Fcepsilon RI control of Ras via inositol (1,4,5) trisphosphate 3-kinase and inositol tetrakisphosphate

The inositol (1,4,5) trisphosphate 3-kinase (ITP3K) phosphorylates Ins (1,4,5) P3 to produce Ins (1,3,4,5) P4. The ITP3K substrate, InsP3, and its product, InsP4, both have the potential to regulate mast cell function. Here, we explore the effects of dominant inhibition of ITP3K upon secretory responses and Ras GTPase activation following antigenic cross-linking of the mast cell immunoreceptor, FcvarepsilonRI. Inhibition of ITP3K potentiates both calcium release from intracellular stores and calcium-dependent secretory responses in mast cells. Moreover, mast cells with dominantly inhibited ITP3K display constitutive activation of Ras and certain Ras effector pathways. We propose three mechanisms by which ITP3K inhibition could influence Ras activation. The protection of InsP3 that results from ITP3K inhibition may lead to enhanced activation of calcium-sensitive Ras-GAPs or -GRFs. Similarly, the deficit in InsP4 may change the behavior of the InsP4 receptor, the GAP1(IP4BP). Our data are inconsistent with calcium-sensitive Ras-GAP activation being the primary consequence of ITP3K inhibition in mast cells. Rather, we observe potentiation of Ras responses in mast cells transfected with dominant negative GAP1(IP4BP). Moreover, shRNA-mediated knockdown of GAP1(IP4BP) potentiates FcvarepsilonRI-mediated Ras activation, indicating that this InsP4-binding GAP protein may be used by the FcvarepsilonRI immunoreceptor to regulate Ras.     

Rat inositol 1,4,5-trisphosphate 3-kinase C is enzymatically specialized for basal cellular inositol trisphosphate phosphorylation and shuttles actively between nucleus and cytoplasm

The calcium-liberating second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is converted to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) by Ins(1,4,5)P3 3-kinases (IP3Ks) that add a fourth phosphate group to the 3-position of the inositol ring. Two isoforms of IP3Ks (named A and B) from different vertebrate species have been well studied. Recently the cloning and examination of a human full-length cDNA encoding a novel isoform, termed human IP3K-C (HsIP3K-C), has been reported. In the present study we report the cloning of a full-length cDNA encoding a rat homologue of HsIP3K-C with a unique mRNA expression pattern, which differs remarkably from the tissue distribution of HsIP3K-C. Of the rat tissues examined, rat IP3K-C (RnIP3K-C) is mainly present in heart, brain, and testis and shows the strongest expression in an epidermal tissue, namely tongue epithelium. RnIP3K-C has a calculated molecular mass of approximately 74.5 kDa and shows an overall identity of approximately 75% with HsIP3K-C. A bacterially expressed, enzymatically active and Ca2+-calmodulin-regulated fragment of this isoform displays remarkable enzymatic properties like a very low Km for Ins(1,4,5)P3 ( approximately 0.2 microm), substrate inhibition by high concentrations of Ins(1,4,5)P3, allosteric product activation by Ins(1,3,4,5)P4 in absence of Ca2+-calmodulin (Ka(app) 0.52 microm), and the ability to efficiently phosphorylate a second InsP3 substrate, inositol 2,4,5-trisphosphate, to inositol 2,4,5,6-tetrakisphosphate in the presence of Ins(1,3,4,5)P4. Furthermore, the RnIP3K-C fused with a fluorescent protein tag is actively transported into and out of the nucleus when transiently expressed in mammalian cells. A leucine-rich nuclear export signal and an uncharacterized nuclear import activity are localized in the N-terminal domain of the protein and determine its nucleocytoplasmic shuttling. These findings point to a particular role of RnIP3K-C in nuclear inositol trisphosphate phosphorylation and cellular growth.     

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.     

Synergistic control of Ca2+ mobilization in permeabilized mouse L1210 lymphoma cells by inositol 2,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate.

