Phosphatidylinositol 4-phosphate 5-kinase Research Articles

Loss of PI5P4Kα slows the progression of a Pten mutant basal cell model of prostate cancer.

While early prostate cancer (PCa) depends on the androgen receptor (AR) signaling pathway, which is predominant in luminal cells, there is much to be understood about the contribution of epithelial basal cells in cancer progression. Herein, we observe cell-type specific differences in the importance of the metabolic enzyme phosphatidylinositol 5-phosphate 4-kinase alpha (PI5P4Kα β ; gene name PIP4K2A) in the prostate epithelium. We report the development of a basal-cell-specific genetically engineered mouse model (GEMM) targeting Pip4k2a alone or in combination with the tumor suppressor phosphatase and tensin homolog (Pten). PI5P4Kα is enriched in basal cells, and no major histopathological changes were detectable following gene deletion. Notably, the combined loss of Pip4k2a slowed the development of Pten mutant mouse prostatic intraepithelial neoplasia (mPIN). Through the inclusion of a lineage tracing reporter, we utilize single-cell RNA sequencing to evaluate changes resulting from in vivo downregulation of Pip4k2a and characterize cell populations influenced in the established Probasin-Cre and Cytokeratin 5 (CK5)-Cre driven GEMMs. Transcriptomic pathway analysis points towards the disruption of lipid metabolism as a mechanism for reduced tumor progression. This was functionally supported by shifts of carnitine lipids in LNCaP PCa cells treated with siPIP4K2A. Overall, these data nominate PI5P4Kα as a target for PTEN mutant PCa. Implications: PI5P4Kα is enriched in prostate basal cells and its targeted loss slows the progression of a model of advanced PCa.

Investigation of the Roles of Phosphatidylinositol 4-Phosphate 5-Kinases 7,9 and Wall-Associated Kinases 1–3 in Responses to Indole-3-Carbinol and Biotic Stress in Arabidopsis Thaliana

Indole-3-carbinol (I3C), a hydrolysis product of indole-3-methylglucosinolate, is toxic to herbivorous insects and pathogens. In mammals, I3C is extensively studied for its properties in cancer prevention and treatment. Produced in Brassicaceae, I3C reversibly inhibits root elongation in a concentration-dependent manner. This inhibition is partially explained by the antagonistic action of I3C on auxin signaling through TIR1. To further elucidate the mode of action of I3C in plants, we have employed a forward-genetic amiRNA screen that circumvents functional redundancy. We identified and characterized two amiRNA lines with impaired I3C response. The first line, ICT2, targets the phosphatidylinositol 4-phosphate 5-kinase family (PIP5K), exhibiting tolerance to I3C, while the second line, ICS1, targets the Wall-Associated Kinases (WAK1–3) family, showing susceptibility to I3C. Both lines maintain I3C-induced antagonism of auxin signaling, indicating that their phenotypes are due to auxin-independent mechanisms. Transcript profiling experiments reveal that both lines are transcriptionally primed to respond to I3C treatment. Physiological, metabolomic, and transcriptomic analysis reveal that these kinases mediate numerous developmental processes and are involved in abiotic and biotic stress responses.

Abstract C044: PI5P4Kα regulates cell fitness through iron homeostasis in pancreatic cancer

