Role of inositol trisphosphate as a second messenger in signal transduction processes: An essay

This essay attempts to summarize some of the best evidence for the role of inositol trisphosphate as a second messenger in signal transduction processes. The following aspects are addressed in the essay: (a) The synthesis of inositol trisphosphate and other inositol lipids, (b) Receptor-phosphatidylinositol bisphosphate phospholipase C coupling and the N-ras protooncogene, (c) Inositol trisphosphate and intracellular calcium, (d) Cell growth and oncogenes, (e) Receptors linked to the phosphatidylinositol cycle, (f) Phototransduction and (g) Interactions between inositol trisphosphate and other second messengers.Abbreviations Cyclic AMP Adenosine 3,5-cyclic monophosphate - Cyclic GMP Guanosine 3,5-cyclic monophosphate - DG sn, 1,2-Diacylglycerol - EGF Epidermal growth factor - GDP Guanosine diphosphate - GTP Guanosine triphosphate - IP Inositol 1-monophosphate - IP₂ Inositol 1,4-diphosphate - IP₃ Inositol 1,4,5-trisphosphate - PA Phosphatidic acid - PDGF Platelet-derived growth factor - PI Phosphatidylinositol - PIP Phosphatidylinositol 4-monophosphate - PIP₂ Phosphatidylinositol 4,5-bisphosphate - PIP₃ Phosphatidylinositol 3,4,5-trisphosphate - PLC Phospholipase C     

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP₃R) are the most widely expressed intracellular Ca²⁺ release channels. Their activation by IP₃ and Ca²⁺ allows Ca²⁺ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca²⁺] may directly regulate cytosolic effectors or fuel Ca²⁺ uptake by other organelles, while the decrease in ER luminal [Ca²⁺] stimulates store-operated Ca²⁺ entry (SOCE). We are close to understanding the structural basis of both IP₃R activation, and the interactions between the ER Ca²⁺-sensor, STIM, and the plasma membrane Ca²⁺ channel, Orai, that lead to SOCE. IP₃Rs are the usual means through which extracellular stimuli, through ER Ca²⁺ release, stimulate SOCE. Here, we review evidence that the IP₃Rs most likely to respond to IP₃ are optimally placed to allow regulation of SOCE. We also consider evidence that IP₃Rs may regulate SOCE downstream of their ability to deplete ER Ca²⁺ stores. Finally, we review evidence that IP₃Rs in the plasma membrane can also directly mediate Ca²⁺ entry in some cells.     

Comparative analysis of inositol phospholipid metabolism in viral and chemical transformation of mammalian cells

1. Inositol phospholipid metabolites were measured from virally and chemically transformed cells.2. Increased levels of PIP and PIP₂ were observed from both transformed cell lines as compared with controls.3. Intracellular levels of IP₃ were also increased approximately three folds in BPV-1 infected ID 13 cells and in 3-MC transformed NIH 3T3 cells.4. The results suggest that phosphorylation of phosphatidylinositols and enhanced generation of IP₃ second messenger molecules are the common signal tranducing process leading to the cell transformation.     

Probes for manipulating and monitoring IP3

Inositol 1,4,5-trisphosphate (IP₃) is an important second messenger produced via G-protein-coupled receptor- or receptor tyrosine kinase-mediated pathways. IP₃ levels induce Ca²⁺ release from the endoplasmic reticulum (ER) via IP₃ receptor (IP₃R) located in the ER membrane. The resultant spatiotemporal pattern of Ca²⁺ signals regulates diverse cellular functions, including fertilization, gene expression, synaptic plasticity, and cell death. Therefore, monitoring and manipulating IP₃ levels is important to elucidate not only the functions of IP₃-mediated pathways but also the encoding mechanism of IP₃R as a converter of intracellular signals from IP₃ to Ca²⁺.     

Alpha-1-adrenergic stimulation of phosphoinositide breakdown in cultured neonatal rat ventricular myocytes

Summary The regulation of and the intracellular events following a,-adrenergic receptor stimulation in the myocardium still remain to be disclosed. The effect of ₁- adrenergic stimulation on phosphoinositide breakdown was studied in cultured neonatal rat ventricular myocytes. Phenylephrine (30 M) stimulated inositolphosphates formation, but only in the presence of 10 mM LiCl this could be measured. The increase was antagonized by prazosin (1 M) but not by propranolol (1 M). The variability in proportional distribution of the three inositolphosphates is discussed.Abbreviations PIP₂ Phosphatidylinositol 4,5-bisphosphate - PI Phosphatidylinositol - IP₃ Inositol 1,4,5,-bisphosphate - IP₂ Inositol 1,4-bisphosphate - IP₁ Inositol 1-monophosphate - DG Diacylglycerol - PKC Calcium/phospholipid-dependent protein kinase - PhE Phenylephrine.     

