Phosphatidylinositol 4-phosphate is associated to extracellular lipoproteic fractions and is detected in tomato apoplastic fluids

We have recently detected phosphatidylinositol-4-phosphate (PI4P) in the extracellular medium of tomato cell suspensions. Extracellular PI4P was shown to trigger the activation of defence responses induced by the fungal elicitor xylanase. In this study, by applying a differential centrifugation technique, we found that extracellular PI4P is associated with fractions composed of diverse phospholipids and proteins, which were pelleted from the extracellular medium of tomato cell suspensions grown under basal conditions. Using mass spectrometry, we identified the proteins present in these pelleted fractions. Most of these proteins have previously been characterised as having a role in defence responses. Next, we evaluated whether PI4P could also be detected in an entire plant system. For this, apoplastic fluids of tomato plants grown under basal conditions were analysed using a lipid overlay assay. Interestingly, PI4P could be detected in intercellular fluids obtained from tomato leaflets and xylem sap of tomato plants. By employing electrospray ionisation tandem mass spectrometry (ESI-MS/MS), other phospholipids were also found in intercellular fluids of tomato plants. These had a markedly different profile from the phospholipid pattern identified in entire leaflets. Based on these results, the potential role of extracellular phospholipids in plant intercellular communication is discussed.     

Phosphatidylinositol kinase,phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [γ-³²P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150–200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4–11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40–70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP₂ in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.     

Phosphatidylinositol 4-phosphate is required for translation initiation in Saccharomyces cerevisiae

The small natural product wortmannin inhibits protein synthesis by modulating several phosphatidylinositol (PI) metabolic pathways. A primary target of wortmannin in yeast is the plasma membrane-associated PI 4-kinase (PI4K) Stt4p, which is required for actin cytoskeleton organization. Here we show that wortmannin treatment or inactivation of Stt4p, but not disorganization of the actin cytoskeleton per se, leads to a rapid attenuation of translation initiation. Interestingly, inactivation of Pik1p, a wortmannin-insensitive, functionally distinct PI4K, implicated in the regulation of Golgi functions and secretion, also results in severe translation initiation defects with a marked increase of the phosphorylation of the translation initiation factor eIF2alpha. Because wortmannin largely phenocopies the effects of rapamycin (e.g. it triggers nuclear accumulation of Gln3p), it likely also inhibits the PI kinase-related, target of rapamycin (TOR) kinases. Importantly, however, neither inactivation of Stt4p nor Pik1p significantly affects TOR-controlled readouts other than translation initiation, indicating that these PI4Ks do not simply function upstream of TOR. Together, our results reveal the existence of a novel translation initiation control mechanism in yeast that is tightly coupled to the synthesis of distinct PI4P pools.     

Phosphatidylinositol 4-phosphate 5-kinase is indispensable for mouse spermatogenesis

The lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, phosphatidylinositol 4,5-bisphosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5k1b-KO sperm were morphologically normal. Notably, the flagellar shape of D-KO sperm was more severely impaired than that of Pip5k1a-KO sperm. These results suggest that PIP5K1A and PIP5K1B may coordinately and/or redundantly function in the maintenance of sperm number and morphology during spermatogenesis.     

Phosphatidylinositol 4-phosphate 5-kinase is essential for ROCK-mediated neurite remodeling

Phosphatidylinositol 4-phosphate 5-kinase (PIP-5kin) regulates actin cytoskeletal reorganization through its product phosphatidylinositol 4,5-bisphosphate. In the present study we demonstrate that PIP-5kin is essential for neurite remodeling, which is regulated by actin cytoskeletal reorganization in neuroblastoma N1E-115 cells. Overexpression of wild-type mouse PIP-5kin-alpha inhibits the neurite formation that is normally stimulated by serum depletion, whereas a lipid kinase-defective mutant of PIP-5kin-alpha, D266A, triggers neurite extension even in the presence of serum and blocks lysophosphatidic acid-induced neurite retraction. These results phenocopy those previously reported for the small GTPase RhoA and its effector p160 Rho-associated coiled coil-forming protein kinase (ROCK). However, the ROCK-specific inhibitor Y-27632 failed to block the inhibition by PIP-5kin-alpha of neurite extension, whereas D266A did block the neurite retraction induced by overexpression of ROCK. These results, taken together, suggest that PIP-5kin-alpha functions as a downstream effector for RhoA/ROCK to couple lysophosphatidic acid signaling to neurite retraction presumably through its product phosphatidylinositol 4,5-bisphosphate.     

