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1
Kawase, Atsushi, Yuta Inoue, Miho Hirosoko, Yuka Sugihara, Hiroaki Shimada, and Masahiro Iwaki. "Decrease in Multidrug Resistance-associated Protein 2 Activities by Knockdown of Phosphatidylinositol 4-phosphate 5-kinase in Hepatocytes and Cancer Cells." Journal of Pharmacy & Pharmaceutical Sciences 22 (November 19, 2019): 576–84. http://dx.doi.org/10.18433/jpps30444.
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Purpose: The plasma membrane localization and transport activity of multidrug resistance-associated protein 2 (MRP2/ABCC2) and P-glycoprotein (P-gp/ABCB1) efflux transporters are governed by transporter-associated proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) formed by phosphatidylinositol 4-phosphate 5-kinase type 1 (PIP5K1) activates the linker function of radixin for efflux transporters. Radixin is involved in the plasma membrane localization of efflux transporters. We examined whether PIP5K1 could be a target for the modulation of transporter activities in hepatocytes and cancer cells. Methods: The effects of PIP5K1 depletion by siRNA in mouse primary hepatocytes, PANC1 human pancreatic carcinoma cells, and HepG2 human hepatocellular carcinoma cells on the intracellular accumulation of MRP2 and P-gp substrates were examined. Results: PIP5K1A depletion resulted in increased intracellular accumulation of carboxydichlorofluorescein, a MRP2 fluorescent substrate, in mouse primary hepatocytes, PANC1 cells, and HepG2 cells. In PANC1 and HepG2 cells, the transport activities of MRP2 were significantly decreased by PIP5K1C depletion. However, the transport activities of P-gp were unchanged by PIP5K1 depletion. PIP2 levels were unchanged between control and PIP5K1A- or PIP5K1C-depleted HepG2 cells. MRP2 mRNA levels showed few changes in HepG2 cells following PIP5K1A or PIP5K1C depletion. The expression of phosphorylated radixin was decreased by PIP5K1A and PIP5K1C depletion, although total radixin levels were unchanged. Conclusions: These data suggest that PIP5K1A and PIP5K1C could be target proteins for modulating MRP2 function, partly because of the resulting changes of the linker function of radixin.
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Wright, Brittany D., Catherine Simpson, Michael Stashko, Dmitri Kireev, Emily A. Hull-Ryde, Mark J. Zylka, and William P. Janzen. "Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C." Journal of Biomolecular Screening 20, no. 5 (December 22, 2014): 655–62. http://dx.doi.org/10.1177/1087057114564057.
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Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes, including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Because small-molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel, high-throughput, microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay uses fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated adenosine triphosphate Michaelis constant (Km) of 15 µM, performed with z’ values >0.7, and was used to screen a kinase-focused library of ~4700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be adapted easily to screen additional lipid kinases.
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Khadka, Bijendra, and Radhey S. Gupta. "Novel Molecular Signatures in the PIP4K/PIP5K Family of Proteins Specific for Different Isozymes and Subfamilies Provide Important Insights into the Evolutionary Divergence of this Protein Family." Genes 10, no. 4 (April 21, 2019): 312. http://dx.doi.org/10.3390/genes10040312.
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Members of the PIP4K/PIP5K family of proteins, which generate the highly important secondary messenger phosphatidylinositol-4,5-bisphosphate, play central roles in regulating diverse signaling pathways. In eukaryotic organisms, multiple isozymes and subfamilies of PIP4K/PIP5K proteins are found and it is of much interest to understand their evolution and species distribution and what unique molecular and biochemical characteristics distinguish specific isozymes and subfamilies of proteins. We report here the species distribution of different PIP4K/PIP5K family of proteins in eukaryotic organisms and phylogenetic analysis based on their protein sequences. Our results indicate that the distinct homologs of both PIP4K and PIP5K are found in different organisms belonging to the Holozoa clade of eukaryotes, which comprises of various metazoan phyla as well as their close unicellular relatives Choanoflagellates and Filasterea. In contrast, the deeper-branching eukaryotic lineages, as well as plants and fungi, contain only a single homolog of the PIP4K/PIP5K proteins. In parallel, our comparative analyses of PIP4K/PIP5K protein sequences have identified six highly-specific molecular markers consisting of conserved signature indels (CSIs) that are uniquely shared by either the PIP4K or PIP5K proteins, or both, or specific subfamilies of these proteins. Of these molecular markers, 2 CSIs are distinctive characteristics of all PIP4K homologs, 1 CSI distinguishes the PIP4K and PIP5K homologs from the Holozoa clade of species from the ancestral form of PIP4K/PIP5K found in deeper-branching eukaryotic lineages. The remaining three CSIs are specific for the PIP5Kα, PIP5Kβ, and PIP4Kγ subfamilies of proteins from vertebrate species. These molecular markers provide important means for distinguishing different PIP4K/PIP5K isozymes as well as some of their subfamilies. In addition, the distribution patterns of these markers in different isozymes provide important insights into the evolutionary divergence of PIP4K/PIP5K proteins. Our results support the view that the Holozoa clade of eukaryotic organisms shared a common ancestor exclusive of the other eukaryotic lineages and that the initial gene duplication event leading to the divergence of distinct types of PIP4K and PIP5K homologs occurred in a common ancestor of this clade. Based on the results gleaned from different studies presented here, a model for the evolutionary divergence of the PIP4K/PIP5K family of proteins is presented.
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Wang, Yanfeng, Lurong Lian, Aae Suzuki, Rustem I. Litvinov, Timothy J. Stalker, Alec A. Schmaier, Lawrence F. Brass, John Weisel, and Charles S. Abrams. "Loss of Individual PIP5KI Isoforms Demonstrate That Spatial PIP2 Synthesis Is Required for Platelet Second Messenger Formation & Integrity of the Actin Cytoskeleton." Blood 112, no. 11 (November 16, 2008): 109. http://dx.doi.org/10.1182/blood.v112.11.109.109.
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Abstract Following thrombin stimulation, platelet PIP5KI synthesizes phosphatidylinositol 4,5-bisphosphate (PIP2), which can be hydrolyzed by phospholipase C to generate second messengers such as IP3. PIP2 also regulates cytoskeletal dynamics by directly interacting with actin-binding proteins. Three isoforms of PIP5KI (α, β, and γ) are all capable of phosphorylating PI4P to synthesize PIP2. However, these isoforms have different primary structures, expression levels in various tissues, and intracellular localization. We have generated and characterized murine lines lacking PIP5KIβ or PIP5KIγ, which are the two predominant platelet isoforms. We also phenotyped platelets lacking PIP5KIα, which is the least abundant isoform. PIP5KIβ-null mice appeared developmentally normal and had normal platelet counts, however they had small defects in aggregation following exposure to all agonists. In contrast, platelets lacking PIP5KIα aggregated normally. Although platelets lacking PIP5KIβ have only a moderate deficiency of PIP2 under basal conditions, they have a striking deficiency in PIP2 synthesis and IP3 formation following thrombin stimulation. We have also observed that platelets lacking both PIP5KIα and PIP5KIβ have a complete loss of thrombin-induced IP3 synthesis, even though they still contain PIP5KIγ, which is the predominant PIP5KI isoform in platelets. Additionally, we found when using a carotid injury model that PIP5KIβ-null platelets failed to properly form arterial thrombi in vivo. This demonstrates that PIP5KIβ, like PIP5KIα, contributes to the rapid synthesis of a pool of PIP2 that is required for second messenger formation and in vivo platelet adhesion. This contrasts the PIP5KIγ-synthesized pool of PIP2 that does not contribute to these processes. We have found that loss of PIP5KIγ null mutation impairs cardiac development and leads to embryonic lethality. PIP5KIγ null megakaryocytes derived from yolk sac progenitor cells have a defect in anchoring their cell membranes to the underlying actin cytoskeleton. To understand the role of this PIP5KI isoform in platelet biology, we conditionally rescued the PIP5KIγ null mutation within myocardiocytes allowing us to obtain living mice. Platelets from these animals lacked PIP5KIγ, yet aggregated normally when exposed to all agonists. To analyze these cells for a failure to anchor their cell membranes, we used laser tweezers to pull the cell membrane apart from the cytoskeleton. Wild type cells had rigid membranes that resisted stretching by trapped fibrinogen-coated beads that were pulled by laser tweezers. In contrast, the PIP5KIγ-null platelets had flexible membranes that were easily stretched, and ultimately allowed membrane tethers to form. Together, these results demonstrate that following stimulation of a G-protein coupled receptor, IP3 is completely derived from a rapidly synthesized discrete pool of PIP2 that is synthesized by PIP5KIα and PIP5KIβ. In contrast, the pool of PIP2 synthesized by PIP5KIγ contributes to preserving the integrity of the membrane cytoskeleton. In conclusion, this work demonstrates that spatially restricted PIP2 synthesis by individual PIP5KI isoforms differentially controls second messenger formation and the integrity of the actin cytoskeleton.
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Padrón, David, Ying Jie Wang, Masaya Yamamoto, Helen Yin, and Michael G. Roth. "Phosphatidylinositol phosphate 5-kinase Iβ recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis." Journal of Cell Biology 162, no. 4 (August 11, 2003): 693–701. http://dx.doi.org/10.1083/jcb.200302051.