L1210 lymphoma cells were permeabilized with digitonin, and the ability of Ins(2,4,5)P3 and Ins(1,3,4,5)P4 to mobilize intracellular Ca2+ was studied. At high doses of Ins(2,4,5)P3 Ca2+ was rapidly released from intracellular stores, and prior or subsequent addition of Ins(1,3,4,5)P4 had no discernible effect. However, the Ca2(+)-mobilizing action of low (threshold or just above) concentrations of Ins(2,4,5)P3 was markedly enhanced by Ins(1,3,4,5)P4, which alone caused no mobilization of Ca2+; this phenomenon was shown not to be due to protection of Ins(2,4,5)P3 by the Ins(1,3,4,5)P4 against hydrolysis. The ability of the pre-addition of Ins(1,3,4,5)P4 to enhance subsequent Ins(2,4,5)P3-induced Ca2+ mobilization was always seen whether or not the free Ca2+ concentration was low (pCa = 7) or high (pCa = 6). However, at low Ca2+, Ins(1,3,4,5)P4 could cause a further mobilization if added after the Ins(2,4,5)P3, whereas at higher Ca2+ values Ins(1,3,4,5)P4 was only able to affect Ca2+ if added before Ins(2,4,5)P3. These effects of Ins(1,3,4,5)P4 were not, at the same concentration, mimicked by a random mixture of InsP4 isomers obtained by partial acid hydrolysis of phytic acid, by Ins(1,3,4)P3 or by Ins(1,3,4,5,6)P5, and they were shown not to be due to enzymic generation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 by (a) the absence of any detectable production of Ins(1,4,5)P3 if radiolabelled Ins(1,3,4,5)P4 was used, or (b) the observation that Ins(1,3,4,5,6)P5 could mimic Ins(1,3,4,5)P4 provided that higher doses were used; this inositol phosphate, when added radiolabelled, yielded only trace quantities of D/L-Ins(1,4,5,6)P4, which itself does not mobilize Ca2+. We interpret these results overall to mean that in these cells there is a small proportion of the Ins(2,4,5)P3-mobilizable Ca2+ pools which can only be mobilized in the presence of Ins(1,3,4,5)P4 [or at the least, Ins(1,3,4,5)P4 can help Ins(2,4,5)P3 to gain access to them]. The significance of this conclusion is discussed in the light of current concepts of the second messenger function of Ins(1,3,4,5)P4.     

Inositol tetrakisphosphate-induced sequestration of Ca2+ replenishes an intracellular pool sensitive to inositol trisphosphate

In a permeable neoplastic rat liver epithelial (261B) cell system, inositol 1,3,4,5-tetrakisphosphate--Ins(1,3,4,5)P4--induces sequestration of Ca2+ released by inositol 2,4,5-trisphosphate--Ins(2,4,5)P3; a non-metabolized inositol trisphosphate (InsP3) isomer--and Ca2+ added exogenously in the form of CaCl2. Studies were performed to identify the Ca2+ pool filled after Ins(1,3,4,5)P4 treatment. Both Ins(2,4,5)P3 and inositol 1,4,5-trisphosphate--Ins(1,4,5)P3--dose-dependently release Ca2+ from permeable 261B cells--Ins(1,4,5)P3 having a threefold greater potency--but differ in that Ca2+ released by Ins(1,4,5)P3 is readily sequestered, while the Ca2+ released by Ins(2,4,5)P3 is not. Maximal release of Ca2+ by 6 microM Ins(2,4,5)P3 blocked the action of Ins(1,4,5)P3, demonstrating that these two isomers influence the same intracellular Ca2+ pool through a shared membrane receptor. Addition of 2 microM Ins(2,4,5)P3 to discharge partially the Ca2+ pool reduced the amount of Ca2+ released by a maximal dose of Ins(1,4,5)P3 (2 microM). Ins(1,3,4,5)P4 combined with Ins(2,4,5)P3 produced a Ca2+ release and sequestration response, which replenished the InsP3-sensitive pool as indicated by a recovery of full Ca2+ release by 2 microM Ins(1,4,5)P3. Induction of Ca2+ sequestration by Ins(1,3,4,5)P4 occurred dose-dependently, with a half-maximal response elicited at a dose of 0.9 microM. Further studies of the effect of Ins(1,3,4,5)P4 apart from the influence of Ins(2,4,5)P3 using a model in which the Ca2+ levels are raised by an exogenous addition of CaCl2 showed that Ins(1,4,5)P3 released twice the amount of Ca2+ from the storage pool following Ins(1,3,4,5)P4-induced Ca2+ sequestration. These results demonstrate that the Ca2+ uptake induced by Ins(1,3,4,5)P4 preferentially replenishes the intracellular Ca2+ storage sites regulated by Ins(1,4,5)P3 and Ins(2,4,5)P3.     