Abstract PI5P4Ks (Phosphatidylinositol 5-phosphate 4-kinases) are a family of lipid kinases that phosphorylate phosphatidylinositol 5-monophosphate (PI-5-P) at the 4-position to generate distinct pools of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) largely on intracellular membranes. These lipid species are involved in cellular and metabolic stress responses, signaling and organelle communication. PI5P4Ks have been implicated in multiple cancers, with compelling evidence showing synthetic lethality when PI5P4Ks are inhibited in TP53-mutant cancer cells. Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive metabolic rewiring but despite the critical roles of PI5P4Ks in cellular metabolism, their role in PDAC remains completely unexplored. We have found that depletion of PI5P4Kα selectively induces apoptotic cell death in PDAC cells. To gain insights into the metabolic mechanisms underlying this apoptotic cell death, we quantified polar metabolites using gas chromatography- mass spectrometry (GC/MS) and found significant reductions in the ratio of monounsaturated to saturated fatty acids, lactate and TCA metabolites. Orthogonal rescue assays demonstrated that exogenous supplementation with fatty acids, pyruvate or lactate fail to rescue the observed apoptosis in PI5P4Kα-knockdown cells; however, TCA cycle metabolites did reverse the extent of apoptosis. Interestingly, we identify that these altered metabolic read-outs and the effects on cell fitness are dependent on iron. Consistent with these findings, intracellular iron levels are reduced upon PI5P4Kα inhibition, and this is directly linked to PI5P4Kα depletion disturbing the intracellular labile iron pools via iron import. Using a heterotopic xenograft mouse model, we demonstrate that PI5P4Kα knockdown leads to the abrogation of PDAC tumor growth due to increased intratumoral cell death. Validating the physiological relevance of PI5P4Kα in human PDAC, we find that PIP4K2A, the gene that encodes PI5P4Kα, is upregulated in human PDAC specimens relative to normal pancreas, and that PIP4K2A gene expression is strongly correlated with iron uptake related gene signatures. This study highlights the critical role of PI5P4Kα in PDAC and suggests that PI5P4Kα inhibition has therapeutic potential in pancreatic cancer. Citation Format: Gurpreet Kaur Arora, Ryan Loughran, Kyanh Ly, Cheska Marie Galapate, Alicia Llorente, Taylor R Anderson, Chantal Pauli, David A Scott, Yoav Altman, Cosimo Commisso, Brooke M Emerling. PI5P4Kα regulates cell fitness through iron homeostasis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C044.

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.

Regulation of the poly(A) Polymerase Star-PAP by a Nuclear Phosphoinositide Signalosome.

Star-PAP is a noncanonical poly(A) polymerase that controls gene expression. Star-PAP was previously reported to bind the phosphatidylinositol 4-phosphate 5-kinase PIPKI⍺ and its product phosphatidylinositol 4,5-bisphosphate, which regulate Star-PAP poly(A) polymerase activity and expression of specific genes. Recent studies have revealed a nuclear PI signaling pathway in which the PI transfer proteins PITP⍺/β, PI kinases and phosphatases bind p53 to sequentially modify protein-linked phosphatidylinositol phosphates and regulate its function. Here we demonstrate that multiple phosphoinositides, including phosphatidylinositol 4-monophosphate and phosphatidylinositol 3,4,5-trisphosphate are also coupled to Star-PAP in response to stress. This is initiated by PITP⍺/β binding to Star-PAP, while the Star-PAP-linked phosphoinositides are modified by PI4KII⍺, PIPKI⍺, IPMK, and PTEN recruited to Star- PAP. The phosphoinositide coupling enhances the association of the small heat shock proteins HSP27/⍺B-crystallin with Star-PAP. Knockdown of the PITPs, kinases, or HSP27 reduce the expression of Star-PAP targets. Our results demonstrate that the PITPs generate Star-PAP-PIPn complexes that are then modified by PI kinases/phosphatases and small heat shock proteins that regulate the linked phosphoinositide phosphorylation and Star-PAP activity in response to stress.

Pip5k1γ promotes anabolism of nucleus pulposus cells and intervertebral disc homeostasis by activating CaMKII-Ampk pathway in aged mice.

Degenerative disc disease (DDD) represents a significant global health challenge, yet its underlying molecular mechanisms remain elusive. This study aimed to investigate the role of type 1 phosphatidylinositol 4-phosphate 5-kinase (Pip5k1) in intervertebral disc (IVD) homeostasis and disease. All three Pip5k1 isoforms, namely Pip5k1α, Pip5k1β, and Pip5k1γ, were detectable in mouse and human IVD tissues, with Pip5k1γ displaying a highest expression in nucleus pulposus (NP) cells. The expression of Pip5k1γ was significantly down-regulated in the NP cells of aged mice and patients with severe DDD. To determine whether Pip5k1γ expression is required for disc homeostasis, we generated a Pip5k1γfl/fl; AggrecanCreERT2 mouse model for the conditional knockout of the Pip5k1γ gene in aggrecan-expressing IVD cells. Our findings revealed that the conditional deletion of Pip5k1γ did not affect the disc structure or cellular composition in 5-month-old adult mice. However, in aged (15-month-old) mice, this deletion led to several severe degenerative disc defects, including decreased NP cellularity, spontaneous fibrosis and cleft formation, and a loss of the boundary between NP and annulus fibrosus. At the molecular level, the absence of Pip5k1γ reduced the anabolism of NP cells without markedly affecting their catabolic or anti-catabolic activities. Moreover, the loss of Pip5k1γ significantly dampened the activation of the protective Ampk pathway in NP cells, thereby accelerating NP cell senescence. Notably, Pip5k1γ deficiency blunted the effectiveness of metformin, a potent Ampk activator, in activating the Ampk pathway and mitigating lumbar spine instability (LSI)-induced disc lesions in mice. Overall, our study unveils a novel role for Pip5k1γ in promoting anabolism and maintaining disc homeostasis, suggesting it as a potential therapeutic target for DDD.

