Phosphatidylinositol 4-phosphate 5-kinase Research Articles

SUMOylation is required for PIPK1γ-driven keratinocyte migration and growth.

PIPKIγ, a key member of the type I phosphatidylinositol 4-phosphate kinase (PIPKI) family that regulates the spatial-temporal generation of PIP2, has been implicated in diverse biological processes including cell survival, cell polarity, and cell migration. An essential role of PIPKIγ in tumor cells and nerve cells has been established in previous studies. However, the function and regulatory mechanism of PIPKIγ remains incompletely understood. Here, we showed that PIPKIγ can specifically associate with the SUMO-conjugating (E2) enzyme UBC9 and can be SUMOylated both invivo and invitro. We further identified that Lys-591 is the critical SUMO-acceptor site of PIPKIγ and that SUMO conjugation at this site is required for PIPKIγ-driven keratinocyte migration and growth. Mechanistically, SUMOylation deficiency significantly disrupts PIPKIγ-mediated generation of intracellular PIP2, rather than the subcellular translocation and protein stability of PIPKIγ. Our findings reveal that PIPKIγ is a novel SUMOylation target and highlight the essential role of PIPKIγ SUMOylation in human keratinocyte function, providing an important orientation for in-depth study of wound repair.

The Caenorhabditis elegans Tubby homolog dynamically modulates olfactory cilia membrane morphogenesis and phospholipid composition.

Plasticity in sensory signaling is partly mediated via regulated trafficking of signaling molecules to and from primary cilia. Tubby-related proteins regulate ciliary protein transport; however, their roles in remodeling cilia properties are not fully understood. We find that the C. elegans TUB-1 Tubby homolog regulates membrane morphogenesis and signaling protein transport in specialized sensory cilia. In particular, TUB-1 is essential for sensory signaling-dependent reshaping of olfactory cilia morphology. We show that compromised sensory signaling alters cilia membrane phosphoinositide composition via TUB-1-dependent trafficking of a PIP5 kinase. TUB-1 regulates localization of this lipid kinase at the cilia base in part via localization of the AP-2 adaptor complex subunit DPY-23. Our results describe new functions for Tubby proteins in the dynamic regulation of cilia membrane lipid composition, morphology, and signaling protein content, and suggest that this conserved family of proteins plays a critical role in mediating cilia structural and functional plasticity.

Membrane Recognition and Binding by the Phosphatidylinositol Phosphate Kinase PIP5K1A: A Multiscale Simulation Study

SummaryPhosphatidylinositol phosphates (PIPs) are lipid signaling molecules that play key roles in many cellular processes. PIP5K1A kinase catalyzes phosphorylation of PI4P to form PIP2, which in turn interacts with membrane and membrane-associated proteins. We explore the mechanism of membrane binding by the PIP5K1A kinase using a multiscale molecular dynamics approach. Coarse-grained simulations show binding of monomeric PIP5K1A to a model cell membrane containing PI4P. PIP5K1A did not bind to zwitterionic or anionic membranes lacking PIP molecules. Initial encounter of kinase and bilayer was followed by reorientation to enable productive binding to the PI4P-containing membrane. The simulations suggest that unstructured regions may be important for the preferred orientation for membrane binding. Atomistic simulations indicated that the dimeric kinase could not bind to the membrane via both active sites at the same time, suggesting a conformational change in the protein and/or bilayer distortion may be needed for dual-site binding to occur.

Phosphatidylinositol Kinases and Phosphatases in Entamoeba histolytica.

Phosphatidylinositol (PtdIns) metabolism is indispensable in eukaryotes. Phosphoinositides (PIs) are phosphorylated derivatives of PtdIns and consist of seven species generated by reversible phosphorylation of the inositol moieties at the positions 3, 4, and 5. Each of the seven PIs has a unique subcellular and membrane domain distribution. In the enteric protozoan parasite Entamoeba histolytica, it has been previously shown that the PIs phosphatidylinositol 3-phosphate (PtdIns3P), PtdIns(4,5)P2, and PtdIns(3,4,5)P3 are localized to phagosomes/phagocytic cups, plasma membrane, and phagocytic cups, respectively. The localization of these PIs in E. histolytica is similar to that in mammalian cells, suggesting that PIs have orthologous functions in E. histolytica. In contrast, the conservation of the enzymes that metabolize PIs in this organism has not been well-documented. In this review, we summarized the full repertoire of the PI kinases and PI phosphatases found in E. histolytica via a genome-wide survey of the current genomic information. E. histolytica appears to have 10 PI kinases and 23 PI phosphatases. It has a panel of evolutionarily conserved enzymes that generate all the seven PI species. However, class II PI 3-kinases, type II PI 4-kinases, type III PI 5-phosphatases, and PI 4P-specific phosphatases are not present. Additionally, regulatory subunits of class I PI 3-kinases and type III PI 4-kinases have not been identified. Instead, homologs of class I PI 3-kinases and PTEN, a PI 3-phosphatase, exist as multiple isoforms, which likely reflects that elaborate signaling cascades mediated by PtdIns(3,4,5)P3 are present in this organism. There are several enzymes that have the nuclear localization signal: one phosphatidylinositol phosphate (PIP) kinase, two PI 3-phosphatases, and one PI 5-phosphatase; this suggests that PI metabolism also has conserved roles related to nuclear functions in E. histolytica, as it does in model organisms.

