Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology

Phosphoinositides play a central role in the control of several cellular events including actin cytoskeleton organization. Here we show that, upon infection of epithelial cells with the Gram-negative pathogen Shigella flexneri, the virulence factor IpgD is translocated directly into eukaryotic cells and acts as a potent inositol 4-phosphatase that specifically dephosphorylates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] into phosphatidylinositol 5-monophosphate [PtdIns(5)P] that then accumulates. Transfection experiments indicate that the transformation of PtdIns(4,5)P(2) into PtdIns(5)P by IpgD is responsible for dramatic morphological changes of the host cell, leading to a decrease in membrane tether force associated with membrane blebbing and actin filament remodelling. These data provide the molecular basis for a new mechanism employed by a pathogenic bacterium to promote membrane ruffling at the entry site.     

PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics during focal adhesions assembly and disassembly in a cancer cell line

Focal adhesions (FAs) are large assemblies of proteins that mediate intracellular signals between the cytoskeleton and the extracellular matrix (ECM). The turnover of FA proteins plays a critical regulatory role in cancer cell migration. Plasma membrane lipids locally generated or broken down by different inositide kinases and phosphatase enzymes to activate and recruit proteins to specific regions in the plasma membrane. Presently, little attention has been given to the use of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) fluorescent biosensors in order to determine the spatiotemporal organisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within and around or during assembly and disassembly of FAs. In this study, specific biosensors were used to detect PtdIns(4,5)P2, PtdIns(3,4,5)P3, and FAs proteins conjugated to RFP/GFP in order to monitor changes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels within FAs. We demonstrated that the localisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were moderately correlated with that of FA proteins. Furthermore, we demonstrate that local levels of PtdIns(4,5)P2 increased within FA assembly and declined within FA disassembly. However, PtdIns(3,4,5)P3 levels remained constant within FAs assembly and disassembly. In conclusion, this study shows that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 localised in FAs may be regulated differently during FA assembly and disassembly.     

The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate

Phosphoinositides (PI) are synthesized and turned over by specific kinases, phosphatases, and lipases that ensure the proper localization of discrete PI isoforms at distinct membranes. We analyzed the role of the yeast synaptojanin-like proteins using a strain that expressed only a temperature-conditional allele of SJL2. Our analysis demonstrated that inactivation of the yeast synaptojanins leads to increased cellular levels of phosphatidylinositol (3,5)-bisphosphate and phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)), accompanied by defects in actin organization, endocytosis, and clathrin-mediated sorting between the Golgi and endosomes. The phenotypes observed in synaptojanin-deficient cells correlated with accumulation of PtdIns(4,5)P(2), because these effects were rescued by mutations in MSS4 or a mutant form of Sjl2p that harbors only PI 5-phosphatase activity. We utilized green fluorescent protein-pleckstrin homology domain chimeras (termed FLAREs for fluorescent lipid-associated reporters) with distinct PI-binding specificities to visualize pools of PtdIns(4,5)P(2) and phosphatidylinositol 4-phosphate in yeast. PtdIns(4,5)P(2) localized to the plasma membrane in a manner dependent on Mss4p activity. On inactivation of the yeast synaptojanins, PtdIns(4,5)P(2) accumulated in intracellular compartments, as well as the cell surface. In contrast, phosphatidylinositol 4-phosphate generated by Pik1p localized in intracellular compartments. Taken together, our results demonstrate that the yeast synaptojanins control the localization of PtdIns(4,5)P(2) in vivo and provide further evidence for the compartmentalization of different PI species.     

Supervised membrane swimming: small G-protein lifeguards regulate PIPK signalling and monitor intracellular PtdIns(4,5)P2 pools

