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Robin F Irvine

Publications and source records attributed to Robin F Irvine.

At least 19 recordsLinked to original sources
PubMed2007

Type II PtdInsP kinases: location, regulation and function.

The regulation of the synthesis of PtdIns(4,5)P2 is emerging as being as complex as we might expect from the multi-functional nature of this lipid. In the present chapter we focus on one aspect of inositide metabolism, which is the functions of the Type II PIPkins (Type II PtdInsP kinases). These are primarily PtdIns5P 4-kinases, although in vitro they will also phosphorylate PtdIns3P to PtdIns(3,4)P2. Thus they have three, not necessarily exclusive, functions: to make PtdIns(4,5)P2 by a quantitatively minor route, to remove PtdIns5P and to make PtdIns(3,4)P2 by a route that does not involve a Class I PtdIns 3-kinase. None of these three possible functions has yet been unambiguously proven or ruled out. Of the three isoforms, alpha and beta are widely expressed, the IIalpha being predominantly cytosolic and the IIbeta primarily nuclear. PIPkin IIgamma has a much more restricted tissue expression pattern, and appears to be localized primarily to intracellular vesicles. Here we introduce in turn each of the three Type II PIPkins, and discuss what we know about their localization, their regulation and their function.

Amino Acid Substitution↗
PubMed2006

The intracellular localisation and mobility of Type Igamma phosphatidylinositol 4P 5-kinase splice variants.

There are three known splice variants of Type Igamma phosphatidylinositol 4-phosphate 5-kinase (PIPkin Igamma): PIPkins Igamma87, Igamma90, and the most recently cloned (Giudici, M.L., Emson, P.C. and Irvine, R.F. (2004) A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma. Biochem. J. 379, 489-496) PIPkin IgammaC (here called PIPkin Igamma93). Here, we have explored the subcellular localisation and mobility of Type I PIPkins in transfected cells by confocal microscopy and flourescence recovery after photobleaching. The unique behaviour shown by PIPkin Igamma93 is consistent with its suggested distinct function. Moreover, the markedly different localisation and mobility of active versus inactive PIPkin Igamma93 provide insights into the factors that dictate cellular targeting of Type Igamma PIPkins.

Animals↗
PubMed2006

The behaviour of myo-inositol hexakisphosphate in the presence of magnesium(II) and calcium(II): protein-free soluble InsP6 is limited to 49 microM under cytosolic/nuclear conditions.

Progress in the biology of myo-inositol hexakisphosphate (InsP(6)) has been delayed by the lack of a quantitative description of its multiple interactions with divalent cations. Our recent initial description of these [J. Torres, S. Dominguez, M.F. Cerda, G. Obal, A. Mederos, R.F. Irvine, A. Diaz, C. Kremer, J. Inorg. Biochem. 99 (2005) 828-840] predicted that under cytosolic/nuclear conditions, protein-free soluble InsP(6) occurs as Mg(5)(H(2)L), a neutral complex that exists thanks to a significant, but undefined, window of solubility displayed by solid Mg(5)(H(2)L).22H(2)O (L is fully deprotonated InsP(6)). Here we complete the description of the InsP(6)-Mg(2+)-Ca(2+) system, defining the solubilities of the Mg(2+) and Ca(2+) (Ca(5)(H(2)L).16H(2)O) solids in terms of K(s0)=[M(2+)](5)[H(2)L(10-)], with pK(s0)=32.93 for M=Mg and pK(s0)=39.3 for M=Ca. The concentration of soluble Mg(5)(H(2)L) at 37 degrees C and I=0.15M NaClO(4) is limited to 49muM, yet InsP(6) in mammalian cells may reach 100muM. Any cytosolic/nuclear InsP(6) in excess of 49muM must be protein- or membrane-bound, or as solid Mg(5)(H(2)L).22H(2)O, and any extracellular InsP(6) (e.g. in plasma) is surely protein-bound.

Calcium↗
PubMed2006

Nuclear inositide signalling -- expansion, structures and clarification.

The extent and content of this review issue highlights how our understanding of lipid signalling in the nucleus has grown, both in what we actually know, and the breadth of signalling pathways that we now have to consider. Here, a few key issues with regard to nuclear inositide signalling are briefly addressed.

Animals↗
PubMed2006

Regulation of type II PIP kinase by PKD phosphorylation.

