Are inositol pyrophosphates signalling molecules?

MINI-REVIEW 8J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology
Are Inositol Pyrophosphates
Signalling Molecules?
ADAM BURTON, XIAOWEN HU, AND ADOLFO SAIARDI*
Medical Research Council (MRC), Cell Biology Unit, Laboratory for Molecular Cell Biology,

Department of Cell and Developmental Biology, University College London, London, UK

The inositol polyphosphate family of small, cytosolic molecules has a prominent place in the field of cell signalling, and inositolpyrophosphates are the most recent addition to this large family. First identified in 1993, they have since been found in all eukaryoticorganisms studied. The defining feature of inositol pyrophosphates is the presence of the characteristic ‘high energy’ pyrophosphate group,which immediately attracted interest in them as possible signalling molecules. In addition to their unique ‘high energy’ pyrophosphate bond,their concentration in the cell is tightly regulated with an extremely rapid turnover. This, together with the history of other inositolpolyphosphates, makes it likely that they have an important role in intracellular signalling involving some basic cellular processes. Thishypothesis is supported by the surprisingly wide range of cellular functions where inositol pyrophosphates seem to be involved. A seminalfinding was that inositol pyrophosphates are able to directly phosphorylate pre-phosphorylated proteins, thereby identifying an entirelynew post-translational protein modification, namely serine-pyrophosphorylation. Rapid progress has been made in characterising themetabolism of these molecules in the 15 years since their first identification. However, their detailed signalling role in specific cellularprocesses and in the context of relevant physiological cues has developed more slowly, particularly in mammalian system. We will discussinositol pyrophosphates from the cell signalling perspective, analysing how their intracellular concentration is modulated, what theirpossible molecular mechanisms of action are, together with the physiological consequences of this novel form of signalling.

J. Cell. Physiol. 220: 8–15, 2009. � 2009 Wiley-Liss, Inc.

*Correspondence to: Adolfo Saiardi, Medical Research Council(MRC), Laboratory for Molecular Cell Biology, University CollegeLondon, Gower Street, London WC1E 6BT, UK.E-mail: [email protected]

Received 13 February 2009; Accepted 17 February 2009

Published online in Wiley InterScience(www.interscience.wiley.com.), 26 March 2009.DOI: 10.1002/jcp.21763

Intracellular signalling is the mechanism by which cells are ableto transduce and coordinate external inputs in order to behaveappropriately in a given environment. In single-celled organismssignalling is of primary importance for responding toenvironmental conditions while in a multicelluar organismsignalling takes on an extra dimension in necessitating thetransfer of information between individual cells in order tocommunicate and integrate the cells into the differentiatedtissues and coordinate these functional units into the organismas whole. The transduction of an external stimulus inside thecell often involves a surprisingly limited number of intracellularsignalling molecules or second messengers (Sutherland and Rall,1958). An intracellular signalling molecule must be able torespond dynamically and rapidly to changes in external stimuliand must achieve a rapid and specific transduction and oftenamplification of this signal into a different form, in order for thecell to respond correctly to particular stimuli.

The inositol polyphosphate family includes several secondmessengers and has a distinguished history in the field of cellularsignalling. The calcium mobilising role ofinositol(1,4,5)trisphosphate (Ins(1,4,5)P3) was revealed in theearly 1980s (Putney, 1987; Berridge et al., 2000). The linkbetween the ‘PI response’ (the rapid incorporation ofradiolabelled phosphate into inositol lipids) and cytosoliccalcium mobilisation was shown to be mediated by Ins(1,4,5)P3

produced by the hydrolysis of PIns(4,5)P2 leaving behinddiacylglycerol (DAG) on the membrane (for review and historicperspective see Michell, 1975; Irvine, 2003). In a short time, thedephosphorylation route of Ins(1,4,5)P3 that allows therecycling of inositol was identified and this led to the descriptionof the ‘inositol-cycle’ or ‘phosphatidylinositol-cycle’ (Divechaet al., 1993; Resnick and Saiardi, 2008). The mechanism of actionfor Ins(1,4,5)P3 was demonstrated before the end of the decadeto be through its direct binding to a receptor on theendoplasmic reticulum membrane, which induces the openingof its intrinsic calcium channel allowing calcium to diffuse intothe cytosol (Ferris et al., 1989; Mikoshiba et al., 1993).

The signalling role for inositol polyphosphates however ismore complex than being merely restricted to the ‘inositolcycle’. In all eukaryotic organisms, Ins(1,4,5)P3 is furtherphosphorylated and represents the precursor of a plethora of

� 2 0 0 9 W I L E Y - L I S S , I N C .

inositol polyphosphates (for review see Hughes and Michell,1993; Irvine and Schell, 2001; Resnick and Saiardi, 2008). Acommon theme of the inositol polyphosphate molecules is theirduality of function: as transient metabolites in the pathways ofsynthesis of further or less phosphorylated inositol species aswell as being signalling molecules themselves (Resnick andSaiardi, 2008; Saiardi and Cockcroft, 2008). The variety ofinositol phosphate species and isomers that are generated bytheir metabolism and their subtle differences has, notsurprisingly, been exploited by evolution. Although the greatproliferation of inositol polyphosphates has implicated a rolepermeating almost all aspects of cell biology, from plasmamembrane ion channel regulation to nuclear mRNA export,it has been difficult to identify detailed signalling pathways forthese molecules (for review see Irvine and Schell, 2001; Shears,2004; Bennett et al., 2006; Michell, 2008). Studies of themetabolism of these molecules have been greatly aided bythe use of yeast genetics. The simple phenotypic analysis ofyeast mutants for the enzymes responsible for inositolpolyphosphate synthesis has enabled the attribution of aspecific function to a particular inositol phosphate. Thisapproach has frequently not been able to define a specificinositol polyphosphate as a second messenger because thesignalling pathways from cellular stimulus to mechanisticcellular response have often remained elusive.