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Overexpression of phosphatidylinositol phosphate 5-kinase (PIP5KI) isoforms α, β, or γ in CV-1 cells increased phosphatidylinositol 4,5-bisphosphate (PIP2) levels by 35, 180, and 0%, respectively. Endocytosis of transferrin receptors, association of AP-2 proteins with membranes, and the number of clathrin-coated pits at the plasma membrane increased when PIP2 increased. When expression of PIP5KIβ was inhibited with small interference RNA in HeLa cells, expression of PIP5KIα was also reduced slightly, but PIP5KIγ expression was increased. PIP2 levels and internalization of transferrin receptors dropped 50% in these cells; thus, PIP5KIγ could not compensate for loss of PIP5KIβ. When expression of PIP5KIα was reduced, expression of both PIP5KIβ and PIP5KIγ increased and PIP2 levels did not change. A similar increase of PIP5KIα and PIP5KIβ occurred when PIP5KIγ was inhibited. These results indicate that constitutive endocytosis in CV-1 and HeLa cells requires (and may be regulated by) PIP2 produced primarily by PIP5KIβ.
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Clarke, Jonathan H., Piers C. Emson, and Robin F. Irvine. "Localization of phosphatidylinositol phosphate kinase IIγ in kidney to a membrane trafficking compartment within specialized cells of the nephron." American Journal of Physiology-Renal Physiology 295, no. 5 (November 2008): F1422—F1430. http://dx.doi.org/10.1152/ajprenal.90310.2008.
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PIP4Ks (type II phosphatidylinositol 4-phosphate kinases) are phosphatidylinositol 5-phosphate (PtdIns5P) 4-kinases, believed primarily to regulate cellular PtdIns5P levels. In this study, we investigated the expression, localization, and associated biological activity of the least-studied PIP4K isoform, PIP4Kγ. Quantitative RT-PCR and in situ hybridization revealed that compared with PIP4Kα and PIP4Kβ, PIP4Kγ is expressed at exceptionally high levels in the kidney, especially the cortex and outer medulla. A specific antibody was raised to PIP4Kγ, and immunohistochemistry with this and with antibodies to specific kidney cell markers showed a restricted expression, primarily distributed in epithelial cells in the thick ascending limb and in the intercalated cells of the collecting duct. In these cells, PIP4Kγ had a vesicular appearance, and transfection of kidney cell lines revealed a partial Golgi localization (primarily the matrix of the cis-Golgi) with an additional presence in an unidentified vesicular compartment. In contrast to PIP4Kα, bacterially expressed recombinant PIP4Kγ was completely inactive but did have the ability to associate with active PIP4Kα in vitro. Overall our data suggest that PIP4Kγ may have a function in the regulation of vesicular transport in specialized kidney epithelial cells.
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Bultsma, Yvette, Willem-Jan Keune, and Nullin Divecha. "PIP4Kβ interacts with and modulates nuclear localization of the high-activity PtdIns5P-4-kinase isoform PIP4Kα." Biochemical Journal 430, no. 2 (August 13, 2010): 223–35. http://dx.doi.org/10.1042/bj20100341.
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The β-isoform of PIP4K (PtdIns5P-4-kinase) regulates the levels of nuclear PtdIns5P, which in turn modulates the acetylation of the tumour suppressor p53. The crystal structure of PIP4Kβ demonstrated that it can form a homodimer with the two subunits arranged in opposite orientations. Using MS, isoform-specific antibodies against PIP4Ks, RNAi (RNA interference) suppression and overexpression studies, we show that PIP4Kβ interacts in vitro and in vivo with the PIP4Kα isoform. As the two isoforms phosphorylate the same substrate to generate the same product, the interaction could be considered to be functionally redundant. However, contrary to expectation, we find that PIP4Kβ has 2000-fold less activity towards PtdIns5P compared with PIP4Kα, and that the majority of PIP4K activity associated with PIP4Kβ comes from its interaction with PIP4Kα. Furthermore, PIP4Kβ can modulate the nuclear localization of PIP4Kα, and PIP4Kα has a role in regulating PIP4Kβ functions. The results of the present study suggest a rationale for the functional interaction between PIP4Kα and PIP4Kβ and provide insight into how the relative levels of the two enzymes may be important in their physiological and pathological roles.
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Chen, Xinsheng, Yanfeng Wang, Tami L. Bach, Lurong Lian, Rustem I. Litvinov, John W. Weisel, and Charles S. Abrams. "Mice Lacking PIP5Kβ or PIP5Kγ Have Unique Cytoskeletal Changes within Their Megakaryocytes & Platelets." Blood 106, no. 11 (November 16, 2005): 380. http://dx.doi.org/10.1182/blood.v106.11.380.380.
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Abstract Phosphatidylinositol-4, 5-bisphosphate (PIP2) is vital for the signaling cascades that are critical for actin dynamics. Although all PIP5K isoforms (α , β , and γ ) synthesize PIP2 by phosphorylating PI4P, the isoforms have different primary structures, expression levels in various tissues, and intracellular localization. To test the hypothesis that the functions of these isoforms are also different, we have generated murine lines that contain null mutations in either of two platelet PIP5K isoforms, PIP5Kβ and PIP5Kγ . PIP5Kβ -null mice were born 42% less than anticipated by Mendelian genetics, and heterozygotes were born 18% less than the expected frequency. Adult PIP5Kβ −/− mice had normal platelet counts and exhibited no spontaneous hemorrhage. However, PIP5Kβ −/− platelets had impaired aggregation in response to thrombin and thromboxane analogues. In addition, following agonist stimulation they failed to properly synthesize additional PIP2. Platelets lacking PIP5Kβ also exhibited a marked defect in spreading upon immobilized fibrinogen. This failure to spread was associated with a striking decrease in filopodia formation and extension of actin rich lamellipodia. In contrast to the phenotype of PIP5Kβ -knockout mice, we found that targeted disruption of PIP5Kγ results in early prenatal mortality due to a cardiovascular developmental defect. This early lethality prevented studies of hematopoietic cells derived from the bone marrow or the liver. However, we were able to analyze yolk sac progenitor cells that were treated with thrombopoietin ex vivo, differentiating them into megakaryocytes. Similar to PIP5Kβ knockout megakaryocytes, PIP5Kγ knockout megakaryocytes had normal basal levels of PIP2, but decreased synthesis following stimulation with thrombin. Using spinning disk video confocal microscopy, we examined membrane dynamics during cell adhesion in real time. Wild type megakaryocytes actively formed and contracted lamellipodia, and rapidly spread upon the fibrinogen matrix. In contrast, PIP5Kγ -null megakaryocytes continuously extended and retracted membrane blebs, rather than lamellipodia, but eventually spread as much as wild type cells. This observation is consistent with the previous suggestion that PIP2 contributes to the stable association of the membrane with the cytoskeleton. Using laser tweezers to pull the cell membrane apart from the cytoskeleton, we found that long tethers of the membrane could easily be drawn from PIP5Kγ −/− megakaryocytes, but not from PIP5Kγ +/− or PIP5Kβ −/− megakaryocytes. This implies that PIP5Kγ −/− megakaryocytes have a defect in their ability to anchor the cell membrane to the cytoskeleton. This defect was attributable to the ability of PIP5Kγ to synthesize PIP2 since it could be rescued by adding back wild type PIP5Kγ , but could not be rescued by a catalytically inactive PIP5Kγ mutant. Accordingly, our data are consistent with the hypothesis that different PIP5K isoforms contribute to compartmentalized pools of PIP2 that contribute to different aspect of platelet &megakaryocyte actin dynamics. PIP5Kβ synthesizes PIP2 that contributes to the formation of filopodia and lamellipodia, while PIP5Kγ generates PIP2 that is required for the stable association of the membrane with the cytoskeleton.
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Wang, Yanfeng, Aae Suzuki, Lurong Lian, Rustem I. Litvinov, Timothy J. Stalker, John K. Choi, John W. Weisel, Lawrence F. Brass, and Charles S. Abrams. "Platelets Lacking PIP5KIγ Have Impaired Cytoskeletal Dynamics and Adhesion, but No Defect in Integrin Activation." Blood 114, no. 22 (November 20, 2009): 772. http://dx.doi.org/10.1182/blood.v114.22.772.772.
