The role of phosphoinositides in mast cell signalling

Signal Transduction 2006, 6, 81– 91 R. Byrne, B. Larijani 81

Review Article

The role of phosphoinositides in mast cell signalling

Richard Byrne, Banafsh� Larijani

Cell Biophysics Laboratory, Cancer Research UK, London Research Institute, London, UK

The release of pre-formed mediators such as histamine from mast cells and basophils is an inte-gral part of the normal immune response to infection by parasites. This exocytosis is also charac-teristic response in a number of disease states including asthma, which, due to their prevalencein western society are becoming of increasing clinical importance. In an effort to tackle thisgrowing problem much work has gone into unlocking the mechanisms through which mastcells function in health and disease. To date we have learned a lot about the various proteinsthat regulate degranulation. However, our knowledge on the contribution of lipids to this pro-cess is less clear. This review will discuss the role of phospholipids, particularly the phosphoino-sitides (PIs) in the processes that regulate mast cell exocytosis.

Keywords: Phosphoinositide 3-kinase / phospholipases / protein kinase C / SHIP / membrane fusion /

Received: August 23, 2005; accepted: October 13, 2005

DOI 10.1002/sita.200500074

Introduction

Mast cells play a critical role in inducing and maintainingthe immune response to allergens and parasitic hel-minths. Foreign antigens, complexed with immunoglo-bulin E (IgE) activate mast cells, in a response charac-terised by the non-cytotoxic exocytosis of pre-formedsecretory granules containing histamine and b-hexosami-nidase [1]. Mast cell activation is further characterised by

the de novo synthesis and release of lipids, cytokines andchemokines. Although mast cell function is an integralpart of the immune response, the hyper-reactivity of mastcells is now recognised to be at the root of allergic disor-ders including anaphylaxis, rhinitis, atopic dermatitisand asthma. The latter disorder in particular is of concern,with almost 20% of US high school attendees, and a simi-lar proportion of UK children and being affected [2, 3].

Under ,normal’ physiological conditions, IgE serumlevels are very low, and are only raised during an immuneresponse to select parasites such as helminths. Here theinfective agent triggers in B cells the production of IgEantibodies recognising this agent. The binding of IgEcross-linked to the immunogen triggers mast cell activa-tion and the release of immunogenic mediators. Thesemediators have various biological effects which togetherhelp fight infection. For example, histamine increases vas-cular permeability, which in conjunction with cytokinesallows other cells of the immune system to migrate to,and access to the site of infection. However, under someconditions the body can stimulate inappropriate produc-tion of IgE after contact with non-parasitic antigens. Theseantigens, termed allergens include pollen, viral particles,pollution fumes, fungal spores and dust mite faeces. Oftenthe response to allergens is localised, especially if theallergy has a genetic component (atopy). For example, in

Correspondence: Dr. Richard Byrne, Cell Biophysics Laboratory, Can-cer Research UK, London Research Institute, 44 Lincoln’s Inn Fields,London WC2A 3PX, UK.E-mail: [email protected]: +44 (0)20 7269-3094

Abbreviations: AA, arachidonic acid; Arf, ADP ribosylation factor;BMMC, bone marrow-derived mast cells; Btk, Bruton’s tyrosine kinase;DAG, 1,2-diacylglycerol; GEF, guanine nucleotide exchange factor; IgE,immunoglobulin E; InsP3, inositol 1,4,5-trisphosphate; ITAM, immunore-ceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; PH, Pleckstrin homology; PI, phosphoinositide;PI3K, phosphoinositide 3-kinase; PIPKI, phosphatidylinositol monophos-phate kinase I; PITP, phosphatidylinositol/phosphatidylcholine transferprotein; PKB, protein kinase B; PKC, protein kinase C; PLA2, phospholi-pase A2; PLC, phospholipase C; PLD, phospholipase D; PtdCho, phos-phatidylcholine; PtdEtn, phosphatidylinositol ethanolamine; PtdIns,phosphatidylinositol; PtdOH, phosphatidic acid; PTEN, phosphatase andtensin homologue; RBL, rat basophilic leukaemic; SH2, Src-homology 2;SHIP, SH2 domain containing inositol 5-phosphatase.