Design and one-pot synthesis of new substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine as potential antitumor agents: in vitro and in vivo studies

A new efficient and versatile one-pot three-component synthesis of substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives has been developed. It is based on a multistep cascade reaction from 2-aminothiophenes and 2-hydroxy-4-oxobut-2-enoic acids, and derivatives of cyanoacetic acid catalyzed by diisopropylethylamine. As a result, novel pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives (21 compounds) were synthesized in a mild reaction conditions with a high yield. The structures of the developed compounds were confirmed by NMR and elemental analysis. The influence of electron-withdrawing or electron-donor substituents on the antitumor activity of the developed compounds has been identified. In vitro screening analysis of 21 compounds revealed six lead candidates (12aa, 12dc, 12hc, 12ic, 12lb, and 12mb) that demonstrated the most significant antitumor activity against B16-F10, 4T1 and CT26 cells. Necrosis/apoptosis assay showed that apoptosis was the predominant mechanism of cell death. Molecular docking analysis revealed several potential targets for tested compounds, i.e. phosphatidylinositol 5-phosphate 4-kinase (PI5P4K2C), proto-oncogene serine/threonine-protein kinase (Pim-1), nicotinamide phosphoribosyltransferase (NAMPT) and dihydrofolate reductase (DHFR). The lead compound (12aa) can effectively induce cell apoptosis, possesses a high yield (98 %) and requires low-cost starting chemicals for its synthesis. In vivo experiments with melanoma-bearing mice confirmed that 12aa compound resulted in the significant tumor inhibition on 15 d after the therapy. In particular, tumor volume was ∼0.19 cm3 for 50 mg/kg versus ∼2.39 cm3 in case of untreated mice and tumor weight was ∼71.6 mg for 50 mg/kg versus ∼452.4 mg when considered untreated mice. Thus, our results demonstrated the high potential of the 12aa compound in the treatment of melanoma and can be recommended for further preclinical studies.

Identification of cotton PIP5K genes and role of GhPIP5K9a in primary root development

Phosphatidylinositol 4 phosphate 5-kinase (PIP5K) is crucial for the phosphatidylinositol (PI) signaling pathway. It plays a significant role in plant growth and development, as well as stress response. However, its effects on cotton are unknown. This study identified PIP5K genes from four cotton species and conducted bioinformatic analyses, with a particular emphasis on the functions of GhPIP5K9a in primary roots. The results showed that cotton PIP5Ks were classified into four subgroups. Analysis of gene structure and motif composition showed obvious conservation within each subgroup. Synteny analysis suggested that the PIP5K gene family experienced significant expansion due to both whole-genome duplication (WGD) and segmental duplication. Transcriptomic data analysis revealed that the majority of GhPIP5K genes had the either low or undetectable levels of expression. Moreover, GhPIP5K9a is highly expressed in the root and was located in plasmalemma. Suppression of GhPIP5K9a transcripts resulted in longer primary roots, longer primary root cells and increased auxin polar transport-related genes expression, and decreased abscisic acid (ABA) content, indicating that GhPIP5K9a negatively regulates cotton primary root growth. This study lays the foundation for further exploration of the role of the PIP5K genes in cotton.