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Abstract Abstract 772 Following thrombin stimulation, platelet PIP5KI synthesizes phosphatidylinositol 4,5-bisphosphate (PIP2), which can be hydrolyzed by phospholipase C to generate second messengers such as IP3. PIP2 also regulates cytoskeletal dynamics by directly interacting with actin-binding proteins such as talin. Three isoforms of PIP5KI (α, β, and γ) are all capable of phosphorylating PI4P to synthesize PIP2. We have generated and characterized murine lines lacking individual PIP5KI isoforms. While mice lacking PIP5KIα and PIP5KIα have absent second messenger formation and partially impaired integrin activation, they are viable. In contrast, mice lacking PIP5KIγ die in utero due to a cardiovascular developmental defect. Megakaryocytes derived from PIP5KIγ-null embryos bleb their membranes due to impaired anchoring of the cell membrane with the underlying cytoskeleton. Since platelets can not be obtained from these embryos, we employed a genetic approach. We used a MLC-2v Cre transgene that targets Cre expression to myocytes, and generated living mice lacking PIP5KIγ by a conditional rescue. PIP5KIγ-/- MLC-2v Cre+ mice expressed PIP5KIγ in the myocardium, but they had absent expression of PIP5KIγ in all other analyzed tissue. These mice had normal appearing hearts, brains, livers, and bone marrow morphology, as well as normal platelet counts. Since mice lacking PIP5KIα and PIP5KIβ have impaired platelet PIP2 production that causes absent IP3 formation, we analyzed platelets lacking PIP5KIγ for second messenger formation. Even though PIP5KIγ an abundant PIP5KI isoform in platelets, loss of PIP5KIγ does not affect IP3 formation or Akt phosphorylation. It has been previously demonstrated that PIP5KIγ can directly bind talin, a protein that regulates the function of integrins. An existing proposed model for integrin activation is that talin-associated PIP5KIγ synthesize PIP2. This newly synthesized PIP2 then binds a FERM domain within talin. The model suggests that this complex of PIP5KIγ-PIP2-talin is critical for the final step that stimulates integrins to bind their ligand. We found three lines of evidence that disprove this model of integrin activation. First, we found that PIP5KIγ-/- platelets had normal integrin-mediated aggregation in response to all analyzed doses of thrombin, ADP, collagen, and a thromboxane analogue (U46619). Second, we observed that PIP5KIγ-null platelets exhibited normal binding of Jon/A, an antibody that recognizes only the activated form of αIIb/β3. Third, we determined that platelets lacking PIP5KIγ spread normally upon adherent fibrinogen. Together, these results disprove the existing model that PIP5KIγ is a critical component of talin-mediated integrin activation. To determine the true function of PIP5KIγ within platelets, we extended our previous studies by analyzing the role PIP5KIγ plays in the regulation of the cytoskeleton. Therefore, we analyzed platelets lacking this enzyme for their ability to anchor the cytoskeleton to the cell membrane. We used optical tweezers to pull the cell membrane apart from the cytoskeleton. Wild type cells had rigid membranes that resisted stretching by trapped fibrinogen-coated beads that were pulled by the optical trap. In contrast, the PIP5KIγ-null platelets had flexible membranes that were easily stretched, and ultimately allowed membrane tethers to form. We further analyzed whether this defect in anchoring the cell membrane to the underlying cytoskeleton causes a defect in vivo using a carotid artery arterial injury model. Mice lacking platelet PIP5KIγ exhibited unstable adhesion in vivo suggesting that impaired cytoskeletal dynamics causes impaired platelet adhesion under flow. Together, our studies demonstrate that the abundant PIP5KI isoform, PIP5KIγ does not contribute to a pool of PIP2 required for second messenger formation or integrin activation. However it does synthesize the pool of PIP2 required to preserve the integrity of the membrane cytoskeleton, and support stable platelet adhesion under conditions of shear. Disclosures: No relevant conflicts of interest to declare.
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Drake, J. M., and J. Huang. "PIP5K1 inhibition as a therapeutic strategy for prostate cancer." Proceedings of the National Academy of Sciences 111, no. 35 (August 12, 2014): 12578–79. http://dx.doi.org/10.1073/pnas.1413363111.
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Aikawa, Yoshikatsu, and Thomas F. J. Martin. "ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis." Journal of Cell Biology 162, no. 4 (August 18, 2003): 647–59. http://dx.doi.org/10.1083/jcb.200212142.
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ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIγ restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIγ with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.
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Fairn, Gregory D., Koji Ogata, Roberto J. Botelho, Philip D. Stahl, Richard A. Anderson, Pietro De Camilli, Tobias Meyer, Shoshana Wodak, and Sergio Grinstein. "An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis." Journal of Cell Biology 187, no. 5 (November 30, 2009): 701–14. http://dx.doi.org/10.1083/jcb.200909025.
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Plasmalemmal phosphatidylinositol (PI) 4,5-bisphosphate (PI4,5P2) synthesized by PI 4-phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-γ prevented the disappearance of PI4,5P2 and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.
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Wang, Yanfeng, Rustem Litvinov, John W. Weisel, John H. Hartwig, and Charles S. Abrams. "PIP5KIγ Knockout Megakaryocytes Have Defects in Their Cytoskeleton & Demarcation Membrane System, yet Form Proplatlets & Platelets." Blood 108, no. 11 (November 16, 2006): 1793. http://dx.doi.org/10.1182/blood.v108.11.1793.1793.
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Abstract Phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical component of intracellular signaling cascades as well as actin dynamics. It is synthesized by the phosphorylation of PI4P by one of three isoforms of PIP5KI (α,β, or γ). We found that the targeted disruption of PIP5KIγ results in early prenatal mortality that prevented studies of bone marrow or liver hematopoietic cells. However, we were able to analyze yolk sac progenitor cells differentiated ex vivo into megakaryocytes. Imaging in the electron microscope showed that PIP5KIγ-null megakaryocytes have normal architectural appearance of their microtubules, and formed proplatelets and platelets. This demonstrates that PIP5KIγ is not essential for the development of megakaryocytes, proplatelets, or platelets. PIP5KIγ knockout megakaryocytes had normal basal levels of PIP2, but decreased synthesis following stimulation with thrombin. Using spinning disk video confocal microscopy, we found that wild type megakaryocytes actively formed and contracted lamellipodia, and rapidly spread upon a fibrinogen matrix. In contrast, PIP5KIγ-null megakaryocytes continuously and slowly extended and retracted membrane blebs, rather than lamellipodia. We analyzed whether PIP5KIγ-null megakaryocytes have a defect in their ability to anchor their membranes to the cytoskeleton accounting for the blebbing phenotype by using laser tweezers to pull the cell membrane apart from the cytoskeleton. Fibrinogen-coated beads bound both wild type and PIP5KIγ-null megakaryocytes. Yet wild type cells had rigid membranes that resisted stretching by trapped beads that were pulled by laser tweezers. In contrast, the majority of PIP5KIγ-null megakaryocytes had flexible membranes that were easily stretched by pulling on the fibrinogen-coated beads, and ultimately allowed membrane tethers to form. We found that the membrane phenotype was completely reverted by adding back wild type PIP5KIγ, but not by a catalytically inactive PIP5KIγ mutant. This shows that the lipid kinase activity of PIP5KIγ regulates the membrane phenotype. Surprisingly, PIP5KIβ-null megakaryocytes have at least as great of a defect in PIP2 synthesis but lack this membrane abnormality. Furthermore, overexpression of PIP5KIβ in PIP5KIγ-null megakaryocytes fails to rescue the cytoskeletal defect. Adding back a splice variant of PIP5KIγ that lacks the binding site for talin, also fails to revert this cytoskeletal defect present in PIP5KIγ-null cells. This suggests that talin binding and PIP2 synthesized specifically by the PIP5KIγ isoform are required for the cytoskeletal organization of megakaryocytes. Given the proposed role of PIP2 on the regulation of the demarcation membrane system (DMS), we expressed GFP-fused to β3-integrin and followed its trafficking from the DMS to the surface membrane. GFP-β3 situated on the DMS externalized to the cellular membrane within minutes of plating a wild type megakaryocyte upon immobilized fibrinogen. In megakaryocytes lacking PIP5KIγ, GFP-β3 situated on the DMS poorly externalized, and was barely detectable on the cell membrane 1 hour after adhering to immobilized fibrinogen. This shows that trafficking of αIIbβ3 from the DMS to the cell membrane requires PIP5KIγ. Together, this demonstrates that spatially restricted production of PIP2 by PIP5KIγ is required to preserve the integrity of the membrane cytoskeleton, and that PIP2 synthesized by PIP5KIβ in megakaryocytes can not contribute to this process.
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Zeng, Xuankun, Arzu Uyar, Dexin Sui, Nazanin Donyapour, Dianqing Wu, Alex Dickson, and Jian Hu. "Structural insights into lethal contractural syndrome type 3 (LCCS3) caused by a missense mutation of PIP5Kγ." Biochemical Journal 475, no. 14 (July 25, 2018): 2257–69. http://dx.doi.org/10.1042/bcj20180326.
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Signaling molecule phosphatidylinositol 4,5-bisphosphate is produced primarily by phosphatidylinositol 4-phosphate 5-kinase (PIP5K). PIP5K is essential for the development of the human neuronal system, which has been exemplified by a recessive genetic disorder, lethal congenital contractural syndrome type 3, caused by a single aspartate-to-asparagine mutation in the kinase domain of PIP5Kγ. So far, the exact role of this aspartate residue has yet to be elucidated. In this work, we conducted structural, functional and computational studies on a zebrafish PIP5Kα variant with a mutation at the same site. Compared with the structure of the wild-type (WT) protein in the ATP-bound state, the ATP-associating glycine-rich loop of the mutant protein was severely disordered and the temperature factor of ATP was significantly higher. Both observations suggest a greater degree of disorder of the bound ATP, whereas neither the structure of the catalytic site nor the Km toward ATP was substantially affected by the mutation. Microsecond molecular dynamics simulation revealed that negative charge elimination caused by the mutation destabilized the involved hydrogen bonds and affected key electrostatic interactions in the close proximity of ATP. Taken together, our data indicated that the disease-related aspartate residue is a key node in the interaction network crucial for effective ATP binding. This work provides a paradigm of how a subtle but critical structural perturbation caused by a single mutation at the ATP-binding site abolishes the kinase activity, emphasizing that stabilizing substrate in a productive conformational state is crucial for catalysis.
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Hassan, Bassem A., Sergei N. Prokopenko, Sebastian Breuer, Bing Zhang, Achim Paululat, and Hugo J. Bellen. "skittles, a Drosophila Phosphatidylinositol 4-Phosphate 5-Kinase, Is Required for Cell Viability, Germline Development and Bristle Morphology, But Not for Neurotransmitter Release." Genetics 150, no. 4 (December 1, 1998): 1527–37. http://dx.doi.org/10.1093/genetics/150.4.1527.
