in a whole animal model, we generated triallelic mice with genotype Fig 4-/-, Pip4k2c+/-; these mice are null for Fig 4 and haploinsufficient for Pip4k2c. The neonatal lethality of Fig 4 null mice in the C57BL/6J strain background was rescued by reduced expression of Pip4k2c. The lysosome enlargement characteristic of Fig 4 null cells was also reduced by heterozygous loss of Pip4k2c. The data demonstrate interaction between these two genes, and suggest that inhibition of the kinase PIPK4C2 could be a target for treatment of FIG4 deficiency disorders such as Charcot-Marie-Tooth Type 4J and Yunis-Varón Syndrome.

Modeling the molecular fingerprint of protein-lipid interactions of MLKL on complex bilayers.

Lipids, the structural part of membranes, play important roles in biological functions. However, our understanding of their implication in key cellular processes such as cell division and protein-lipid interaction is just emerging. This is the case for molecular interactions in mechanisms of cell death, where the role of lipids for protein localization and subsequent membrane permeabilization is key. For example, during the last stage of necroptosis, the mixed lineage kinase domain-like (MLKL) protein translocates and, eventually, permeabilizes the plasma membrane (PM). This process results in the leakage of cellular content, inducing an inflammatory response in the microenvironment that is conducive to oncogenesis and metastasis, among other pathologies that exhibit inflammatory activity. This work presents insights from long all-atom molecular dynamics (MD) simulations of complex membrane models for the PM of mammalian cells with an MLKL protein monomer. Our results show that the binding of the protein is initially driven by the electrostatic interactions of positively charged residues. The protein bound conformation modulates lipid recruitment to the binding site, which changes the local lipid environment recruiting PIP lipids and cholesterol, generating a unique fingerprint. These results increase our knowledge of protein-lipid interactions at the membrane interface in the context of molecular mechanisms of the necroptotic pathway, currently under investigation as a potential treatment target in cancer and inflamatory diseases.

Modulation of the IKS channel by PIP2 requires two binding sites per monomer

The phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipid has been shown to be crucial for the coupling between the voltage sensor and the pore of the potassium voltage-gated KV7 channel family, especially the KV7.1 channel. Expressed in the myocardium membrane, KV7.1 forms a complex with KCNE1 auxiliary subunits to generate the IKS current. Here we present molecular models of the transmembrane region of this complex in its three known states, namely the Resting/Closed (RC), the Intermediate/Closed (IC), and the Activated/Open (AO), robustness of which is assessed by agreement with a range of biophysical data. Molecular Dynamics (MD) simulations of these models embedded in a lipid bilayer including phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipids show that in presence of KCNE1, two PIP2 lipids are necessary to stabilize each state. The simulations also show that KCNE1 interacts with both PIP2 binding sites, forming a tourniquet around the pore and preventing its opening. The present investigation provides therefore key molecular elements that govern the role of PIP2 in KCNE1 modulation of IKS channels, possibly a common mechanism by which auxiliary KCNE subunits might modulate a variety of other ion channels.

Modulation of TRPV2 by endogenous and exogenous ligands: A computational study.

Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C-terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor-like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includesa clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating.

Binding of active Ras and its mutants to the Ras binding domain of PI-3-kinase: A quantitative approach to KD measurements

Ras family GTPases (H/K/N-Ras) modulate numerous effectors, including the lipid kinase PI3K (phosphatidylinositol-3-kinase) that generates growth signal lipid PIP3 (phosphatidylinositol-3,4,5-triphosphate). Active GTP-Ras binds PI3K with high affinity, thereby stimulating PIP3 production. We hypothesize the affinity of this binding interaction could be significantly increased or decreased by Ras mutations at PI3K contact positions, with clinical implications since some Ras mutations at PI3K contact positions are disease-linked. To enable tests of this hypothesis, we have developed an approach combining UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the KD for binding. The approach measures the total Ras concentration, the fraction of Ras in the active state, and the affinity of active Ras binding to its docking site on PI3K Ras binding domain (RBD) in solution. The approach is illustrated by KD measurements for the binding of active H-Ras and representative mutants, each loaded with GTP or GMPPNP, to PI3Kγ RBD. The findings demonstrate that quantitation of the Ras activation state increases the precision of KD measurements, while also revealing that Ras mutations can increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding affinity. Significant Ras affinity changes are predicted to alter PI3K regulation and PIP3 growth signals.

