Faculty Opinions recommendation of Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate levels.

We aimed to understand how spatial compartmentalization in the plasma membrane might contribute to the functions of the ubiquitous class IA phosphoinositide 3-kinase (PI3K) isoforms, p110α and p110β. We found that p110β localizes to membrane rafts in a Rac1-dependent manner. This localization potentiates Akt activation by G-protein-coupled receptors (GPCRs). Thus genetic targeting of a Rac1 binding-deficient allele of p110β to rafts alleviated the requirement for p110β-Rac1 association for GPCR signaling, cell growth and migration. In contrast, p110α, which does not play a physiological role in GPCR signaling, is found to reside in nonraft regions of the plasma membrane. Raft targeting of p110α allowed its EGFR-mediated activation by GPCRs. Notably, p110β dependent, PTEN null tumor cells critically rely upon raft-associated PI3K activity. Collectively, our findings provide a mechanistic account of how membrane raft localization regulates differential activation of distinct PI3K isoforms and offer insight into why PTEN-deficient cancers depend on p110β.

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PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

10.1101/2021.06.22.449474 ◽

2021 ◽

Author(s):

Max Gass ◽

Sarah Borkowsky ◽

Marie-Luise Lotz ◽

Rita Schroeter ◽

Pavel Nedvetsky ◽

...

Keyword(s):

Plasma Membrane ◽

Model System ◽

Dominant Negative ◽

Slit Diaphragm ◽

Phosphatidylinositol 3 Kinase ◽

Ectopic Activation ◽

Phosphatidylinositol 4 ◽

Asymmetrically Distributed ◽

Phosphatidylinositol 3

Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

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The dynamics of plasma membrane PtdIns(4,5)P2 at fertilization of mouse eggs

Journal of Cell Science ◽

10.1242/jcs.115.10.2139 ◽

2002 ◽

Vol 115 (10) ◽

pp. 2139-2149 ◽

Cited By ~ 2

Author(s):

Guillaume Halet ◽

Richard Tunwell ◽

Tamas Balla ◽

Karl Swann ◽

John Carroll

Keyword(s):

Plasma Membrane ◽

De Novo ◽

Confocal Imaging ◽

Cortical Granule ◽

Cortical Granules ◽

Vegetal Cortex ◽

Living Mouse ◽

Cortical Granule Exocytosis ◽

Phosphatidylinositol 4 ◽

Mouse Eggs

A series of intracellular Ca2+ oscillations are responsible for triggering egg activation and cortical granule exocytosis at fertilization in mammals. These Ca2+ oscillations are generated by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which results from the hydrolysis of phosphatidylinositol 4,5-bisphosphate[PtdIns(4,5)P2]. Using confocal imaging to simultaneously monitor Ca2+ and plasma membrane PtdIns(4,5)P2in single living mouse eggs we have sought to establish the relationship between the kinetics of PtdIns(4,5)P2 metabolism and the Ca2+ oscillations at fertilization. We report that there is no detectable net loss of plasma membrane PtdIns(4,5)P2either during the latent period or during the subsequent Ca2+oscillations. When phosphatidylinositol 4-kinase is inhibited with micromolar wortmannin a limited decrease in plasma membrane PtdIns(4,5)P2 is detected in half the eggs studied. Although we were unable to detect a widespread loss of PtdIns(4,5)P2, we found that fertilization triggers a net increase in plasma membrane PtdIns(4,5)P2 that is localized to the vegetal cortex. The fertilization-induced increase in PtdIns(4,5)P2 follows the increase in Ca2+, is blocked by Ca2+ buffers and can be mimicked, albeit with slower kinetics, by photoreleasing Ins(1,4,5)P3. Inhibition of Ca2+-dependent exocytosis of cortical granules, without interfering with Ca2+ transients, inhibits the PtdIns(4,5)P2 increase. The increase appears to be due to de novo synthesis since it is inhibited by micromolar wortmannin. Finally,there is no increase in PtdIns(4,5)P2 in immature oocytes that are not competent to extrude cortical granules. These studies suggest that fertilization does not deplete plasma membrane PtdIns(4,5)P2 and that one of the pathways for increasing PtdIns(4,5)P2 at fertilization is invoked by exocytosis of cortical granules.

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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 ◽

10.18433/jpps30444 ◽

2019 ◽

Vol 22 ◽

pp. 576-584 ◽

Cited By ~ 1

Author(s):

Atsushi Kawase ◽

Yuta Inoue ◽

Miho Hirosoko ◽

Yuka Sugihara ◽

Hiroaki Shimada ◽

...

Keyword(s):

Plasma Membrane ◽

Multidrug Resistance ◽

Cancer Cells ◽

Hepg2 Cells ◽

Carcinoma Cells ◽

Efflux Transporters ◽

Membrane Localization ◽

Intracellular Accumulation ◽

Primary Hepatocytes ◽

Phosphatidylinositol 4

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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