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82 R. Byrne, B. Larijani Signal Transduction 2006, 6, 81 –91

the case of allergic rhinitis (hayfever) pollen is detected inthe eyes and nasal cavity and triggers a mast cell responsethere, hence the symptoms of watery eyes and sneezing.On the other hand, for sufferers of allergic asthma, air-borne particles including pollen, dust and pollution arefirst detected in the lower respiratory tract, and as suchthe immune response takes place there, producing vari-ous effects including bronchoconstriction, mucus secre-tion and inflammatory cell recruitment.

Given the prevalence of these diseases, mast cell func-tion in immunity is currently the subject of much scru-tiny. In such studies pharmacological agents includingcompound 48/80, steel factor and IgE are routinely usedto trigger mast cell degranulation and hence elucidatetheir regulation. These tools have greatly aided ourunderstanding of the plethora of proteins that regulatemast cell degranulation, which have been extensivelyreviewed elsewhere [1, 4]. The focus of this review will beinstead to look at the role of phospholipids, with a spe-cial focus on the PI lipids in mast cell degranulation.

Introduction to phosphoinositide signalling

PI lipids exist in the plasma membrane, various organel-lar membranes, and structures within the nucleus ofcells. Within these structures they are subject to tightspatial and temporal regulation. One method of PI lipidregulation is the generation of multiple PI species by thephosphorylation/dephosphorylation of the inositol ringby PI kinases and phosphatases respectively, illustratedin Figure 1. PI lipids can be reversibly phosphorylated atthe 3, 4 and 5 position of the inositol ring in various com-binations, leading to the eight species described so far [5].Alternatively, phosphatidylinositol 4,5-bisphosphate(PtdIns(4,5)P2) can be hydrolysed by the phospholipase C(PLC) family of enzymes to generate the fusogenic/secondmessenger lipid 1,2-diacylglycerol (DAG) and the solubleinositol 1,4,5-trisphosphate (InsP3) headgroup. The solu-ble inositol headgroup can be further modified at all 6positions of the inositol ring to produce in the region of30 soluble inositol species [6]. Finally there exists a familyof proteins, the PtdIns/phosphatidylcholine (PtdCho)transfer proteins (PITP)s, whose role is to shuttle phos-pholipids between different subcellular compartments,thus enabling the alteration of organellar lipid composi-tions [7]. Thus, PI lipids exert control over biologicalevents by the regulation of their individual levels in theplasma membrane and within sub-cellular compart-ments. The intricate details of PI lipid regulation and adescription of the diverse biological processes they regu-late have been reviewed extensively elsewhere [5, 6, 8].

This review will focus on the role of PIs, and other lipids,and their mediators in mast cell function, and describethe recent advances made in elucidating the role of thesesecond messengers.

Regulation of degranulation – role of lipid raftsThe plasma membrane of cells is asymmetric. The outerleaflet is believed to differ from the cytoplasmic leaflet interms of lipid composition. For example the outer, exo-plasmic leaflet is enriched in sphingolipids and choles-terol, which are thought to form domains termed

,

rafts’surrounded by PtdCho molecules [9]. The physical proper-ties of rafts described to date include their low buoyantdensity on sucrose gradients, enrichment of sphingoli-pids and cholesterol and lack of solubility in the non-ionic detergent triton X-100 at 48C [9]. Such domains inmast cells are also thought to contain PtdIns(4,5)P2 [10,11]. Rafts are perceived to facilitate cell signalling eventsby creating a signalling ,platform’, whereby transmem-brane receptors are localised in rafts, and this enhancestheir interaction with protein molecules attached to theinner leaflet of the plasma membrane. Such a scheme isthought to play a role in cellular responses to immuno-genic mediators including IgE, and various growth fac-tors. The role of lipid rafts and mast cell activation arediscussed below.


Figure 1. Modification of phosphoinositide species by reversiblephosphorylation and hydrolysis. The 8 potential PI species arehighlighted. For PI species described in more detail in the text,modifying proteins are included in bold. Kinase pathways areindicated by a red arrow, phosphatase pathways by a bluearrow, and phospholipases by a green arrow. Soluble inositidespecies (italicised) can be reversibly phosphorylated on all posi-tions of the inositol ring to yield numerous species [6]. The inosi-tol headgroup is eventually recycled and incorporated into newlysynthesised PtdIns molecules. It should be noted that this figureis not exhaustive, and other PI phosphatase steps exist, but areexcluded for brevity as they are not described in the text.