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Abstract The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. We have cloned and generated mutations in a Drosophila PIP5K type I (skittles). Our analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, we found no evidence for PIP5KI involvement in neural secretion.
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Kuroda, Ryo, Mariko Kato, Tomohiko Tsuge, and Takashi Aoyama. "Arabidopsis phosphatidylinositol 4‐phosphate 5‐kinase genes PIP5K7 , PIP5K8 , and PIP5K9 are redundantly involved in root growth adaptation to osmotic stress." Plant Journal 106, no. 4 (April 5, 2021): 913–27. http://dx.doi.org/10.1111/tpj.15207.
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Parkhitko, Andrey A., Arashdeep Singh, Sharon Hsieh, Yanhui Hu, Richard Binari, Christopher J. Lord, Sridhar Hannenhalli, Colm J. Ryan, and Norbert Perrimon. "Cross-species identification of PIP5K1-, splicing- and ubiquitin-related pathways as potential targets for RB1-deficient cells." PLOS Genetics 17, no. 2 (February 16, 2021): e1009354. http://dx.doi.org/10.1371/journal.pgen.1009354.
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The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. We identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. We validated 38 mammalian orthologs of Rbf1 modifiers as RB1 SL partners in human cancer cell lines with defective RB1 alleles. We further show that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer.
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Wang, Xiaoxiang, Lan Yu, Xing Xiong, Yao Chen, and Bo Men. "Bone Marrow Mesenchymal Stem Cells (BMSCs) Transplantation Alleviates Acute Pancreatitis Through Inhibiting Inflammation and Promoting Caspase-8 Apoptosis Pathway." Journal of Biomaterials and Tissue Engineering 12, no. 5 (May 1, 2022): 1034–39. http://dx.doi.org/10.1166/jbt.2022.2969.
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Bone marrow mesenchymal stem cells (BMSCs) are capable of multipolar differentiation and repairing injured tissues. Herein, we aimed to investigate the mechanism by how BMSCs modulate the apoptotic pathway in the acute pancreatitis (AP). In this study, primary BMSCs were cultured and administrated into 10 AP mice while 10 healthy mice were taken as a blank group and 10 AP mice as a control group. The mouse pancreatic tissues were assessed by HE staining and evaluated by pancreatitis score and serum amylase detection. Level of inflammatory factors CRP and TNF-α was measured by ELISA and PIPK1, PIPK3, MLKL and Caspase-8 expression was detected by RT-qPCR and Western blot. The pancreatitis score (7.29±1.36) and the serum amylase score of (453.66±103.67) mu/ml of BMSCs group was significantly higher than that of control group, indicating increased tissue repair after BMSCs treatment. BMSCs group exhibited a higher level of CRP (711.01±115.31) and TNF-α (132.81±22.13) in serum compared to control group (p < 0.05). PIPK1, PIPK3, and MLKL expression in BMSCs group decreased (p < 0.05) whereas Caspase-8 was increased (p < 0.05). On the other hand, BMSCs group presented upregulated PIPK1, PIPK3, and MLKL (p < 0.05) and downregulated Caspase-8 (p < 0.05). In conclusion, BMSCs regulate cell apoptosis by upregulating Caspase-8 expression, and downregulating PIPK1, PIPK3 and MLKL level, thereby alleviating the inflammation in AP.
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Wang, Yanfeng, Lurong Lian, Tami L. Bach, Xinsheng Chen, Qing-Min Chen, and Charles S. Abrams. "PIP5Kγ-Null Mutation Induces Cytoskeletal Changes within Megakaryocytes." Blood 104, no. 11 (November 16, 2004): 629. http://dx.doi.org/10.1182/blood.v104.11.629.629.
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Abstract All eukaryotic cells contain the phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2) that serves multiple roles in signaling cascades critical for actin dynamics. Phosphatidylinositol-4-phosphate 5 kinase (PIP5K) catalyzes the synthesis of PIP2 by phosphorylating PI4P. Although the classical PIP5K isoforms (α, β, and γ) generate the same phospholipid product, the isoforms have different primary structures, are expressed at different levels in various tissues, and localize in different intracellular compartments. Therefore, it is likely that the functions of these isoforms are different as well. PIP5Kγ is unique because it associates directly with talin and consequently localizes in focal adhesion. Given the role of PIP2 in actin dynamics, we generated a murine line containing a null mutation in the PIP5Kγ gene. We found that the targeted disruption of PIP5Kγ results in early prenatal mortality that is associated with pleotropic effects involving the cardiovascular, neurologic, and hematopoietic systems. This early lethality prevented studies of hematopoietic cells derived from the bone marrow or the liver. However, we were able to analyze yolk sac progenitor cells that were treated with thrombopoietin ex vivo, differentiating them into megakaryocytes. After 5 days in culture, many of the non-adherent yolk sac progenitor cells were multinucleated, and approximately 70% expressed CD41 (αIIb), a marker of the megakaryocyte lineage. We then examined their cytoskeletal content and dynamics. Absence of PIP5Kγ had no effect on the quantity of F-actin in unstimulated or thrombin-stimulated cells that were adherent to fibrinogen or were maintained in suspension in the absence of stimulation. Next, we examined membrane dynamics during cell adhesion in real time using spinning disk video confocal microscopy. Wild type megakaryocytes actively formed and contracted lamellipodia and rapidly spread upon the fibrinogen matrix. In contrast, PIP5Kγ-null megakaryocytes continuously extended and retracted membrane blebs, rather than lamellipodia, but eventually spread as much as wild type cells. This observation is consistent with the previous suggestion that PIP2 contributes to the stable association of the membrane with the cytoskeleton. It is also noteworthy that a similar phenotype has been described in cells with defective anchoring of the membrane to the cytoskeleton because of a lack of filamin. Accordingly, our data are consistent with the hypothesis that PIP5Kγ generates compartmentalized pools of PIP2 that contribute to the association of the membrane with the cytoskeleton.
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Chen, Xinsheng, Yanfeng Wang, Edward K. Williamson, Timothy J. Stalker, Lawrence F. Brass, Morris J. Birnbaum, John H. Harwig, and Charles S. Abrams. "Loss of PIP5KIβ Causes a Defect in Lamellipodia Formation and Shear Resistant Adhesion." Blood 108, no. 11 (November 16, 2006): 141. http://dx.doi.org/10.1182/blood.v108.11.141.141.
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Abstract Phosphatidylinositol 4,5-bisphosphate (PIP2) is widely known for the production of lipid second messengers after its hydrolysis by phospholipase C or phosphorylation by phosphatidylinositol 3-kinase. PIP2 also regulates cytoskeletal dynamics by directly interacting with actin-binding proteins. Three isoforms of PIP5KI (α, β, and γ) are all capable of phosphorylating PI4P to synthesize PIP2. However, these isoforms have different primary structures, expression levels in various tissues, and intracellular localization. Our previous studies have demonstrated that PIP5KIβ and PIP5KIγ are the dominant isoforms present in platelets. We generated and bred mice heterozygous for a null mutation into the murine PIP5KIβ gene, and crossed these mice to determine the phenotype of mice lacking this protein. PIP5KIβ-null mice were born, appeared developmentally normal, had normal platelet counts, and exhibited no spontaneous hemorrhage. Compared to platelets derived from wild type littermates, platelets lacking PIP5KIβ had PIP2 concentrations that were 61% of normal under basal conditions (p<0.01), and 51% of normal 45 seconds following thrombin stimulation (p<0.01). Similarly, maximum IP3 levels were only 65% of normal in the knockout platelets (p<0.01). Consistent with this second messenger defect, PIP5KIβ −/− platelets had impaired aggregation in response to submaximal doses of thrombin, ADP, collagen, and a thromboxane analogue (U46619). PIP5KIβ-null platelets exhibited disaggregation suggesting that sustained second messenger formation is critical for a sustained aggregation response. Since PIP2 can directly associate with, and thereby regulate actin-binding proteins, we analyzed platelet spreading upon fibrinogen. PIP5KIβ knockout platelets start to spread, but ultimately spread less well than platelets derived from wild type littermates. Imaging this process with real time differential interference contrast microscopy, we found that PIP5KIβ-null platelets extend filopodia as efficiently as wild type platelets, but have difficulty anchoring down these extended membranes. When a filopod on a PIP5KIβ −/− platelet does ultimately adhere to the matrix, a normal lamellipod is rapidly formed. The cytoskeletal organization of PIP5KIβ knockout platelets spread upon fibrinogen was further studied in the electron microscope. This higher resolution analysis verified the profound defect in lamellipodia formation. We speculated that this process of lamellipodia formation is critical for adhesion under the shear conditions found within the arterial system. To test this hypothesis, we analyzed the ability of PIP5KIβ knockout platelets to adhere to collagen in a flow chamber. At all shear conditions between 200 and 1100/s, platelets lacking PIP5KIβ consistently adhered less than wild type platelets. To further analyze the necessity of PIP5KIβ in adhesion of platelets under conditions of arterial shear, we compared PIP5KIβ −/− and PIP5KIβ +/+ mice in a ferric chloride carotid injury model. Under conditions that induced thrombosis in 75% of wild type mice (n=4), we only detected thrombi in 20% of PIP5KIβ-null mice (n=5). Together, these data demonstrate that PIP5KIβ is required for sustained PIP2 and second messenger synthesis, the formation of actin-rich lamellipodia, and stable ex vivo and in vivo platelet adhesion under shear.