In comparison with the lipid phosphatidylinositides the toolsto study the function of soluble inositol polyphosphateshave lagged behind, particularly in mammalian cells. The small,water-soluble nature of these molecules has made localisationdifficult, whereas the use of GFP-tagged lipids probes has greatly

I N O S I T O L P Y R O P H O S P H A T E S 9

aided the study of phosphatidylinositols (PIns; Halet, 2005;Lemmon, 2008). A particularly useful tool for the study of bothsoluble inositol phosphates (InsP) and membrane-bound PIns islabelling the total inositide content of a cell to equilibrium withradiolabelled inositol, which is readily taken up by cells. Highperformance liquid chromatography (HPLC) is then used toseparate and determine the concentration in the cell of thedifferentially phosphorylated inositol species (Irvine et al., 1985;Balla et al., 1989; Azevedo and Saiardi, 2006). It is this techniquethat led to the next breakthrough in inositol polyphosphatessignalling, the identification of pyrophosphorylated forms ofinositol that are the subject of this review.

The inositol polyphosphate field was not prepared for thediscovery in 1993, just a decade after the function of Ins(1,4,5)P3

ignited inositol polyphosphate research, that more than sixphosphates can be bound to the six-carbon inositol ring, clearlyindicating the presence of pyrophosphate groups (Menniti et al.,1993; Stephens et al., 1993). In the 15 years since theirdiscovery, soluble inositol pyrophosphates have been found inevery eukaryotic system studied from yeast to mammalianneurons, along with the widespread conservation of theenzymes responsible for their synthesis (Bennett et al., 2006).The best characterised are diphosphoinositolpentakisphosphate (PP-InsP5, commonly known as InsP7 or IP7)and bis-diphopshoinositol tetrakisphosphate ((PP)2-InsP4,commonly known as InsP8 or IP8) (Fig. 1), although there is

Fig. 1. Chemical nature of inositol pyrophosphates. The figureshows the structure of the most studied inositol pyrophosphates.The six carbon group of the inositol ring is represented in black,the phosphates groups are represented in grey. The isomerIns(1,3,4,5,6)P5 represents the most abundant cellular inositolpentakisphosphate and from it derive a series of inositolpyrophosphate such 5PP-InsP4 (top left) possessing the free axialhydroxyl group of myo-inositol (position two). From the fullyphosphorylated ring of InsP6 derive the inositol pyrophosphates InsP7

or 5PP-InsP5 (top right) and the double phosphorylated form of ‘InsP8’5,6(PP)2InsP4 (bottom left), this isomer has been identified inDictyostelium (Albert et al., 1997); while the isomer 1/3,5(PP)2InsP4 ispredicted to be present in mammalian cells (Lin et al., 2009). A triplephosphorylated form of ‘InsP8’ 5PPP-InsP5 (bottom right) has alsobeen recently identified (Draskovic et al., 2008).

JOURNAL OF CELLULAR PHYSIOLOGY

clearly a greater potential for diversity of form and functionin pyrophosphorylated derivatives of the many inositolpolyphosphates. Indeed derivatives of Ins(1,3,4,5,6)P5, suchas diphosphoinositol tetrakisphosphate PP-InsP4 (Fig. 1) andbis-diphopshoinositol triskisphosphate (PP)2-InsP3 have beenobserved, as well as a triphosphoinositol isomer of ‘InsP8’PPP-InsP5 (Fig. 1) (Draskovic et al., 2008).

The defining feature of inositol pyrophosphates immediatelysuggests a distinctive role due to the presence of thischaracteristic ‘high energy’ pyrophosphate group. It is thischaracteristic feature of inositol pyrophosphates that generatedan immediate interest in their possible role as intracellularsignalling molecules. The standard free energy of hydrolysis ofthe pyrophosphate bond in InsP7 has been calculated at 6.6 Kcal/mol, higher than that of ADP (Stephens et al., 1993), while thevalue for InsP8 is estimated to be higher than that of ATP(7.3 Kcal/mol) due to the release of electrostatic and stericconstraints (Laussmann et al., 1996). This led to the idea thatinositol pyrophosphates could be important for drivingphosphotransfer reactions and when tested this proved to betrue, introducing an entirely new mechanism of action for theinositol polyphosphates family. Strikingly, the phosphotransferreaction from InsP7 was shown to be kinase-independent itselfand therefore represents an entirely new mechanism of proteinphosphorylation (Saiardi et al., 2004). It also defines a new kindof protein post-translational modification because a subsequentstudy has demonstrated that inositol pyrophosphates donatethe phosphate group to a pre-phosphorylated serine, thusgenerating a pyrophosphorylated protein (Bhandari et al., 2007).