The type II PIP kinases phosphorylate the poorly understood inositol lipid PtdIns5P, producing the multi-functional lipid product PtdIns(4,5)P(2). To investigate the regulation of these enzymes by phosphorylation, we partially purified a protein kinase from pig platelets that phosphorylated type IIalpha PIP kinase on an activation loop threonine residue, T376. Pharmacological studies suggested this protein kinase was protein kinase D (PKD), and in vitro experiments confirmed this identification. A phospho-specific antibody was developed and used to demonstrate phosphorylation of T376 in living cells, and its enhancement under conditions in which PKD was activated. Although we were unable to determine the effects of phosphorylation on PIP kinase activity directly, mutation of T376 to aspartate significantly inhibited enzyme activity. We conclude that the type II PIP kinases are physiological targets for PKD phosphorylation, and that this modification is likely to regulate inositol lipid turnover by inhibition of these lipid kinases.

1-Phosphatidylinositol 4-Kinase↗
PubMed2006

Calcium-triggered exit of F-actin and IP(3) 3-kinase A from dendritic spines is rapid and reversible.

The structure of the actin cytoskeleton in dendritic spines is thought to underlie some forms of synaptic plasticity. We have used fixed and live-cell imaging in rat primary hippocampal cultures to characterize the synaptic dynamics of the F-actin binding protein inositol trisphosphate 3-kinase A (IP3K), which is localized in the spines of pyramidal neurons derived from the CA1 region. IP3K was intensely concentrated as puncta in spine heads when Ca(2+) influx was low, but rapidly and reversibly redistributed to a striated morphology in the main dendrite when Ca(2+) influx was high. Glutamate stimulated the exit of IP3K from spines within 10 s, and re-entry following blockage of Ca(2+) influx commenced within a minute; IP3K appeared to remain associated with F-actin throughout this process. Ca(2+)-triggered F-actin relocalization occurred in about 90% of the cells expressing IP3K endogenously, and was modulated by the synaptic activity of the cultures, suggesting that it is a physiological process. F-actin relocalization was blocked by cytochalasins, jasplakinolide and by the over-expression of actin fused to green fluorescent protein. We also used deconvolution microscopy to visualize the relationship between F-actin and endoplasmic reticulum inside dendritic spines, revealing a delicate microorganization of IP3K near the Ca(2+) stores. We conclude that Ca(2+) influx into the spines of CA1 pyramidal neurons triggers the rapid and reversible retraction of F-actin from the dendritic spine head. This process contributes to changes in spine F-actin shape and content during synaptic activity, and might also regulate spine IP3 signals.

Actins↗
PubMed2005

Regulation of the localization and activity of inositol 1,4,5-trisphosphate 3-kinase B in intact cells by proteolysis.

IP3K (inositol 1,4,5-trisphosphate 3-kinase) catalyses the Ca2+-regulated phosphorylation of the second messenger Ins(1,4,5)P3, thereby inactivating the signal to release Ca2+ and generating Ins(1,3,4,5)P4. Here we have investigated the localization and activity of IP3KB and its modulation by proteolysis. We found that the N- and C-termini (either side of residue 262) of IP3KB localized predominantly to the actin cytoskeleton and ER (endoplasmic reticulum) respectively, both in COS-7 cells and in primary astrocytes. The functional relevance of this was demonstrated by showing that full-length (actin-localized) IP3KB abolished the histamine-induced Ca2+ response in HeLa cells more effectively than truncated constructs localized to the ER or cytosol. The superior efficacy of full-length IP3KB was also attenuated by disruption of the actin cytoskeleton. By transfecting COS-7 cells with double-tagged IP3KB, we show that the translocation from actin to ER may be a physiologically regulated process caused by Ca2+-modulated constitutive proteolysis in intact cells.

Animals↗
PubMed2005

Agonist-induced regulation of mitochondrial and endoplasmic reticulum motility.