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Semenas, J., A. Hedblom, R. R. Miftakhova, M. Sarwar, R. Larsson, L. Shcherbina, M. E. Johansson, P. Harkonen, O. Sterner, and J. L. Persson. "The role of PI3K/AKT-related PIP5K1 and the discovery of its selective inhibitor for treatment of advanced prostate cancer." Proceedings of the National Academy of Sciences 111, no. 35 (July 28, 2014): E3689—E3698. http://dx.doi.org/10.1073/pnas.1405801111.
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Liu, Aizhuo, Dexin Sui, Dianqing Wu, and Jian Hu. "The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing." Science Advances 2, no. 11 (November 2016): e1600925. http://dx.doi.org/10.1126/sciadv.1600925.
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Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K’s activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K. Although the activation loop is severely disordered in the crystal structure of PIP5K, the NMR experiments showed that the largely unstructured peptide folded into an amphipathic helix upon its association with the 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellar surface. Systematic mutagenesis and functional assays further demonstrated the crucial roles of the amphipathic helix and its hydrophobic surface in kinase activity and membrane-sensing function, supporting a working model in which the activation loop is a critical structural module conferring a membrane-sensing mechanism on PIP5K. The activation loop, surprisingly functioning as a membrane sensor, represents a new paradigm of kinase regulation by the activation loop through protein-membrane interaction, which also lays a foundation on the regulation of PIP5K (and other PIPKs) by membrane lipids for future studies.
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Carpenter, C. L. "Btk-dependent regulation of phosphoinositide synthesis." Biochemical Society Transactions 32, no. 2 (April 1, 2004): 326–29. http://dx.doi.org/10.1042/bst0320326.
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Activation of the BCR (B cell antigen receptor) stimulates the production of both PtdIns(3,4,5)P3 and Ins(1,4,5)P3. PtdIns(3,4,5)P3 and Ins(1,4,5)P3 are generated from a common substrate, PtdIns(4,5)P2. In some systems, continuous PtdIns(4,5)P2 synthesis is necessary for maximal Ins(1,4,5)P3 production, but whether this is true for the BCR, and whether PtdIns(4,5)P2 synthesis is regulated following BCR activation, are not known. We found that Btk (Bruton's tyrosine kinase), a member of the Tec family of cytoplasmic protein tyrosine kinases, is constitutively associated with PIP5Ks (phosphatidylinositol 4-phosphate 5-kinases), the enzymes that synthesize PtdIns(4,5)P2. Btk functions as a shuttle to bring PIP5K to the plasma membrane as a means of stimulating PtdIns(4,5)P2 synthesis. The Btk–PIP5K complex appears to localize to lipid rafts. This complex provides a novel shuttling mechanism that allows Btk to regulate the production of the substrate required by both its upstream activator phosphoinositide 3-kinase and its downstream target phospholipase Cγ2.
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El Sayegh, T. Y., P. D. Arora, K. Ling, C. Laschinger, P. A. Janmey, R. A. Anderson, and C. A. McCulloch. "Phosphatidylinositol-4,5 Bisphosphate Produced by PIP5KIγ Regulates Gelsolin, Actin Assembly, and Adhesion Strength of N-Cadherin Junctions." Molecular Biology of the Cell 18, no. 8 (August 2007): 3026–38. http://dx.doi.org/10.1091/mbc.e06-12-1159.
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Phosphoinositides regulate several actin-binding proteins but their role at intercellular adhesions has not been defined. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was generated at sites of N-cadherin–mediated intercellular adhesion and was a critical regulator of intercellular adhesion strength. Immunostaining for PI(4,5)P2 or transfection with GFP-PH-PLCδ showed that PI(4,5)P2 was enriched at sites of N-cadherin adhesions and this enrichment required activated Rac1. Isoform-specific immunostaining for type I phosphatidylinositol 4-phosphate 5 kinase (PIP5KI) showed that PIP5KIγ was spatially associated with N-cadherin–Fc beads. Association of PIP5KIγ with N-cadherin adhesions was in part dependent on the activation of RhoA. Transfection with catalytically inactive PIP5KIγ blocked the enrichment of PI(4,5)P2 around beads. Catalytically inactive PIP5KIγ or a cell-permeant peptide that mimics and competes for the PI(4,5)P2-binding region of the actin-binding protein gelsolin inhibited incorporation of actin monomers in response to N-cadherin ligation and reduced intercellular adhesion strength by more than twofold. Gelsolin null fibroblasts transfected with a gelsolin severing mutant containing an intact PI(4,5)P2 binding region, demonstrated intercellular adhesion strength similar to wild-type transfected controls. We conclude that PIP5KIγ-mediated generation of PI(4,5)P2 at sites of N-cadherin contacts regulates intercellular adhesion strength, an effect due in part to PI(4,5)P2-mediated regulation of gelsolin.
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Zarza, Xavier, Ringo Van Wijk, Lana Shabala, Anna Hunkeler, Matthew Lefebvre, Antia Rodriguez‐Villalón, Sergey Shabala, Antonio F. Tiburcio, Ingo Heilmann, and Teun Munnik. "Lipid kinases PIP5K7 and PIP5K9 are required for polyamine‐triggered K + efflux in Arabidopsis roots." Plant Journal 104, no. 2 (August 19, 2020): 416–32. http://dx.doi.org/10.1111/tpj.14932.
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Wang, Ying Jie, Wen Hong Li, Jing Wang, Ke Xu, Ping Dong, Xiang Luo, and Helen L. Yin. "Critical role of PIP5KIγ87 in InsP3-mediated Ca2+ signaling." Journal of Cell Biology 167, no. 6 (December 20, 2004): 1005–10. http://dx.doi.org/10.1083/jcb.200408008.
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Phosphatidylinositol 4,5-bisphosphate (PIP2) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP3 or IP3) and is therefore critical to intracellular Ca2+ signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase γ (PIP5KIγ87) as the major contributor of the PIP2 pool that supports G protein–coupled receptor (GPCR)-mediated IP3 generation. PIP5KIγ87 RNAi decreases the histamine-induced IP3 response and Ca2+ flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP2 mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KIγ87's PIP2 pool that supports GPCR-mediated Ca2+ signaling is functionally compartmentalized from those generated by the other PIP5KIs.
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Zhao, Xiaoying, Penglei Cui, Guoli Hu, Chuandong Wang, Lei Jiang, Jingyu Zhao, Jiake Xu, and Xiaoling Zhang. "PIP5k1β controls bone homeostasis through modulating both osteoclast and osteoblast differentiation." Journal of Molecular Cell Biology 12, no. 1 (April 16, 2019): 55–70. http://dx.doi.org/10.1093/jmcb/mjz028.
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Abstract PIP5k1β is crucial to the generation of phosphotidylinosotol (4, 5)P2. PIP5k1β participates in numerous cellular activities, such as B cell and platelet activation, cell phagocytosis and endocytosis, cell apoptosis, and cytoskeletal organization. In the present work, we aimed to examine the function of PIP5k1β in osteoclastogenesis and osteogenesis to provide promising strategies for osteoporosis prevention and treatment. We discovered that PIP5k1β deletion in mice resulted in obvious bone loss and that PIP5k1β was highly expressed during both osteoclast and osteoblast differentiation. Deletion of the gene was found to enhance the proliferation and migration of bone marrow-derived macrophage-like cells to promote osteoclast differentiation. PIP5k1β−/− osteoclasts exhibited normal cytoskeleton architecture but stronger resorption activity. PIP5k1β deficiency also promoted activation of mitogen-activated kinase and Akt signaling, enhanced TRAF6 and c-Fos expression, facilitated the expression and nuclear translocation of NFATC1, and upregulated Grb2 expression, thereby accelerating osteoclast differentiation and function. Finally, PIP5k1β enhanced osteoblast differentiation by upregulating master gene expression through triggering smad1/5/8 signaling. Therefore, PIP5k1β modulates bone homeostasis and remodeling.
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Ren, X. D., G. M. Bokoch, A. Traynor-Kaplan, G. H. Jenkins, R. A. Anderson, and M. A. Schwartz. "Physical association of the small GTPase Rho with a 68-kDa phosphatidylinositol 4-phosphate 5-kinase in Swiss 3T3 cells." Molecular Biology of the Cell 7, no. 3 (March 1996): 435–42. http://dx.doi.org/10.1091/mbc.7.3.435.
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Our previous work showed that post-translationally modified Rho in its GTP-bound state stimulated phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity in mouse fibroblast lysates. To investigate whether Rho physically interacts with PIP5K, we incubated immobilized Rho-GST with Swiss 3T3 cell lysates and tested for retained PIP5K activity. Rho-GST, but not Ras-GST or GST alone, bound significant PIP5K activity. The binding of PIP5K was independent of whether Rho was in a GTP- or GDP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognized a single 68-kDa protein eluted from Rho-GST column. The Rho-associated PIP5K responded to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a distinct isoform not reported previously. Rho co-immunoprecipitated with the 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous Rho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzyme enhanced PIP5K binding by approximately eightfold, consistent with the ADP-ribosylated Rho functioning as a dominant negative inhibitor. These results demonstrate that Rho physically interacts with a 68-kDa PIP5K, although whether the association is direct or indirect is unknown.