Type Iγ phosphatidylinositol phosphate kinase promotes tumor growth by facilitating Warburg effect in colorectal cancer

BackgroundEmerging evidence suggests that metabolic alterations are a hallmark of cancer cells and contribute to tumor initiation and development. Cancer cells primarily utilize aerobic glycolysis (the Warburg effect) to produce energy and support anabolic growth. The type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) is profoundly implicated in tumorigenesis, however, little is known about its role in reprogrammed energy metabolism. MethodsLoss- and gain-of-function studies were applied to determine the oncogenic roles of PIPKIγ in colorectal cancer. Transcriptome analysis, real-time qPCR, immunohistochemical staining, Western blotting, and metabolic analysis were carried out to uncover the cellular mechanism of PIPKIγ. FindingsIn this study, we showed that PIPKIγ was frequently upregulated in colorectal cancer and predicted a poor prognosis. Genetic silencing of pan-PIPKIγ suppressed cell proliferation and aerobic glycolysis of colorectal cancer. In contrast, the opposite effects were observed by overexpression of PIPKIγ_i2. Importantly, PIPKIγ-induced prolific effect was largely glycolysis-dependent. Mechanistically, PIPKIγ facilitated activation of PI3K/Akt/mTOR signaling pathways to upregulate c-Myc and HIF1α levels, which regulate expression of glycolytic enzymes to enhance glycolysis. Moreover, pharmacological inhibition by PIPKIγ activity with the specific inhibitor UNC3230 significantly inhibited colorectal cancer glycolysis and tumor growth. InterpretationOur findings reveal a new regulatory role of PIPKIγ in Warburg effect and provide a key contributor in colorectal cancer metabolism with potential therapeutic potentials. FundNational Key Research and Development Program of China, Outstanding Clinical Discipline Project of Shanghai Pudong, Natural Science Foundation of China, and Science and Technology Commission of Shanghai Municipality.

The Small GTPase Arf6: An Overview of Its Mechanisms of Action and of Its Role in Host⁻Pathogen Interactions and Innate Immunity.

The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens. Arf6 is also used in the signaling of Toll-like receptors and in the activation of NADPH oxidases. In this review, we first give an overview of the different roles and mechanisms of action of Arf6 and then focus on its role in innate immunity and host–pathogen interactions.

Phosphatidylinositol 5 Phosphate 4-Kinase Regulates Plasma-Membrane PIP3 Turnover and Insulin Signaling

SummaryPhosphatidylinositol 3,4,5-trisphosphate (PIP3) generation at the plasma membrane is a key event during activation of receptor tyrosine kinases such as the insulin receptor required for normal growth and metabolism. We report that in Drosophila, phosphatidylinositol 5 phosphate 4-kinase (PIP4K) is required to limit PIP3 levels during insulin receptor activation. Depletion of PIP4K increases the levels of PIP3 produced in response to insulin stimulation. We find that PIP4K function at the plasma membrane enhances class I phosphoinositide 3-kinase (PI3K) activity, although the catalytic ability of PIP4K to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5) P2] at the plasma membrane is dispensable for this regulation. Animals lacking PIP4K show enhanced insulin signaling-dependent phenotypes and are resistant to the metabolic consequences of a high-sugar diet, highlighting the importance of PIP4K in normal metabolism and development. Thus, PIP4Ks are key regulators of receptor tyrosine kinase signaling with implications for growth factor-dependent processes including tumor growth, T cell activation, and metabolism.

