Phosphoinositide Lipids Research Articles

The scaffolding protein IQGAP1 enhances EGFR signaling by promoting oligomerization and preventing degradation

IQGAP1 is a large, multi-domain scaffold that connects and modulates different signaling networks including the one initiated by epidermal growth factor (EGF). In this study, we have used live cell fluorescence imaging methods along with other biochemical techniques to follow the mechanisms used by IQGAP1 to enhance EGF signaling. We show that IQGAP1 enhances EGF signaling by promoting the oligomerization of its receptor, EGFR, upon EGF addition along with concurrent IQGAP oligomerization. Using cellular markers, we find that IQGAP1 promotes the plasma membrane localization of EGFR and promotes association to one of its phosphoinositide lipid pathway ligands, PI(3,4,5)P3. Additionally, we find that binding of EGFR to IQGAP1 protects EGFR from lysosomal degradation. Taken together, our results show that IQGAP1 enhances EGF-mediated pathway progression through mechanisms that augment simple scaffolding activities.

Admixture mapping of cognitive function in diverse Hispanic and Latino adults: Results from the Hispanic Community Health Study/Study of Latinos.

We conducted admixture mapping and fine-mapping analyses to identify ancestry-of-origin loci influencing cognitive abilities. We estimated the association of local ancestry intervals across the genome with five neurocognitive measures in 7140 diverse Hispanic and Latino adults (mean age 55 years). We prioritized genetic variants in associated loci and tested them for replication in four independent cohorts. We identified nine local ancestry-associated regions for the five neurocognitive measures. There was strong biological support for the observed associations to cognitive function at all loci and there was statistical evidence of independent replication at 4q12, 9p22.1, and 13q12.13. Our study identified multiple novel loci harboring genes implicated in cognitive functioning and dementia, and uncovered ancestry-relevant genetic variants. It adds to our understanding of the genetic architecture of cognitive function in Hispanic and Latino adults and demonstrates the power of admixture mapping to discover unique haplotypes influencing cognitive function, complementing genome-wide association studies. We identified nine ancestry-of-origin chromosomal regions associated with five neurocognitive traits. In each associated region, we identified single nucleotide polymorphisms (SNPs) that explained, at least in part, the admixture signal and were tested for replication in independent samples of Black, non-Hispanic White, and Hispanic/Latino adults with the same or similar neurocognitive tests. Statistical evidence of independent replication of the prioritized SNPs was observed for three of the nine associations, at chr4q12, chr9p22.1, and chr13q12.13. At all loci, there was strong biological support for the observed associations to cognitive function and dementia, prioritizing genes such as KIT, implicated in autophagic clearance of neurotoxic proteins and on mast cell and microglial-mediated inflammation; SLC24A2, implicated in synaptic plasticity associated with learning and memory; and MTMR6, implicated in phosphoinositide lipids metabolism.

Structural basis of TRPV1 modulation by endogenous bioactive lipids

TRP ion channels are modulated by phosphoinositide lipids, but the underlying structural mechanisms remain unclear. The capsaicin- and heat-activated receptor, TRPV1, has served as a model for deciphering lipid modulation, which is relevant to understanding how pro-algesic agents enhance channel activity in the setting of inflammatory pain. Identification of a pocket within the TRPV1 transmembrane core has provided initial clues as to how phosphoinositide lipids bind to and regulate the channel. Here we show that this regulatory pocket in rat TRPV1 can accommodate diverse lipid species, including the inflammatory lipid lysophosphatidic acid, whose actions are determined by their specific modes of binding. Furthermore, we show that an empty-pocket channel lacking an endogenous phosphoinositide lipid assumes an agonist-like state, even at low temperature, substantiating the concept that phosphoinositide lipids serve as negative TRPV1 modulators whose ejection from the binding pocket is a critical step toward activation by thermal or chemical stimuli.

Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

Abstract 4468: Elucidating the phosphoinositide-modulatory function of INPP5B in regulating cancer stemness and tumorigenesis in liver cancer

Abstract Hepatocellular carcinoma (HCC) represents a major global health challenge, characterized by its heterogeneity and aggressive nature. Solid evidence has now suggested that tumor growth is driven by subpopulations of tumor cells with less differentiated cell states, called cancer stem cells (CSCs), which govern therapeutic resistance and tumor recurrence. Therefore, characterization of CSCs and their associated oncogenic signaling pathways is crucial to the development of more specific and sensitive HCC prognostic markers and to the design of novel targeted therapies against CSCs for HCC treatment. In search of the altered genes and pathways that are tightly associated with cancer stemness trait in HCC, we exploited the stemness and oncogenic dedifferentiation signature to mine the HCC dataset of the Cancer Genome Atlas. Phosphoinositide signaling was identified to be enriched and downregulated in the transcriptomes of HCC patients with strong stemness and dedifferentiated signature. Subsequent clinical correlation analysis found inositol polyphosphate-5-phosphatase B (INPP5B) to be the top downregulated gene in phosphoinositide signaling with its expression correlated with poor survival in HCC patients. Functional studies showed that INPP5B overexpression reversed the oncogenic and stemness properties while INPP5B knockout promoted tumor growth in immunocompetent mice. Subsequent RNA sequencing and molecular assays revealed INPP5B deregulation to be associated with lipid metabolism remodeling. Collectively, our results suggest an intricate relationship between phosphoinositide signaling and lipid metabolism through the regulation of INPP5B in HCC. The findings may open new avenue for future research aimed at devising targeted therapies to interfere with liver CSCs and enhance the prognosis of HCC patients. Citation Format: Linglin Liu, Yifei Wang, Yunong Xie, Man Tong. Elucidating the phosphoinositide-modulatory function of INPP5B in regulating cancer stemness and tumorigenesis in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4468.

Asymmetric tethering by exocyst in vitro requires a Rab GTPase, an R-SNARE and a Sac1-sensitive phosphoinositide lipid.

Tethering factors play a critical role in deciphering the correct combination of vesicle and target membrane, before SNARE complex formation and membrane fusion. The exocyst plays a central role in tethering post-Golgi vesicles to the plasma membrane, although the mechanism by which this occurs is poorly understood. We recently established an assay for measuring exocyst-mediated vesicle tethering in vitro and we have adapted this assay to examine the ability of exocyst to tether vesicles in an asymmetric manner. We demonstrate that exocyst differs from another post-Golgi vesicle tethering protein, Sro7, in that it is fully capable of tethering vesicles with a functional Rab GTPase, Sec4, to vesicles lacking a functional Rab GTPase. Using this assay, we show that exocyst requires both the Rab and R-SNARE, Snc1, to be present on the same membrane surface. Using Sac1 phosphatase treatment, we demonstrate a likely role for phosphoinositides on the opposing Rab-deficient membrane. This suggests a specific model for exocyst orientation and its points of contact between membranes during heterotypic tethering of post-Golgi vesicles with the plasma membrane.

PRL1 and PRL3 promote macropinocytosis via its lipid phosphatase activity.

PRL1 and PRL3, members of the protein tyrosine phosphatase family, have been associated with cancer metastasis and poor prognosis. Despite extensive research on their protein phosphatase activity, their potential role as lipid phosphatases remains elusive. Methods: We conducted comprehensive investigations to elucidate the lipid phosphatase activity of PRL1 and PRL3 using a combination of cellular assays, biochemical analyses, and protein interactome profiling. Functional studies were performed to delineate the impact of PRL1/3 on macropinocytosis and its implications in cancer biology. Results: Our study has identified PRL1 and PRL3 as lipid phosphatases that interact with phosphoinositide (PIP) lipids, converting PI(3,4)P2 and PI(3,5)P2 into PI(3)P on the cellular membranes. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis, facilitating nutrient extraction, cell migration, and invasion, thereby contributing to tumor development. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis. Additionally, we found a correlation between PRL1/3 expression and glioma development, suggesting their involvement in glioma progression. Conclusions: Combining with the knowledge that PRLs have been identified to be involved in mTOR, EGFR and autophagy, here we concluded the physiological role of PRL1/3 in orchestrating the nutrient sensing, absorbing and recycling via regulating macropinocytosis through its lipid phosphatase activity. This mechanism could be exploited by tumor cells facing a nutrient-depleted microenvironment, highlighting the potential therapeutic significance of targeting PRL1/3-mediated macropinocytosis in cancer treatment.