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Abajy, Mohammad Y., Jolanta Kopeć, Katarzyna Schiwon, Michal Burzynski, Mike Döring, Christine Bohn, and Elisabeth Grohmann. "A Type IV-Secretion-Like System Is Required for Conjugative DNA Transport of Broad-Host-Range Plasmid pIP501 in Gram-Positive Bacteria." Journal of Bacteriology 189, no. 6 (January 5, 2007): 2487–96. http://dx.doi.org/10.1128/jb.01491-06.
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ABSTRACT Plasmid pIP501 has a very broad host range for conjugative transfer among a wide variety of gram-positive bacteria and gram-negative Escherichia coli. Functionality of the pIP501 transfer (tra) genes in E. coli was proven by pIP501 retrotransfer to Enterococcus faecalis (B. Kurenbach, C. Bohn, J. Prabhu, M. Abudukerim, U. Szewzyk, and E. Grohmann, Plasmid 50:86-93, 2003). The 15 pIP501 tra genes are organized in a single operon (B. Kurenbach, J. Kopeć, M. Mägdefrau, K. Andreas, W. Keller, C. Bohn, M. Y. Abajy, and E. Grohmann, Microbiology 152:637-645, 2006). The pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA. Three of the 15 pIP501-encoded Tra proteins show significant sequence similarity to the Agrobacterium type IV secretion system proteins VirB1, VirB4, and VirD4. Here we report a comprehensive protein-protein interaction map of all of the pIP501-encoded Tra proteins determined by the yeast two-hybrid assay. Most of the interactions were verified in vitro by isolation of the protein complexes with pull-down assays. In conjunction with known or postulated functions of the pIP501-encoded Tra proteins and computer-assisted prediction of their cellular location, we propose a model for the first type IV-secretion-like system encoded by a conjugative plasmid from gram-positive bacteria.
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Mao, Yuntao S., Masaki Yamaga, Xiaohui Zhu, Yongjie Wei, Hui-Qiao Sun, Jing Wang, Mia Yun, et al. "Essential and unique roles of PIP5K-γ and -α in Fcγ receptor-mediated phagocytosis." Journal of Cell Biology 184, no. 2 (January 19, 2009): 281–96. http://dx.doi.org/10.1083/jcb.200806121.
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The actin cytoskeleton is dynamically remodeled during Fcγ receptor (FcγR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP2)-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) γ and α isoforms, which synthesize PIP2, during phagocytosis. PIP5K-γ−/− bone marrow–derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcγR clustering. Delivery of exogenous PIP2 rescued these defects. PIP5K-γ knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-γ by RNA interference inhibits particle attachment. In contrast, PIP5K-α knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-γ but not PIP5K-α is transiently activated by spleen tyrosine kinase–mediated phosphorylation. We propose that PIP5K-γ acts upstream of Rac/Rho and that the differential regulation of PIP5K-γ and -α allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.
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Poli, Alessandro, Shidqiyyah Abdul-Hamid, Antonio Enrico Zaurito, Francesca Campagnoli, Valeria Bevilacqua, Bhavwanti Sheth, Roberta Fiume, Massimiliano Pagani, Sergio Abrignani, and Nullin Divecha. "PIP4Ks impact on PI3K, FOXP3, and UHRF1 signaling and modulate human regulatory T cell proliferation and immunosuppressive activity." Proceedings of the National Academy of Sciences 118, no. 31 (July 26, 2021): e2010053118. http://dx.doi.org/10.1073/pnas.2010053118.
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Regulatory T cells (Tregs) play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses, a feature hijacked in tumor microenvironments in which the recruitment of Tregs often extinguishes immune surveillance through suppression of T-effector cell signaling and tumor cell killing. The pharmacological tuning of Treg activity without impacting on T conventional (Tconv) cell activity would likely be beneficial in the treatment of various human pathologies. PIP4K2A, 2B, and 2C constitute a family of lipid kinases that phosphorylate PtdIns5P to PtdIns(4,5)P2. They are involved in stress signaling, act as synthetic lethal targets in p53-null tumors, and in mice, the loss of PIP4K2C leads to late onset hyperinflammation. Accordingly, a human single nucleotide polymorphism (SNP) near the PIP4K2C gene is linked with susceptibility to autoimmune diseases. How PIP4Ks impact on human T cell signaling is not known. Using ex vivo human primary T cells, we found that PIP4K activity is required for Treg cell signaling and immunosuppressive activity. Genetic and pharmacological inhibition of PIP4K in Tregs reduces signaling through the PI3K, mTORC1/S6, and MAPK pathways, impairs cell proliferation, and increases activation-induced cell death while sparing Tconv. PIP4K and PI3K signaling regulate the expression of the Treg master transcriptional activator FOXP3 and the epigenetic signaling protein Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). Our studies suggest that the pharmacological inhibition of PIP4K can reprogram human Treg identity while leaving Tconv cell signaling and T-helper differentiation to largely intact potentially enhancing overall immunological activity.
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Shimada, Takashi L., Shigeyuki Betsuyaku, Noriko Inada, Kazuo Ebine, Masaru Fujimoto, Tomohiro Uemura, Yoshitaka Takano, Hiroo Fukuda, Akihiko Nakano, and Takashi Ueda. "Enrichment of Phosphatidylinositol 4,5-Bisphosphate in the Extra-Invasive Hyphal Membrane Promotes Colletotrichum Infection of Arabidopsis thaliana." Plant and Cell Physiology 60, no. 7 (April 15, 2019): 1514–24. http://dx.doi.org/10.1093/pcp/pcz058.
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Abstract 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.
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Goessweiner-Mohr, Nikolaus, Markus Eder, Gerhard Hofer, Christian Fercher, Karsten Arends, Ruth Birner-Gruenberger, Elisabeth Grohmann, and Walter Keller. "Structure of the double-stranded DNA-binding type IV secretion protein TraN fromEnterococcus." Acta Crystallographica Section D Biological Crystallography 70, no. 9 (August 29, 2014): 2376–89. http://dx.doi.org/10.1107/s1399004714014187.
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Conjugative transfer through type IV secretion multiprotein complexes is the most important means of spreading antimicrobial resistance. Plasmid pIP501, frequently found in clinicalEnterococcus faecalisandEnterococcus faeciumisolates, is the first Gram-positive (G+) conjugative plasmid for which self-transfer to Gram-negative (G−) bacteria has been demonstrated. The pIP501-encoded type IV secretion system (T4SS) protein TraN localizes to the cytoplasm and shows specific DNA binding. The specific DNA-binding site upstream of the pIP501 origin of transfer (oriT) was identified by a novel footprinting technique based on exonuclease digestion and sequencing, suggesting TraN to be an accessory protein of the pIP501 relaxase TraA. The structure of TraN was determined to 1.35 Å resolution. It revealed an internal dimer fold with antiparallel β-sheets in the centre and a helix–turn–helix (HTH) motif at both ends. Surprisingly, structurally related proteins (excisionases from T4SSs of G+ conjugative transposons and transcriptional regulators of the MerR family) resembling only one half of TraN were found. Thus, TraN may be involved in the early steps of pIP501 transfer, possibly triggering pIP501 TraA relaxase activity by recruiting the relaxosome to the assembled mating pore.
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Xie, Zhongjian, Sandra M. Chang, Sally D. Pennypacker, Er-Yuan Liao, and Daniel D. Bikle. "Phosphatidylinositol-4-phosphate 5-kinase 1α Mediates Extracellular Calcium-induced Keratinocyte Differentiation." Molecular Biology of the Cell 20, no. 6 (March 15, 2009): 1695–704. http://dx.doi.org/10.1091/mbc.e08-07-0756.
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Extracellular calcium (Cao) is a major regulator of keratinocyte differentiation, but the mechanism is unclear. Phosphatidylinositol-4-phosphate 5-kinase 1α (PIP5K1α) is critical in synthesizing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In this study, we sought to determine whether PIP5K1α plays a role in mediating the ability of Cao to induce keratinocyte differentiation. We found that treatment of human keratinocytes in culture with Cao resulted in increased PIP5K1α level and activity, as well as PI(4,5)P2 level, binding of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] to and activation of phospholipase C-γ1 (PLC-γ1), with the resultant increase in inositol 1,4,5-trisphosphate (IP3) and intracellular calcium (Cai). Knockdown of PIP5K1α in human keratinocytes blocked Cao-induced increases in the binding of PI(3,4,5)P3 to PLC-γ1; PLC-γ1 activity; levels of PI(4,5)P2, IP3, and Cai; and induction of keratinocyte differentiation markers. Coimmunoprecipitation and confocal studies revealed that Cao stimulated PIP5K1α recruitment to the E-cadherin–catenin complex in the plasma membrane. Knockdown of E-cadherin or β-catenin blocked Cao-induced activation of PIP5K1α. These results indicate that after Cao stimulation PIP5K1α is recruited by the E-cadherin–catenin complex to the plasma membrane where it provides the substrate PI(4,5)P2 for both PI3K and PLC-γ1. This signaling pathway is critical for Cao-induced generation of the second messengers IP3 and Cai and keratinocyte differentiation.
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Zhang, Jiping, Ruihua Luo, Heqing Wu, Shunhui Wei, Weiping Han, and GuoDong Li. "Role of Type Iα Phosphatidylinositol-4-Phosphate 5-Kinase in Insulin Secretion, Glucose Metabolism, and Membrane Potential in INS-1 β-Cells." Endocrinology 150, no. 5 (December 30, 2008): 2127–35. http://dx.doi.org/10.1210/en.2008-0516.