Using fluorescently tagged markers for organelles in conjunction with confocal microscopy, we have studied the effects of agonist-induced Ca2+ signals on the motility of mitochondria and the ER (endoplasmic reticulum). We observed that the muscarinic agonist carbachol produced a rapid, simultaneous and reversible cessation of the movements of both organelles, which was dependent on a rise in cytosolic Ca2+. This rise in Ca2+ was shown to cause a fall in cellular ATP levels, and the effect of carbachol on organelle movement could be mimicked by depleting ATP with metabolic inhibitors in the absence of any such rise in Ca2+. However, a Ca2+-sensing process independent of ATP appears also to be involved, because we identified conditions where the ATP depletion was blocked (by inhibitors of the Ca2+ pumps), but the organelle movements still ceased following a rise in cytosolic Ca2+. We conclude that the co-ordinated cessation of mitochondria and ER motility is a process regulated by the cytosolic concentration of both Ca2+ and ATP, and that these two parameters are likely to synergize to regulate the localization of the two organelles, and to facilitate the transfer of Ca2+ between them.

Adenosine Triphosphate↗
PubMed2005

Phospholipase D2 stimulates integrin-mediated adhesion via phosphatidylinositol 4-phosphate 5-kinase Igamma b.

Cellular adhesion can be regulated by, as yet, poorly defined intracellular signalling events. Phospholipase D enzymes generate the messenger lipid phosphatidate and here we demonstrate that suppression of this reaction inhibits cellular adhesion. This effect was reversed by the addition of cell-permeable analogues of either phosphatidate or phosphatidylinositol 4,5-bisphosphate. By contrast, neither diacylglycerol nor lysophosphatidic acid were able to reverse this effect suggesting that phosphatidate itself acts directly on a target protein(s) to regulate adhesion rather than as the result of its conversion to either of these metabolite lipids. Antibodies that block beta1 and beta2 integrin-substrate interactions inhibited adhesion stimulated by both phosphatidate and phosphatidylinositol 4,5-bisphosphate indicating that these lipids regulate beta1 and beta2 integrin-mediated adhesion. In vivo, these lipids can be generated by phospholipase D2 and phosphatidylinositol 4-phosphate 5-kinase Igamma b, respectively, and over-expression of catalytically-functional forms of these enzymes dose-dependently stimulated adhesion while siRNA depletion of PLD2 levels inhibited adhesion. Furthermore the ability of over-expressed phospholipase D2 to stimulate adhesion was inhibited by a dominant-negative version of phosphatidylinositol 4-phosphate 5-kinase Igamma b. Consistent with this, phosphatidylinositol 4-phosphate 5-kinase Igamma b-mediated adhesion was dependent upon phospholipase D2's product, phosphatidate indicating that phosphatidylinositol 4-phosphate 5-kinase Igamma b is downstream of, and necessary for, phospholipase D2's regulation of adhesion. It is likely that this phospholipase D2-generated phosphatidate directly stimulates phosphatidylinositol 4-phosphate 5-kinase Igamma b to generate phosphatidylinositol 4,5-bisphosphate as this mechanism has previously been demonstrated in vitro. Thus, our data indicates that during the initial stages of adhesion, phospholipase D2-derived phosphatidate stimulates phosphatidylinositol 4-phosphate 5-kinase Igamma b to generate phosphatidylinositol 4,5-bisphosphate and that consequently this inositol phospholipid promotes adhesion through its regulation of cell-surface integrins.

Animals↗
PubMed2005

Mobility of proteins associated with the plasma membrane by interaction with inositol lipids.

Translocation of a protein to the plasma membrane in response to the generation of polyphosphoinositol lipids is believed to be an important component of cellular regulation, in part because it increases the effective concentration of that protein relative to other proteins in the same membrane by restricting it to a two-dimensional space. However, such a concept assumes that, once translocated, a protein retains the free mobility it had in the cytoplasm, and also that the possible existence of partitioned pools of inositol lipids does not restrict its sphere of influence. We have explored by fluorescence recovery after photobleaching (FRAP) the mobility of four green-fluorescent-protein-tagged proteins, GAP1(IP4BP) and GAP1(m), when they are either cytoplasmic or attached to the plasma membrane, and the PH domain of PI-PLCdelta(1) and ICAM as representative of, respectively, another inositol-lipid-anchored protein and a single-transmembrane-span-domain protein. The data from GAP1(m) and the PI-PLCdelta(1) PH domain show that, when proteins associate with inositol lipids in the plasma membrane, they retain a mobility similar to that in the cytoplasm, and probably also similar to the inositol lipid to which they are attached, suggesting a free diffusion within the plane of the membrane. Moreover, this free diffusion is similar whether they are bound to PtdIns(3,4,5)P(3) or to PtdIns(4,5)P(2), and no evidence was found by these criteria for restricted pools of PtdIns(4,5)P(2). The mobility of GAP1(IP4BP), which has been reported to associate with PtdIns(4,5)P(2) in the plasma membrane, is much lower, suggesting that it might interact with other cellular components. Moreover, the mobility of GAP1(IP4BP) is not detectably altered by the generation of either of its two potential regulators, Ins(1,3,4,5)P(4) or PtdIns(3,4,5)P(3).

