Fission yeast Duc1 links to ER-PM contact sites and influences PM lipid composition and cytokinetic ring anchoring.

Cytokinesis is the final stage of the cell cycle that results in the physical separation of daughter cells. To accomplish cytokinesis, many organisms build an actin- and myosin-based cytokinetic ring (CR) anchored to the plasma membrane (PM). Defects in CR-PM anchoring can arise when the PM lipid, phosphatidylinositol-4,5- bisphosphate [PI(4,5)P2], is depleted. In Schizosaccharomyces pombe, reduced PM PI(4,5)P2 results in a CR that cannot maintain its medial position and slides toward one cell end, resulting in two differently sized daughter cells. S. pombe PM PI(4,5)P2 is synthesized by the PI5-kinase Its3, but what regulates this enzyme to maintain appropriate PM PI(4,5)P2 levels is not known in S. pombe. To identify Its3 regulators, we used proximity-based biotinylation and the uncharacterized protein Duc1 was specifically detected. We discovered that Duc1 decorates the PM except at the cell division site and that its unique localization pattern is dictated by binding to the ER-PM contact site proteins, Scs2 and Scs22. Our evidence suggests Duc1 also binds PI(4,5)P2 and helps enrich Its3 at the lateral PM, thereby promoting PM PI(4,5)P2 synthesis and robust CR-PM anchoring.

Targeting phosphoinositide signaling in cancer: relevant techniques to study lipids and novel avenues for therapeutic intervention.

Phosphoinositides serve as essential players in numerous biological activities and are critical for overall cellular function. Due to their complex chemical structures, localization, and low abundance, current challenges in the phosphoinositide field include the accurate measurement and identification of specific variants, particularly those with acyl chains. Researchers are intensively developing innovative techniques and approaches to address these challenges and advance our understanding of the impact of phosphoinositide signaling on cellular biology. This article provides an overview of recent advances in the study of phosphoinositides, including mass spectrometry, lipid biosensors, and real-time activity assays using fluorometric sensors. These methodologies have proven instrumental for a comprehensive exploration of the cellular distribution and dynamics of phosphoinositides and have shed light on the growing significance of these lipids in human health and various pathological processes, including cancer. To illustrate the importance of phosphoinositide signaling in disease, this perspective also highlights the role of a family of lipid kinases named phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), which have recently emerged as exciting therapeutic targets for cancer treatment. The ongoing exploration of phosphoinositide signaling not only deepens our understanding of cellular biology but also holds promise for novel interventions in cancer therapy.

25 Years of PI5P.

The accidental discovery of PI5P (phosphatidylinositol-5-phosphate) was published 25 years ago, when PIP5K type II (phosphoinositide-4-phosphate 5-kinase) was shown to actually be a 4-kinase that uses PI5P as a substrate to generate PI(4,5)P2. Consequently, PIP5K type II was renamed to PI5P4K, or PIP4K for short, and PI5P became the last of the 7 signaling phosphoinositides to be discovered. Much of what we know about PI5P comes from genetic studies of PIP4K, as the pathways for PI5P synthesis, the downstream targets of PI5P and how PI5P affects cellular function all remain largely enigmatic. Nevertheless, PI5P and PI5P-dependent PI(4,5)P2 synthesis have been clearly implicated in metabolic homeostasis and in diseases such as cancer. Here, we review the past 25 years of PI5P research, with particular emphasis on the impact this small signaling lipid has on human health.

Multimodal action of KRP203 on phosphoinositide kinases in vitro and in cells.