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Insulin secretion from β-cells is regulated by a complex signaling network. Our earlier study has reported that Rac1 participates in glucose- and cAMP-induced insulin secretion probably via maintaining a functional actin structure for recruitment of insulin granules. Type Iα phosphatidylinositol-4-phosphate 5-kinase (PIP5K-Iα) is a downstream effector of Rac1 and a critical enzyme for synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2). By using an RNA interference technique, PIP5K-Iα in INS-1 β-cells could be specifically knocked down by 70–75%. PIP5K-Iα knockdown disrupted filamentous actin structure and caused changes in cell morphology. In addition, PIP2 content in the plasma membrane was reduced and the glucose effect on PIP2 was abolished but without affecting glucose-induced formation of inositol 1,4,5-trisphosphate. At basal conditions (2.8 mm glucose), PIP5K-Iα knockdown doubled insulin secretion, elevated glucose metabolic rate, depolarized resting membrane potential, and raised cytoplasmic free Ca2+ levels ([Ca2+]i). The total insulin release at high glucose was increased upon PIP5K-Iα knockdown. However, the percent increment of insulin secretion by high glucose and forskolin over the basal release was significantly reduced, an effect more apparent on the late phase of insulin secretion. Metabolism and [Ca2+]i rises at high glucose were also attenuated in cells after PIP5K-Iα knockdown. In contrast, PIP5K-Iα knockdown had no effect on cell growth and viability. Taken together, our data suggest that PIP5K-Iα may play an important role in both the proximal and distal steps of signaling cascade for insulin secretion in β-cells.
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Chakrabarti, Rajarshi, Sulagna Sanyal, Amit Ghosh, Kaushik Bhar, Chandrima Das, and Anirban Siddhanta. "Phosphatidylinositol-4-phosphate 5-Kinase 1α Modulates Ribosomal RNA Gene Silencing through Its Interaction with Histone H3 Lysine 9 Trimethylation and Heterochromatin Protein HP1-α." Journal of Biological Chemistry 290, no. 34 (July 7, 2015): 20893–903. http://dx.doi.org/10.1074/jbc.m114.633727.
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Phosphoinositide signaling has been implicated in the regulation of numerous cellular processes including cytoskeletal dynamics, cellular motility, vesicle trafficking, and gene transcription. Studies have also shown that nuclear phosphoinositide(s) regulates processes such as mRNA export, cell cycle progression, gene transcription, and DNA repair. We have shown previously that the nuclear form of phosphatidylinositol-4-phosphate 5-kinase 1α (PIP5K), the enzyme responsible for phosphatidylinositol 4,5-bisphosphate synthesis, is modified by small ubiquitin-like modifier (SUMO)-1. In this study, we have shown that due to the site-specific Lys to Ala mutations of PIP5K at Lys-244 and Lys-490, it is unable to localize in the nucleus and nucleolus, respectively. Furthermore, by using chromatin immunoprecipitation assays, we have observed that PIP5K associates with the chromatin silencing complex constituted of H3K9me3 and heterochromatin protein 1α at multiple ribosomal DNA (rDNA) loci. These interactions followed a definite cyclical pattern of occupancy (mostly G1) and release from the rDNA loci (G1/S) throughout the cell cycle. Moreover, the immunoprecipitation results clearly demonstrate that PIP5K SUMOylated at Lys-490 interacts with components of the chromatin silencing machinery, H3K9me3 and heterochromatin protein 1α. However, PIP5K does not interact with the gene activation signature protein H3K4me3. This study, for the first time, demonstrates that PIP5K, an enzyme actively associated with lipid modification pathway, has additional roles in rDNA silencing.
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Serror, Pascale, Golnar Ilami, Hichem Chouayekh, S. Dusko Ehrlich, and Emmanuelle Maguin. "Transposition in Lactobacillus delbrueckii subsp. bulgaricus: identification of two thermosensitive replicons and two functional insertion sequences." Microbiology 149, no. 6 (June 1, 2003): 1503–11. http://dx.doi.org/10.1099/mic.0.25827-0.
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In this report, it is shown that the rolling circle replicon pG+host and the theta replicon pIP501 are thermosensitive in Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus). Using a pIP501 derivative as a delivery vector for six insertion sequences originating from lactic acid bacteria, it is shown that IS1223 and IS1201 transpose in L. bulgaricus.
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Kurenbach, Brigitta, Jolanta Kopeć, Marion Mägdefrau, Kristin Andreas, Walter Keller, Christine Bohn, Mouhammad Y. Abajy, and Elisabeth Grohmann. "The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501." Microbiology 152, no. 3 (March 1, 2006): 637–45. http://dx.doi.org/10.1099/mic.0.28468-0.
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The conjugative multiple antibiotic resistance plasmid pIP501 can be transferred and stably maintained in a variety of Gram-positive genera, including multicellular Streptomyces lividans, as well as in Gram-negative Escherichia coli. The 15 putative pIP501 transfer (tra) genes are organized in an operon-like structure terminating in a strong transcriptional terminator. This paper reports co-transcription of the pIP501 tra genes in exponentially growing Enterococcus faecalis JH2-2 cells, as shown by RT-PCR. The tra genes are expressed throughout the life cycle of Ent. faecalis, and the expression level is independent of the growth phase. Electrophoretic mobility shift assays indicated that the TraA relaxase, the first gene of the tra operon, binds to the tra promoter P tra , which partially overlaps with the origin of transfer (oriT). DNase I footprinting experiments further delimited the TraA binding region and defined the nucleotides bound by TraA. β-Galactosidase assays with P tra–lacZ fusions proved P tra promoter activity, which was strongly repressed when TraA was supplied in trans. Thus, it is concluded that the pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA.
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Wong, Ka-Wing, and Ralph R. Isberg. "Arf6 and Phosphoinositol-4-Phosphate-5-Kinase Activities Permit Bypass of the Rac1 Requirement for β1 Integrin–mediated Bacterial Uptake." Journal of Experimental Medicine 198, no. 4 (August 18, 2003): 603–14. http://dx.doi.org/10.1084/jem.20021363.
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Efficient entry of the bacterium Yersinia pseudotuberculosis into mammalian cells requires the binding of the bacterial invasin protein to β1 integrin receptors and the activation of the small GTPase Rac1. We report here that this Rac1-dependent pathway involves recruitment of phosphoinositol-4-phosphate-5-kinase (PIP5K) to form phosphoinositol-4,5-bisphosphate (PIP2) at the phagocytic cup. Reducing the concentration of PIP2 in the target cell by using a membrane-targeted PIP2-specific phosphatase lowered bacterial uptake proportionately. PIP2 formation is regulated by Arf6. An Arf6 derivative defective for nucleotide binding (Arf6N122I) interfered with uptake and decreased the level of PIP2 around extracellular bacteria bound to host cells. This reduction in PIP2 occurred in spite of fact that PIP5K appeared to be recruited efficiently to the site of bacterial binding, indicating a role for Arf6 in activation of the kinase. The elimination of the Rac1-GTP–bound form from the cell by the introduction of the Y. pseudotuberculosis YopE RhoGAP protein could be bypassed by the overproduction of either PIP5K or Arf6, although the degree of bypass was greater for Arf6 transfectants. These results indicate that both Arf6 and PIP5K are involved in integrin-dependent uptake, and that Arf6 participates in both activation of PIP5K as well as in other events associated with bacterial uptake.
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SANTONI, Véronique, Joëlle VINH, Delphine PFLIEGER, Nicolas SOMMERER, and Christophe MAUREL. "A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots." Biochemical Journal 373, no. 1 (July 1, 2003): 289–96. http://dx.doi.org/10.1042/bj20030159.
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Aquaporins are channel proteins that facilitate the diffusion of water across cell membranes. The genome of Arabidopsis thaliana encodes 35 full-length aquaporin homologues. Thirteen of them belong to the plasma membrane intrinsic protein (PIP) subfamily and predominantly sit at the plasma membrane (PM). In the present work we combine separations of membrane proteins (by one- and two-dimensional gel electrophoresis) with identification by MS (matrix-assisted laser-desorption ionization–time-of-flight and electrospray-ionization tandem MS) to take an inventory of aquaporin isoforms expressed in the PM of Arabidopsis thaliana roots. Our analysis provides direct evidence for the expression of five PIPs (PIP1;1, PIP1;5, PIP2;1, PIP2;2 and PIP2;7) in the root PM and suggests the presence of at least three other PIP isoforms. In addition, we show that the same PIP isoform can be present under several forms with distinct isoelectric points. More specifically, we identify phosphorylated aquaporins in the PIP1 and PIP2 subgroups and suggest the existence of other post-translational modifications. Their identification should provide clues to reveal novel molecular mechanisms for aquaporin regulation.
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Kumari, Aastha, Avishek Ghosh, Sourav Kolay, and Padinjat Raghu. "Septins tune lipid kinase activity and PI(4,5)P2 turnover during G-protein–coupled PLC signalling in vivo." Life Science Alliance 5, no. 6 (March 11, 2022): e202101293. http://dx.doi.org/10.26508/lsa.202101293.