Microcolin H, a novel autophagy inducer, exerts potent antitumour activity by targeting PITPα/β

The identification of effective drug targets and the development of bioactive molecules are areas of high need in cancer therapy. The phosphatidylinositol transfer protein alpha/beta isoform (PITPα/β) has been reported to play an essential role in integrating phosphoinositide trafficking and lipid metabolism in diverse cellular processes but remains unexplored as a potential target for cancer treatment. Herein, data analysis of clinical cancer samples revealed that PITPα/β expression is closely correlated with the poor prognosis. Target identification by chemical proteomic methods revealed that microcolin H, a naturally occurring marine lipopeptide, directly binds PITPα/β and displays antiproliferative activity on different types of tumour cell lines. Furthermore, we identified that microcolin H treatment increased the conversion of LC3I to LC3II, accompanied by a reduction of the level of p62 in cancer cells, leading to autophagic cell death. Moreover, microcolin H showed preeminent antitumour efficacy in nude mouse subcutaneous tumour models with low toxicity. Our discoveries revealed that by targeting PITPα/β, microcolin H induced autophagic cell death in tumours with efficient anti-proliferating activity, which sheds light on PITPα/β as a promising therapeutic target for cancer treatment.

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.

Proteomic and yeast 2-hybrid screens to identify PTEN binding partners

PTEN is a phosphoinositide lipid phosphatase and an important tumour suppressor protein. PTEN function is reduced or lost in around a third of all human cancers through diverse mechanisms, from gene deletion to changes in the function of proteins which regulate PTEN through direct protein binding. Here we present data from SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic screens to identify proteins which bind to PTEN. These experiments using untransformed epithelial cells and glioma cells identified several novel candidate proteins in addition to many previously identified PTEN binding partners and many proteins which are recognised as common false positives using these methods. From subsequent co-expression pull-down experiments we provide further evidence supporting the physical interaction of PTEN with MMP1, Myosin 18A and SHROOM3. We also performed yeast two-hybrid screens which identify the previously recognised PTEN binding partner MSP58 in addition to the nuclear import export receptor TNPO3. These experiments identify several novel candidate binding partners of PTEN and provide further data addressing the set of proteins that interact with this important tumour suppressor.

Comprehensive classification of proteins based on structures that engage lipids by COMPOSEL

Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the β barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2.

Direct interactions between picornavirus 3CD protein and phosphoinositide lipids in virus replication organelles.

Positive-strand RNA viruses disrupt host membrane systems and reconstitute these materials into so-called replication organelles, which serve as sites for virus replication and genome packaging. In picornaviruses, these new membrane systems are enriched in phosphoinositide lipids, including phosphoinositide-4-phosphate (PI4P). The 3CD polyprotein is central to membrane biogenesis, as it was previously shown that ectopic expression of 3CD leads to PI4P induction and redistribution, a phenotype similar to that of poliovirus-infected cells. Here, we have shown direct interaction between poliovirus 3CD and PI4P using nuclear magnetic resonance (NMR) spectroscopy, especially providing atomic-level insights into PI4P interaction with both the 3C protease and 3D polymerase derived domains of 3CD. Our studies using paramagnetically-labeled lipid nanodiscs demonstrated that 3C interacts peripherally with the membrane through its positively charged N-terminal helix, which also interacts with genome RNA sequences important for coordinating replication and translation processes. Extension of the C-terminus of 3C increases its affinity to PI4P-containing membranes, suggesting a means by which interactions may change between 3CD and its processed counterparts. Adjustments to virus protein-membrane interactions may be an important component of the switch from replication to packaging modes on these membranes.

Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3.

Arap3, a dual GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA, plays key roles in regulating a wide range of biological processes, including cancer cell invasion and metastasis. It is known that Arap3 is a PI3K effector that can bind directly to PI(3,4,5)P3, and the PI(3,4,5)P3-mediated plasma membrane recruitment is crucial for its function. However, the molecular mechanism of how the protein recognizes PI(3,4,5)P3 remains unclear. Here, using liposome pull-down and surface plasmon resonance (SPR) analysis, we found that the N-terminal first pleckstrin homology (PH) domain (Arap3-PH1) can interact with PI(3,4,5)P3 and, with lower affinity, with PI(4,5)P2. To understand how Arap3-PH1 and phosphoinositide (PIP) lipids interact, we solved the crystal structure of the Arap3-PH1 in the apo form and complex with diC4-PI(3,4,5)P3. We also characterized the interactions of Arap3-PH1 with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 in solution by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we found overexpression of Arap3 could inhibit breast cancer cell invasion in vitro, and the PIPs-binding ability of the PH1 domain is essential for this function.

Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling.

Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purifiedLCVs indicate the presence of membrane contact sites (MCS)proteins possibly implicated in lipid exchange. Using duallyfluorescence-labeled Dictyostelium discoideum amoeba, we revealthat VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L.pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L.pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation.

Abstract 151: Direct interactions between picornavirus 3CD protein and phosphoinositide lipids found in replication organelles

Abstract 151: Direct interactions between picornavirus 3CD protein and phosphoinositide lipids found in replication organelles

Transcription-independent functions of p53 in DNA repair pathway selection.

Recently discovered transcription-independent features of p53 involve the choice of DNA damage repair pathway after PARylation, and p53's complex formation with phosphoinositide lipids, PI(4,5)P2 . PARylation-mediated rapid accumulation of p53 at DNA damage sites is linked to the recruitment of downstream repair factors and tumor suppression. This links p53's capability to sense damaged DNA in vitro and its relevant functions in cells. Further, PI(4,5)P2 rapidly accumulates at damage sites like p53 and complexes with p53, while it is required for ATR recruitment. These findings help explain how p53 and PI(4,5)P2 maintain genome stability by directing DNA repair pathway choice. Additionally, there is a strong correlation between p53 sequence homology, genome mutation rates as well as lifespans across various mammalian species. Further investigation is required to better understand the connections between genome stability, tumor suppression, longevity and the transcriptional-independent function of p53.

Cofilin-Membrane Interactions: Electrostatic Effects in Phosphoinositide Lipid Binding.

The actin cytoskeleton interacts with the cell membrane primarily through the indirect interactions of actin-binding proteins such as cofilin-1. The molecular mechanisms underlying the specific interactions of cofilin-1 with membrane lipids are still unclear. Here, we performed coarse-grain molecular dynamics simulations of cofilin-1 with complex lipid bilayers to analyze the specificity of protein-lipid interactions. We observed the maximal interactions with phosphoinositide (PIP) lipids, especially PIP2 and PIP3 lipids. A good match was observed between the residues predicted to interact and previous experimental studies. The clustering of PIP lipids around the membrane bound protein leads to an overall lipid demixing and gives rise to persistent membrane curvature. Further, through a series of control simulations, we observe that both electrostatics and geometry are critical for specificity of lipid binding. Our current study is a step towards understanding the physico-chemical basis of cofilin-PIP lipid interactions.

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover.

Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2β, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2β. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch.

Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome “fusion-fission” cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.

Metabolic routing maintains the unique fatty acid composition of phosphoinositides.

Phosphoinositide lipids (PPIn) are enriched in stearic- and arachidonic acids (38:4) but how this enrichment is established and maintained during phospholipase C (PLC) activation is unknown. Here we show that the metabolic fate of newly synthesized phosphatidic acid (PA), the lipid precursor of phosphatidylinositol (PI), is influenced by the fatty acyl-CoA used with preferential routing of the arachidonoyl-enriched species toward PI synthesis. Furthermore, during agonist stimulation the unsaturated forms of PI(4,5P)2 are replenished significantly faster than the more saturated ones, suggesting a favored recycling of the unsaturated forms of the PLC-generated hydrolytic products. Cytidine diphosphate diacylglycerol synthase 2 (CDS2) but not CDS1 was found to contribute to increased PI resynthesis during PLC activation. Lastly, while the lipid transfer protein, Nir2 is found to contribute to rapid PPIn resynthesis during PLC activation, the faster re-synthesis of the 38:4 species does not depend on Nir2. Therefore, the fatty acid side-chain composition of the lipid precursors used for PI synthesis is an important determinant of their metabolic fates, which also contributes to the maintenance of the unique fatty acid profile of PPIn lipids.