Regulation of PIPK (phosphatidylinositol phosphate kinase) and PtdIns(4,5)P2 signalling by small G-proteins and their effectors is key to many biological functions. Through selective recruitment and activation of different PIPK isoforms, small G-proteins such as Rho, Rac and Cdc42 modulate actin dynamics and cytoskeleton-dependent cellular events in response to extracellular signalling. These activities affect a number of processes, including endocytosis, bacterial penetration into host cells and cytolytic granule-mediated targeted cell killing. Small G-proteins and their modulators are also regulated by phosphoinositides through translocation and conformational changes. Arf family small G-proteins act at multiple sites as regulators of membrane trafficking and actin cytoskeletal remodelling, and regulate a feedback loop comprising phospholipase D, phosphatidic acid, PIPKs and PtdIns(4,5)P2, contributing to enhancement of PtdIns(4,5)P2-mediated cellular events and receptor signalling. Na+, Kir (inwardly rectifying K+), Ca2+ and TRP (transient receptor potential) ion channels are regulated by small G-proteins and membrane pools of PtdIns(4,5)P2. Yeast phosphatidylinositol 4-phosphate 5-kinases Mss4 and Its3 are involved in resistance against disturbance of sphingolipid biosynthesis and maintenance of cell integrity through the synthesis of PtdIns(4,5)P2 and downstream signalling through the Rom2/Rho2 and Rgf1/Rho pathways. Here, we review models for regulated intracellular targeting of PIPKs by small G-proteins and other modulators in response to extracellular signalling. We also describe the spatial and temporal cross-regulation of PIPKs and small G-proteins that is critical for a number of cellular functions.     

Differential roles of phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in plasma membrane targeting of C2 domains. Molecular dynamics simulation, membrane binding, and cell translocation studies of the PKCalpha C2 domain

Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca(2+)-dependent C2 domain of protein kinase Calpha (PKCalpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCalpha-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), but that PS and Ca(2+) binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) augments the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca(2+)-dependent PS binding is essential for membrane interactions of PKCalpha-C2. PtdIns(4,5)P(2) alone cannot drive the membrane attachment of the domain but further stabilizes the Ca(2+)- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCalpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P(2) and/or PtdIns(3,4,5)P(3) from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) at the PM slowed membrane dissociation of PKCalpha-C2. Collectively, these studies show that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) augment the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCalpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.     

Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreas in vitro.

We studied the possibility that hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] may be the initiating event for the increase in [32P]Pi incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) during carbachol and pancreozymin (cholecystokinin-octapeptide) action in the rat pancreas. After prelabelling acini for 2h, [32P]Pi incorporation into PtdA, PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P) had reached equilibrium. Subsequent addition of carbachol or pancreozymin caused 32P in PtdIns(4,5)P2 to decrease by 30-50% within 10-15 s, and this was followed by sequential increases in [32P]Pi incorporation into PtdA and PtdIns. Similar changes in 32P-labelling of PtdIns4P were not consistently observed. Confirmation that the decrease in 32P in chromatographically-purified PtdIns(4,5)P2 reflected an actual decrease in this substance was provided by the fact that similar results were obtained (a) when PtdIns(4,5)P2 was prelabelled with [2-3H]inositol, and (b) when PtdIns(4,5)P2 was measured as its specific product (glycerophosphoinositol bisphosphate) after methanolic alkaline hydrolysis and ion-exchange chromatography. The secretogogue-induced breakdown of PtdIns(4,5)P2 was not inhibited by Ca2+ deficiency (severe enough to inhibit amylase secretion and Ca2+-dependent hydrolysis of PtdIns), and ionophore A23187 treatment did not provoke PtdIns(4,5)P2 hydrolysis. The increase in the hydrolysis of PtdIns(4,5)P2 and the increase in [32P]Pi incorporation into PtdA commenced at the same concentration of carbachol in dose-response studies. Our findings suggest that the hydrolysis of PtdIns(4,5)P2 is an early event in the action of pancreatic secretogogues that mobilize Ca2+, and it is possible that this hydrolysis may initiate the Ca2+-independent labelling of PtdA and PtdIns. Ca2+ mobilization may follow these responses, and subsequently cause Ca2+-dependent hydrolysis of PtdIns and exocytosis.     