Increased phosphoinositide signaling is commonly associated with cancers. While "one-drug one-target" has been a major drug discovery strategy for cancer therapy, a "one-drug multi-targets" approach for phosphoinositide enzymes has the potential to offer a new therapeutic approach. In this study, we sought a new way to target phosphoinositides metabolism. Using a high-throughput phosphatidylinositol 5-phosphate 4-kinase-alpha (PI5P4Kα) assay, we have identified that the immunosuppressor KRP203/Mocravimod induces a significant perturbation in phosphoinositide metabolism in U87MG glioblastoma cells. Despite high sequence similarity of PI5P4K and PI4K isozymes, in vitro kinase assays showed that KRP203 activates some (e.g., PI5P4Kα, PI4KIIß) while inhibiting other phosphoinositide kinases (e.g., PI5P4Kß, γ, PI4KIIα, class I PI3K-p110α, δ, γ). Furthermore, KRP203 enhances PI3P5K/PIKFYVE's substrate selectivity for phosphatidylinositol (PI) while preserving its selectivity for PI(3)P. At cellular levels, 3 h of KRP203 treatment induces a prominent increase of PI(3)P and moderate increase of PI(5)P, PI(3,5)P2, and PI(3,4,5)P3 levels in U87MG cells. Collectively, the finding of multimodal activity of KRP203 towards multi-phosphoinositide kinases may open a novel basis to modulate cellular processes, potentially leading to more effective treatments for diseases associated with phosphoinositide signaling pathways.

Targeting the Dark Lipid Kinase PIP4K2C with a Potent and Selective Binder and Degrader.

Phosphatidylinositol 5-phosphate 4-kinase, type II, gamma (PIP4K2C) remains a poorly understood lipid kinase with minimal enzymatic activity but potential scaffolding roles in immune modulation and autophagy-dependent catabolism. Achieving potent and selective agents for PIP4K2C while sparing other lipid and non-lipid kinases has been challenging. Here, we report the discovery of the highly potent PIP4K2C binder TMX-4102, which shows exclusive binding selectivity for PIP4K2C. Furthermore, we elaborated the PIP4K2C binder into TMX-4153, a bivalent degrader capable of rapidly and selectively degrading endogenous PIP4K2C. Collectively, our work demonstrates that PIP4K2C is a tractable and degradable target, and that TMX-4102 and TMX-4153 are useful leads to further interrogate the biological roles and therapeutic potential of PIP4K2C.

Development of potent and selective degraders of PI5P4Kγ.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), a family of three members in mammals (α, ß and γ), have emerged as potential therapeutic targets due to their role in regulating many important cellular signaling pathways. In comparison to the PI5P4Kα and PI5P4Kß, which usually have similar expression profiles across cancer cells, PI5P4Kγ exhibits distinct expression patterns, and pathological functions for PI5P4Kγ have been proposed in the context of cancer and neurodegenerative diseases. PI5P4Kγ has very low kinase activity and has been proposed to inhibit the PI4P5Ks through scaffolding function, providing a rationale for developing a selective PI5P4Kγ degrader. Here, we report the development and characterization of JWZ-1-80, a first-in-class PI5P4Kγ degrader. JWZ-1-80 potently degrades PI5P4Kγ via the ubiquitin-proteasome system and exhibits proteome-wide selectivity and is therefore a useful tool compound for further dissecting the biological functions of PI5P4Kγ.

The GTP responsiveness of PI5P4Kß evolved from a compromised trade-off between activity and specificity.

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kß has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kß use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kß has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.

Expanding role of PI5P4Ks in cancer: A promising druggable target.

Cancer cells are challenged by a myriad of microenvironmental stresses, and it is their ability to efficiently adapt to the constantly changing nutrient, energy, oxidative, and/or immune landscape that allows them to survive and proliferate. Such adaptations, however, result in distinct vulnerabilities that are attractive therapeutic targets. Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are a family of druggable stress-regulated phosphoinositide kinases that become conditionally essential as a metabolic adaptation, paving the way to targeting cancer cell dependencies. Further, PI5P4Ks have a synthetic lethal interaction with the tumor suppressor p53, the loss of which is one of the most prevalent genetic drivers of malignant transformation. PI5P4K's emergence as a crucial axis in the expanding landscape of phosphoinositide signaling in cancer has already stimulated the development of specific inhibitors. Thus, a better understanding of the biology of the PI5P4Ks will allow for targeted and effective therapeutic interventions. Here, we attempt to summarize the mounting roles of the PI5P4Ks in cancer, including evidence that targeting them is a therapeutic vulnerability and promising next-in-line treatment for multiple cancer subtypes.