The role of the membrane skeleton in concanavalin A-mediated agglutination of human erythrocytes

In the erythrocyte membrane, the mobility of band 3 protein, the receptor for concanavalin A (Con A), is drastically reduced by the membrane skeleton. Yet, the vesicles free of membrane skeletal proteins, isolated from the highly agglutinable proteinase-treated cells, are found to be devoid of Con A agglutinability. The vesicles bind Con A in normal amounts, and remain agglutinable with the wheat germ and Ricinus agglutinins. Intracellular entrapment of monospecific antibodies to spectrin and 4.1 protein (two of the major skeletal components of the membrane) is also found to inhibit agglutination by 30-50%. Thus the membrane skeleton appears to play a positive role in the agglutination of the cells with Con A. The anti-ankyrin antibodies are found to be without any effect. The anti-band 3 (cytoplasmic domain) antibodies are also inhibitory to agglutination. Since Con A binding to cells alters the shape responses and deformability of the cells, and the cells resist fragmentation at 49 degrees C, the properties of the whole skeleton, especially spectrin, appear to be changed. The Con A-bound membranes also do not release the complex of spectrin-band 4.1-actin when extracted with a hypotonic medium. It appears that Con A binding leads to interaction of the cytoplasmic domain of the receptor with a skeletal component, possibly spectrin. Subsequent to this, the receptor molecules and the skeletal proteins undergo aggregation in the membrane, which is detected by their crosslinking by an 8.6-A span bifunctional reagent. The contractility believed to be associated with the membrane skeleton may be responsible for the aggregation.     

Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation

Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.     

Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase.

The tyrosine kinase activity intrinsic to the insulin receptor is thought to be important in eliciting the intracellular responses to insulin; however, it has been difficult to determine the biochemical functions of the proteins which are substrates for this receptor. Treatment of Chinese hamster ovary (CHO) cells overexpressing the human insulin receptor (CHO.T) with insulin results in a 38 +/- 11 (mean +/- S.E., n = 9)-fold increase in a phosphatidylinositol (PtdIns) kinase activity in anti-phosphotyrosine immunoprecipitates of whole cell lysates. One minute of treatment of cells with insulin causes a dramatic increase in the PtdIns kinase activity in the anti-phosphotyrosine immunoprecipitates; the activity peaks within 5 min and remains elevated for at least 60 min after addition of insulin to the cells. This response is only slightly delayed compared with the time course we observe for activation of the insulin receptor tyrosine kinase. The insulin dose-response curves are also very similar for the activation of the insulin receptor tyrosine kinase activity and for the appearance of PtdIns kinase in the anti-phosphotyrosine immunoprecipitates. Stimulation of the endogenous insulin receptor of CHO cells also results in the association of PtdIns kinase activity with phosphotyrosine-containing proteins. However, CHO cells are less sensitive to insulin than CHO.T cells, and the maximal PtdIns kinase activity in antiphosphotyrosine immunoprecipitates from CHO cells is one-sixth that of CHO.T cells. In contrast, immunoprecipitates from CHO.T cells made with anti-insulin receptor antibodies do not contain significant levels of PtdIns kinase activity. This demonstrates that the PtdIns kinase is either a substrate for the insulin receptor tyrosine kinase or is tightly associated with another tyrosine phosphoprotein, which is not the insulin receptor.     

Phosphatidylinositol 3-phosphate is generated in phagosomal membranes.

Phagocytic cells such as neutrophils and macrophages engulf and destroy invading microorganisms. After internalization, material captured within the phagosomal membrane is destroyed by a complex process of coordinated delivery of digestive enzymes and reactive oxygen species. Several endosomal, lysosomal, and oxidase components expected to participate in these events have recently been shown to bind PtdIns3P, suggesting that this lipid may play a role in this process. We used live, digital fluorescence imaging of RAW 264.7 cells stably expressing either a PtdIns3P binding GFP-PX domain or a GFP-FYVE domain to visualize changes in the levels and subcellular localization of PtdIns3P during phagocytic uptake of IgG-opsonized zymosan particles. Very similar results were obtained using both PtdIns3P probes. The basal distribution of each PtdIns3P probe was partially cytosolic and partially localized to EEA-1-positive endosomal structures. Within about 2-3 min of zymosan attachment and concomitant with the closure of the phagosomal membrane, GFP-positive vesicles moved toward and attached to a localized area of the phagosome. A dramatic, transient accumulation of GFP probe around the entire phagosome rapidly ensued, accompanied by a transient drop in cytosolic GFP fluorescence. The magnitude and timing of this rise in PtdIns3P clearly suggest that it is an ideal candidate for controlling the early stages of phagosomal maturation.     