PIP4Ks Suppress Insulin Signaling through a Catalytic-Independent Mechanism

SUMMARYInsulin stimulates the conversion of phosphatidylino-sitol-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-phos-phate 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 VMLϕFPDD 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.

Enrichment of Phosphatidylinositol 4,5-Bisphosphate in the Extra-Invasive Hyphal Membrane Promotes Colletotrichum Infection of Arabidopsis thaliana.

Pathogenic fungi from the genus Colletotrichum form invasive hyphae; the hyphae are surrounded by an extra-invasive hyphal membrane (EIHM), which is continuous with the plant plasma membrane. Although the EIHM plays a crucial role as the interface between plant and fungal cells, its precise function during Colletotrichum infection remains elusive. Here, we show that enrichment of phosphoinositides (PIs) has a crucial role in Colletotrichum infection. We observed the localization of PIs in Arabidopsis thaliana cells infected by A. thaliana-adapted Colletotrichum higginsianum (Ch), and found that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] was extremely enriched in the EIHM during Ch infection. We also found that phosphatidylinositol 4-phosphate-5 kinase (PIP5K), which catalyzes production of PI(4,5)P2, also accumulated at the EIHM. The overexpression of PIP5K3 in A. thaliana increased hyphal invasion by Ch. An exocytic factor, EXO84b, was targeted to the EIHM during Ch infection, although endocytic factors such as CLATHRIN LIGHT CHAIN 2 and FLOTILLIN 1 did not. Intriguingly, the interfacial membranes between A. thaliana and powdery mildew- or downy mildew-causing pathogens did not accumulate PI(4,5)P2. These results suggest that Ch could modify the PI(4,5)P2 levels in the EIHM to increase the exocytic membrane/protein supply of the EIHM for successful infection. Our results also suggest that PI(4,5)P2 biosynthesis is a promising target for improved defense against Colletotrichum infection.

PtdIns(4,5)P2 is generated by a novel phosphatidylinositol 4-phosphate 5-kinase in the protist parasite Entamoebahistolytica.

Entamoebahistolytica is an intestinal protist parasite that causes amoebiasis, a major source of morbidity and mortality in developing countries. Phosphoinositides are involved in signalling systems that have a role in invasion and pathogenesis of this parasite. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyses the generation of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2 ), a key species of phosphoinositide that regulates various cellular processes. However, phosphatidylinositol phosphate kinase (PIPK) family of enzymes have not been characterized in E.histolytica. Here, we report the identification and characterization of type I PIPK (EhPIPKI) of E.histolytica. Computational analysis revealed homologs of type I and III PIPK family in E.histolytica and the absence of type II PIPK. In spite of low overall sequence identity, the kinase domain was found to be highly conserved. Interestingly, a unique insertion of a tandem repeat motif was observed in EhPIPKI distinguishing it from existing PIPKs of other organisms. Substrate profiling showed that EhPIPKI could phosphorylate at third and fifth hydroxyl positions of phosphatidylinositols, though the predominant substrate was phosphatidylinositol 4-phosphate (PtdIns(4)P). Furthermore, EhPIPKI underwent intracellular cleavage close to the amino-terminal, generating twodistinct fragments Nter-EhPIPKI (27p) and Cter-EhPIPKI (47p). Immunofluorescence and cellular fractionation revealed that the full-length EhPIPKI and the Cter-EhPIPKI containing carboxyl-terminal activation loop were present in the plasma membrane while the Nter-EhPIPKI was observed in the cytosolic region. In conclusion, E.histolytica has a single EhPIPKI gene that displays novel properties of post-translational processing, the presence of a repeat domain and substrate specificity not observed in any PIPK enzyme so far.

A nuclear phosphoinositide kinase complex regulates p53.

The tumour suppressor p53 (encoded by TP53) protects the genome against cellular stress and is frequently mutated in cancer. Mutant p53 acquires gain-of-function oncogenic activities that are dependent on its enhanced stability. However, the mechanisms by which nuclear p53 is stabilized are poorly understood. Here, we demonstrate that the stability of stress-induced wild-type and mutant p53 is regulated by the type I phosphatidylinositol phosphate kinase (PIPKI-α (also known as PIP5K1A)) and its product phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Nuclear PIPKI-α binds to p53 upon stress, resulting in the production and association of PtdIns(4,5)P2 with p53. PtdIns(4,5)P2 binding promotes the interaction between p53 and the small heat shock proteins HSP27 (also known as HSPB1) and αB-crystallin (also known as HSPB5), which stabilize nuclear p53. Moreover, inhibition of PIPKI-α or PtdIns(4,5)P2 association results in p53 destabilization. Our results point to a previously unrecognized role of nuclear phosphoinositide signalling in regulating p53 stability and implicate this pathway as a promising therapeutic target in cancer.