Ionization properties of monophosphoinositides in mixed model membranes.

Phosphoinositides are found in low concentration in cellular membranes but perform numerous functions such as signaling, membrane trafficking, protein recruitment and modulation of protein activity. Spatiotemporal regulation by enzymes that phosphorylate and dephosphorylate the inositol ring results in the production of seven distinct and functionally diverse derivatives. Ionization properties of the phosphorylated headgroups of anionic lipids have been shown to impact how they interact with proteins and lipids in the membrane. While the ionization properties of the three bis and one tris phosphorylated forms have been studied in physiologically relevant model membranes, that of the monophosphorylated forms (i.e., phosphatidylinositol-3-phosphate (PI3P), phosphatidylinositol-4-phosphate (PI4P), phosphatidylinositol-5-phosphate (PI5P)) has received less attention. Here, we used 31P MAS NMR to determine the charge of 5 mol% of the monophosphorylated derivatives in pure dioleoylphosphatidylcholine (DOPC) and DOPC/dioleoylphosphatidylethanolamine (DOPE) bilayers as a function of pH. We find that PI3P, PI4P and PI5P each have unique pKa2 values in a DOPC bilayer, and each is reduced in DOPC/DOPE model membranes through the interaction of their headgroups with DOPE according to the electrostatic-hydrogen bond switch model. In this study, using model membranes mimicking the plasma membrane (inner leaflet), Golgi, nuclear membrane, and endosome (outer leaflet), we show that PI3P, PI4P or PI5P maximize their charge at neutral pH. Our results shed light on the electrostatics of the monophosphorylated headgroups of PI3P, PI4P, and PI5P and form the basis of their intracellular functions.

YPIBP: A repository for phosphoinositide-binding proteins in yeast.

Phosphoinositides (PIs) are a family of eight lipids consisting of phosphatidylinositol (PtdIns) and its seven phosphorylated forms. PIs have important regulatory functions in the cell including lipid signaling, protein transport, and membrane trafficking. Yeast has been recognized as a eukaryotic model system to study lipid-protein interactions. Hundreds of yeast PI-binding proteins have been identified, but this research knowledge remains scattered. Besides, the complete PI-binding spectrum and potential PI-binding domains have not been interlinked. No comprehensive databases are available to support the lipid-protein interaction research on phosphoinositides. Here we constructed the first knowledgebase of Yeast Phosphoinositide-Binding Proteins (YPIBP), a repository consisting of 679 PI-binding proteins collected from high-throughput proteome-array and lipid-array studies, QuickGO, and a rigorous literature mining. The YPIBP also contains protein domain information in categories of lipid-binding domains, lipid-related domains and other domains. The YPIBP provides search and browse modes along with two enrichment analyses (PI-binding enrichment analysis and domain enrichment analysis). An interactive visualization is given to summarize the PI-domain-protein interactome. Finally, three case studies were given to demonstrate the utility of YPIBP. The YPIBP knowledgebase consolidates the present knowledge and provides new insights of the PI-binding proteins by bringing comprehensive and in-depth interaction network of the PI-binding proteins. YPIBP is available at http://cosbi7.ee.ncku.edu.tw/YPIBP/.

Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner.

Autophagy is an essential catabolic process induced to provide cellular energy sources in response to nutrient limitation through the activation of kinases, like AMP-activated protein kinase (AMPK) and ULK1. Although glucose starvation induces autophagy, the exact mechanism underlying this signaling has yet to be elucidated. Here, we reveal a role for ULK1 in non-canonical autophagy signaling using diverse cell lines. ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on S1548, thereby increasing its activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P2. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels. Our study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve.

PI5P4Ks drive metabolic homeostasis through peroxisome-mitochondria interplay.

