Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-α

Cytosolic phospholipase A₂-α (cPLA₂) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA₂ is dependent on a rise in cytosolic Ca²⁺ concentration, membrane association via the Ca²⁺-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA₂ via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP₂), including the role of a “pleckstrin homology (PH)-like” region of cPLA₂ (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate + the Ca²⁺ ionophore, A23187, or epidermal growth factor + A23187, expression of the PH domain of phospholipase C-δ1 (which sequesters membrane PIP₂) attenuated cPLA₂ activity. Stimulated cPLA₂ activity was also attenuated by the expression of cPLA₂ 135-366, or cPLA₂ 2-366, and expression of a PIP₂-specific 5′-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP₂, with high affinity, only cPLA₂ 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA₂s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA₂ activity can be modulated by sequestration or depletion of cellular PIP₂, although the interaction of cPLA₂ with membrane PIP₂ appears to be indirect, or of weak affinity.     

Hypercholesterolemia induces up-regulation of KACh cardiac currents via a mechanism independent of phosphatidylinositol 4,5-bisphosphate and Gβγ

Hypercholesterolemia is a well-known risk factor for cardiovascular disease. In the heart, activation of K(ACh) mediates the vagal (parasympathetic) negative chronotropic effect on heart rate. Yet, the effect of cholesterol on K(ACh) is unknown. Here we show that cholesterol plays a critical role in modulating K(ACh) currents (I(K,ACh)) in atrial cardiomyocytes. Specifically, cholesterol enrichment of rabbit atrial cardiomyocytes led to enhanced channel activity while cholesterol depletion suppressed I(K,ACh). Moreover, a high-cholesterol diet resulted in up to 3-fold increase in I(K,ACh) in rodents. In accordance, elevated currents were observed in Xenopus oocytes expressing the Kir3.1/Kir3.4 heteromer that underlies I(K,ACh). Furthermore, our data suggest that cholesterol affects I(K,ACh) via a mechanism which is independent of both PI(4,5)P(2) and Gβγ. Interestingly, the effect of cholesterol on I(K,ACh) is opposite to its effect on I(K1) in atrial myocytes. The latter are suppressed by cholesterol enrichment and by high-cholesterol diet, and facilitated following cholesterol depletion. These findings establish that cholesterol plays a critical role in modulating I(K,ACh) in atrial cardiomyocytes via a mechanism independent of the channel's major modulators.     

Immunocytochemical detection of the intranuclear variations of phosphatidylinositol 4,5-bisphosphate amount associated with changes of activity and amount of phospholipase C β1 in cells exposed to mitogenic or differentiating agonists

The intracellular localizations of phosphatidylinositol 4,5-bisphosphate (PIP₂) and of its hydrolyzing enzyme phospholipase C (PLC; in this case the β₁ isoform) have been evaluated by electron microscope immunocytochemistry in cells exposed to mitogenic or differentiating agents. These cells have been previously demonstrated to present a signal transduction system based on the polyphosphoinositide hydrolysis localized at the nuclear level, which can be specifically modulated by agonists. The results demonstrate that in Swiss 3T3 mouse fibroblasts mitogenically stimulated by insulin-like growth factor I (IGF-I), a rapid and transient decrease of the PIP₂ detectable by immunogold labeling occurs at the nuclear interior. This effect appears due to the activation of the PLC β₁ isozyme already present in the nucleus, since no significant variations of the enzyme amount and distribution can be detected by immunolabeling. However, after 30 min of exposure to IGF-I, when the PLC β₁ activity is returned to basal level, a slight but significant increase of the enzyme amount is detected both in the nucleus and in the cytoplasm. On the other hand, an increased accumulation of PIP₂ in the nucleus, accompanied by a decrease of the intranuclear amount of PLC β₁ isozyme, have been observed in mouse erythroleukemia Friend cells, induced to erythroid differentiation by dimethylsulfoxide (DMSO). These results indicate that quantitative immunocytochemistry represents an increment in the available methodologies to investigate the complex regulation of nuclear PI-signalling.     