Functional analysis of the biochemical activity of mammalian phosphatidylinositol 5 phosphate 4-kinase enzymes.

Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) are enzymes that catalyse the phosphorylation of phosphatidylinositol 5-phosphate (PI5P) to generate PI(4,5)P2. Mammalian genomes contain three genes, PIP4K2Α, 2B and 2C and murine knockouts for these suggested important physiological roles in vivo. The proteins encoded by PIP4K2A, 2B and 2C show widely varying specific activities in vitro; PIP4K2A is highly active and PIP4K2C 2000-times less active, and the relationship between this biochemical activity and in vivo function is unknown. By contrast, the Drosophila genome encodes a single PIP4K (dPIP4K) that shows high specific activity in vitro and loss of this enzyme results in reduced salivary gland cell size in vivo. We find that the kinase activity of dPIP4K is essential for normal salivary gland cell size in vivo. Despite their highly divergent specific activity, we find that all three mammalian PIP4K isoforms are able to enhance salivary gland cell size in the Drosophila PIP4K null mutant implying a lack of correlation between in vitro activity measurements and in vivo function. Further, the kinase activity of PIP4K2C, reported to be almost inactive in vitro, is required for in vivo function. Our findings suggest the existence of unidentified factors that regulate PIP4K enzyme activity in vivo.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α, β, γ in the three major salivary glands insitu of mice and their response to β-adrenoceptor stimulation.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which is composed of three isozymes (α, β and γ), catalyzes the production of phosphatidylinositol bisphosphate (PIP2). This phospholipid functions in membrane trafficking, as an anchor for actin cytoskeletons and as a regulator of intramembranous channels/transporters. It is also a precursor of such second messengers as diacylglycerol, inositol triphosphate and phosphatidylinositol (3,4,5)-triphosphate. In the present study, the expression and localization of endogenous PIP5Ks were examined in the three major salivary glands of young adult mice insitu. In western blotting of normal control glands, immunoreactive bands for individual PIP5Ks were detectable, with the highest density in the parotid gland and the weakest density in the submandibular gland. In immuno-light microscopy under non-stimulated condition, weak immunoreactivity for PIP5Kα was confined to the apical plasmalemma in parotid, but not sublingual or submandibular, acinar cells. Immunoreactivity for PIP5Kβ was weak to moderate and confined to ductal cells but not acinar cells, whereas that for PIP5Kγ was selectively and intensely detected in myoepithelial cells but not acinar cells, and it was weak in ductal cells in the three glands. In western blot of the parotid gland stimulated by isoproterenol, a β-adrenoceptor agonist, no changes were seen in the intensity of immunoreactive bands for any of the PIP5Ks. In contrast, in immuno-light microscopy, the apical immunoreactivity for PIP5Kα in parotid acinar cells was transiently and distinctly increased after the stimulation. The increased immunoreactivity was ultrastructurally localized on most apical microvilli and along contiguous plasma membrane, where membranous invaginations of various shapes and small vesicles were frequently found. It was thus suggested that PIP5Kα is involved in post-exocytotic membrane dynamics via microvillous membranes. The present finding further suggests that each of the three isoforms of PIP5K functions through its product PIP2 discretely in different cells of the glands to regulate saliva secretion.

Osh Proteins Control Nanoscale Lipid Organization Necessary for PI(4,5)P <sub>2</sub> Synthesis

The plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP/Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate, PI(4,5)P2, a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both ORP/Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Our biophysical and simulation analyses further reveal an unconventional co-segregation of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, we show that Osh protein-mediated unsaturated PI4P/PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP/Osh family members create an unsaturated phospholipid nanoscale environment that drives PIP5K activity and PI(4,5)P2 synthesis that ultimately controls global PM organization and dynamics.