The CDC4 gene product is associated with the yeast nuclear skeleton

The CDC4 gene product of Saccharomyces cerevisiae is required at the late G1/S phase boundary of the cell cycle. In an attempt to better understand the function of CDC4, we performed experiments to localize this protein in the yeast cell. Using antisera, directed against a TrpE-CDC4 fusion protein, to analyze immuno-blots of different subcellular fractions from yeast, we demonstrated that the CDC4 gene product localizes in the nucleus by two different biochemical preparations of the yeast nucleoskeletal proteins. Immunofluorescence microscopy further confirmed its nuclear localization. These data support a model that includes the CDC4 gene product as a component of the yeast nuclear skeleton. The significance of this association in relationship to the biological role of CDC4 is discussed.     

Association of phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities with the cytoskeleton in human platelets

The inositol lipid kinases were investigated in the cytoskeletons of human platelets. In the absence of added lipids the kinases were only barely detectable in the Triton-soluble fractions and undetectable in cytoskeletons of resting cells. However at least 30% of the total phosphatidylinositol kinase was present in the cytoskeleton as revealed by saturation of the enzyme. Phosphatidylinositol 4-phosphate kinase was also found in significant amounts in the cytoskeletons. On the other hand, both enzymes being only recovered in the particulate fraction of the cells, we suggest that inositol lipid kinases may be present near the anchoring points of the cytoskeletons at the membranes.     

Phosphatidylinositol 3-phosphate recognition and membrane docking by the FYVE domain

The FYVE domain is a small zinc binding module that recognizes phosphatidylinositol 3-phosphate [PtdIns(3)P], a phospholipid enriched in membranes of early endosomes and other endocytic vesicles. It is usually present as a single module or rarely as a tandem repeat in eukaryotic proteins involved in a variety of biological processes including endo- and exocytosis, membrane trafficking and phosphoinositide metabolism. A number of FYVE domain-containing proteins are recruited to endocytic membranes through the specific interaction of their FYVE domains with PtdIns(3)P. Structures and PtdIns(3)P binding modes of several FYVE domains have recently been characterized, shedding light on the molecular basis underlying multiple cellular functions of these proteins. Here, structural and functional aspects and the current mechanism of the multivalent membrane anchoring by monomeric or dimeric FYVE domain are reviewed. This mechanism involves stereospecific recognition of PtdIns(3)P that is facilitated by non-specific electrostatic contacts and modulated by the histidine switch, and is accompanied by hydrophobic insertion. Contributions of each component to the FYVE domain specificity and affinity for PtdIns(3)P-containing membranes are discussed.     

Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg(2+)-dependent and is stimulated by Mn(2+) and F(-) ions. The initial reaction is rapid, with a broad pH profile and a ;transition' temperature for its activation energy at 27 degrees C. The apparent K(m) for ATP is 5mum. ATP, N-ethylmaleimide, Cu(2+) ions and NaIO(4) are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2-3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo.     

Phosphatidylinositol 3-phosphate is found in microdomains of early endosomes

Phosphatidylinositol 3-phosphate [PI(3)P] is a phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P₂, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling.David J. Gillooly and Camilla Raiborg contributed equally     

Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton.

Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes.     

Chemoattractants stimulate phosphatidylinositol-4-phosphate kinase in human polymorphonuclear leukocytes

Chemoattractant receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C is instrumental for leukocyte activation. Previous studies have demonstrated that chemoattractant treatment of intact polymorphonuclear leukocytes (PMN) causes a transient decrease in PIP2 due to phospholipase C activation, followed by an increase in cellular PIP2 levels. The present study determined whether chemoattractants altered the activities of the two enzymes responsible for the synthesis of PIP2, phosphatidylinositol kinase, and phosphatidylinositol-4-phosphate (PIP) kinase. Incubation of intact PMN with the N-formylated peptide chemoattractant formyl-methionyl-leucyl-phenylalanine at 37 degrees C caused a rapid (3 min), 2-fold stimulation of PIP kinase activity isolated from a particulate membrane fraction. The increase in PIP kinase was dose-dependent for a variety of N-formylated chemoattractants as well as leukotriene B4. Lineweaver-Burk analysis showed that the Vmax of PIP kinase was increased 2-fold by formyl-methionyl-leucyl-phenylalanine, without a significant change in the apparent Km of the enzyme for ATP. Phosphatidylinositol kinase was, however, not altered by any chemoattractants tested. Nonchemotactic activators of the oxidative burst in leukocytes such as phorbol myristate acetate and ionophore A23187 did not significantly alter PIP kinase, suggesting a specificity for chemotactic agents. These findings demonstrate direct, chemoattractant-induced stimulation of PMN PIP kinase which may serve to replenish the important phospholipid, PIP2, in the membrane following its hydrolysis by phospholipase C.     

Subtypes of protein kinase C in isolated nerve growth cones: only type II is associated with the membrane skeleton from growth cones.

The growth cone particle (GCP) fraction was isolated from fetal and neonatal rat brains and the distribution of protein kinase C subtypes in the fraction was examined by using subtype-specific antibodies. The main subtype in the GCP fraction from fetal forebrain was type II, and type III was also present, but not type I. The pattern was not altered from embryonic day 17 to postnatal day 5. The membrane skeleton subfraction from the GCP fraction contained type II, but far less amount of type III. Our results suggest that type II and type III may be closely related to the functions of growth cones but that they appear to be associated with distinct signal transduction processes.     

Phosphatidylinositol 4-phosphate 5-kinase type I is regulated through phosphorylation response by extracellular stimuli

Phosphatidylinositol 4-phosphate 5-kinase (PIPK) catalyzes a final step in the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), a lipid signaling molecule. Strict regulation of PIPK activity is thought to be essential in intact cells. Here we show that type I enzymes of PIPK (PIPKI) are phosphorylated by cyclic AMP-dependent protein kinase (PKA), and phosphorylation of PIPKI suppresses its activity. Serine 214 was found to be a major phosphorylation site of PIPK type Ialpha (PIPKIalpha) that is catalyzed by PKA. In contrast, lysophosphatidic acid-induced protein kinase C activation increased PIPKIalpha activity. Activation of PIPKIalpha was induced by dephosphorylation, which was catalyzed by an okadaic acid-sensitive phosphatase, protein phosphatase 1 (PP1). In vitro dephosphorylation of PIPKIalpha with PP1 increased PIPK activity, indicating that PP1 plays a role in lysophosphatidic acid-induced dephosphorylation of PIPKIalpha. These results strongly suggest that activity of PIPKIalpha in NIH 3T3 cells is regulated by the reversible balance between PKA-dependent phosphorylation and PP1-dependent dephosphorylation.     

IL-4 receptor signal transduction in human monocytes is associated with protein kinase C translocation.

IL-4 regulates B cell differentiation and monocyte functions. Protein kinases, such as kinase C (PKC), transduce receptor signals. The involvement of PKC in IL-4R signaling was investigated in human monocytes. Treatment with IL-4 (10 ng/ml) for 10 min resulted in a significant redistribution of the PKC activity from cytosol to nuclear fraction. Total PKC activity localized in the nuclear fraction of IL-4-treated and control monocytes was, respectively, 68 and 19%. In contrast, similar PKC activity was found in membrane fraction of IL-4-treated and control cells. The kinetics of IL-4-mediated redistribution of PKC activity to the nuclear fraction were rapid. Within 30 s of IL-4 exposure, 29% of the total PKC activity localized in the nuclear fraction as compared to 15% in control monocytes and increased to 69% at 10 min. The PKC activity in the nuclear fraction appears to be a sequestered form. Extraction with Triton X-100 and additional sonication were required for functional assay of PKC activity. Additional support for PKC involvement in IL-4R signaling is provided by the dose-dependent effect of IL-4 on PKC activity and the abrogation of this effect after heat denature and immunoabsorption of IL-4. Furthermore, electron microscope examination and subcellular marker enzyme assays excluded significant contamination of the nuclear fraction by plasma membranes or subcellular organelles and IL-4 altering membrane disruption. The data presented indicate that IL-4R signaling in human monocytes involves PKC translocation to a nuclear fraction.