Phosphatidylinositol 4,5-bisphosphate and calcium at ER-PM junctions — Complex interplay of simple messengers

Endoplasmic reticulum-plasma membrane contact sites (ER-PM MCS) are a specialised domain involved in the control of Ca²⁺ dynamics and various Ca²⁺-dependent cellular processes. Intracellular Ca²⁺ signals are broadly supported by Ca²⁺ release from intracellular Ca²⁺ channels such as inositol 1,4,5-trisphosphate receptors (IP₃Rs) and subsequent store-operated Ca²⁺ entry (SOCE) across the PM to replenish store content. IP₃Rs sit in close proximity to the PM where they can easily access newly synthesised IP₃, interact with binding partners such as actin, and localise adjacent to ER-PM MCS populated by the SOCE machinery, STIM1–2 and Orai1–3, to possibly form a locally regulated unit of Ca²⁺ influx. PtdIns(4,5)P₂ is a multiplex regulator of Ca²⁺ signalling at the ER-PM MCS interacting with multiple proteins at these junctions such as actin and STIM1, whilst also being consumed as a substrate for phospholipase C to produce IP₃ in response to extracellular stimuli. In this review, we consider the mechanisms regulating the synthesis and turnover of PtdIns(4,5)P₂ via the phosphoinositide cycle and its significance for sustained signalling at the ER-PM MCS. Furthermore, we highlight recent insights into the role of PtdIns(4,5)P₂ in the spatiotemporal organization of signalling at ER-PM junctions and raise outstanding questions on how this multi-faceted regulation occurs.     

Unbalancing the Phosphatidylinositol-4,5-bisphosphate–Cofilin Interaction Impairs Cell Steering

Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P₂ and slower release from the plasma membrane to study the role of the PI(4,5)P₂–cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P₂ is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P₂ regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.     

Function of the phosphatidylinositol transfer protein gene family: is phosphatidylinositol transfer the mechanism of action?

Phosphatidylinositol transfer proteins (PITPs) bind and facilitate the transport of phosphatidylinositol (PI) and phosphatidylcholine between membrane compartments. They are highly conserved proteins, are found in both unicellular and multicellular organisms, and can be present as a single domain or as part of a larger, multi-domain protein. The hallmark of PITP proteins is their ability to sequester PI in their hydrophobic pocket. Ablation or knockdown of specific isoforms in vivo has wide ranging effects such as defects in signal transduction via phospholipase C and phosphoinositide 3-kinase, membrane trafficking, stem cell viability, Drosophila phototransduction, neurite outgrowth, and cytokinesis. In this review, we identify the common mechanism underlying each of these phenotypes as the cooperation between PITP proteins and lipid kinases through the provision of PI for phosphorylation. We propose that recruitment and concentration of PITP proteins at specific membrane sites are required for PITP proteins to execute their function rather than lipid transfer.     

Inhibition of D-ribulose 1,5-bisphosphate carboxylase by pyridoxal 5′-phosphate

Homogeneous D-ribulose 1,5-bisphosphate carboxylase from $\text{Rhodospirillum rubrum}$, $\text{Chlamydomonas reinhardtii}$, and $\text{Hydrogenomonas eutropha}$ are inhibited by low concentrations of pyridoxal 5′-phosphate. In the case of the enzyme from $\text{Rhodospirillum rubrum}$, this inhibition is strongly antagonized by the substrate, D-ribulose 1,5-bisphosphate. These results suggest that pyridoxal 5′-phosphate may act close to or at the ribulose 1,5-bisphosphate binding site of the enzyme from $\text{Rhodospirillum rubrum}$.     