CRISPR/Cas9 Unsettle PIP4K2A and α, β, and γ Globin Genes Expression

Phosphatidylinositol-phosphate kinases (PIPKins) belong to a family of lipid kinase enzymes that generate lipid messengers derivate from inositol, including the second messenger phosphatidylinositol-4,5-biphosphate [PI(4,5)P2], which participates in several cell regulation processes including gene expression. Previous studies developed in our laboratory suggested that the enzyme phosphatidylinositol-4-phosphate kinase-II-alpha (PIP4KIIα) may be related with the expression of the globin genes. Thus, the aim of this work was to evaluate the expression levels of the α, β and γ globin genes after disruption and overexpression of the PIP4KIIα gene (PIP4KIIA) in KU812 erythroleukemic cell line. The PIP4KIIα gene disruption was performed by using CRISPR/Cas9 and CRISPR/dCas9 techniques: KU812 cells were electro-transfected (Lonza 4D Nucleofector) with CRISPR/Cas9 lentiviral plasmid construct containing 3 gRNA targets to exon 8 of PIP4KIIA to knockout the gene; RNA samples from the pool of cells were collected and quantified by qPCR after 9 (t1) and 14 days (t2) of transfection. To overexpress PIP4K2A, CRISPR/dCas9 system (five lentiviral plasmids - 3 gRNAs targeting 214 nt upstream the transcriptional start site of PIP4K2A and 2 SAM accessorial factors) was transfected (Lipofectamine 3000) and RNA quantified by qPCR 24 (h1), 48 (h2) and 72 (h3) hours after the procedure. β-ACTIN and GAPDH genes were used as endogenous controls for qPCR. Our results with transfected cells without selection revealed, as expected, a reduction of PIP4KIIA compared to the control (no transfected cells - t1 and t2). The α globin genes presented a variation of 42% (increase in t1) and a 60% reduction in t2 (RQ = 1.42; RQ = 0.40, respectively), while β globin genes presented a 123% increase in t1 and remained similar to the control at t2 (RQ = 2.23; RQ = 1.01). The γ globin genes presented variation of 18% and 59% reduction in t1 and t2, respectively (RQ = 0.82; RQ = 0.41). The overexpression results suggested that PIP4KIIA leads to a higher γ globin expression in h2 and h3 (RQ = 3.26; RQ = 2.01) as well as a lower β globin expression in h1, h2 and h3 (RQ = 0.54; RQ = 0.002; RQ = 0.18), while a globin remained similar to the control at all analysed times (RQ variation of 1.26; 0.82 and 1.25, respectively). These results, although preliminary, suggest a possible relationship between PIP4KIIα and the regulation of expression of the globin genes. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp. DisclosuresNo relevant conflicts of interest to declare.

PtdIns(3,5)P2 mediates root hair shank hardening in Arabidopsis.

Root hairs elongate by tip growth and simultaneously harden the shank by constructing the inner secondary cell wall layer. While much is known about the process of tip growth1, almost nothing is known about the mechanism by which root hairs harden the shank. Here we show that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), the enzymatic product of FORMATION OF APLOID AND BINUCLEATE CELLS 1 (FAB1), is involved in the hardening of the shank in root hairs in Arabidopsis. FAB1 and PtdIns(3,5)P2 localize to the plasma membrane along the shank of growing root hairs. By contrast, phosphatidylinositol 4-phosphate 5-kinase 3 (PIP5K3) and PtdIns(4,5)P2 localize to the apex of the root hair where they are required for tip growth. Reduction of FAB1 function results in the formation of wavy root hairs while those of the wild type are straight. The localization of FAB1 in the plasma membrane of the root hair shank requires the activity of Rho-related GTPases from plants 10 (ROP10) and localization of ROP10 requires FAB1 activity. Computational modelling of root hair morphogenesis successfully reproduces the wavy root hair phenotype. Taken together, these data demonstrate that root hair shank hardening requires PtdIns(3,5)P2/ROP10 signalling.

Apical PtdIns(4,5)P2is required for ciliogenesis and suppression of polycystic kidney disease

Cilia are hair-like structures that function like antennae to detect chemical and mechanical signals in the environment. Recently, phosphoinositides were shown to play an important role in cilia assembly and disassembly. However, the precise molecular and cellular mechanisms underlying this process remain unknown. Here, we report that suppression of apical phosphatidylinositol 4,5- bisphosphate [PtdIns(4,5)P2], by overexpressing apically targeted PtdIns(4,5)P2 phosphatase or by knocking down type I phosphatidylinositol 4-phosphate 5-kinase (Pip5k1), leads to ciliogenesis defects and polycystic kidney disease (PKD) in zebrafish embryos that phenocopied inpp5e mutant, a Joubert syndrome model. We further demonstrate that decreased expression of apical PtdIns(4,5)P2 disrupted apical ezrin recruitment, F-actin organization, and basal body docking. Moreover, the ciliogenesis and polycystic kidney defects in PtdIns(4,5)P2-depleted embryos can be rescued by overexpression of ezrin. Finally, Pip5k1a overexpression rescued the ciliogenesis defects and PKD phenotypes in Inpp5e-depleted embryos. Taken together, our results reveal that apical PtdIns(4,5)P2 is essential for ciliogenesis and the prevention of PKD and suggest a novel possibility for treating PKD and other human ciliopathies.-Xu, W., Jin, M., Huang, W., Wang, H., Hu, R., Li, J., Cao, Y. Apical PtdIns(4,5)P2 is required for ciliogenesis and suppression of polycystic kidney disease.