In mammalian cells inositol pyrophosphates are present in thesub-micromolar range representing less than 5% of their highlyabundant precursor InsP6 (Bennett et al., 2006). Although thelevels of inositol pyrophosphate species are relatively low andapparently stable this conceals a remarkably rapid turnover thathas been estimated (using fluoride inhibition of phosphates inmammalian cells) to convert up to 50% of the large InsP6 poolevery hour to its pyrophosphorylated derivatives (Menniti et al.,1993). These derivatives are presumably carrying out animportant signalling function inside the cell. The significance ofthis is made all the more clear when the considerable energyinput expended by the cell in terms of the number of moleculesof ATP required for their synthesis is recognised.

There are clearly potentially important roles for inositolpyrophosphates as intracellular signalling molecules, but it hasbeen difficult to elucidate their function in a clearly definedsignalling pathway from extracellular stimulus to physiologicalresponse. Although there are a number of reports of inositolpyrophosphates levels changing in response to stimuli, these arerelatively isolated and lacking cellular mechanism and context.Downstream of inositol pyrophosphates, there are also anumber of seemingly isolated reports of involvement inparticular cellular functions, but again lacking physiologicalcontext.

Excellent reviews on the metabolic pathways that lead toinositol pyrophosphate biosynthesis have been published(Irvine and Schell, 2001; Shears, 2004); and a comprehensivereview summarising this exciting field of research is available(Bennett et al., 2006). Here, our aim is to examine inositolpyrophosphate literature from the cell signalling standpoint. Wewill discuss the role of inositol pyrophosphates as secondmessengers, focusing on the three main features of cellularsignalling: how the level of a second messenger is regulated, themolecular mechanism of action and the physiologicalconsequence of its signalling.

Stimuli Controlling Inositol Pyrophosphates Levels

A defining feature of intracellular signalling molecules is theirability to respond rapidly and dynamically to a particular

10 B U R T O N E T A L .

stimulus or stimuli so that the cell is able to swiftly recognise notonly the presence of extracellular signals, but also how thestimulus is changing over time. Therefore, in order to place aparticular molecule within the signalling network, it is vital thatthe upstream signals that it is able to recognise and respond toare understood. The levels of second messenger in the cellor particular subcellular localisation are thus crucial and arecontrolled by the tight regulation of the synthetic enzyme(s) aswell as by the ‘catabolism’ of the signal.

There have been a number of reports of inositolpyrophosphate species responding dynamically to a number ofstimuli. In fact the identification of inositol pyrophosphates inmammalian cells in 1993 was made possible through the use offluoride treatment that dramatically modulates the levels of thisclass of molecules (Glennon and Shears, 1993; Menniti et al.,1993). When pancreatoma cells were incubated with sodiumfluoride, the levels of the pyrophosphorylated derivatives ofboth InsP5 and InsP6 were substantially increased, allowing theirinitial discovery by HPLC. These experiments also immediatelyrevealed that up to 50% of the InsP6 pool is converted everyhour to its pyrophosphorylated derivative InsP7 (Glennon andShears, 1993; Menniti et al., 1993). Due to its relatively lowsteady-state level this indicates that the cellular InsP7 is turningover itself many times during the same time. In primaryhepatocytes it was calculated that the pool of InsP7 turns overten times in a 40-min time period (Glennon and Shears, 1993).This rapid turnover implicates an important signalling role forthese molecules in their capacity to allow a swift and dynamicresponse to the ever-changing upstream physiologicalenvironment. Although these early observations introducedthe inositol pyrophosphates as novel signalling molecules, theirphysiological significance remains unknown. Fluoride inhibitsthe activity of diphosphoinositol polyphosphatephosphohydrolase (DIPP) that specifically cleaves theb-phosphate of the pyrophosphate moiety of InsP7 and InsP8

(Safrany et al., 1998). However, fluoride is a commonly usedphosphatase inhibitor and has multiple effects on cell signalling.Consequently, the fluoride effect on inositol pyrophosphatemetabolism is not fully understood and in addition fluoride doesnot elevate inositol pyrophosphate concentration in the simpleand genetically tractable yeast experimental system (A. Saiardi,unpublished observation).

The most dramatic modulation of the levels of inositolpyrophosphates has been seen, perhaps not surprisingly, inDictyostelium where the extremely high levels of the inositolpyrophosphates allows easier analysis (Stephens et al., 1993).The steady-state levels of these molecules show a correlationwith the stage of the life cycle, perhaps one of the mostfundamental of cellular processes, but also a very particularuniqueness of this model organism. Although the levels ofinositol pyrophosphates show similar ratios to the precursor IP6

levels in the vegetative state, compared to mammalian cells, theconcentrations of both IP7 and IP8 increase considerably duringstarvation-induced aggregation and chemotaxis as stationaryphase is reached (Laussmann et al., 2000). When starvation issensed in Dictyostelium the second messenger cAMP is thesignalling sensor and is able to induce aggregation (VanHaastert, 1995). It was demonstrated in a later study that cAMPalone is sufficient to induce a rapid and considerable elevation inthe levels of InsP7 and InsP8 in Dictyostelium (Luo et al., 2003).

Interestingly, it has also been observed that inositolpyrophosphate concentrations are regulated by changes incAMP levels in mammalian cells. In the DTT1MF-2 smoothmuscle cell line, raising cAMP levels, either through the use ofphosphodiesterase inhibitors or via the activation ofb2-adrenergic receptors, was able to reduce IP8 levels,providing the first link between receptor activation and inositolpyrophosphate turnover (Safrany and Shears, 1998). Thisphenomenon was independent of either PKA or PKG signalling


and it seems likely, therefore, that a non-canonical downstreameffect of cAMP is the mechanism mediating the specific decreasein IP8.

The observations linking cAMP signalling with inositolpyrophosphates turnover differ substantially betweenDictyostelium and mammalian cells however, in that an increaseof both IP7 and IP8 in response to cAMP signalling is observed inDictyostelium, while a specific decrease of IP8, is observed inmammalian cells. This is likely to be due to the fact thatDictyostelium is evolutionary relatively distant to mammals andcAMP signalling is quite different in character to other modelorganisms (cAMP is a secreted chemoattractant) and inositolpyrophosphate signalling may also be quite different betweenDictyostelium and higher eukaryotes. It is clear that thisinteresting signalling relationship needs to be studied in furtherdetail to determine the role of inositol pyrophosphates in thecAMP signalling responses in mammalian cells. It would also beinteresting to determine whether this observed association isactually part of a more general relationship between nucleotidemetabolism and inositol pyrophosphate signalling. In contrast,it was soon realised that there is an apparent independence ofinositol pyrophosphate turnover from the most wellcharacterised response of the inositol phosphates, theactivation of phospholipase C (Safrany and Shears, 1998). Thesestudies are complemented by the recent overexpression of aconstitutively active Galpha(q)QL, where phospholipase C ishyper-stimulated but fails to induce an increase in inositolpyrophosphate levels (Otto et al., 2007).

Although the life cycle of Dictyostelium is also very different incharacter to the mammalian cell cycle, a correlation betweeninositol pyrophosphate levels and the cell cycle has also beenobserved in mammalian cells, where twofold higher levels wereobserved in G1 compared to S phase (Barker et al., 2004).Importantly, it remains to be discovered whether inositolpyrophosphate dynamics during the cell cycle are a response tothe particular phase (and are therefore able to act as molecularsensors) or whether inositol pyrophosphates levels are part of aparticular upstream signalling pathway to induce or inhibit cellcycle progression or differentiation. Unfortunately thispotentially highly significant relationship between inositolpyrophosphates and the interface between growth/self-renewaland differentiation has not been studied further as yet.

The levels of InsP7 in mammalian cells are controlled bystimuli of particular relevance to multicellular eukaryotes. Itappears that a number of apoptosis-inducing agents such asstaurosporine, cisplatin lead to an increase in cellular IP7 and amore modest increase in InsP8 (Nagata et al., 2005). In smoothmuscle cells, the levels of IP8 have been observed to specificallyincrease in response to environmental stresses (Pesesse et al.,2004; Choi et al., 2005). Both heat shock and osmotic stressinduce a rapid accumulation of InsP8, but not of InsP7 orPP-InsP4 in DTT1MF-2 cells. A 15-fold increase in InsP8 levelsafter a 30-min challenge with sorbitol was observed, while aless-pronounced three- to fourfold increase of InsP8 was alsoobserved upon heat shock at 428C or cold shock at 308C(Pesesse et al., 2004; Choi et al., 2005). Although the MAPkinase pathway was initially suggested to be involved (throughthe use of pharmacological inhibitors), it has more recentlybeen found that it was the off-target effect of the inhibitors oncellular energetic status that was responsible for the observedeffects upon InsP8 levels. The induction of bioenergetic stressreduces the rate of InsP8 synthesis in order to conserve energyand this is dominant over the increased level normally observedupon induction of osmotic stress (Choi et al., 2008).

Although important and providing insight into the stimuli thatcontrol individual inositol pyrophosphates, the physiologicalrelevance of the effects of cellular stresses on InsP8 levels seemmore applicable to yeast in their environmental context.It is unclear to what extent individual mammalian cells in a

Fig. 2. Possible mechanisms of signal transduction by inositolpyrophosphates. The upper panel depicts the direct bindingmechanism in which a protein target possesses a specific pocket ableto recognise InsP7. The lower panel shows the pyrophosphorylationmodification of a protein carried out by InsP7. A pre-phosphorylatedserine (yellow circle) is the recipient of theb-phosphate (red circle) ofthe pyrophosphate moiety of InsP7.


physiological context are challenged by either heat or osmoticimbalance, except in the cases of kidney cells as subject to saltstress and neurons of the hypothalamus responding to changesin temperature. In yeast such as S. cerevisiae, challenges such asheat and osmotic stress are a constant environmental pressure;however, it appears that levels of InsP8 do not respond to eitherstress in S. cerevisiae (Choi et al., 2005). Also of particularrelevance to yeast is the observation that intracellular inositolpyrophosphate levels may be controlled by levels of phosphatein the extracellular environment. It was recently reported that adecrease in extracellular inorganic phosphate concentrationled to a rise in the intracellular inositol pyrophosphates (Muluguet al., 2007). Unfortunately we have been unable to reproducethis data (Onnebo and Saiardi, 2007) and have instead observedthe opposite response (A. Saiardi, unpublished observation),which seems more physiologically coherent as a decrease ininorganic phosphate in the extracellular medium leads toreduced amounts of ATP available for inositol pyrophosphatebiosynthesis.