Transfer of IP₃ through gap junctions is critical, but not sufficient, for the spread of apoptosis

Decades of research have indicated that gap junction channels contribute to the propagation of apoptosis between neighboring cells. Inositol 1,4,5-trisphosphate (IP₃) has been proposed as the responsible molecule conveying the apoptotic message, although conclusive results are still missing. We investigated the role of IP₃ in a model of gap junction-mediated spreading of cytochrome C-induced apoptosis. We used targeted loading of high-molecular-weight agents interfering with the IP₃ signaling cascade in the apoptosis trigger zone and cell death communication zone of C6-glioma cells heterologously expressing connexin (Cx)43 or Cx26. Blocking IP₃ receptors or stimulating IP₃ degradation both diminished the propagation of apoptosis. Apoptosis spread was also reduced in cells expressing mutant Cx26, which forms gap junctions with an impaired IP₃ permeability. However, IP₃ by itself was not able to induce cell death, but only potentiated cell death propagation when the apoptosis trigger was applied. We conclude that IP₃ is a key necessary messenger for communicating apoptotic cell death via gap junctions, but needs to team up with other factors to become a fully pro-apoptotic messenger.     

Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

IP3-mediated cytosolic and nuclear calcium elevation in NRK-52E cells using ‘caged’ GPIP2

Alterations in calcium homeostasis play a pivotal role in the cellular response to injury. Increases in the concentration of cytosolic free calcium ([Ca²⁺]i) result in a variety of calcium mediated toxic responses such as cytoskeletal alterations, mitochondrial damage, and over-expression of gene products. Inositol trisphosphate is a second messenger that links external cell surface signals to [Ca²⁺]i elevation. The present study explored the use of caged glycerophosphoryl-myo-inositol-1,4,5-bisphosphate (GPIP₂) to mediate a rapid and prolonged increase in [Ca²⁺]i in a normal rat kidney epithelial cell line (NRK-52E). In intact NRK-52E cells, UV photolysis of microinjected GPIP₂ resulted in a 3–4-fold sustained increase in [Ca²⁺]_i. Graded photolytic release of GPIP2 also resulted in calcium-mediated morphologic alterations, as shown by confocal microscopy, with cellular blebs apparent within 30 min. There was no apparent increase in [Ca²⁺]i or morphologic alterations in control cells microinjected with calcium indicator and equally exposed to UV light. Subsequent application of thapsigargin or ionomycin (1.0 μM) produced a rapid and transient increase in [Ca²⁺]i. In addition, we show that activation of IN stores results in increased concentration of ionized nuclear calcium, ([Ca²⁺]n) which persists longer than the increase in [Ca²⁺]i. These findings indicate that GPIP₂ mediates a rapid and sustained elevation in [Ca²⁺]n and [Ca²⁺]i and this IP₃-mediated calcium elevation is translated to the nucleus in rat kidney epithelial cells.     

Inositol bisphosphate and inositol trisphosphate inhibit cell-to-cell passage of carboxyfluorescein in staminal hairs ofSetcreasea purpurea

pH-buffered carboxyfluorescein (Buffered-CF) alone (control), or Buffered-CF solutions containing one of the following: (1)d-myo-inositol (I); (2)d-myo-inositol 2-monophosphate (IP₁); (3)d-myo-inositol 1,4-bisphosphate (IP₂); (4)d-myo-inositol 1,4,5-trisphosphate (IP₃); (5)d-fructose 2,6-diphosphate (F-2,6P₂) were microinjected into the terminal cells of staminal hairs ofSetcreasea purpurea Boom. Passage of the CF from this terminal cell along the chain of cells towards the filament was monitored for 5 min using fluorescence microscopy and quantified using computer-assisted fluorescence-intensity video analysis. Cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either I, IP₁ or F-2,6P₂ was similar to that in hairs microinjected with Buffered-CF only. On the other hand, cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either IP₂ or IP₃ was inhibited. These results indicate that polyphosphoinositols may be involved in the regulation of intercellular transport of low-molecular-weight, hydrophilic molecules in plants.Abbreviations CF 5(6)Carboxyfluorescein - DG diacylglycerol - F2, 6P₂ d-fructose 2,6-diphosphate - I d-myo-inositol - IP₁ d-myo-inositol 2-monophosphate - IP₂ d-myo-inositol 1,4-bisphosphate - IP₃ d-myo-inositol 1,4,5-trisphosphate     

Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation

Hydrogen peroxide (H₂O₂) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H₂O₂ are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca²⁺) from intracellular stores or influx of extracellular Ca²⁺. One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP₃) mobilizes intracellular Ca²⁺ by binding to a family of receptors (IP₃Rs) on the endoplasmic–sarcoplasmic reticulum that act as ligand-gated Ca²⁺ channels. IP₃Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP₃R content. In this study we show that IP₃R₁ and IP₃R₃ are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H₂O₂, through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP₃R by H₂O₂ is accompanied by a reduction in calcium efflux induced by IP₃ in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP₃R by activation of NADPH oxidase and that preincubation with H₂O₂ decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP₃ receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H₂O₂. Altogether, these results suggest that H₂O₂ mediates IP₃R down-regulation via proteasome activity.     

Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells

We have shown previously that inositol-1,4,5-trisphosphate (IP₃) stimulates an efflux of ⁴⁵Ca²⁺ from fusogenic carrot protoplasts (M Rincón, WF Boss [1987] Plant Physiol 83: 395-398). In light of these results, we suggested that IP₃ might serve as a second messenger for the mobilization of intracellular Ca²⁺ in higher plant cells. To determine whether or not IP₃ and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-[2-³H]inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that [³H]inositol metabolites coeluted with inositol bisphosphate (IP₂) and IP₃ when separated by anion exchange chromatography. However, we could not detect IP₂ or IP₃ when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP₂ and IP₃, were present in these cells. Thus, [³H] inositol metabolites other than IP₂ and IP₃ had coeluted on the anion exchange columns. The data indicate that either IP₃ is rapidly metabolized or that it is not present at a detectable level in the carrot cells.     

The involvement of the cytoskeleton in regulating IP3 receptor-mediated internal Ca2+ release in human blood platelets.

In this study we have used saponin to permeabilize platelet membranes in order to test directly the involvement of IP₃ in regulating internal Ca²⁺ release, and to measure IP₃ binding to its receptor. Our results indicate that platelet vesicles release Ca²⁺ as early as 3 seconds after IP₃ addition. Using [³H]IP₃, we have found that platelets contain a single class of high affinity IP₃ binding sites with a Kd of ～0.20 (± 0.01) nM. Immuno-blotting shows that platelets contain a 260 kDa polypeptide which shares immunological cross reactivity with brain IP₃ receptor. Immunofluorescence staining data indicate that the IP₃ receptor is preferentially located at the periphery of the platelet plasma membrane. Most importantly, both IP₃ binding and IP₃-induced Ca²⁺ release activities are significantly inhibited by cytochalasin D (a microfilament inhibitor) and colchicine (a microtubule inhibitor). These findings suggest that the cytoskeleton is involved in the regulation of IP₃ binding and IP₃ receptor-mediated Ca²⁺ release during platelet activation.     

IP6: A novel anti-cancer agent

Inositol hcxaphosphate (InsP₆ or IP₆) is ubiquitous. At 10 μM to 1 mM concentrations, IP₆ and its lower phosphorylated forms (IP_1–5) as well as inositol (Ins) are contained in most mammalian cells, wherein they are important in regulating vital cellular functions such as signal transduction, cell proliferation and differentiation. A striking anti-cancer action of IP₆ has been demonstrated both in vivo and in vitro, which is based on the hypotheses that exogenously administered IP₆ may be internalized, dephosphorylated to IP_1–5, and inhibit cell growth. There is additional evidence that Ins alone may further enhance the anti-cancer effect of IP₆. Besides decreasing cellular proliferation, IP₆ also causes differentiation of malignant cells often resulting in a reversion to normal phenotype. These data strongly point towards the involvement of signal transduction pathways, cell cycle regulatory genes, differentiation genes, oncogenes and perhaps, tumor suppressor genes in bringing about the observed anti-neoplastic action of IP₆.     