Fluconazole-induced actin cytoskeleton remodeling requires phosphatidylinositol 3-phosphate 5-kinase in the pathogenic yeast Candida glabrata.

SummaryKnown azole antifungal resistance mechanisms include mitochondrial dysfunction and overexpression of the sterol biosynthetic target enzyme and multidrug efflux pumps. Here, we identify, through a genetic screen, the vacuolar membrane‐resident phosphatidylinositol 3‐phosphate 5‐kinase (CgFab1) to be a novel determinant of azole tolerance. We demonstrate for the first time that fluconazole promotes actin cytoskeleton reorganization in the emerging, inherently less azole‐susceptible fungal pathogen Candida glabrata, and genetic or chemical perturbation of actin structures results in intracellular sterol accumulation and azole susceptibility. Further, CgFAB1 disruption impaired vacuole homeostasis and actin organization, and the F‐actin‐stabilizing compound jasplakinolide rescued azole toxicity in cytoskeleton defective‐mutants including the Cgfab1Δ mutant. In vitro assays revealed that the actin depolymerization factor CgCof1 binds to multiple lipids including phosphatidylinositol 3,5‐bisphosphate. Consistently, CgCof1 distribution along with the actin filament‐capping protein CgCap2 was altered upon both CgFAB1 disruption and fluconazole exposure. Altogether, these data implicate CgFab1 in azole tolerance through actin network remodeling. Finally, we also show that actin polymerization inhibition rendered fluconazole fully and partially fungicidal in azole‐susceptible and azole‐resistant C. glabrata clinical isolates, respectively, thereby, underscoring the role of fluconazole‐effectuated actin remodeling in azole resistance.

Multiple Aspects of PIP2 Involvement in C. elegans Gametogenesis.

One of the most studied phosphoinositides is phosphatidylinositol 4,5-bisphosphate (PIP2), which localizes to the plasma membrane, nuclear speckles, small foci in the nucleoplasm, and to the nucleolus in mammalian cells. Here, we show that PIP2 also localizes to the nucleus in prophase I, during the gametogenesis of C. elegans hermaphrodite. The depletion of PIP2 by type I PIP kinase (PPK-1) kinase RNA interference results in an altered chromosome structure and leads to various defects during meiotic progression. We observed a decreased brood size and aneuploidy in progeny, defects in synapsis, and crossover formation. The altered chromosome structure is reflected in the increased transcription activity of a tightly regulated process in prophase I. To elucidate the involvement of PIP2 in the processes during the C. elegans development, we identified the PIP2-binding partners, leucine-rich repeat (LRR-1) protein and proteasome subunit beta 4 (PBS-4), pointing to its involvement in the ubiquitin–proteasome pathway.

Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability

In human cancers, oncogenic mutations commonly occur in the RAS genes KRAS, NRAS, or HRAS, but there are no clinical RAS inhibitors. Mutations are more prevalent in KRAS, possibly suggesting a unique oncogenic activity mediated by KRAS-specific interaction partners, which might be targeted. Here, we determine the specific protein interactomes of each RAS isoform by BirA proximity-dependent biotin identification. The combined interactomes are screened by CRISPR-Cas9 loss-of-function assays for proteins required for oncogenic KRAS-dependent, NRAS-dependent, or HRAS-dependent proliferation and censored for druggable proteins. Using this strategy, we identify phosphatidylinositol phosphate kinase PIP5K1A as a KRAS-specific interactor and show that PIP5K1A binds to a unique region in KRAS. Furthermore, PIP5K1A depletion specifically reduces oncogenic KRAS signaling and proliferation, and sensitizes pancreatic cancer cell lines to a MAPK inhibitor. These results suggest PIP5K1A as a potential target in KRAS signaling for the treatment of KRAS-mutant cancers.