Intracellular cholesterol stimulates ENaC by interacting with phosphatidylinositol‑4,5‑bisphosphate and mediates cyclosporine A-induced hypertension

We have previously shown that blockade of ATP-binding cassette transporter A1 (ABCA1) with cyclosporine A (CsA) stimulates the epithelial sodium channel (ENaC) in cultured distal nephron cells. Here we show that CsA elevated systolic blood pressure in both wild-type and apolipoprotein E (ApoE) knockout (KO) mice to a similar level. The elevated systolic blood pressure was completely reversed by inhibition of cholesterol (Cho) synthesis with lovastatin. Inside-out patch-clamp data show that intracellular Cho stimulated ENaC in cultured distal nephron cells by interacting with phosphatidylinositol?4,5?bisphosphate (PIP₂), an ENaC activator. Confocal microscopy data show that both α?ENaC and PIP₂ were localized in microvilli via a Cho-dependent mechanism. Deletion of membrane Cho reduced the levels of γ?ENaC in the apical membrane. Reduced ABCA1 expression and elevated intracellular Cho were observed in old mice, compared to young mice. In parallel, cell-attached patch-clamp data from the split-open cortical collecting ducts (CCD) show that ENaC activity was significantly increased in old mice. These data suggest that elevation of intracellular Cho due to blockade of ABCA1 stimulates ENaC, which may contribute to CsA-induced hypertension. This study also implies that reduced ABCA1 expression may mediate age-related hypertension by increasing ENaC activity via elevation of intracellular Cho.     

Occurrence of adenosine 2′,5′-bisphosphate in rat liver

Adenosine 2′,5′-bisphosphate (pAp) is present in liver from 2-day-fasted rats, at a concentration of around 1 μM. pAp was obtained through perchloric acid extraction of the liver followed by two successive DEAE-cellulose chromatographies and an ion-pair high-pressure liquid chromatography. Both pAp extracted from liver and that obtained from a commercial source showed the same pattern of hydrolysis by alkaline phosphatase, i.e., more 5′-AMP than 2′-AMP was obtained as an intermediate of the reaction.     

Phylogenetic analysis of plant multi-domain SEC14-like phosphatidylinositol transfer proteins and structure–function properties of PATELLIN2

Plant Molecular Biology - SEC14L-PITPs guide membrane recognition and signaling. An increasingly complex modular structure of SEC14L-PITPs evolved in land plants compared to green algae....     

Two phosphatidylinositol 4-kinases control lysosomal delivery of the Gaucher disease enzyme, β-glucocerebrosidase

Gaucher disease is a lysosomal storage disorder caused by a defect in the degradation of glucosylceramide catalyzed by the lysosomal enzyme β-glucocerebrosidase (GBA). GBA reaches lysosomes via association with its receptor, lysosomal integral membrane protein type 2 (LIMP-2). We found that distinct phosphatidylinositol 4-kinases (PI4Ks) play important roles at multiple steps in the trafficking pathway of the LIMP-2/GBA complex. Acute depletion of phosphatidylinositol 4-phosphate in the Golgi caused accumulation of LIMP-2 in this compartment, and PI4KIIIβ was found to be responsible for controlling the exit of LIMP-2 from the Golgi. In contrast, depletion of PI4KIIα blocked trafficking at a post-Golgi compartment, leading to accumulation of LIMP-2 in enlarged endosomal vesicles. PI4KIIα depletion also caused secretion of missorted GBA into the medium, which was attenuated by limiting LIMP-2/GBA exit from the Golgi by PI4KIIIβ inhibitors. These studies identified PI4KIIIβ and PI4KIIα as important regulators of lysosomal delivery of GBA, revealing a new element of control to sphingolipid homeostasis by phosphoinositides.     

Regulation of type IIα phosphatidylinositol phosphate kinase localisation by the protein kinase CK2

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIα PIP kinase at a single site unique to that isoform – Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

In vitro and in vivo analyses of the role of the carboxysomal β-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate

The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 μmol mg^?1 protein min^?1) was only 30 % of that observed for the wild-type carboxysomes (0.71 μmol mg^?1 protein min^?1), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO₂ in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO₂ for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO₂ by the cytoplasmically localized Rubisco by counteracting the action of the CO₂ hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO₂ fixation by the cyanobacterium at CO₂ levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.     