The metabolism of tris- and tetraphosphates of inositol by 5-phosphomonoesterase and 3-kinase enzymes

Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate to form both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,2-cyclic 4,5-trisphosphate (cInsP3). The further metabolism of these inositol trisphosphates is determined by two enzymes: a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate (InsP4), while the latter forms inositol 1,4-bisphosphate and inositol 1,2-cyclic 4-bisphosphate from Ins(1,4,5)P3 and cInsP3, respectively. The current studies show that the 3-kinase is unable to phosphorylate cInsP3. Also, the 5-phosphomonoesterase hydrolyzes InsP4 with an apparent Km of 0.5-1.0 microM to form inositol 1,3,4-trisphosphate at a maximal velocity approximately 1/30 that for Ins(1,4,5)P3. The apparent affinity of the enzyme for the three substrates is InsP4 greater than Ins(1,4,5)P3 greater than cInsP3; however, the rate at which the phosphatase hydrolyzes these substrates is Ins(1,4,5)P3 greater than cInsP3 greater than InsP4. The 5-phosphomonoesterase and 3-kinase enzymes may control the levels of inositol trisphosphates in stimulated cells. The 3-kinase has a low apparent Km for Ins(1,4,5)P3 as does the 5-phosphomonoesterase for InsP4, implying that the formation and breakdown of InsP4 may proceed when both it and its precursor are present at low levels. Ins(1,4,5)P3 is utilized by both the 3-kinase and 5-phosphomonoesterase, while cInsP3 is utilized relatively poorly only by the 5-phosphomonoesterase. These findings imply that inositol cyclic trisphosphate may be metabolized slowly after its formation in stimulated cells.     

Identification of a novel inositol phosphate recognition site: specific [3H]inositol hexakisphosphate binding to brain regions and cerebellar membranes

[3H]Inositol hexakisphosphate (InsP6) binds with a heterogeneous distribution to frozen sections of unfixed rat brain and is displaced by unlabelled InsP6. The pattern of binding correlates with binding to neuronal cell bodies. [3H]InsP6 binding to cerebellar membranes has been further characterised, is reversible, and saturable, and exhibits high specificity for inositol polyphosphates. The IC50 for competition by unlabelled InsP6 is approximately 100nM, whereas inositol 1,3,4,5,6 pentakisphosphate (Ins(13456)P5), inositol 1,3,4,5 tetrakisphosphate (Ins(1345)P4), and inositol 1,4,5 trisphosphate (Ins(145)P3) bind with an affinity at least one order of magnitude lower. [3H]InsP6 binding is clearly distinct from previously characterised Ins(145)P3 (ref. 1, 2) and Ins(1345)P4 (ref. 3) binding, both in terms of pharmacology and brain distribution.     

Metabolic relations of inositol 3,4,5,6-tetrakisphosphate revealed by cell permeabilization. Identification of inositol 3,4,5, 6-tetrakisphosphate 1-kinase and inositol 3,4,5,6-tetrakisphosphate phosphatase activities in mesophyll cells