Cell Adhesion Molecules↗
PubMed2005

Effects of lipid kinase expression and cellular stimuli on phosphatidylinositol 5-phosphate levels in mammalian cell lines.

Phosphatidylinositol 5-phosphate (PtdIns5P) is a relatively recently discovered inositol lipid whose metabolism and functions are not yet clearly understood. We have transfected cells with a number of enzymes that are potentially implicated in the synthesis or metabolism of PtdIns5P, or subjected cells to a variety of stimuli, and then measured cellular PtdIns5P levels by a specific mass assay. Stable or transient overexpression of Type IIalpha PtdInsP kinase, or transient overexpression of Type Ialpha or IIbeta PtdInsP kinases caused no significant change in cellular PtdIns5P levels. Similarly, subjecting cells to oxidative stress or EGF stimulation had no significant effect on PtdIns5P, but stimulation of HeLa cells with a phosphoinositide-specific PLC-coupled agonist, histamine, caused a 40% decrease within 1 min. Our data question the degree to which inositide kinases regulate PtdIns5P levels in cells, and we discuss the possibility that a significant part of both the synthesis and removal of this lipid may be regulated by phosphatases and possibly phospholipases.

Animals↗
PubMed2005

Inositide evolution - towards turtle domination?

When viewing the changes in our understanding of inositides over the last 20 years, it is difficult to know whether to be more impressed by the proliferation in the number of inositides themselves (e.g. seven polyphosphoinositol lipids, more than 30 inositol phosphates), or by the number of functions for each. This review will focus on two specific aspects of this diversity: the evolution of the polyphosphoinositides, and the synthesis and functions of the higher inositol phosphates.

Animals↗
PubMed2005

The structural integrity of the endoplasmic reticulum, and its possible regulation by inositol 1,3,4,5-tetrakisphosphate.

The endoplasmic reticulum (ER) is a dynamic organelle thought to consist of a single interconnected network of membranes. Using fluorescence recovery after photobleaching (FRAP) of HEK-293 cells dually transfected with soluble fluorescent proteins targeted to the ER (GFP) and mitochondria (DsRed), we have confirmed this continuity, which contrasts that of the mitochondria, which behave as a population of discrete organelles. The degree of ER integrity (interconnected versus fragmented) has been suggested to be regulated in some cells by inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In HEK-293 and freshly isolated murine lacrimal acinar cells, we manipulated ER structure by disrupting cellular Ca(2+) homeostasis with the Ca(2+) ionophore ionomycin, and by permeabilisation of the plasma membrane, protocols known to cause ER fragmentation. However, we were subsequently unable to detect by FRAP any significant effect of Ins(1,3,4,5)P(4) on ER integrity.

Animals↗
PubMed2005

Solution behaviour of myo-inositol hexakisphosphate in the presence of multivalent cations. Prediction of a neutral pentamagnesium species under cytosolic/nuclear conditions.

myo-Inositol hexakisphosphate (InsP6) is an ubiquitous and abundant molecule in the cytosol and nucleus of eukaryotic cells whose biological functions are incompletely known. A major hurdle for studying the biology of InsP6 has been a deficiency of a full understanding of the chemistry of its interaction with divalent and trivalent cations. This deficiency has limited our appreciation of how it remains in solution within cells, and the likely degree to which it might interact in vivo with physiologically important cations such as Ca2+ and Fe3+. We report here the initial part of the description of the InsP6-multivalent cation chemistry, including its solution equilibria studied by high resolution potentiometry and (for the Fe(III)/Fe(II) couple) cyclic voltammetry. InsP6 forms anionic complexes of high affinities and 1:1 stoichiometry with Mg(II), Ca(II), Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II). Of particular importance is the observation that, in the exceptional case of Mg(II), InsP6 forms the species [Mg5(H2L)] (L representing fully deprotonated InsP6); this soluble neutral species is predicted to be the predominant form of InsP6 under nuclear or cytosolic conditions in animal cells. Contrary to previous suggestions, InsP6 is predicted not to interact with cytosolic calcium even when calcium is increased during signalling events. In vitro, InsP6 also forms high affinity 1:1 complexes with Fe(III) and Al(III). However, our data predict that in the biological context of excess free Mg(II), neither Fe(III) nor Fe(II) are complexed by InsP6.