The Inositol Trisphosphate Receptor in the Control of Autophagy

The second messenger myo-inositol-1,4,5-trisphosphate (IP₃) acts on the IP₃ receptor (IP₃R), an IP3-activated Ca²⁺ channel of the endoplasmic reticulum (ER). The IP₃R agonist IP3 inhibits starvation-induced autophagy. The IP₃R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-X_L, two proteins that physically interact with IP₃R. Autophagy can also be induced by depletion of the IP₃R by small interfering RNAs. Autophagy induction by IP3R blockade cannot be explained by changes in steady state levels of Ca²⁺ in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP₃R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

Addendum to:

Regulation of Autophagy by the Inositol Trisphosphate Receptor

A. Criollo, M.C. Maiuri, E. Tasdemir, I. Vitale, A.A. Fiebig, D. Andrews, J. Molgo, J. Diaz, S. Lavandero, F. Harper, G. Pierron, D. di Stefano, R. Rizzuto, G. Szabadkai and G. Kroemer

Cell Death Differ 2007; In press     

Dibutyryl cyclic adenosine monophosphate enhancement of α-methyl-d-glucoside accumulation by kidney cortex slices

Cyclic adenosine 3′,5′-monophosphate and N⁶-2′-O-dibutyryl cyclic adenosine 3′,5′-monophosphate increase the accumulation of α-methyl-d-glucoside by cortical slices from rat, rabbit, dog and human kidney. The characteristics of the effect have been studied in rat tissue. At least 90 min of exposure of the tissue to cyclic nucleotide prior to onset of glucoside accumulation is required as well as presence of the cyclic nucleotide during the accumulation phase. Inhibition of protein synthesis does not abolish the effect of N⁶-2′-O-dibutyryl cyclic adenosine 3′,5′-monophosphate. The cyclic nucleotide causes an increase in the initial entry rate of α-methyl-d-glucoside into cells and an increase in the intracellular steady state concentration. The cyclic nucleotide does not affect the apparent K_m of the glucoside entry process but increases the maximum velocity of accumulation.     

Melatonin Receptor-Mediated Stimulation of Phosphoinositide Breakdown in Chick Brain Slices

Abstract: The direct effect of melatonin and related agonists on Li⁺-amplified phosphoinositide breakdown was studied in chick brain slices prelabeled with myo-[2-³H]-inositol. The melatonin receptor agonist 6-chloromelatonin (10–100 µM) increased, in a concentration-dependent manner, the accumulation of inositol phosphates (IP) in chick brain slices. This effect of 6-chloromelatonin (10 µM) was rapid as transient increases in IP₃/IP₄ (maximal increase, 29% at 20 s) and IP₂ levels (maximal increase, 36% at 1 min) were observed, followed by a slower but sustained increase in IP₁ level (30% at 5 min), when the amount of IP₃/IP₄ and IP₂ had already been decreased to the control level. The phosphoinositide response elicited by 6-chloromelatonin (10 µM) was dependent on the presence of extracellular calcium. Direct stimulation of membrane phospholipase C by 6-chloromelatonin (10 µM) in isolated myo-[2-³H]inositol-prelabeled optic tectum membranes was dependent on the presence of guanosine-5′-O-(3-thio)triphosphate (1 µM), thus suggesting that G protein(s) link melatonin receptor activation to phospholipase C stimulation. The competitive melatonin receptor antagonist luzindole (10–100 µM) inhibited in a concentration-dependent manner the IP₁ accumulation stimulated by 6-chloromelatonin (10–100 µM); however, it did not affect the accumulation stimulated by 5-hydroxytryptamine (10 µM). By contrast, methysergide (10 µM) completely inhibited 5-hydroxytryptamine (10 µM)-, but not 6-chloromelatonin (10 µM)-, induced IP₁ accumulation. Melatonin receptor agonists increased IP₁ accumulation in a concentration-dependent manner reaching different maximal responses. N-Acetyl-5-hydroxytryptamine was more potent than melatonin in increasing IP₁ accumulation, suggesting activation of a melatonin receptor site other than the ML-1 melatonin receptor (i.e., N-acetyl-5-hydroxytryptamine ≥ melatonin). In conclusion, these results demonstrate that activation of a melatonin receptor with pharmacological characteristics different from those of the ML-1 subtype leads to activation of the phospholipase C-mediated signal transduction pathway.     