Signal Transduction 2006, 6, 81– 91 The role of phosphoinositides in mast cell signalling 83

The mechanistic steps leading to mast cell degranula-tion are initiated at the plasma membrane, by the bind-ing of IgE cross-linked to antigen to the hetero-tetramericIgE receptor, FceRI. Under conditions where the IgE-anti-gen interaction is a high affinity one, multiple IgE mole-cules bind to allergens, thus FceRI receptors aggregate atthe plasma membrane. The very first step toward degra-nulation is under the control of membrane lipids. It hasbeen postulated that FceRI receptors, when aggregatingwill do so in rafts [12]. The translocation of aggregatedFceRI receptors into rafts is thought to co-localise themwith the tyrosine kinase Lyn, which by phosphorylatingthe b and c subunits of the FceRI receptor generatesimmunoreceptor tyrosine-based activation motifs(ITAMS). These ITAM motifs act as docking sites for src-homology 2 (SH2) domain containing proteins that pro-pagate the immune response. Accordingly, when ratbasophilic leukaemic (RBL) cells are depleted of choles-terol with methyl-b-cyclodextrin, and hence rafts are dis-rupted, the tyrosine phosphorylation of FceRI is inhib-ited [13]. Thus, lipid rafts could be seen to control theinitial events in mast cell activation. However, data onthe role of rafts in mast cell signalling is conflicting, andrafts themselves remain poorly characterised in vivo in

terms of their size and dynamics [14]. For example,methyl-b-cyclodextrin is widely used in the study of raftsbut is a non-specific tool, and will deplete all areas of theplasma membrane of cholesterol and not just rafts [15].One cannot therefore distinguish between raft and non-raft mediated events that are cholesterol dependent. Thisis highlighted in studies on RBL cells, where tyrosinephosphorylation and calcium signalling events thoughtto be critical to the degranulation process are differen-tially affected by methyl-b-cyclodextrin [15]. Indeed theformer event is induced by treatment with methyl-b-cyclodextrin alone [15]. Progesterone is now an alterna-tive agent to deplete cholesterol from biological mem-branes, and is thought to be more specific to membranemicro-domains [16]. An improved ability to manipulateand study rafts will allow their role in biological eventssuch as mast cell signalling to become clearer.

Phosphoinositide generation during mast celldegranulationDownstream of FceRI activation, our knowledge of PIlipids in mast cell degranulation is better understood.The role of PIs in mast cell function has expanded frominitial observations where changes in PI hydrolysis and


Figure 2. Schematic Representation of Phospholipid Regulated Mast Cell Degranulation Pathways. Activation of the FceRI receptorwith IgE bound antigen (green circles) results in the generation of ITAMs (P) on the b and c subunits of the receptor by the Lyn tyro-sine kinase. ITAMs directly or indirectly recruit PI3K p110d and PLCc, which generate PtdIns(3,4,5)P3 and DAG on the plasma mem-brane (grey) respectively. PLCc also produces soluble InsP3 leading to calcium mobilisation and the activation of calcium-dependentpathways. p110d leads to the exocytosis of adenosine (black circles), and to the recruitment of other signalling mediators to theplasma membrane, including Btk, PKB and potentially PLCc. Adenosine leads to the activation of PI3K p110c and calcium signalling,which, along with DAG leads indirectly to production of arachidonic acid and via its metabolism, and the release of pre-formed inflam-matory mediators. Cytokine transcription is also initiated, partly under the control of PKB. Lipid kinases are coloured red, phospholi-pases green, receptors blue, lipid phosphatases in black and other signalling molecules yellow. Numerous proteins have beenomitted from the scheme for brevity, but have been described here in the text or elsewhere [1, 4].