Although as described above, a number of stimuli thatregulate the levels of inositol pyrophosphates have now beenidentified, the observations still seem disconnected. A clearerpicture of how inositol pyrophosphate levels are regulated isneeded in order to help place them in the intracellular signallingnetwork. It is also unclear how directly all of the stimulimentioned above regulate the levels of inositol pyrophosphatesand, in particular no stimulus has been shown to directlyregulate the enzymes responsible for their synthesis. Similarly,no regulatory mechanism has been identified for thephosphatase responsible for inositol pyrophosphatesdegradation DIPP (yeast gene called DDP1) (Safrany et al., 1998;Ingram et al., 2003). This phosphatase must have a primary, yetnot fully understood, role in regulating inositol pyrophosphatesmetabolism. In fact, the analysis of the double yeast mutants forthe IP6-kinase and DIPP genes (kcs1Dddp1D) unexpectedlyreveals higher levels of InsP7 (Seeds et al., 2005) and this led tothe discovery of a new class of enzymes able to synthesiseinositol pyrophosphates, namely VIP1 (Mulugu et al., 2007); themammalian homologue is referred to as PP-IP5-kinase for itsprimary enzymatic ability to convert InsP7 to InsP8 (Choi et al.,2007; Fridy et al., 2007).

Conceivably, inositol pyrophosphates are involved in varioussignalling pathways responding to diverse stimuli in different celltypes, and the specificity required for the appropriatedownstream response is achieved through subtle routes, whichwill make understanding the signalling role of inositolpyrophosphates more difficult. This is indeed likely if theirmechanism of action is via a fundamental biochemical processthat can play a role in the regulation of several different signallingpathways.

Mechanisms of Action

An intracellular signalling pathway is defined by the transductionof an extracellular signal to its interior. The signalling pathwayoften enables the amplification and integration of the stimulusinto the cellular signalling machinery so that the cell is able tocoordinate its behaviour appropriately for the particularenvironmental context. There are two general mechanismsthat cells have evolved to transduce signals. Firstly, signals canbe transmitted by the covalent modifications of proteins, suchas the kinase-dependent phosphorylation cascades. The secondpredominant mechanism used in signal transduction isthrough the binding of second messenger molecules to specificprotein targets, such as cAMP to protein kinase A or calcium toprotein kinase C.

A well-documented mechanism of action for signaltransduction by the inositol phosphates family is via binding to aparticular receptor, such as the paradigmatic binding of InsP3 to


the IP3 receptor (Mikoshiba et al., 1993), which causes aconformational change in the structure of the integral calciumchannel, opening the channel to allow calcium to flow from theendoplasmic reticulum into the cytoplasm. Binding partners formany other inositol phosphates have also been identified,especially for the lipid phosphatidylinositols such as the bindingto pleckstrin homology (PH), phagocyte oxidase homology(PX) or FYVE (for Fab1, YOTB, Vac1 and EEA1) proteindomains (Lemmon, 2008). It seems logical, therefore thatinositol pyrophosphates may also signal through allostericinteractions with proteins, and indeed binding partners of IP7

have been identified (Fig. 2). However the peculiar presence of ahighly energetic pyrophosphate bond has also suggested thepossibility to covalently modify proteins as a mechanism ofaction for this class of molecules. Both mechanisms are nowdiscussed.

InsP7 has been observed to bind to various proteins, mostnotably those involved in trafficking, including AP-2, AP-180 andthe Golgi Coatamer protein (Fleischer et al., 1994; Ali et al.,1995; Ye et al., 1995). Binding of InsP7 to the adaptins in vitroresulted in an inhibition of clathrin lattice formation (Ye et al.,1995). However these binding studies were often conductedusing inappropriate experimental conditions, as reviewed(Shears, 2001). Furthermore, all the proteins that bind to InsP7

also bind to InsP6, albeit to a modest extent, andphosphatidylinositol lipids have recently been suggested to bethe more important ligands in vivo (Cremona and De Camilli,2001). This highlights the major problem encountered whenperforming any binding experiments, particularly with InsP7 and


the other inositol pyrophosphates. The dramatically lowercellular levels of inositol pyrophosphates species necessitates astrict specificity of binding for InsP7 over InsP6 or the inositollipids, before any physiological relevance can be attributed to it.Such a level of specificity for any inositol pyrophosphates has notbeen observed thus far. In addition, such a dramatic difference inbinding affinity for InsP7 over InsP6 may not be practicallypossible due to their relatively similar structures, although it isconceptually more likely if InsP8 is the functional bindingpartner. A conceivable possibility to overcome the problem ofbinding specificity may involve a precise intracellular spatialcontrol of the local levels of inositol pyrophosphates. A specificlocalisation of InsP6 kinase activity would, for example, induce alocalised increase in the InsP7/InsP6 ratio and therefore aspecificity of binding to InsP7 could potentially be viable in such aparticular microlocalisation in vivo. This would also allow amore specific regulation of this signalling mechanism as both theactivity and localisation of the InsP6 kinase enzyme could becontrolled by upstream signals in order to achieve a localisedhigh concentration of IP7 under specific physiologicalconditions.

In Dictyostelium, InsP7 and InsP8 levels increase dramaticallyduring aggregation (Laussmann et al., 2000) and therefore amore modest selective binding affinity for InsP7 over InsP6

presents InsP7 as a viable intracellular binding partner during theaggregation stage of the life cycle. Indeed, the relevance ofbinding as a mechanism of action has been demonstrated viaInsP7-mediated competition for the binding of PIns(3,4,5)P3 tothe PH-domain containing CRAC and is therefore inhibitory tochemotaxis (Luo et al., 2003). It was also shown that InsP6 isonly 1–2% as potent as InsP7, and therefore InsP7 is more likelyto be the physiological binding partner during aggregation whenthe combined InsP7 and InsP8 levels reach more that half ofthe levels of their precursor InsP6 (Laussmann et al., 2000).The in vivo relevance was supported by the demonstrationthat IP6-kinase deletion increases sensitivity to cAMP-inducedaggregation (Luo et al., 2003). Although InsP7 was found to bindto various other PH-domain containing proteins, includingmammalian Akt in vitro (Luo et al., 2003), the significance of thisbinding is less clear in mammalian cells where the ratio of theinositol pyrophosphates to InsP6 levels have not so far beenobserved to show such dramatic modulation. Furthermore, amore recent and rigorous study demonstrated the inability ofthe PH-domain of phosphoinositide-dependent protein kinase1 (PDK1) to bind InsP7 (Komander et al., 2004).

In direct contrast to the well-documented mechanism ofInsPs acting as signalling molecules via receptor binding, anentirely novel mechanism of signal transduction is used byinositol pyrophosphates. It has been demonstrated that theb-phosphate of the pyrophosphate group is donated to proteinsin a reaction that is independent of a protein kinase (Saiardiet al., 2004), in contrast to the ATP-mediated proteinphosphorylation event that operates in classical proteinphosphorylation cascades. Despite this break from thetraditional mode of signalling by inositol polyphosphates, thisnew mechanism is not so surprising and was in fact predictedupon their original discovery (Stephens et al., 1993) andthereafter (Voglmaier et al., 1996). This is due to their uniquebiochemistry brought about by the presence of an additionalfunctional group: the pyrophosphate group. It is this group thatclassifies the inositol pyrophosphates and mediates this novelsignalling event (Fig. 2) (Saiardi et al., 2004). Theoreticalcomputation studies attribute a high phosphorylation potentialto InsP7 due to the sterically and electronically packedenvironment of the pyrophosphate moiety (Hand andHonek, 2007). InsP7-mediated protein phosphorylation is aeukaryotic-specific process and the reason for this becameclear when it was observed that a priming event is requiredfor phosphorylation to occur (Saiardi et al., 2004; Bhandari


et al., 2007). This priming was characterised as a traditionalkinase-dependent protein phosphorylation and that proteinphosphorylation mediated by InsP7 is actually apyrophosphorylation event as the phosphate donated by InsP7 isadded to an existing phosphorylated serine (Fig. 2) (Bhandariet al., 2007).

Interestingly, the substrates of IP7-mediated proteinpyrophosphorylation that have been identified so far have allbeen found to contain stretches of serine residues surroundedby acidic residues. The acidic residues may play a role incoordinating magnesium ions that are an absolute requirementfor the reaction to take place (Saiardi et al., 2004; Bhandari et al.,2007). However, it is clear that substrates containing longerstretches of serines are more easily experimentally detected astargets if multiple serines can be pyrophosphorylated, and it isconceivable that only one phosphorylated serine residue withsurrounding acidic residues may serve as a target. In the absenceof a specific antibody to detect pyrophosphorylated proteins,this remains a hypothesis that may be difficult to substantiate.

This protein pyrophosphorylation mechanism of signalling byinositol pyrophosphates eliminates the potential problemsencountered in the binding model where InsP6 may often be themore physiologically relevant binding partner due to itssignificantly higher cellular concentration. As InsP6 does notcontain a pyrophosphate group it is obviously unable to carryout the phosphotransfer reaction. The relatively lowintracellular InsP7 concentration then becomes less relevantand the extremely high turnover becomes more significant andsuggests a high level of InsP7-mediated proteinpyrophosphorylation occurring in vivo. However, as with anysignalling mechanism a potential for regulation must exist, and inthe absence of a defined protein involved in InsP7-mediatedpyrophosphorylation step, the question of how this process maybe regulated arises. The kinase-dependent pre-phosphorylation step would act as an initial regulatory step, butit is also likely that the IP7-mediated pyrophosphorylation itselfcan also be controlled separately through some, as yetunknown, mechanism.

The precise spatial control of signalling events is a commonmethod for the achievement of the necessary specificity. Themechanism described earlier for the generation of specificity ofaction for InsP7 through its localised synthesis can also applyto allow specificity of the pyrophosphorylation reaction. Onepotential model might involve a particular stimulus leading toactivation and an increase in the interaction between theInsP6-kinase enzyme and a specific substrate ofphosphorylation. In addition, it is conceivable that an InsP7

phosphatase such as DIPP would also be present in thevicinity to prevent diffusion of the InsP7 and maintain itsmicrolocalisation. This process would allow the more subtleregulation of IP7-dependent protein pyrophosphorylation andmaintain specificity for a particular substrate in a given cellularmicroenvironment.

Pyrophosphorylation mediated by InsP7 is expected to becapable of inducing the same functional changes at the proteinlevel as traditional phosphorylation. For example it is likely toinduce further structural changes in protein conformation andtherefore may give rise to differences in the protein’sinteractions, activity or localisation. A question that arisestherefore, asks why would an entirely different means ofprotein phosphorylation coexist if it has the sameconsequences? It is likely that protein pyrophosphorylation hasdistinctive biochemical and physiological roles. In fact there isevidence that the pyrophosphorylated protein may have uniqueproperties. InsP7 pyrophosphorylated peptides are more acidlabile and more resistant to phosphatases than ATPphosphorylated peptides (Bhandari et al., 2007). If this applies tocellular proteins, then the dominant effect of IP7-mediatedprotein pyrophosphorylation would allow the protein to remain


transducing the signal even in the presence of an active OFFsignal from a general phosphatase. In the context of cellularsignalling events this is of enormous potential significancewhere the precise temporal control of an active signallingpathway is of paramount importance.

It must be stressed that the possible mechanisms forsignalling through IP7 by binding and pyrophosphorylation arenot mutually exclusive and there is now good evidence for both.The control of any signalling event must be tightly regulatedboth temporally and spatially and it remains an intriguingprospect to discover how specificity is achieved for this novelsignalling event. Further investigation into the mechanism ofaction of InsP7 will hopefully provide insight into its regulation inthe cell and coordination with other signalling pathways.

Physiological Role

An insight into the physiological role of inositol pyrophosphatesignalling has been greatly aided by the use of yeast genetics.In S. cerevisiae there is a single homologue of the mammalianIP6-kinases enzymes called KCS1 and kcs1D null yeast havebeen created in which the levels of inositol pyrophosphatesare close to zero (Dubois et al., 2002; Saiardi et al., 2002).Phenotypic studies of the yeast kcs1D mutant have implicatedthese signalling molecules in a diverse range of cellularprocesses including DNA recombination, the regulation oftelomere length and endocytosis. On a morphological level,kcs1D yeast display considerable abnormalities, suggestingimportant functions for the inositol pyrophosphates products ofthe KCS1 protein. The cells are larger that wild-type and growsignificantly slower. They are growth impaired at 378C andhypersensitive to salt stress but appear unaffected by osmoticchallenge with sorbitol (Dubois et al., 2002). A fragmentedvacuolar morphology (Saiardi et al., 2000, 2002) and problemsin cell wall integrity were also observed (Dubois et al., 2002).The catalytically active KCS1 protein alone was able to rescuethese defects, implicating the inositol pyrophosphate productsof KCS1 in the cellular processes involved in growth,endocytosis, and the response to stress.

The KCS1 gene in yeast was initially identified as encoding anovel leucine zipper protein in a screen for mutations in genesthat were able to suppress the observed increase inrecombination rate caused by mutations in the PKC1 gene(Huang and Symington, 1995). It was later shown that it was theinositol pyrophosphate products of KCS1 that are responsiblefor the DNA hyper-recombination in PKC1 mutants (Luo et al.,2002). Studies in yeast have also implicated the inositolpyrophosphates in another nuclear function, the regulation oftelomere length (Saiardi et al., 2005; York et al., 2005).Telomere maintenance in yeast is controlled by the kinasesMec1 and Tel1. Kcs1D yeast are resistant to the inhibitors ofthese kinases, caffeine and wortmannin, indicating that inositolpyrophosphates may be responsible for mediating the lethalactions of these drugs (Saiardi et al., 2005). Furthermore, kcs1Dmutant yeast also displayed an increased telomere length,while both tel1D and tel1kcs1D double mutants had shortertelomere lengths, placing inositol pyrophosphates upstreamof Tel1 in the same signalling pathway controlling telomeremaintenance (Saiardi et al., 2005; York et al., 2005).

An important link between phosphate metabolism andinositol pyrophosphate signalling is emerging in the literature.The screening of yeast deletion mutants based on acidphosphatase activity revealed that KCS1 is a negative regulatorof the phosphate sensing PHO regulon (Auesukaree et al.,2005). A subsequent study suggested that the InsP7 isomersynthesised by the VIP1 enzyme was able to regulate thecyclin-CDK–CDK inhibitor complex, which is important forthe transcriptional control of the PHO genes (Lee et al., 2007).However, as mentioned previously, we are unable to


reproduce the increase of InsP7 observed by the authorsfollowing phosphate starvation (Onnebo and Saiardi, 2007).Consequently, further studies are required to clarify the linkbetween inositol pyrophosphates and phosphate metabolism,which appears very complex as indicated by a recent reportwhere KCS1 expression is regulated by an intragenic antisenseRNA through a novel transcriptional mechanism controlled bythe medium inorganic phosphate concentration (Nishizawaet al., 2008).

Due to the lack of experimental tools to specifically reducethe levels of the inositol pyrophosphates in higher eukaryotes,together with the likely greater complexity of signallingfunctions, the determination of the role of inositolpyrophosphates in the mammalian system has been moredifficult. The presence of three isoforms of IP6-kinase inmammalian cells (IP6K1-3) (Saiardi et al., 1999, 2001) and twogenes for the PP-IP5 kinases (Huang et al., 1998; Fridy et al.,2007) has made studying the functional effects of inositolpyrophosphates more complex. Despite this, some excellentstudies exist that have begun to define the role of inositolpyrophosphates in mammalian cells.

In ovarian carcinoma cells, the apoptotic actions ofg-irradiation and IFN-b were enhanced or decreased byoverexpression or down-regulation of IP6K2 respectively(Morrison et al., 2001). Although the intracellularconcentrations of inositol pyrophosphates were not measuredin this study, IFN-b was able to post-transcriptionally elevatelevels of IP6K2. A role for inositol pyrophosphates specifically inthe Apo2L/TRAIL cell death signalling pathway is suggested dueto the requirement for expression of the Apo2L/TRAIL ligandfor the above effects to be observed (Morrison et al., 2005). Theapoptotic-promoting actions of IP6K2 have been extended tomultiple cell lines and in response to multiple apoptotic stimuli,including cisplatin and staurosporine which were also shown tomarkedly increase the levels of endogenous inositolpyrophosphates (Nagata et al., 2005). The apoptotic effect ofIP6K2 is also specific to this isoform as only the knockdown ofthis particular isoform conferred protection against apoptosis(Nagata et al., 2005). In this study IP6K2 was shown totranslocate to apoptotic mitochondria from the nucleus afterapoptosis induction. More recently, IP6K2 has been shown tobind to the heat shock protein HSP90, which has anti-apoptoticactions in cells, and this binding was able to physiologicallyinhibit the catalytic activity of IP6K2 (Chakraborty et al., 2008).In addition, a number of apoptotic drugs, including cisplatin andstaurosporine, reduced the binding between the two proteinsproviding a possible mechanism for the increase in cellularinositol pyrophosphates levels stimulated by these drugs(Chakraborty et al., 2008).