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Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] hydrolysis by phospholipase C (PLC) is a conserved mechanism of signalling. Given the low abundance of PI(4,5)P2, its hydrolysis needs to be coupled to resynthesis to ensure continued PLC activity; however, the mechanism by which depletion is coupled to resynthesis remains unknown. PI(4,5)P2 synthesis is catalyzed by the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K). In Drosophila photoreceptors, photon absorption is transduced into PLC activity and during this process, PI(4,5)P2 is resynthesized by a PIP5K. However, the mechanism by which PIP5K activity is coupled to PI(4,5)P2 hydrolysis is unknown. In this study, we identify a unique isoform dPIP5KL, that is both necessary and sufficient to mediate PI(4,5)P2 synthesis during phototransduction. Depletion of PNUT, a non-redundant subunit of the septin family, enhances dPIP5KL activity in vitro and PI(4,5)P2 resynthesis in vivo; co-depletion of dPIP5KL reverses the enhanced rate of PI(4,5)P2 resynthesis in vivo. Thus, our work defines a septin-mediated mechanism through which PIP5K activity is coupled to PLC-mediated PI(4,5)P2 hydrolysis.
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Wang, Y., X. Chen, L. Lian, T. Tang, T. J. Stalker, T. Sasaki, L. F. Brass, J. K. Choi, J. H. Hartwig, and C. S. Abrams. "Loss of PIP5KI demonstrates that PIP5KI isoform-specific PIP2 synthesis is required for IP3 formation." Proceedings of the National Academy of Sciences 105, no. 37 (September 4, 2008): 14064–69. http://dx.doi.org/10.1073/pnas.0804139105.
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Horaud, T., G. de Céspèdes, and P. Trieu-Cuot. "Chromosomal gentamicin resistance transposon Tn3706 in Streptococcus agalactiae B128." Antimicrobial Agents and Chemotherapy 40, no. 5 (May 1996): 1085–90. http://dx.doi.org/10.1128/aac.40.5.1085.
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Streptococcus agalactiae B128 is the only highly gentamicin-resistant group B streptococcal (GBS) strain described so far. This strain carries a chromosomal gentamicin resistance transposon, designated Tn3706, which is similar, if not identical, to the Tn4001 and Tn5281 transpons detected in Staphylococcus aureus and Enterococcus faecalis, respectively. Transposition of Tn3706 occurred onto the GBS plasmid pIP501 in two different loci of its 7.5-kb AvaII fragment carrying the genes for chloramphenicol and erythromycin resistance. Molecular analysis of pIP501 derivatives showed that Tn3706 is composed of a central fragment containing the aac6'-aph2" gene; this fragment is flanked by two tandemly repeated copies of IS256 at the 5' extremity of the resistance gene and a single inverted copy of IS256 at its 3' extremity. The two tandemly repeated copies of IS256 were separated by a 6-bp segment identical to that found, in the same orientation, in the IS256-aac6'-aph2" junction. The hybrid replicons pIP501::Tn3706 were found to be structurally unstable following conjugative transfer between GBS strains. Numerous individual copies of IS256 were detected in B128, but this insertion sequence was not found in the 11 wild-type, gentamicin-susceptible GBS strains studied.
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Yamamoto, Masaya, Donald H. Hilgemann, Siyi Feng, Haruhiko Bito, Hisamitsu Ishihara, Yoshikazu Shibasaki, and Helen L. Yin. "Phosphatidylinositol 4,5-Bisphosphate Induces Actin Stress-Fiber Formation and Inhibits Membrane Ruffling in Cv1 Cells." Journal of Cell Biology 152, no. 5 (February 26, 2001): 867–76. http://dx.doi.org/10.1083/jcb.152.5.867.
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Phosphatidylinositol 4,5 bisphosphate (PIP2) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP2 effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase α (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP2 signaling. PIP5KI overexpression increased PIP2 and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP2 and PI4P synthesis in cells. However, Y-27632 had no effect on PIP2 synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP2 synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP2 in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP2 in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP2.
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van den Bout, Iman, David R. Jones, Zahid H. Shah, Jonathan R. Halstead, Willem-Jan Keune, Shabaz Mohammed, Clive S. D’Santos, and Nullin Divecha. "Collaboration of AMPK and PKC to induce phosphorylation of Ser413 on PIP5K1B resulting in decreased kinase activity and reduced PtdIns(4,5)P2 synthesis in response to oxidative stress and energy restriction." Biochemical Journal 455, no. 3 (October 10, 2013): 347–58. http://dx.doi.org/10.1042/bj20130259.
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Karlsson, Richard, Per Larsson, Regina Miftakhova, Azharuddin Sajid Syed Khaja, Martuza Sarwar, Julius Semenas, Sa Chen, et al. "Establishment of Prostate Tumor Growth and Metastasis Is Supported by Bone Marrow Cells and Is Mediated by PIP5K1α Lipid Kinase." Cancers 12, no. 9 (September 22, 2020): 2719. http://dx.doi.org/10.3390/cancers12092719.
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Cancer cells facilitate growth and metastasis by using multiple signals from the cancer-associated microenvironment. However, it remains poorly understood whether prostate cancer (PCa) cells may recruit and utilize bone marrow cells for their growth and survival. Furthermore, the regulatory mechanisms underlying interactions between PCa cells and bone marrow cells are obscure. In this study, we isolated bone marrow cells that mainly constituted populations that were positive for CD11b and Gr1 antigens from xenograft PC-3 tumor tissues from athymic nu/nu mice. We found that the tumor-infiltrated cells alone were unable to form tumor spheroids, even with increased amounts and time. By contrast, the tumor-infiltrated cells together with PCa cells formed large numbers of tumor spheroids compared with PCa cells alone. We further utilized xenograft athymic nu/nu mice bearing bone metastatic lesions. We demonstrated that PCa cells were unable to survive and give rise to colony-forming units (CFUs) in media that were used for hematopoietic cell colony-formation unit (CFU) assays. By contrast, PC-3M cells survived when bone marrow cells were present and gave rise to CFUs. Our results showed that PCa cells required bone marrow cells to support their growth and survival and establish bone metastasis in the host environment. We showed that PCa cells that were treated with either siRNA for PIP5K1α or its specific inhibitor, ISA-2011B, were unable to survive and produce tumor spheroids, together with bone marrow cells. Given that the elevated expression of PIP5K1α was specific for PCa cells and was associated with the induced expression of VEGF receptor 2 in PCa cells, our findings suggest that cancer cells may utilize PIP5K1α-mediated receptor signaling to recruit growth factors and ligands from the bone marrow-derived cells. Taken together, our study suggests a new mechanism that enables PCa cells to gain proliferative and invasive advantages within their associated host microenvironment. Therapeutic interventions using PIP5K1α inhibitors may not only inhibit tumor invasion and metastasis but also enhance the host immune system.
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Yamamoto, A., D. B. DeWald, I. V. Boronenkov, R. A. Anderson, S. D. Emr, and D. Koshland. "Novel PI(4)P 5-kinase homologue, Fab1p, essential for normal vacuole function and morphology in yeast." Molecular Biology of the Cell 6, no. 5 (May 1995): 525–39. http://dx.doi.org/10.1091/mbc.6.5.525.
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The FAB1 gene of budding yeast is predicted to encode a protein of 257 kDa that exhibits significant sequence homology to a human type II PI(4)P 5-kinase (PIP5K-II). The recently cloned human PIP5K-II specifically converts PI(4)P to PI(4,5)P2 (Boronenkov and Anderson, 1995). The region of highest similarity between Fab1p and PIP5K-II includes a predicted nucleotide binding motif, which is likely to correspond to the catalytic domain of the protein. Interestingly, neither PIP5K-II nor Fab1p exhibit significant homology with cloned PI 3-kinases or PI 4-kinases. fab1 mutations result in the formation of aploid and binucleate cells (hence the name FAB). In addition, loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth. Experiments with a temperature conditional fab1 mutant revealed that their vacuoles rapidly (within 30 min) enlarge to more than double the size upon shifting cells to the nonpermissive temperature. Additional experiments with the fab1 ts mutant together with results obtained with fab1 vps (vacuolar protein sorting defective) double mutants indicate that the nuclear division and cell surface integrity defects observed in fab1 mutants are secondary to the vacuole morphology defects. Based on these data, we propose that Fab1p is a PI(4)P 5-kinase and that the product of the Fab1p reaction, PIP2, functions as an important regulator of vacuole homeostasis perhaps by controlling membrane flux to and/or from the vacuole. Furthermore, a comparison of the phenotypes of fab1 mutants and other yeast mutants affecting PI metabolism suggests that phosphoinositides may serve as general regulators of several different membrane trafficking pathways.
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Gough, N. R. "Inhibition of PIP5K by Apoptotic Stresses." Science's STKE 2006, no. 354 (September 19, 2006): tw332. http://dx.doi.org/10.1126/stke.3542006tw332.
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Toda, Atsushi, Hisataka Kayahara, Hitomi Yasuhira, and Junichi Sikigichi. "Conjugal Transfer of pIP501 fromEnterococcus faecalistoPediococcus halophilus." Agricultural and Biological Chemistry 53, no. 12 (December 1989): 3317–18. http://dx.doi.org/10.1080/00021369.1989.10869865.
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Sarwar, Martuza, Azharuddin Sajid Syed Khaja, Mohammed Aleskandarany, Richard Karlsson, Maryam Althobiti, Niels Ødum, Nigel P. Mongan, et al. "The role of PIP5K1α/pAKT and targeted inhibition of growth of subtypes of breast cancer using PIP5K1α inhibitor." Oncogene 38, no. 3 (August 13, 2018): 375–89. http://dx.doi.org/10.1038/s41388-018-0438-2.
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