Molecular dynamics simulations reveal membrane lipid interactions of the full-length lymphocyte specific kinase (Lck)

The membrane-bound lymphocyte-specific protein-tyrosine kinase (Lck) triggers T cell antigen receptor signalling to initiate adaptive immune responses. Despite many structure–function studies, the mode of action of Lck and the potential role of plasma membrane lipids in regulating Lck’s activity remains elusive. Advances in molecular dynamics simulations of membrane proteins in complex lipid bilayers have opened a new perspective in gathering such information. Here, we have modelled the full-length Lck open and closed conformations using data available from different crystalographic studies and simulated its interaction with the inner leaflet of the T cell plasma membrane. In both conformations, we found that the unstructured unique domain and the structured domains including the kinase interacted with the membrane with a preference for PIP lipids. Interestingly, our simulations suggest that the Lck-SH2 domain interacts with lipids differently in the open and closed Lck conformations, demonstrating that lipid interaction can potentially regulate Lck’s conformation and in turn modulate T cell signalling. Additionally, the Lck-SH2 and kinase domain residues that significantly contacted PIP lipids are found to be conserved among the Src family of kinases, thereby potentially representing similar PIP interactions within the family.

IFITM3 Acts As a PIP3 Scaffold in TCR-PI3K Signaling

Background: Phosphatidylinositol 3-kinase (PI3K) signaling is an indispensable element of T-cell receptor (TCR), co-stimulatory receptor, and cytokine receptor activation in T cells and in the oncogenic signaling underlying T-cell malignancies. We recently identified interferon-inducible transmembrane protein 3 (IFITM3) as an unexpected but essential component of antigen receptor signaling and malignant transformation in B cells. In contrast to its previously characterized role as an interferon-stimulated gene which restricts viral entry through endosomal membranes, IFITM3 also directly binds to the lipid PIP3 - the product of PI3K - at the B cell surface and amplifies PI3K-mediated signal transduction. Whether IFITM3 serves an analogous function in other lymphocytes is currently unknown. Here, we demonstrate that IFITM3 is also required for robust PI3K-driven signaling and proliferation in primary and transformed human T cells. Results: We found that resting primary human T cells express low levels of IFITM3 but robustly upregulate IFITM3 protein following CD3/CD28 stimulation. To test whether IFITM3 participates in TCR signaling, we analyzed IFITM3 and PIP3 subcellular localization in Jurkat cells. Proximity ligation assays revealed that IFITM3 co-localizes with CD3δ at the cell surface, indicating recruitment of IFITM3 to TCR signaling complexes. Moreover, in the absence of IFITM3, a fluorescent reporter of PIP3 localization failed to redistribute from a diffuse cytosolic pattern to discrete foci at the cell surface upon TCR stimulation. We further explored whether these aberrant PIP3 dynamics were associated with broader defects in PIP3-dependent signaling axes important in T cells. In accord with prior findings in B cells, both Jurkat and primary T cells lacking IFITM3 expression exhibited diminished TCR signaling, including Ca2+ flux and PI3K-dependent AKT phosphorylation, and impaired cell proliferation relative to controls. These results demonstrate that T cells require IFITM3 for optimal antigen receptor- and PI3K-driven signaling. Mechanistically, IFITM3 is phosphorylated by Src family kinases at Y20, promoting retention at the plasma membrane, and specifically binds to PIP3, a highly negatively charged phospholipid, through basic residues in its conserved intracellular loop (CIL). We assessed the functional relevance of these residues in T cells by comparing the effects of wild-type and mutant IFITM3 variants. Overexpression of wild-type IFITM3 or a mutant with constitutive plasma membrane localization (Y20E) increased TCR-induced AKT phosphorylation, but a CIL mutant incapable of binding to PIP3 (K83A/K104A) lacked this activity and instead compromised cell viability and growth. Primary T cells expressing CIL-mutant IFITM3 were more apoptotic and demonstrated impaired cell cycle progression and proliferation, highlighting PIP3 binding as an essential feature of IFITM3 in T cell biology. Beyond normal T cells, PI3K signaling also enables the transformation of malignant T cells, with PI3K inhibitors emerging as promising agents in the treatment of peripheral T cell lymphomas (PTCLs). To determine whether IFITM3 contributes to the oncogenic signaling in PTCLs, we developed disease models incorporating both the expression of PTCL-derived oncogenes in T cells and the capability to assess candidate genes as functional players in pathogenesis. In a mouse bone marrow chimera system, conditional expression (via CD4-Cre) of a lentivirus-delivered oncogene (e.g., ITK-SYK) gave rise to circulating transgenic donor T cells and oncogene-dependent morbidity and mortality. In a humanized mouse system, a T cell-specific promoter controlled transgene expression upon transduction of CD34+ cord blood progenitors. In a separate approach, transduction of mature human T cells differentiated in vitro from CD34+ cells permitted a constitutive promoter. These results establish tractable systems to assess IFITM3 as a requisite factor for oncogenic signaling in PTCLs. Conclusion: We conclude that IFITM3 is essential for antigen receptor and oncogenic signaling not only in B cells but also in T cells. These findings identify a novel component of TCR signaling and suggest a shared mechanism for lymphocyte dependency on IFITM3 through scaffolding the lipid PIP3, a mechanism which can be leveraged to amplify oncogenic signaling in T cell malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal