How do inositol phosphates regulate calcium signaling?

Activation of a variety of cell surface receptors results in the phospholipase C-catalyzed hydrolysis of the minor plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate, with concomitant formation of inositol 1,4,5-trisphosphate and diacylglycerol. There is strong evidence that inositol 1,4,5-trisphosphate stimulates Ca2+ release from intracellular stores. The Ca2+-releasing actions of inositol 1,4,5-trisphosphate are terminated by its metabolism through two distinct pathways. Inositol 1,4,5-trisphosphate is dephosphorylated by a 5-phosphatase to inositol 1,4-bisphosphate; alternatively, inositol 1,4,5-trisphosphate can also be phosphorylated to inositol 1,3,4,5-tetrakisphosphate by a 3-kinase. Although the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known; the proposal that inositol 1,4,5-trisphosphate secondarily elicits Ca2+ entry by emptying an intracellular Ca2+ pool is considered.     

Inhibition of inositol phospholipids metabolism and calcium mobilization by cyclic AMP-increasing agents and phorbol ester in neutrophils

Cyclic AMP-increasing agents such as PGE2 and dibutyryl cAMP inhibited the fMLP-induced inositol phospholipids metabolism mainly through the suppression of the conversion of phosphatidylinositol(PI) to phosphatidylinositol 4,5-bisphosphate(PIP2). A part of this inhibition was found to be caused by the inhibitory effect of cAMP on PI kinase using isolated plasma membranes. On the other hand, 12-O-tetradecanoyl phorbol acetate(TPA) mainly inhibited the conversion of phosphatidylinositol 4-phosphate(PIP) to PIP2 without a significant effect on the fMLP-induced breakdown of PIP2, though direct effect of TPA on PI and PIP kinases was not demonstrated in isolated plasma membranes. Concerning Ca2+ mobilization, both cAMP-increasing agents and TPA inhibited the fMLP-induced second phase of Ca2+ elevation, while they did not affect the first phase of Ca2+ rapid increase. However, Ca2+ ionophore ionomycin-induced Ca2+ elevation was only inhibitable by TPA but not PGE2. These results suggest that cAMP inhibits the fMLP-induced Ca2+ influx, while TPA stimulates Ca2+ removal from cytosol.     

Receptor-mediated net breakdown of phosphatidylinositol 4,5-bisphosphate in parotid acinar cells.

The metabolism of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] in rat parotid acinar cells was investigated, particularly with regard to the effects of receptor-active agonists. Stimulation of cholinergic-muscarinic receptors with methacholine provoked a rapid disappearance of 40--50% of [32P]PtdIns(4,5)P2, but had no effect on PtdIns4P. Adrenaline, acting on alpha-adrenoceptors, and Substance P also stimulated net loss of PtdIns(4,5)P2. The beta-adrenoceptor agonist, isoprenaline, and the Ca2+ ionophore, ionomycin, failed to affect labelled PtdIns(4,5)P2 or PtdIns4P. By chelation of extracellular Ca2+ with excess EGTA, and by an experimental protocol that eliminates cellular Ca2+ release, it was demonstrated that the agonist-induced decrease in PtdIns(4,5)P2 is independent of both Ca2+ influx and Ca2+ release. These results may suggest that net PtdIns(4,5)P2 breakdown is an early event in the stimulus-response pathway of the parotid acinar cell and could be directly involved in the mechanism of agonist-induced Ca2+ release from the plasma membrane.     

The effects of bombesin on polyphosphoinositide and calcium metabolism in Swiss 3T3 cells

Bombesin, a peptide mitogen for a variety of cell types, acts as a typical Ca2+-mobilizing hormone in Swiss 3T3 fibroblasts. At its mitogenic concentrations (1-25 nM), bombesin stimulates polyphosphoinositide turnover, i.e. breakdown of phosphatidylinositol 4,5-bisphosphate and a concomitant increase in inositol phosphates in a time- and dose-dependent manner. In particular, bombesin induces an initial transient increase in inositol 1,4,5-trisphosphate concentration, followed by an increase in the concentration of inositol 1,3,4-trisphosphate. Also, within 30 s of bombesin addition, the mass of 1,2-diacylglycerol nearly doubles and remains at this level for up to 60 min. Intracellular [Ca2+] measurements with a photoprotein, aequorin, demonstrate that bombesin stimulates a transient rise in cytosolic free Ca2+ concentration. A mobilization of Ca2+ from an intracellular pool is observed as a dose-dependent, transient increase in 45Ca2+ efflux from prelabeled cells, both in the presence and absence of extracellular Ca2+. Bombesin also induces a sustained increase in Ca2+ influx rate and stimulates 3-O-methyl-D-glucose transport across the plasma membrane. These composite results indicate that the mitogenic effect of bombesin is mediated through an activation of the Ca2+ messenger system.     

Phosphoinositide synthesis and Ca2+ gating in blowfly salivary glands exposed to 5-hydroxytryptamine.

Blowfly salivary glands, previously exposed to 10 microM-5-hydroxytryptamine for 30 min, demonstrated a rapid compensatory resynthesis of [3H]inositol-labelled phosphatidylinositol 4,5-bisphosphate when allowed to recover in medium containing 3-5 microM-inositol. Phosphatidylinositol 4,5-bisphosphate comprised 70% of the total [3H]-phosphoinositide, and there was a corresponding decrease in the formation of [3H]-phosphatidylinositol. Subsequent addition of 5-hydroxytryptamine produced an equivalent breakdown of the newly synthesized phosphoinositides but little 45Ca2+ gating. Increasing the inositol concentration in the medium to 300 microM produced a 14-fold stimulation of phosphatidylinositol synthesis but only a 5-fold increase in phosphatidylinositol 4,5-bisphosphate synthesis. Increasing the inositol concentration in the medium from 3 microM to 300 microM resulted in a progressively greater recovery of the 45Ca2+-gating response. At 300 microM-inositol there was an 85% recovery of 45Ca2+-gating response. These results indicate that conversion of phosphatidylinositol into phosphatidylinositol 4,5-bisphosphate occurs in blowfly salivary glands and is secondary to an initial breakdown of the phosphoinositides. Recovery of Ca2+ gating is dependent on the restoration of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate to appropriate concentrations.     

Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane

Synaptotagmin-1 (syt), the putative Ca2+ sensor for exocytosis, is anchored to the membrane of secretory organelles. Its cytoplasmic domain is composed of two Ca2+-sensing modules, C2A and C2B. Syt binds phosphatidylinositol 4,5-bisphosphate (PIP2), a plasma membrane lipid with an essential role in exocytosis and endocytosis. We resolved two modes of PIP2 binding that are mediated by distinct surfaces on the C2B domain of syt. A novel Ca2+-independent mode of binding predisposes syt to penetrate PIP2-harboring target membranes in response to Ca2+ with submillisecond kinetics. Thus, PIP2 increases the speed of response of syt and steers its membrane-penetration activity toward the plasma membrane. We propose that syt-PIP2 interactions are involved in exocytosis by facilitating the close apposition of the vesicle and target membrane on rapid time scales in response to Ca2+.     

Metabolism and functions of inositol phosphates

Activation of Ca2+-mobilizing receptors rapidly increases the cytoplasmic Ca2+ concentration both by releasing Ca2+ stored in endoplasmic reticulum and by stimulating Ca2+ entry into the cells. The mechanism by which Ca2+ release occurs has recently been elucidated. Receptor activation of phospholipase C results in the hydrolysis of the plasma membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to yield two intracellular messengers, diacylglycerol (DAG) and (1,4,5)inositol trisphosphate [(1,4,5)IP3]. DAG remains in the plasma membrane where it stimulates protein phosphorylation via the phospholipid-dependent protein kinase C. (1,4,5)IP3 diffuses to and interacts with specific sites on the endoplasmic reticulum to release stored Ca2+. Receptor stimulation of phospholipase C appears to be mediated by one or more guanine nucleotide-dependent regulatory proteins by a mechanism analogous to hormonal activation of adenylyl cyclase. The actions of (1,4,5)IP3 on Ca2+ mobilization are terminated by two metabolic pathways, sequential dephosphorylation to inositol bisphosphate (IP2), inositol monophosphate (IP) and inositol or by phosphorylation to inositol tetrakisphosphate (IP4) and sequential dephosphorylation to different inositol phosphates. A sustained cellular response also requires Ca2+ entry into the cell from the extracellular space. The mechanism by which hormones increase Ca2+ entry is not known; a recent proposal involving movement of Ca2+ through the endoplasmic reticulum, possibly regulated by IP4, will be considered here.     

Membrane-associated phospholipase C of Drosophila retina

Phospholipase C activities against phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol have been examined using head homogenate of Drosophila visual mutants. In many mutants the enzyme activities were found to be reduced. The activities against both phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol were always affected in parallel among the mutants, while the activities of other enzymes related to phosphatidylinositol metabolism, such as diacylglycerol kinase, were not. The enzyme was concluded to be membrane-associated and was activated maximally at low Ca2+ concentration (10(-7) M), when phosphatidylinositol 4,5-bisphosphate was used as a substrate, while the activity obtained with phosphatidylinositol increased with the Ca2+ concentration up to 10(-4) M. The effects of pH on these two enzyme activities differed to some extent.     

The ATP-dependent membrane localization of protein kinase Calpha is regulated by Ca2+ influx and phosphatidylinositol 4,5-bisphosphate in differentiated PC12 cells

Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKCalpha localization by using a model system of neural growth factor (NGF)-differentiated pheochromocytoma (PC12) cells and extracellular stimulation with ATP. Strikingly, the Ca2+ influx, initiated by the ATP stimulation of P2X receptors, rather than the Ca2+ released from the intracellular stores, was the driving force behind the translocation of PKCalpha to the plasma membrane. Furthermore, the localization process depended on two regions of the C2 domain: the Ca2+-binding region and the lysine-rich cluster, which bind Ca2+ and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], respectively. It was demonstrated that diacylglycerol was not involved in the localization of PKCalpha through its C1 domain, and in lieu, the presence of PtdIns(4,5)P2 increased the permanence of PKCalpha in the plasma membrane. Finally, it also was shown that ATP cooperated with NGF during the differentiation process of PC12 cells by increasing the length of the neurites, an effect that was inhibited when the cells were incubated in the presence of a specific inhibitor of PKCalpha, suggesting a possible role for this isoenzyme in the neural differentiation process. Overall, these results show a novel mechanism of PKCalpha activation in differentiated PC12 cells, where Ca2+ influx, together with the endogenous PtdIns(4,5)P2, anchor PKCalpha to the plasma membrane through two distinct motifs of its C2 domain, leading to enzyme activation.     

Annexin VI is attached to transverse-tubule membranes in isolated skeletal muscle triads

Annexin VI is a 68-kDa protein of the Annexin family, a group of Ca2+-dependent phospholipid-binding proteins widely distributed in mammalian tissues including skeletal muscle. We investigated a) which membrane system contributes Annexin VI to skeletal muscle triads, and b) whether Annexin VI removal affects triad integrity or function. Annexin VI was present in isolated triads and transverse tubules but not in heavy sarcoplasmic reticulum vesicles, indicating that Annexin VI binds to either free or triad-attached transverse tubules. Extraction with EGTA of Annexin VI from triads did not alter their migration as a single band in sucrose density gradients or their ouabain binding-site density, indicating that triad integrity does not require Annexin VI. Caffeine-induced Ca2+ release kinetics and Ca2+ uptake rates were likewise not affected by Annexin VI removal from triads, suggesting that Annexin VI is not involved in these functions. Annexin VI purified from rabbit skeletal muscle displayed Ca2+-dependent binding to liposomes containing phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Binding saturated at 1/20 molar ratio phosphatidylinositol 4,5-bisphosphate/phosphatidylcholine and was optimal at free [Ca2+] > or = 20 mM. Extraction of Annexin VI from triads did not affect the generation of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, or phosphatidic acid by endogenous lipid kinases, suggesting that despite its capacity to bind to negatively charged phospholipids, Annexin VI does not affect the kinase activities responsible for their generation.     

Gonadotropin releasing hormone stimulates the formation of inositol phosphates in rat anterior pituitary tissue.

The production of inositol phosphates in response to gonadotropin releasing hormone (GnRH) was studied in rat anterior pituitary tissue preincubated with [3H]inositol. Prelabelled paired hemipituitaries from prepubertal female rats were incubated in the presence or absence of GnRH in medium containing 10 mM-Li+ X Li+, which inhibits myo-inositol-1-phosphatase, greatly amplified the stimulation of inositol phosphate production by GnRH (10(-7) M) to 159, 198 and 313% of paired control values for inositol 1-phosphate, inositol bisphosphate and inositol trisphosphate respectively after 20 min. The percentage distribution of [3H]inositol within the phosphoinositides was 91.3, 6.3 and 2.4 for phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively and was unaffected by GnRH. The stimulation of inositol trisphosphate production by GnRH was evident after 5 min incubation, was dose-dependent with a half-maximal effect around 11 nM, and was not inhibited by removal of extracellular Ca2+. Elevation of cytosolic Ca2+ by membrane depolarization with 50 mM-K+ had no significant effect on inositol phosphate production. These findings are consistent with the hypothesis that GnRH action in the anterior pituitary involves the hydrolysis of phosphatidylinositol 4,5-bisphosphate. The resulting elevation of inositol trisphosphate may in turn lead to intracellular Ca2+ mobilization and subsequent stimulation of gonadotropin secretion.     

Phosphatidyl inositol-4,5-bisphosphate bound to bovine cardiac Na+/Ca2+ exchanger displays a MgATP regulation similar to that of the exchange fluxes.

This work shows the existence of a phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross-immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns-4,5-P2. In addition, PtdIns-4,5-P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100-130% when membrane vesicles are incubated (15-20 s at 37 degrees C) with 1 mM MgATP and 1 microM Ca2+ (b) in the presence of 100 microM Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns-4,5-P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns-4,5-P2 is greatly reduced compared with that in the presence of 1 microM Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns-4,5-P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP-stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+-dependent levels of PtdIns-4,5-P2 bound to the exchanger molecule.     

Simulations of Kindlin-2 PIP binding domains reveal protonation-dependent membrane binding modes

Kindlin-2, a member of the Kindlin family of peripheral membrane proteins, is important for integrin activation and stabilization of epidermal growth factor receptor. It associates with the cytoplasmic face of the plasma membrane via dedicated phosphatidylinositol phosphate binding domains located in the N-terminal F0 and Pleckstrin Homology domains. These domains have binding affinity for phosphatidylinositol 4,5-bisphosphate and, to a greater degree, phosphatidylinositol 3,4,5-trisphosphate. The biological significance of the differential binding of these phosphatidylinositol phosphates to Kindlin-2 and the mechanism by which they activate Kindlin-2 are not well understood. Recently, ssNMR identified the predominant protonation states of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate near physiological pH in the presence of anionic lipids. Here, we perform atomistic simulation of the bound state of the Pleckstrin Homology and F0 domains of Kindlin-2 at membranes containing phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate with differing protonation states. This computational approach demonstrates that these two phosphatidylinositol phosphates differently modulate Kindlin-2 subdomain binding in a protonation-state-dependent manner. We speculate these variations in binding mode provide a mechanism for intracellular pH and Ca²⁺ influx to control the membrane binding behavior and activity of Kindlin-2.     

Lipopolysaccharide and phorbol esters induce differentiation but have opposite effects on phosphatidylinositol turnover and Ca2+ mobilization in 70Z/3 pre-B lymphocytes

We have recently shown that both lipopolysaccharide (LPS) and the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) induce differentiation in the transformed murine pre-B lymphocyte cell line 70Z/3 by enhancing Na+-H+ exchange across the plasma membrane through an amiloride-sensitive transport system (Rosoff, P.M., Stein, L.F., and Cantley, L.C. (1984) J. Biol. Chem. 259, 7056-7060). These data suggested that the activation of protein kinase C indirectly by LPS and directly by TPA was the critical step in the initiation of differentiation in these cells. We extend these observations to show that LPS rapidly stimulates an increase in phosphatidylinositol turnover, leading to a rise in the levels of diacylglycerol and inositol 1,4,5-trisphosphate and a concomitant decrease in the amount of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. There is also a rapid elevation of intracellular free [Ca2+] which is independent of the presence of extracellular Ca2+ or Na+. These results suggest that the increase in cytosolic [Ca2+] is due to release of cation from internal stores. TPA, which also causes differentiation in these cells, and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, have opposite effects from LPS on both phosphatidylinositol turnover and cellular Ca+ mobilization. These data suggest that protein kinase C inhibits the activity of phospholipase C. Thus protein kinase C plays a pivotal role in the regulation of mitogen-induced differentiation in these cells by both transducing a positive stimulus to the Na+-H+ exchange system as well as feedback regulating its own stimulatory pathway.     

Subcellular localisation of inositol lipid kinases in rat liver

The subcellular distribution of the enzymes which phosphorylate phosphatidylinositol sequentially to form phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was investigated in rat liver. We demonstrate that whilst phosphatidylinositol kinase is present in Golgi, lysosomes and plasma membranes, the kinase that forms phosphatidylinositol 4,5-bisphosphate is localised predominantly at the plasma membrane. The role of the inositol lipid kinases in cell function is discussed.     

Inhibition of inositol trisphosphate-stimulated calcium mobilization by calmodulin antagonists in rat liver epithelial cells

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), an intracellular second messenger produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate, interacts with cytoplasmic membrane structures to elicit the release of stored Ca2+. Ins(1,4,5)P3-induced Ca2+ mobilization is mediated through high affinity receptor binding sites; however, the biochemical mechanism coupling receptor occupation with Ca2+ channel opening has not been identified. In studies presented here, we examined the effects of naphthalenesulfonamide calmodulin antagonists, W7 and W13, and a new selective antagonist, CGS 9343B, on Ca2+ mobilization stimulated by Ins(1,4,5)P3 in neoplastic rat liver epithelial (261B) cells. Intact fura-2 loaded cells stimulated by thrombin, a physiological agent that causes phosphatidylinositol 4,5-bisphosphate hydrolysis and Ins (1,4,5)P3 release, responded with a rise in cytoplasmic free Ca2+ levels that was dose dependently inhibited by W7(Ki = 25 microM), W13 (Ki = 45 microM), and CGS 9343B (Ki = 110 microM). Intracellular Ca2+ release stimulated by the addition of Ins(1,4,5)P3 directly to electropermeabilized 261B cells was similarly inhibited by pretreatment with anti-calmodulin agents. W7 and CGS 9343B, which potently blocked Ca2+/calmodulin-dependent protein kinase, had no significant effect on protein kinase A or C in dose range required for complete inhibition of Ca2+ mobilization. Ca2+ release channels and Ca2+-ATPase pump activity were also unaffected by calmodulin antagonist treatment. These results indicate that calmodulin is tightly associated with the intracellular membrane mechanism coupling Ins(1,4,5)P3 receptors to Ca2+ release channels     

Phosphatidylinositol 4,5-bisphosphate formation in rabbit skeletal and heart muscle membranes

Incubation of rabbit skeletal muscle microsomes or isolated triads with gamma 32P-ATP/Mg2+ in the absence and in the presence of added phosphatidylinositol resulted in the formation of phosphatidylinositol 4-phosphate catalyzed by phosphatidylinositol kinase. When phosphatidylinositol 4-phosphate was added as exogenous substrate, phosphatidylinositol 4,5-bisphosphate was also formed demonstrating the presence of a membrane bound phosphatidylinositol 4-phosphate kinase. Triads were broken mechanically in a French press and separated on a continuous sucrose gradient. Incubation of these fractions with gamma 32P-ATP/Mg2+ resulted in a rapid labeling of phospholipid in a membrane fraction banding between transverse tubules and the terminal cisternae. Partial triad breakage and triad reformation experiments indicated that this phosphatidylinositol kinase was associated with T-tubules. When exogenous phosphatidylinositol 4-phosphate was employed as substrate phosphatidylinositol 4,5-bisphosphate and phosphatidic acid were formed, indicating the presence of all the enzymes of the polyphosphoinositide signaling system in this special membrane fraction. In contrast, heart muscle microsomes or plasma membranes can catalyze this reaction sequence from endogenous formed phosphatidylinositol 4-phosphate.     

Annexin A8 displays unique phospholipid and F-actin binding properties

Annexin A8 is a poorly characterized member of the annexin family of Ca2+-regulated membrane binding proteins. Initially only identified at the cDNA level it had been tentatively linked to acute promyelocytic leukaemia (APL) due to its high and regulated expression in APL-derived cells. Here we identify unique properties of the annexin A8 protein. We show that it binds Ca2+-dependently and with high specificity to phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) and is also capable of interacting with F-actin. In line with these characteristics annexin A8 is recruited to F-actin-associated PtdIns(4,5)P2-rich membrane domains formed in HeLa cells upon infection with non-invading enteropathogenic Escherichia coli. These properties suggest a role of annexin A8 in the organization of certain actin-associated membrane domains.