Animals↗
PubMed2004

Structure of a human inositol 1,4,5-trisphosphate 3-kinase: substrate binding reveals why it is not a phosphoinositide 3-kinase.

Mammalian cells produce a variety of inositol phosphates (InsPs), including Ins(1,4,5)P3 that serves both as a second messenger and as a substrate for inositol polyphosphate kinases (IPKs), which further phosphorylate it. We report the structure of an IPK, the human Ins(1,4,5)P3 3-kinase-A, both free and in complexes with substrates and products. This enzyme catalyzes transfer of a phosphate from ATP to the 3-OH of Ins(1,4,5)P3, and its X-ray crystal structure provides a template for understanding a broad family of InsP kinases. The catalytic domain consists of three lobes. The N and C lobes bind ATP and resemble protein and lipid kinases, despite insignificant sequence similarity. The third lobe binds inositol phosphate and is a unique four-helix insertion in the C lobe. This lobe embraces all of the phosphates of Ins(1,4,5)P3 in a positively charged pocket, explaining the enzyme's substrate specificity and its inability to phosphorylate PtdIns(4,5)P2, the membrane-resident analog of Ins(1,4,5)P3.

Adenosine Triphosphate↗
PubMed2004

A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma.

Type I PIPkins (phosphatidylinositol 4-phosphate 5-kinases) are the enzymes that catalyse the major cellular route of synthesis of PtdIns(4,5) P2, and three isoforms (alpha, beta and gamma) with several splice variants have been found to date. In the present paper, we describe the discovery of a novel splice variant of the gamma isoform, which we call PIPkin Igammac, and which is characterized by the inclusion of a 26-amino-acid insert near the C-terminus. Its transcript appears to be selectively expressed in brain, where it locates in the neurons of restricted regions, such as cerebellum, hippocampus, cortex and olfactory bulb, as indicated by in situ hybridization studies. Overexpression of two different catalytically inactive constructs of PIPkin Igammac in rat cerebellar granule cells causes a progressive loss of their neuronal processes, whereas equivalent kinase-dead versions of PIPkin Igammaa did not induce any such effect, suggesting the possible existence of a specific PtdIns(4,5) P2 pool synthesized by PIPkin Igammac, which is involved in the maintenance of some neuronal cellular processes.

Alternative Splicing↗
PubMed2003

Kinetic model of the inositol trisphosphate receptor that shows both steady-state and quantal patterns of Ca2+ release from intracellular stores.

The release of Ca(2+) from intracellular stores via InsP(3) receptors shows anomalous kinetics. Successive additions of low concentrations of InsP(3) cause successive rapid transients of Ca(2+) release. These quantal responses have been ascribed to all-or-none release from stores with differing sensitivities to InsP(3) or, alternatively, to a steady-state mechanism where complex kinetic properties of the InsP(3) receptor allow partial emptying of all the stores. We present here an adaptive model of the InsP(3) receptor that can show either pattern, depending on the imposed experimental conditions. The model proposes two interconvertible conformational states of the receptor: one state binds InsP(3) rapidly, but with low affinity, whereas the other state binds slowly, but with high affinity. The model shows repetitive increments of Ca(2+) release in the absence of a Ca(2+) gradient, but more pronounced incremental behaviour when released Ca(2+) builds up at the mouth of the channel. The sensitivity to Ins P (3) is critically dependent on the density of InsP(3) receptors, so that different stores can respond to different concentration ranges of Ins P (3). Since the model generates very high Hill coefficients (h approximately 7), it allows all-or-none release of Ca(2+) from stores of differing receptor density, but questions the validity of the use of h values as a guide to the number of InsP(3) molecules needed to open the channel. The model presents a mechanism for terminating Ca(2+) release in the presence of positive feedback from released Ca(2+), thereby providing an explanation of why elementary Ca(2+) signals ('blips' and 'puffs') do not inevitably turn into regenerative waves.

Calcium↗