Using a permeabilization strategy to introduce Ins(3,4,5,6) P(4) into mesophyll protoplasts of Commelina communis, we have identified Ins(3,4,5,6) P(4) 1-kinase activity in mesophyll cells. Multiple InsP(3) isomers were identified in Spirodela polyrhiza and Arabidopsis. Only two of these, Ins(1,2,3) P(3) and Ins(3,4,6) P(3), have previously been identified in plants and only in monocots. The isomers detected in S. polyrhiza included D- and/or L-Ins(3,4,5) P(3), D- and/or L-Ins(3,5,6) P(3), and D- and/or L-Ins(2,4,5) P(3). Ins(1,4,5) P(3), if present, was only a tiny fraction of total InsP(3) species. We have also identified inositol polyphosphate phosphatase activities, Ins(3,4,5,6) P(4) 6-phosphatase and Ins(3,4, 5, 6) P(4) 4-phosphatase, whose action on endogenous inositol polyphosphates explains the presence of D- and/or L-Ins(3,4,5) P(3) and D- and/or L-Ins(3,5,6) P(3) in mesophyll cells. Inositol trisphosphates identified in Arabidopsis include Ins(1,2,3) P(3) and D- and/or L-Ins(3,4,6) P(3), suggesting that dicots may share pathways of InsP(6) biosynthesis and breakdown in common with monocots.     

Kinetics of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate generation in dog-thyroid primary cultured cells stimulated by carbachol

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog-thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse-phase high pressure liquid chromatography. The structure of inositol phosphates co-eluting with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co-eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co-eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5-10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20s. The level of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] increased continuously for 2 min after the addition of carbachol. Inositol-phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog-thyroid adenylate cyclase and cyclic-AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2(+)-calmodulin sensitivity of the Ins(1,4,5)P3 3-kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacyglycerol on Ins(1,4,5)P3 generation.     

Different patterns of agonist-stimulated increases of 3H-inositol phosphate isomers and cytosolic Ca2+ in bovine adrenal chromaffin cells: comparison of the effects of histamine and angiotensin II

Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3-4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2(+)-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+]i changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

Inositol 1,3,4,5-tetrakisphosphate stimulates calcium release from bovine adrenal microsomes by a mechanism independent of the inositol 1,4,5-trisphosphate receptor.

In bovine adrenal microsomes, Ins(1,4,5)P3 binds to a specific high-affinity receptor site (Kd = 11 nM) with low affinity for two other InsP3 isomers, Ins(1,3,4)P3 and Ins(2,4,5)P3. In the same subcellular fractions Ins(1,4,5)P3 was also the most potent stimulus of Ca2+ release of all the inositol phosphates tested. Of the many inositol phosphates recently identified in angiotensin-II-stimulated adrenal glomerulosa and other cells, Ins(1,3,4,5)P4 has been implicated as an additional second messenger that may act in conjunction with Ins(1,4,5)P3 to elicit Ca2+ mobilization. In the present study, an independent action of Ins(1,3,4,5)P4 was observed in bovine adrenal microsomes. Heparin, a sulphated polysaccharide which binds to Ins(1,4,5)P3 receptors in several tissues, inhibited both the binding of radiolabelled Ins(1,4,5)P3 and its Ca2(+)-releasing activity in adrenal microsomes. In contrast, heparin did not inhibit the mobilization of Ca2+ by Ins(1,3,4,5)P4, even at doses that abolished the Ins(1,4,5)P3 response. Such differential inhibition of the Ins(1,4,5)P3- and Ins(1,3,4,5)P4-induced Ca2+ responses by heparin indicates that Ins(1,3,4,5)P4 stimulates the release of Ca2+ from a discrete intracellular store, and exerts this action via a specific receptor site that is distinct from the Ins(1,4,5)P3 receptor.     

Effects of bombesin and insulin on inositol (1,4,5)trisphosphate and inositol (1,3,4)trisphosphate formation in Swiss 3T3 cells

The effects of bombesin and insulin, separately and in combination, have been studied in Swiss mouse 3T3 cells. Bombesin caused a rapid transfer of 3H from the lipid inositol pool of prelabeled cells into inositol phosphates. Label in inositol tetrakisphosphate (InsP4) and in Ins1,4,5P3 and Ins1,3,4P3 rose within 10 sec of stimulation and that in Ins1,4P2, another InsP2 and InsP1, more slowly. Insulin, which had little effect on its own, increased the turnover of inositol lipids due to acute bombesin stimulation and also enhanced the DNA synthesis evoked by prolonged bombesin treatment. The results suggest that bombesin acting as a growth factor, uses inositol lipids as part of its transduction mechanism and that insulin acts synergistically to enhance both inositol phosphate formation and DNA synthesis.     