The recent generation of the knock-out mouse of IP6K1 hasprovided insight into the functional role of this particularisoform. The mice are smaller than wild-type despite normalfood intake and they have markedly lower levels of circulatingblood insulin (Bhandari et al., 2008). A link between IP6K1 andinsulin production is supported by an earlier study in whichdepletion of IP6K1 in pancreatic beta cells impaired insulinsecretion (Illies et al., 2007). This elegant electrophysiologicalstudy demonstrated that inositol pyrophosphates modulate theexocytotic capacity in the insulin-secreting pancreatic beta cells(Illies et al., 2007). In addition a putative disruption of the IP6K1gene has been described in a family with type 2 diabetes(Kamimura et al., 2004). The male knock-out mice are alsosterile with major defects observed in spermatogenesis and itwas suggested that these defects could also be consistent with arole for IP6K1 in secretion or endocytosis in general (Bhandariet al., 2008).

The physiological roles of inositol pyrophosphates aremultiple and diverse. The evidence is clearly stronger in theyeast model system where inositol pyrophosphates can be


almost completely ablated and resulting phenotypes analysed.The functional studies in mammalian cells have revealedspecificity for the two most widely expressed IP6-kinaseisoforms. A role for IP6K1 in insulin secretion, possibly due toan involvement in endocytic trafficking, has emerged (Illies et al.,2007). The IP6K2 isoform appears to play a specific role inapoptosis (Nagata et al., 2005). It is likely that a completeablation of inositol pyrophosphates in mammalian cells wouldresult in severe phenotypic abnormalities, as observed in theyeast system. It is clear from the few studies performed so far inthe mammalian system that inositol pyrophosphates haveevolved to mediate additional and more complex signallingfunctions.

Outlook and Future Perspectives

The inositol pyrophosphate field has made considerableprogress in the 15 years since their discovery and it has becomeclear that inositol pyrophosphates are indeed intracellularsignalling molecules. Studies have addressed independently allthree aspects of the functionality of signalling molecules fromexternal stimuli via mechanism of action to physiologicalresponse. It is hoped that the evidence accumulated thus far willprovide a framework for a more thorough understanding ofinositol pyrophosphate function. It is vitally important tointegrate the independently studied aspects thus far in order toincorporate the role of these molecules in the cellular signallingnetwork. It is likely that inositol pyrophosphate signalling willhave evolved distinct roles in separate physiological contexts.The integration of inositol pyrophosphate signalling with otherwell known signalling pathways is therefore of undoubtedimportance for further study.

It will also be important to determine the specificity offunction for particular types of inositol pyrophosphates as thereis increasing evidence for a multiplicity of inositol pyrophosphatespecies (Fig. 1) and the likelihood of defined functions carriedout by specific inositol pyrophosphates. Following on from thetheme of inositol polyphosphate signalling (Resnick and Saiardi,2008), evolution is likely to have exploited the potential presentin these structurally distinct inositol pyrophosphates forparticular signalling purposes in different organisms and evenspecific tissues of higher eukaryotes. In order to do this, a betterunderstanding of the regulatory mechanisms controlling themetabolism of inositol pyrophosphates is required.Furthermore, to fully elucidate the role of inositolpyrophosphates in cell physiology, more experimental tools arerequired in order to modulate and detect inositolpyrophosphates levels in vivo, particularly in the mammaliansystem.

It is evident that inositol pyrophosphate signalling has takenon extra roles in more complex multicellular organisms.However, it has become increasingly clear that inositolpyrophosphates have a very basic signalling function inside alleukaryotic cells. It is probable that the inositol pyrophosphateshave evolved dedicated roles due to their unique biochemistry.This, combined with the observed rapid turnover andconsiderable energy expenditure in their synthesis, suggests aprimary cellular function. The prediction that the IP6-kinaseenzyme evolved before other members of the inositolpolyphosphate kinase family (Bennett et al., 2006), combinedwith the signalling role of inositol pyrophosphates in yeast,suggests that inositol pyrophosphate signalling actually predatesthe InsP3 control of calcium signalling, which is restricted tohigher eukaryotes (Irvine, 2005). The future certainly holdsunexpected discoveries, as our understanding of inositolpyrophosphate function might lead to a better comprehensionof the driving forces behind inositide signalling in general andmay also lead to an understanding of why such a simple sugar asinositol has been selected to become the backbone of a


multitude of signalling molecules controlling the most disparateaspects of cell physiology.

Acknowledgments

We thank Dr Anne Mudge, Dr Cristina Azevedo andDr Antonella Riccio for reading the manuscript and themembers of Saiardi lab for discussion. The authors also thankMatthew Bennet for helping with figure one artwork. This workwas supported by the MRC founding of the Cell Biology Unit.

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Are inositol pyrophosphates signalling molecules?

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