Inositol lipid turnover and adenosine 3,5 cyclic monophosphate in the salt-secreting rectal gland of the dogfish (Scyliorhinus canicula)

The rectal gland of the dogfish is rich in inositol lipids. Total phospholipids from the gland contained 9.1 mol% of phosphatidylinositol (PtdIns), 1.0 mol% of phosphatidylinositol 4-phosphate (PtdIns4P) and 0.9 mol% of phosphatidylinositol 4,5-biphosphate (PtdIns4,5P2). [32P]Orthophosphate was readily incorporated into PtdIns, phosphatidic acid (PtdA) and especially into PtdIns4P and PtdIns4,5P2 in salt gland slices incubated in elasmobranch Ringer with glucose and no other additions over a 2 hr period. The calcium ionophore A23187 stimulated incorporation into PtdIns and PtdA, but not into PtdIns4P or PtdIns4,5P2. Oxygen uptake by rectal gland slices was maximally stimulated by 0.08mM forskolin, 2.5mM 8-chlorophenylthio cyclic AMP, 2.0mM dibutyryl cyclic AMP and 0.25mM theophylline. Stimulated oxygen uptake was inhibited by 0.1mM ouabain in all cases. Incorporation of [32P]orthophosphate into PtdIns, PtdA, PtdIns4P and PtdIns4,5P2 was inhibited by 0.08mM forskolin and 2.0mM dibutyryl cyclic AMP over a 2 hr period. The results are discussed in relation to the control of salt secretion by the rectal gland.     

Integrin adhesion and force coupling are independently regulated by localized PtdIns(4,5)2 synthesis

The 90-kDa isoform of the lipid kinase PIP kinase Type I γ (PIPKIγ) localizes to focal adhesions (FAs), where it provides a local source of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Although PtdIns(4,5)P(2) regulates the function of several FA-associated molecules, the role of the FA-specific pool of PtdIns(4,5)P(2) is not known. We report that the genetic ablation of PIPKIγ specifically from FAs results in defective integrin-mediated adhesion and force coupling. Adhesion defects in cells deficient in FAPtdIns(4,5)P(2) synthesis are corrected within minutes while integrin-actin force coupling remains defective over a longer period. Talin and vinculin, but not kindlin, are less efficiently recruited to new adhesions in these cells. These data demonstrate that the specific depletion of PtdIns(4,5)P(2) from FAs temporally separates integrin-ligand binding from integrin-actin force coupling by regulating talin and vinculin recruitment. Furthermore, it suggests that force coupling relies heavily on locally generated PtdIns(4,5)P(2) rather than bulk membrane PtdIns(4,5)P(2).     

Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains

Epsin and AP180/CALM are endocytotic accessory proteins that have been implicated in the formation of clathrin-coated pits. Both proteins have phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding domains in their N termini, but these domains are structurally and functionally different. To understand the basis of their distinct properties, we measured the PtdIns(4,5)P2-dependent membrane binding of the epsin N-terminal homology (ENTH) domain and the AP180 N-terminal homology (ANTH) domain by means of surface plasmon resonance and monolayer penetration techniques and also calculated the effect of PtdIns(4,5)P2 on the electrostatic potential of these domains. PtdIns(4,5)P2 enhances the electrostatic membrane association of both domains; however, PtdIns(4,5)P2 binding exerts distinct effects on their membrane dissociation. Specifically, PtdIns(4,5)P2 induces the membrane penetration of the N-terminal alpha-helix of the ENTH domain, which slows the membrane dissociation of the domain and triggers the membrane deformation. These results provide the biophysical explanation for the membrane bending activity of epsin and its ENTH domain.     

Coupled inositide phosphorylation and phospholipase D activation initiates clathrin-coat assembly on lysosomes.

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis. PtdIns(4,5)P2 is generated on lysosomes by the sequential action of a lysosome-associated type II phosphatidylinositol 4-kinase and a soluble type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate 5-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4, 5)P2 and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P2 functions, at least in part, to activate the PtdIns(4,5)P2-dependent phospholipase D1. These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators.     

FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P

The molecular mechanisms underlying the formation of carriers trafficking from the Golgi complex to the cell surface are still ill-defined; nevertheless, the involvement of a lipid-based machinery is well established. This includes phosphatidylinositol 4-phosphate (PtdIns(4)P), the precursor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). In yeast, PtdIns(4)P exerts a direct role, however, its mechanism of action and its targets in mammalian cells remain uncharacterized. We have identified two effectors of PtdIns(4)P, the four-phosphate-adaptor protein 1 and 2 (FAPP1 and FAPP2). Both proteins localize to the trans-Golgi network (TGN) on nascent carriers, and interact with PtdIns(4)P and the small GTPase ADP-ribosylation factor (ARF) through their plekstrin homology (PH) domain. Displacement or knockdown of FAPPs inhibits cargo transfer to the plasma membrane. Moreover, overexpression of FAPP-PH impairs carrier fission. Therefore, FAPPs are essential components of a PtdIns(4)P- and ARF-regulated machinery that controls generation of constitutive post-Golgi carriers.     