MicroRNA-384-5p regulates ischemia-induced cardioprotection by targeting phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta (PI3K p110δ)

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. This study identified 16 differentially expressed miRNAs in ischemic myocardium of rats using TaqMan Low Density Array. In addition, bioinformatics analyses, such as Gene ontology and Pathway assays, were applied to determine the apoptosis pathway, only regulated by miR-384-5p, and all the associated target genes (PIK3CD, PPP3CA, PPP3CB, PPP3R1, CASP3 and IL1A). These target genes, besides PIK3CB, were shown to be significantly up-regulated by qRT-PCR assay, which further suggested that PIK3CD, PPP3CA, PPP3R1, CASP3, IL1A could be regulated by miR-384-5p. MTT, Western blot, qRT-PCR and luciferase assays were used to investigate the role of miR-384-5p in myocardial ischemia. We found that cleaved caspase3 expression was up-regulated by miR-384-5p and down-regulated by miR-384-5p inhibitor suggesting that apoptosis pathway was regulated by miR-384-5p. We also found that miR-384-5p suppressed cell viability while miR-384-5p inhibitor improved it, confirming H9c2 cell survival was affected by miR-384-5p. In addition, the PIK3CD protein level in H9c2 cells was up-regulated by miR-384-5p inhibitor. We found that miR-384-5p expression level decreased and PIK3CD protein level increased in both ischemic myocardium of rats and hypoxic H9c2 cells, and that miR-384-5p suppress PIK3CD expression through a miR-384-5p binding site within the 3′ untranslational region of PIK3CD. These results show that miR-384-5p, an important protecting factor, plays a significant role in cardioprotection by regulating PIK3CD in myocardial ischemia.     

The small subunit of ribulose-1,5-bisphosphate carboxylase is plastid-encoded in the chlorophyll c-containing alga Cryptomonas Φ

The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) is located in the large single-copy region of the plastid genome of the chlorophyll c-containing alga Cryptomonas . The coding sequence is 417 base pairs long, encoding a protein of 139 amino acids, considerably longer than most other small subunit proteins. It is found 83 base pairs downstream from the gene for the large subunit and is cotranscribed with it. An 18 base pair perfect inverted repeat is located 8 base pairs beyond the termination codon. Sequence analysis shows the gene to be more closely related to cyanobacterial and cyanelle small-subunit genes than to those of green algae or land plants. This is the first reported sequence of a Rubisco small-subunit gene which is plastid-encoded and it exhibits a number of unique features. The derived amino acid sequence shows extensive similarity to a partial amino acid sequence from a brown alga, indicating that this gene will be of major interest as a probe for the small subunit genes in other algae and for determining possible evolutionary ancestors of algal plastids.     

Oxysterol binding protein-dependent activation of sphingomyelin synthesis in the golgi apparatus requires phosphatidylinositol 4-kinase IIα

Cholesterol and sphingomyelin (SM) associate in raft domains and are metabolically coregulated. One aspect of coordinate regulation occurs in the Golgi apparatus where oxysterol binding protein (OSBP) mediates sterol-dependent activation of ceramide transport protein (CERT) activity and SM synthesis. Because CERT transfer activity is dependent on its phosphatidylinositol 4 phosphate [PtdIns(4)P]-specific pleckstrin homology domain, we investigated whether OSBP activation of CERT involved a Golgi-associated PtdIns 4-kinase (PI4K). Cell fractionation experiments revealed that Golgi/endosome-enriched membranes from 25-hydroxycholesterol-treated Chinese hamster ovary cells had increased activity of a sterol-sensitive PI4K that was blocked by small interfering RNA silencing of OSBP. Consistent with this sterol-requirement, OSBP silencing also reduced the cholesterol content of endosome/trans-Golgi network (TGN) fractions containing PI4KIIα. PI4KIIα, but not PI4KIIIβ, was required for oxysterol-activation of SM synthesis and recruitment of CERT to the Golgi apparatus. However, neither PI4KIIα nor PI4KIIIβ expression was required for 25-hydroxycholesterol-dependent translocation of OSBP to the Golgi apparatus. The presence of OSBP, CERT, and PI4KIIα in the TGN of oxysterol-stimulated cells suggests that OSBP couples sterol binding or transfer activity with regulation of PI4KIIα activity, leading to CERT recruitment to the TGN and increased SM synthesis.