IP3R3 silencing induced actin cytoskeletal reorganization through ARHGAP18/RhoA/mDia1/FAK pathway in breast cancer cell lines

Cell morphology is altered in the migration process, and the underlying cytoskeleton remodeling is highly dependent of intracellular Ca²⁺ concentration. Many calcium channels are known to be involved in migration. Inositol 1,4,5-trisphosphate receptor (IP₃R) was demonstrated to be implicated in breast cancer cells migration, but its involvement in morphological changes during the migration process remains unclear. In the present work, we showed that IP₃R3 expression was correlated to cell morphology. IP₃R3 silencing induced rounding shape and decreased adhesion in invasive breast cancer cell lines. Moreover, IP₃R3 silencing decreased ARHGAP18 expression, RhoA activity, Cdc42 expression and ^Y861FAK phosphorylation. Interestingly, IP₃R3 was able to regulate profilin remodeling, without inducing any myosin II reorganization. IP₃R3 silencing revealed an oscillatory calcium signature, with a predominant oscillating profile occurring in early wound repair. To summarize, we demonstrated that IP₃R3 is able to modulate intracellular Ca²⁺ availability and to coordinate the remodeling of profilin cytoskeleton organization through the ARHGAP18/RhoA/mDia1/FAK pathway.     

Regulation of inositol phosphate levels by prostaglandins in cultured endometrial cells

Stimulation of cultured rabbit endometrial cells by one of the rabbit endometrial cell culture proliferation factors, prostaglandin F_2α (PGF_2α), resulted in a very rapid increase in the intracellular levels of [³H]-inositol triphosphate (IP₃), [³H]-inositol biphosphate (IP₂), and [³H]-inositol monophosphate (IP₁) in cells prelabeled with [³H]-inositol. These increases in inositol phosphate levels were detected in periods of stimulation as short as 30 seconds, reached a maximum by 1 1/2?2 min and declined to control levels by 6–10 min. The stimulation was dose-dependent with maximal increases observed near 10^?6 M PGF_2α. The cholinergic agent, carbachol, also led to time and dose-independent increases in IP₃. Lithium, cadmium, silver, copper, and zinc ions had no effect either on the breakdown of IP₃ or on the accumulation of IP₁. In contrast, vanadate at 10^?6 or 10^?5 M did lead to a decrease in the breakdown of IP₁ and a concomitant increase in IP₁, IP₂, and IP₃. PGF_2α was found previously to induce an increase in rabbit endometrial cell DNA synthesis which was inhibited by concomitant or prior addition of prostaglandin E₁ (PGE₁). PGE₁, in a dose-dependent manner, was found to inhibit the observed IP₃ increase by PGF_2α at 1 1/2 min of stimulation. PGF_2α treated and control cultures did not differ in cAMP or cGMP levels, cellular ⁴⁵Ca uptake, nor cellular ²²Na uptake. We propose that IP₃ may be one of the intracellular messenger(s) synthesized following the treatment of rabbit endometrial cell cultures with the proliferation agent PGF_2α and that it may play a crucial role with cAMP in growth regulation.     

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP₇, is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP₇ regulation in β-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP₇. Although the inhibitors have diverse targets, they all perturbed IP₇ levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP₇ levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K_m for ATP makes IP₇ synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP₇ concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP₇ indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic β-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP₇’s role in cellular signal transduction is likely to have been underestimated.     

Reactive Oxygen Species Regulate a Slingshot-Cofilin Activation Pathway

Cellular stimuli generate reactive oxygen species (ROS) via the local action of NADPH oxidases (Nox) to modulate cytoskeletal organization and cell migration through unknown mechanisms. Cofilin is a major regulator of cellular actin dynamics whose activity is controlled by phosphorylation/dephosphorylation at Ser3. Here we show that Slingshot-1L (SSH-1L), a selective cofilin regulatory phosphatase, is involved in H₂O₂-induced cofilin dephosphorylation and activation. SSH-1L is activated by its release from a regulatory complex with 14-3-3ζ protein through the redox-mediated oxidation of 14-3-3ζ by H₂O₂. The ROS-dependent activation of the SSH-1L-cofilin pathway stimulates the SSH-1L–dependent formation of cofilin-actin rods in cofilin-GFP–expressing HeLa cells. Similarly, the formation of endogenous ROS stimulated by angiotensin II (AngII) also activates the SSH-1L-cofilin pathway via oxidation of 14-3-3ζ to increase AngII-induced membrane ruffling and cell motility. These results suggest that the formation of ROS by NADPH oxidases engages a SSH-1L-cofilin pathway to regulate cytoskeletal organization and cell migration.