Type Igamma PIP kinase is a novel uropod component that regulates rear retraction during neutrophil chemotaxis

Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility. Here, we demonstrate that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma661), which generates PtdIns(4,5)P(2), is enriched in the uropod during chemotaxis of primary neutrophils and differentiated HL-60 cells (dHL-60). Using time-lapse microscopy, we show that enrichment of PIPKIgamma661 at the cell rear occurs early upon chemoattractant stimulation and is persistent during chemotaxis. Accordingly, we were able to detect enrichment of PtdIns(4,5)P(2) at the uropod during chemotaxis. Overexpression of kinase-dead PIPKIgamma661 compromised uropod formation and rear retraction similar to inhibition of ROCK signaling, suggesting that PtdIns(4,5)P(2) synthesis is important to elicit the backness response during chemotaxis. Together, our findings identify a previously unknown function for PIPKIgamma661 as a novel component of the backness signal that regulates rear retraction during chemotaxis.     

A role for PtdIns(4,5)P2 and PIP5Kalpha in regulating stress-induced apoptosis

The phosphoinositide phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) is essential for many cellular processes and is linked to the etiology of numerous human diseases . PtdIns(4,5)P(2) has been indirectly implicated as a negative regulator of apoptosis ; however, it is unclear if apoptotic stimuli negatively regulate PtdIns(4,5)P(2) levels in vivo. Here, we show that two apoptotic-stress stimuli, hydrogen peroxide (H(2)O(2)) and UV irradiation, cause PtdIns(4,5)P(2) depletion during programmed cell death independently of and prior to caspase activation. Depletion of PtdIns(4,5)P(2) is essential for apoptosis because maintenance of PtdIns(4,5)P(2) levels by overexpression of PIP5Kalpha rescues cells from H(2)O(2)-induced apoptosis. PIP5Kalpha expression promotes both basal and sustained ERK1/2 activation after H(2)O(2) treatment, and importantly, pharmacological inhibition of ERK1/2 signaling blocks PIP5Kalpha-mediated cell survival. H(2)O(2) induces tyrosine phosphorylation and translocation of PIP5Kalpha away from its substrate at the plasma membrane, and both are dependent upon the activity of c-src family kinases. Furthermore, constitutively active c-src enhances tyrosine phosphorylation of PIP5Kalpha in vivo and is sufficient for the translocation of PIP5Kalpha away from the plasma membrane. These observations demonstrate that certain apoptotic stimuli initiate an essential signaling pathway during cell death, and this pathway leads to caspase-independent downregulation of PIP5Kalpha and its product PtdIns(4,5)P(2).     

Analysis of intact phosphoinositides in biological samples

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.     

Schwann Cell Proliferation Is Accompanied by Enhanced Inositol Phospholipid Metabolism

Inositol phospholipid metabolism during mitogen-induced Schwann cell proliferation has been examined. Addition of axolemma- and myelin-enriched membrane fractions (AXL and MYE, respectively) to cultured Schwann cells stimulated 32P incorporation into phosphatidylinositol 4-monophosphate [PtdIns(4)P] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. During the first 5 min of incubation with the mitogens, the amount of 32P incorporated into PtdIns(4)P and PtdIns(4,5)P2 was four- to fivefold above control values. The phosphorylation of the inositol phospholipids was dependent on the concentration of membrane mitogens and was maximal within 1 h. Schwann cells that were prelabeled with [3H]glycerol and then stimulated with AXL and MYE displayed a 30-70% increase in the amounts of [3H]PtdIns(4)P and [3H]PtdIns(4,5)P2 and a 60-80% increase in the amount of [3H]phosphatidic acid. A concomitant 20% decrease in the content of [3H]PtdIns was observed after stimulation. These results suggest that the increased metabolism of PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 may be one of the initial molecular events in the transduction of the mitogenic signal across the Schwann cell plasma membrane.     

Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones.

Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2 (and PtdIns4P?), and that the simultaneous disappearance of phosphatidylinositol might be a result of its consumption for the continuing synthesis of polyphosphoinositides.     

Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases.

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.     

Osmotic cell shrinkage activates ezrin/radixin/moesin (ERM) proteins: activation mechanisms and physiological implications

Hyperosmotic shrinkage induces multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. Here we investigated the possible roles of ezrin/radixin/moesin (ERM) proteins in these events. Osmotic shrinkage of Ehrlich Lettre ascites cells elicited the formation of long microvillus-like protrusions, rapid translocation of endogenous ERM proteins and green fluorescent protein-tagged ezrin to the cortical region including these protrusions, and Thr(567/564/558) (ezrin/radixin/moesin) phosphorylation of cortical ERM proteins. Reduced cell volume appeared to be the critical parameter in hypertonicity-induced ERM protein activation, whereas alterations in extracellular ionic strength or intracellular pH were not involved. A shrinkage-induced increase in the level of membrane-associated phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] appeared to play an important role in ERM protein activation, which was prevented after PtdIns(4,5)P(2) depletion by expression of the synaptojanin-2 phosphatase domain. While expression of constitutively active RhoA increased basal ERM phosphorylation, the Rho-Rho kinase pathway did not appear to be involved in shrinkage-induced ERM protein phosphorylation, which was also unaffected by the inhibition or absence of Na(+)/H(+) exchanger isoform (NHE1). Ezrin knockdown by small interfering RNA increased shrinkage-induced NHE1 activity, reduced basal and shrinkage-induced Rho activity, and attenuated the shrinkage-induced formation of microvillus-like protrusions. Hyperosmolarity-induced cell death was unaltered by ezrin knockdown or after phosphatidylinositol 3-kinase (PI3K) inhibition. In conclusion, ERM proteins are activated by osmotic shrinkage in a PtdIns(4,5)P(2)-dependent, NHE1-independent manner. This in turn mitigates the shrinkage-induced activation of NHE1, augments Rho activity, and may also contribute to F-actin rearrangement. In contrast, no evidence was found for the involvement of an NHE1-ezrin-PI3K-PKB pathway in counteracting shrinkage-induced cell death.     

Secretagogue-induced phosphoinositide metabolism in human leucocytes.

The relationship between receptor binding of the formylated peptide chemoattractant formylmethionylleucylphenylalanine (fMet-Leu-Phe), lysosomal enzyme secretion and metabolism of membrane phospholipids was evaluated in both human polymorphonuclear leucocytes (PMN) and the dimethyl sulphoxide (Me2SO)-stimulated human myelomonocytic HL-60 leukaemic cell line. In both cell types, exposure to fMet-Leu-Phe (100 nM) induced rapid lysosomal enzyme secretion (maximal release less than 30 s) and marked changes in the 32P-labelling of the inositol lipids phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as phosphatidic acid (PtdA). Specifically, levels of [32P]PtdIns and [32P]PtdIns(4,5)P2 decreased rapidly (peak decrease at 10-15s), with a subsequent increase at 30 s and later. PtdIns4P and PtdA showed only an increase. In Me2SO-differentiated HL-60 cells prelabelled with [3H]inositol for 20 h, fMet-Leu-Phe caused a net increase in the cellular content of [3H]inositol phosphates, including a rapid increase in [3H]inositol 1,4,5-trisphosphate, suggesting that PtdIns(4,5)P2 breakdown occurs by a phospholipase C mechanism. Both lysosomal enzyme secretion and changes in phospholipid metabolism occur over the same agonist concentration range with a similar time course. Binding of [3H]fMet-Leu-Phe, although occurring over the same concentration range, exhibited markedly slower kinetics. Although depletion of extracellular Ca2+ had no effect on ligand-induced polyphosphoinositide turnover, PtdIns turnover, PtdA labelling and lysosomal enzyme secretion were severely curtailed. These studies demonstrate a receptor-mediated enhancement of phospholipid turnover that correlates with a specific biological response to fMet-Leu-Phe. Further, the results are consistent with the idea that phospholipase C-mediated degradation of PtdIns(4,5)P2, which results in the formation of inositol trisphosphate, is an early step in the stimulus-secretion coupling pathway of the neutrophil. The lack of correlation between these two responses and the equilibrium-binding condition suggests that either these parameters are responsive to the rate of ligand-receptor interaction or only fractional occupation is required for a full biological response.