PI5P4Ks are a class of phosphoinositide kinases that phosphorylate PI-5-P to PI-4,5-P2. Distinct localization of phosphoinositides is fundamental for a multitude of cellular functions. Here, we identify a role for peroxisomal PI-4,5-P2 generated by the PI5P4Ks in maintaining energy balance. We demonstrate that PI-4,5-P2 regulates peroxisomal fatty acid oxidation by mediating trafficking of lipid droplets to peroxisomes, which is essential for sustaining mitochondrial metabolism. Using fluorescent-tagged lipids and metabolite tracing, we show that loss of the PI5P4Ks significantly impairs lipid uptake and ß-oxidation in the mitochondria. Further, loss of PI5P4Ks results in dramatic alterations in mitochondrial structural and functional integrity, which under nutrient deprivation is further exacerbated, causing cell death. Notably, inhibition of the PI5P4Ks in cancer cells and mouse tumor models leads to decreased cell viability and tumor growth, respectively. Together, these studies reveal an unexplored role for PI5P4Ks in preserving metabolic homeostasis, which is necessary for tumorigenesis.

Emerging cell biological functions of phosphatidylinositol 5 phosphate 4 kinase.

The generation of phosphoinositides (PIs) with spatial and temporal control is a key mechanism in cellular organization and signaling. The synthesis of PIs is mediated by PI kinases, proteins that are able to phosphorylate unique substrates at specific positions on the inositol headgroup to generate signaling molecules. Phosphatidylinositol 5 phosphate 4 kinase (PIP4K) is one such lipid kinase that is able to specifically phosphorylate phosphatidylinositol 5 phosphate, the most recently discovered PI to generate the well-known and abundant PI, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. PIP4K appears to be encoded only in metazoan genomes, and several genetic studies indicate important physiological functions for these enzymes in metabolism, immune function, and growth control. PIP4K has recently been reported to localize to multiple cellular compartments, including the nucleus, plasma membrane, endosomal systems, and autophagosome. However, the biochemical activity of these enzymes that is relevant to these physiological functions remains elusive. We review recent developments in this area and highlight emerging roles for these enzymes in cellular organization.

Simultaneous Detection of Phosphoinositide Lipids by Radioactive Metabolic Labeling.

Phosphoinositide (PPI) lipids are a crucial class of low-abundance signaling molecules that regulate many processes within cells. Methods that enable simultaneous detection of all PPI lipid species provide a wholistic snapshot of the PPI profile of cells, which is critical for probing PPI biology. Here we describe a method for the simultaneous measurement of cellular PPI levels by metabolically labeling yeast or mammalian cells with myo-3H-inositol, extracting radiolabeled glycerophosphoinositides, and separating lipid species on an anion exchange column via HPLC.

Targeting the PI5P4K Lipid Kinase Family in Cancer Using Covalent Inhibitors.

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/ß/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

Pi5 and Pii Paired NLRs Are Functionally Exchangeable and Confer Similar Disease Resistance Specificity.

Effector-triggered immunity (ETI) is an effective layer of plant defense initiated upon recognition of avirulence (Avr) effectors from pathogens by cognate plant disease resistance (R) proteins. In rice, a large number of R genes have been characterized from various cultivars and have greatly contributed to breeding programs to improve resistance against the rice blast pathogen Magnaporthe oryzae. The extreme diversity of R gene repertoires is thought to be a result of co-evolutionary history between rice and its pathogens including M. oryzae. Here we show that Pii is an allele of Pi5 by DNA sequence characterization and complementation analysis. Pii-1 and Pii-2 cDNAs were cloned by reverse transcription polymerase chain reaction from the Pii -carrying cultivar Fujisaka5 . The complementation test in susceptible rice cultivar Dongjin demonstrated that the rice blast resistance mediated by Pii , similar to Pi5 , requires the presence of two nucleotide-binding leucine-rich repeat genes, Pii-1 and Pii-2 . Consistent with our hypothesis that Pi5 and Pii are functionally indistinguishable, the replacement of Pii-1 by Pi5-1 and Pii-2 by Pi5-2 , respectively, does not change the level of disease resistance to M. oryzae carrying AVR-Pii. Surprisingly, Exo70F3, required for Pii-mediated resistance, is dispensable for Pi5-mediated resistance. Based on our results, despite similarities observed between Pi5 and Pii, we hypothesize that Pi5 and Pii pairs require partially distinct mechanisms to function.