1,2,3-Triazole-totarol conjugates as potent PIP5K1α lipid kinase inhibitors

The human phosphatidylinositol 4-phosphate 5-kinase type I α (hPIP5K1α) plays a key role in the development of prostate cancer. In this work, seventeen derivatives of the natural diterpene totarol were prepared by copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reaction of the correspondingO-propargylated totarol with aryl or alkyl azides and screened for their inhibitory activities toward hPIP5K1α. Five compounds, 3a, 3e, 3f, 3i, and 3r, strongly inhibited the enzyme activity with IC50 values of 1.44, 0.46, 1.02, 0.79, and 3.65 µM, respectively, with the most potent inhibitor 3e 13-[(1-(3-nitrophenyl)triazol-4yl)methoxy]-totara-8,11,13-triene). These compounds were evaluated on their antiproliferative effects in a panel of prostate cancer cell lines. Compound 3r inhibited the proliferation of LNCaP, PC3 and DU145 cells at 20 µM, strongly, but also has strong cytotoxic effects on all tested cells.

Discovery of Novel Bicyclic Pyrazoles as Potent PIP5K1C Inhibitors.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) is generated by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) from phosphatidylinositol 4-phosphate (PI4P). Structurally diverse and selective inhibitors against PIP5Ks are required to further elucidate the therapeutic potential for PIP5K inhibition, although the effects of PIP5K inhibition on various diseases and their symptoms, such as cancer and chronic pain, have been reported. Our medicinal chemistry efforts led to novel and potent PIP5K1C inhibitors. Compounds 30 and 33 not only showed potent activity but also demonstrated low total clearance in mice and high levels of kinase selectivity. These compounds might serve as tools to further elucidate the complex biology and therapeutic potential of PIP5K inhibition.

Type I gamma phosphatidylinositol phosphate 5-kinase i5 controls cell sensitivity to interferon

The interferon signaling pathway is critical for host defense by serving diverse functions in both innate and adaptive immune responses. Here, we show that type I gamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme that synthesizes phosphatidylinositol-4,5-bisphosphate (PI4,5P2), controls the sensitivity to interferon in both human and mouse cells. PIPKIγi5 directly binds to the interferon-gamma (IFN-γ) downstream effector signal transducer and activator of transcription 1 (STAT1), which suppresses the STAT1 dimerization, IFN-γ-induced STAT1 nuclear translocation, and transcription of IFN-γ-responsive genes. Depletion of PIPKIγi5 significantly enhances IFN-γ signaling and strengthens an antiviral response. In addition, PIPKIγi5-synthesized PI4,5P2 can bind to STAT1 and promote the PIPKIγi5-STAT1 interaction. Similar to its interaction with STAT1, PIPKIγi5 is capable of interacting with other members of the STAT family, including STAT2 and STAT3, thereby suppressing the expression of genes mediated by these transcription factors. These findings identify the function of PIPKIγi5 in immune regulation.

The Silencing of GhPIP5K2 and GhPIP5K22 Weakens Abiotic Stress Tolerance in Upland Cotton (Gossypium hirsutum).

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks), essential enzymes in the phosphatidylinositol signaling pathway, are crucial for the abiotic stress responses and the overall growth and development of plants. However, the GhPIP5Ks had not been systematically studied, and their function in upland cotton was unknown. This study identified a total of 28 GhPIP5Ks, and determined their chromosomal locations, gene structures, protein motifs and cis-acting elements via bioinformatics analysis. A quantitative real-time PCR (qRT‒PCR) analysis showed that most GhPIP5Ks were upregulated under different stresses. A virus-induced gene silencing (VIGS) assay indicated that the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were significantly decreased, while malondialdehyde (MDA) content were significantly increased in GhPIP5K2- and GhPIP5K22-silenced upland cotton plants under abiotic stress. Furthermore, the expression of the stress marker genes GhHSFB2A, GhHSFB2B, GhDREB2A, GhDREB2C, GhRD20-1, GhRD29A, GhBIN2, GhCBL3, GhNHX1, GhPP2C, GhCBF1, GhSnRK2.6 and GhCIPK6 was significantly decreased in the silenced plants after exposure to stress. These results revealed that the silencing of GhPIP5K2 and GhPIP5K22 weakened the tolerance to abiotic stresses. These discoveries provide a foundation for further inquiry into the actions of the GhPIP5K gene family in regulating the response and resistance mechanisms of cotton to abiotic stresses.

Temporal involvement of phosphatidylinositol 4-phosphate 5-kinase γ in differentiation of Z-bands and myofilament bundles as well as intercalated discs in mouse heart at mid-gestation.

Considering the occurrence of serious heart failure in a gene knockout mouse of PIP5Kγ and in congenital abnormal cases in humans in which the gene was defective as reported by others, the present study attempted to localize PIP5Kγ in the heart during prenatal stages. It was done on the basis of the supposition that phenotypes caused by gene mutation of a given molecule are owed to the functional deterioration of selective cellular sites normally expressing it at significantly higher levels in wild mice. PIP5Kγ-immunoreactivity was the highest in the heart at E10 in contrast to almost non-significant levels of the immunoreactivity in surrounding organs and tissues such as liver. The immunoreactivity gradually weakened in the heart with the prenatal age, and it was at non-significant levels at newborn and postnatal stages. Six patterns in localization of distinct immunoreactivity for PIP5Kγ were recognized in cardiomyocytes: (1) its localization on the plasma membranes and subjacent cytoplasm without association with short myofibrils and (2) its localization on them as well as short myofibrils in association with them in cardiomyocytes of early differentiation at E10; (3) its spot-like localization along long myofibrils in cardiomyocytes of advanced differentiation at E10; (4) rare occurrences of such spot-like localization along long myofibrils in cardiomyocytes of advanced differentiation at E14; (5) its localization at Z-bands of long myofibrils; and (6) its localization at intercellular junctions including the intercalated discs in cardiomyocytes of advanced differentiation at E10 and E14, especially dominant at the latter stage. No distinct localization of PIP5Kγ-immunoreactivity of any patterns was seen in the heart at E18 and P1D. The present finding suggests that sites of PIP5Kγ-appearance and probably of its high activity in cardiomyocytes are shifted from the plasma membranes through short myofibrils subjacent to the plasma membranes and long myofibrils, to Z-bands as well as to the intercalated discs during the mid-term gestation. It is further suggested that PIP5Kγ is involved in the differentiation of myofibrils as well as intercellular junctions including the intercalated discs at later stages of the mid-term gestation. Failures in its involvement in the differentiation of these structural components are thus likely to cause the mid-term gestation lethality of the mutant mice for PIP5Kγ.

Marked alteration of phosphoinositide signaling-associated molecules in postmortem prefrontal cortex with bipolar disorder.

The etiology of bipolar disorder (BD) remains unknown; however, lipid abnormalities in BD have received increasing attention in recent years. In this study, we examined the expression levels of enzyme proteins associated with the metabolic pathway of phosphoinositides (PIs) and their downstream effectors, protein kinase B (Akt1) and glycogen synthase kinase 3β (GSK3β), which have been assumed to be the targets of mood stabilizers such as lithium, in the postmortem brains of patients with BD. The protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), Akt1, and GSK3β were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays in the prefrontal cortex (PFC). Specifically, PTEN, Akt1, GSK3β, and PIP5K1C were measured in seven BD patients and 48 controls. Additionally, PIK4CA was analyzed in 10 cases and 34 controls. PTEN expression levels were markedly decreased in the PFCs of patients with BD, whereas those of Akt and GSK3β were prominently elevated. Moreover, patients medicated with lithium exhibited higher Akt1 expression levels and lower PTEN expression levels in comparison with the untreated group. Our results suggest that the expression levels of Akt1/GSK3β and its upstream regulator PTEN are considerably altered.

Identification and Analysis of the Expression of the PIP5K Gene Family in Tomatoes.

To explore the function of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) in tomatoes, members of the tomato PIP5K family were identified and characterized using bioinformatic methods, and their expression patterns were also analyzed under salt stress and in different tissues. Twenty-one PIP5K members-namely, SlPIP5K1-SlPIP5K21-were identified from ten chromosomes, and these were divided into three groups according to a phylogenetic analysis. Further bioinformatic analysis showed four pairs of collinear relationships and fragment replication events among the SlPIP5K family members. To understand the possible roles of the SlPIP5Ks, a cis-acting element analysis was conducted, which indicated that tomato PIP5Ks could be associated with plant growth, hormones, and stress responses. We further validated the results of the in silico analysis by integrating RNA-seq and qRT-PCR techniques for salt- and hormone-treated tomato plants. Our results showed that SlPIP5K genes exhibited tissue- and treatment-specific patterns, and some of the SlPIP5Ks exhibited significantly altered expressions after our treatments, suggesting that they might be involved in these stresses. We selected one of the SlPIP5Ks that responded to our treatments, SlPIP5K2, to further understand its subcellular localization. Our results showed that SlPIP5K2 was located on the membrane. This study lays a foundation for the analysis of the biological functions of the tomato SlPIP5K genes and can also provide a theoretical basis for the selection and breeding of new tomato varieties and germplasm innovation, especially under salt stress.