Formation of inositol phosphate isomers in GH3 pituitary tumour cells stimulated with thyrotropin-releasing hormone. Acute effects of lithium ions.

With a h.p.l.c. system, the inositol mono-, bis- and tris-phosphate isomers found in [3H]inositol-labelled GH3 cells were resolved and identified. These cells possess at least ten distinct [3H]inositol-containing substances when acid-soluble extracts are analysed by anion-exchange h.p.l.c. These substances were identified by their co-elution with known inositol phosphate standards and, to a limited extent, by examining their chemical structure. Two major inositol monophosphate (InsP) isomers were identified, namely Ins1P and Ins4P, both of which accumulate after stimulation with the hypothalamic releasing factor (TRH) (thyrotropin-releasing hormone). Three inositol bisphosphate (InsP2) isomers were resolved, of which two were positively identified, i.e. Ins(1,4)P2 and Ins(3,4)P2. TRH treatment increases both of these isomers, with Ins(1,4)P2 being produced at a faster rate than Ins(3,4)P2. The third InsP2 isomer has yet to be fully identified, although it is co-eluted with an Ins(4,5)P2 standard. This third InsP2 is also increased after TRH stimulation. In common with other cell types, the GH3 cell contains two inositol trisphosphate (InsP3) isomers: Ins(1,4,5)P3, which accumulates rapidly, and Ins(1,3,4)P3, which is formed more slowly. The latter substance appears simultaneously with its precursor, inositol 1,3,4,5-tetrakisphosphate. We also examined the effects of acute Li+ treatment on the rates of accumulation of these isomers, and demonstrated that Li+ augments TRH-mediated accumulation of Ins1P, Ins4P, Ins(1,4)P2, the presumed Ins(4,5)P2 and Ins(1,3,4)P3. These results suggest that the effects of Li+ on inositol phosphate metabolism are more complex than was originally envisaged, and support work carried out by less sophisticated chromatographic analysis.     

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.     

Leukotriene B4 stimulation of phagocytes results in the formation of inositol 1,4,5-trisphosphate. A second messenger for Ca2+ mobilization.

Inositol trisphosphate (InsP3) production and cytosolic free Ca2+ ([Ca2+]i) elevations induced by leukotriene B4 (LTB4)-receptor activation were studied in the human promyelocytic-leukaemia cell line HL60, induced to differentiate by retinoic acid. The myeloid-differentiated HL60 cells respond to LTB4 by raising their [Ca2+]i with a dose-response relationship similar to that shown by normal human neutrophils. The observations of the LTB4 transduction mechanism were compared with those of the transduction mechanism of the chemotactic peptide fMet-Leu-Phe in HL60 cells differentiated with dimethyl sulphoxide. Both LTB4 and fMet-Leu-Phe triggered a rapid (less than 5 s) elevation of [Ca2+]i, which occurred in parallel with the InsP3 production from myo-[3H]inositol-labelled cells. The threshold concentrations of the agonists, for InsP3 production, were found at 10(-9) M, a slightly higher concentration than that required to detect [Ca2+]i elevations. No significant changes were noted in the phosphoinositide levels upon stimulation with LTB4. Exposure to Bordetella pertussis toxin before LTB4 stimulation abolished both the increased formation of InsP3 and the rise of [Ca2+]i. LTB4 and fMet-Leu-Phe elicited elevations of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with no detectable lag time, followed by slower and more sustained inositol 1,3,4-trisphosphate elevations. Stimulation with various leukotriene analogues revealed a good correlation between both total InsP3 as well as Ins(1,4,5)P3 formation and elevations of [Ca2+]1. Thus LTB4 receptor activation results in an increased production of Ins(1,4,5)P3 via a transduction mechanism also involving a nucleotide regulatory protein, as previously described for the fMet-Leu-Phe transduction mechanism.     

Inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate are minor components of total mass of inositol trisphosphate in thrombin-stimulated platelets. Rapid formation of inositol 1,3,4-trisphosphate

Stimulation of human platelets by thrombin leads to rises of both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) within 10 s. The mass of Ins(1,4,5)P3 was measured in platelet extracts after conversion to [3-32P]Ins(1,3,4,5)P4 with Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Basal levels were equivalent to 0.2 microM and rose to 1 microM within 10 s of stimulation by thrombin. The mass of Ins(1,3,4)P3 was more than 10-fold greater than that of Ins(1,4,5)P3 between 10 and 60 s of thrombin stimulation. These results indicate that the majority of InsP3 liberated by phospholipase C in stimulated platelets must be the non-cyclic Ins(1,4,5)P3 in order to allow rapid phosphorylation by Ins(1,4,5)P3 3-kinase to Ins(1,3,4,5)P4 and then dephosphorylation to Ins(1,3,4)P3 by 5-phosphomonoesterase. A significant proportion of the InsP3 extracted from thrombin-stimulated platelets under neutral conditions is resistant to Ins(1,4,5)P3 3-kinase but susceptible after acid treatment, implying the presence of inositol 1,2-cyclic 4,5-trisphosphate (Ins(1,2cyc4,5)P3. The relative proportion of Ins(1,2cyc4,5)P3 increases with time. We suggest that such gradual accumulation is attributable to the relative insensitivity of this compound to hydrolytic and phosphorylating enzymes. Therefore, early Ca2+ mobilization in platelets is more likely to be effected by Ins(1,4,5)P3 than by Ins(1,2cyc4,5)P3.     

Ca2(+)-stimulatable and protein kinase C-inhibitable accumulation of inositol 1,3,4,6-tetrakisphosphate in human platelets.

Thrombin-stimulated (10 s) human platelets produce Ins(1,4,5)P3 and an additional inositol trisphosphate (InsP3), in approximately a 1:20 ratio. The major InsP3 co-migrates with Ins(1,3,4)P3 on strong-anion-exchange h.p.l.c. To identify this species unequivocally, we treated putative Ins(1,3,4)P3 obtained from thrombin-stimulated myo-[3H]inositol-labelled platelets with NaIO4/NaBH4 or 4-phosphomonoesterase. The products indicate that the major InsP3 is at least 90% D-Ins(1,3,4)P3. D-[3H]Ins(1,3,4)P3 added to saponin-permeabilized platelets is hydrolysed to an InsP2 (7.8%) and phosphorylated by a kinase to yield an inositol polyphosphate (0.9%) in 5 min. The phosphorylation product co-migrates with Ins(1,3,4,6)P4 on Partisphere WAX h.p.l.c. Under similar conditions, L-[3H]Ins(1,3,4)P3 is dephosphorylated but not phosphorylated. Relative phosphatase:kinase ratios are 8.7:1 (Vmax. values) and 0.86:1 (Km values) with respect to D-Ins(1,3,4)P3. The kinase activity is predominantly cytosolic (96.8% of total activity) in freeze-thaw-disrupted platelets, and the accumulation of its product is Ca2(+)-dependent. The activity is identified as a 6-kinase on the basis of its product's insensitivity to 5-phosphomonoesterase, resistance to periodate oxidation and co-migration with standard Ins(1,3,4,6)P4 on h.p.l.c. Incubation of platelets with beta-phorbol dibutyrate (beta-PDBu, 76 nM), causing activation of protein kinase C, results in a 57.5% inhibition (reversible by the protein kinase C inhibitor staurosporine) of Ins(1,3,4,6)P4 accumulation. alpha-PDBu, which does not stimulate protein kinase C, has no effect. Stimulation of intact platelets with thrombin results in the production of Ins(1,3,4,6)P4 (1.4-fold rise in 30 s) and Ins(1,3,4,5)P4, with the latter being the major InsP4 species. Accumulation of Ins(1,3,4,6)P4 is slightly delayed in comparison with Ins(1,3,4)P3 and is relatively small. We propose that the major route of Ins(1,3,4)P3 metabolism in stimulated human platelets is via phosphatase action.     

Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase.

An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 x 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5'-[gamma-thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.     

GAP1IP4BP contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).