PIP4Ks Suppress Insulin Signaling through a Catalytic-Independent Mechanism.

Insulin stimulates the conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), which mediates downstream cellular responses. PI(4,5)P2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Here, we show that the loss of PIP4Ks (PIP4K2A, PIP4K2B, and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P2 and a concomitant increase in insulin-stimulated production of PI(3,4,5)P3. The reintroduction of either wild-type or kinase-dead mutants of the PIP4Ks restored cellular PI(4,5)P2 levels and insulin stimulation of the PI3K pathway, suggesting a catalytic-independent role of PIP4Ks in regulating PI(4,5)P2 levels. These effects are explained by an increase in PIP5K activity upon the deletion of PIP4Ks, which normally suppresses PIP5K activity through a direct binding interaction mediated by the N-terminal motif VMLΦPDD of PIP4K. Our work uncovers an allosteric function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3 and suggests that the pharmacological depletion of PIP4K enzymes could represent a strategy for enhancing insulin signaling.

Phosphatidylinositol 5 Phosphate (PI5P): From Behind the Scenes to the Front (Nuclear) Stage.

Phosphatidylinositol (PI)-related signaling plays a pivotal role in many cellular aspects, including survival, cell proliferation, differentiation, DNA damage, and trafficking. PI is the core of a network of proteins represented by kinases, phosphatases, and lipases which are able to add, remove or hydrolyze PI, leading to different phosphoinositide products. Among the seven known phosphoinositides, phosphatidylinositol 5 phosphate (PI5P) was the last to be discovered. PI5P presence in cells is very low compared to other PIs. However, much evidence collected throughout the years has described the role of this mono-phosphoinositide in cell cycles, stress response, T-cell activation, and chromatin remodeling. Interestingly, PI5P has been found in different cellular compartments, including the nucleus. Here, we will review the nuclear role of PI5P, describing how it is synthesized and regulated, and how changes in the levels of this rare phosphoinositide can lead to different nuclear outputs.

Phosphatidylinositol Monophosphates Regulate Optimal Vav1 Signaling Output.

Phosphatidylinositol-5 phosphate (PI5P) and other mono-phosphoinositides (mono-PIs) play second messenger roles in both physiological and pathological conditions. Despite this, their intracellular targets and mechanisms of action remain poorly characterized. Here, we show that Vav1, a protein that exhibits both Rac1 GDP/GTP exchange and adaptor activities, is positively modulated by PI5P and, possibly, other mono-PIs. Unlike other phospholipid-protein complexes, the affinity and specificity of the Vav1-lipid interaction entail a new structural solution that involves the synergistic action of the Vav1 C1 domain and an adjacent polybasic tail. This new regulatory layer, which is not conserved in the Vav family paralogs, favors the engagement of optimal Vav1 signaling outputs in lymphocytes.

Structural Basis for Regulation of Phosphoinositide Kinases and Their Involvement in Human Disease.

Lipid phosphoinositides play fundamental roles in virtually all pathways that control a cell's decision to grow, move, divide, and die. Because of this, kinases that phosphorylate phosphoinositide lipids are critically involved in myriad essential functions including growth, development, and membrane trafficking. The misregulation of phosphoinositide kinases is critical in human diseases, including cancer, primary immunodeficiencies, and developmental disorders. Phosphoinositide kinases also play a role in mediating bacterial and viral infections for many potent human pathogens. Furthermore, inhibitors of parasite phosphoinositide kinases are in development as therapies for both malaria and cryptosporidiosis. Therefore, understanding how phosphoinositide kinases are regulated has implications for the treatment of many devastating human diseases. Recent structures of phosphoinositide kinases have revealed unique molecular insight into their regulation. This review will summarize our current molecular knowledge on phosphoinositide kinase regulation, and how this information is being used to generate novel small molecule inhibitors as potential therapeutics.