Discovery of Potent and Selective Phosphatidylinositol 3-Phosphate 5-Kinase (PIKfyve) Inhibitors as Methuosis Inducers.

Cytoplasmic vacuolation-associated cell death, known as methuosis, offers a promising nonapoptotic approach for cancer treatment. In this study, we outline the synthesis and evaluation of potent methuosis-inducing compounds. These compounds selectively induce cell death, characterized by extensive cytoplasmic vacuolation in HeLa and MDA-MB-231 cells. Notably, compound L22 exhibited a remarkable interaction with PIKfyve kinase, boasting a Kd value of 0.47 nM, surpassing the positive controls D-13 and MOMIPP in potency. Furthermore, it is important to highlight that cell death induced by compound L22 is unequivocally attributed to methuosis as it differs from apoptosis, necrosis, or autophagy. Importantly, when administered orally, L22 effectively inhibited tumor growth in a HeLa xenograft model without any apparent signs of toxicity. These results underscore the potential of L22 as a valuable tool for in-depth investigations into the mechanisms of methuosis and as a promising lead compound to guide structural optimization.

Lipid kinases PIP5Ks and PIP4Ks: potential drug targets for breast cancer.

Phosphoinositides, a small group of lipids found in all cellular membranes, have recently garnered heightened attention due to their crucial roles in diverse biological processes and different diseases. Among these, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), the most abundant bis-phosphorylated phosphoinositide within the signaling system, stands notably connected to breast cancer. Not only does it serve as a key activator of the frequently altered phosphatidylinositol 3-kinase (PI3K) pathway in breast cancer, but also its conversion to phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3) is an important direction for breast cancer research. The generation and degradation of phosphoinositides intricately involve phosphoinositide kinases. PI(4,5)P2 generation emanates from the phosphorylation of PI4P or PI5P by two lipid kinase families: Type I phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and Type II phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). In this comprehensive review, we focus on these two lipid kinases and delineate their compositions and respective cellular localization. Moreover, we shed light on the expression patterns and functions of distinct isoforms of these kinases in breast cancer. For a deeper understanding of their functional dynamics, we expound upon various mechanisms governing the regulation of PIP5Ks and PIP4Ks activities. A summary of effective and specific small molecule inhibitors designed for PIP5Ks or PIP4Ks are also provided. These growing evidences support PIP5Ks and PIP4Ks as promising drug targets for breast cancer.

MARCKS and PI(4,5)P2 reciprocally regulate actin-based dendritic spine morphology.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P2. We observed that the concentration of free PI(4,5)P2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P2.

FAB1C, a phosphatidylinositol 3-phosphate 5-kinase, interacts with PIN-FORMEDs and modulates their lytic trafficking in Arabidopsis

PIN-FORMEDs (PINs) are auxin efflux carriers that asymmetrically target the plasma membrane (PM) and are critical for forming local auxin gradients and auxin responses. While the cytoplasmic hydrophilic loop domain of PIN (PIN-HL) is known to include some molecular cues (e.g., phosphorylation) for the modulation of PIN's intracellular trafficking and activity, the complexity of auxin responses suggests that additional regulatory modules may operate in the PIN-HL domain. Here, we have identified and characterized a PIN-HL-interacting protein (PIP) called FORMATION OF APLOID AND BINUCLEATE CELL 1C (FAB1C), a phosphatidylinositol-3-phosphate 5-kinase, which modulates PIN's lytic trafficking. FAB1C directly interacts with PIN-HL and is required for the polarity establishment and vacuolar trafficking of PINs. Unphosphorylated forms of PIN2 interact more readily with FAB1C and are more susceptible to vacuolar lytic trafficking compared to phosphorylated forms. FAB1C also affected lateral root formation by modulating the abundance of periclinally localized PIN1 and auxin maximum in the growing lateral root primordium. These findings suggest that a membrane-lipid modifier can target the cargo-including vesicle by directly interacting with the cargo and modulate its trafficking depending on the cargo's phosphorylation status.

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.