reversible phosphorylation were correlated with degra-nulation, and as such were inferred to play a role in theexocytosis of inflammatory mediators [17–19]. Thesestudies were followed by the application of the PI 3-kinase (PI3Ks) inhibitor wortmannin to bone marrow-derived mast cells (BMMCs). Wortmannin blocked therelease of b-hexoaminidase, inferring that 3-phosphoryl-ated PIs were directly involved in this process [20]. Wenow understand how these 3-PIs are generated. The gen-eration of ITAMs on the b and c subunits of the FceRIreceptor subunits recruits signalling intermediariesdirectly, or indirectly, including PI3K family membersand PLCc (Figure 2). Thus PI3Ks will be brought into thevicinity of their substrate, PtdIns(4,5)P2 and generatePtdIns(3,4,5)P3. The PI3K isoform responsible for linkingthe early tyrosine phosphorylation events to PI signallingand degranulation is in the main the class IA PI3K p110d,an isoform with a primarily leukocyte expression [21]. Inelegant experiments applying a selective p110d inhibitorand p110d kinase-dead BMMCs Ali et al. showedPtdIns(3,4,5)P3 generation, b-hexosaminidase and cyto-kine release were all impaired when p110d signallingwas abrogated. Thus, the lipid products of p110d areclearly a mediator of mast cell signalling. But, it is alsoapparent that p110d is not the only PI3K involved in thedegranulation process. In what appears to be an amplifi-cation step, the class IB PI3K, p110c also regulates cal-cium signalling and b-hexoaminidase release [22]. A ser-ies of events is now envisaged whereby these two PI3Ksact in concert. Initially, FceRI activation leads toPtdIns(3,4,5)3 generation by p110d, whose downstreamactivity leads to release of the autocrine mediator adeno-sine. Adenosine in turn binds to and activates G-proteincoupled adenosine A3 receptors [22, 23], whose Gi bc sub-units activate PI3K p110c. Supporting this scheme, it hasbeen observed that bc subunits will activate permeabi-lised mast cells, and the secretagogues mastoparan andcompound 48/80 [24, 25] trigger histamine release via Gi3

proteins [26, 27].Why does this systematic PI generation exist? One pos-

sibility is that each wave of 3-PIs regulates a different partof the degranulation process. For example, the initialwave could act to activate 3-PI effectors whose activationis a pre-requisite for the exocytosis of pre-stored media-tors and adenosine. The activation of the A3 receptor andp110c on the other hand may be linked to a prolongedrise in calcium required for a more long-term response,including the release of pre-formed agents and the initia-tion of de novo synthesis of mediators such as platelet acti-vating factor, prostaglandin D2 and interleukins.

In addition to the generation of PtdIns(3,4,5)P3 shortlyafter mast cell activation, PtdIns(4,5)P2 is also modified by

hydrolysis to DAG and InsP3. The most likely PLC under-taking this is PLCc, recruited to ITAMs via its SH2domains, and activated by tyrosine phosphorylation byBruton’s tyrosine kinase (Btk) [28]. The translocation ofPLCc from the cytoplasm to the plasma membrane maybe further enhanced by the above-described generationof PtdIns(3,4,5)P3; by binding to the Pleckstrin homology(PH) domain of PLCc. However, this is a point of contro-versy, with conflicting reports on the importance ofPtdIns(3,4,5)P3 on PLCc function. For example, Tkaczyk etal., reported that in human-derived mast cells devoid ofthe class I PI3K adapter subunits p85a and p85b, PLCc

activation was normal [29]. Conversely, Smith et al.,showed that the class I PI3Ks p110d and p110c partici-pated in PLCc activation [30]. Similar data has beenobtained in platelets where a PI3K activity is necessaryfor PLCc2 activation [31]. Nonetheless, PLCc will generateInsP3 from PtdIns(4,5)P2, the InsP3 in turn mobilises cal-cium from intracellular stores. The raising of intracellu-lar calcium levels is a critical part of the mast cell degra-nulation process, having a number of effectors. Theseinclude PKC (see below) and components of the exocyto-tic machinery. This calcium necessity is highlighted bythe sensitivity of mast cell degranulation to pharmacolo-gical inhibition of PLC and the chelation of calcium byBAPTA [32, 33].

In addition to the InsP3 mediated calcium release fromintracellular stores, mast cell calcium signalling mightalso be influenced by extracellular calcium. A novel leu-kocyte expressed PtdIns(3,4,5)P3-regulated calcium chan-nel has been described, which provides an influx of extra-cellular calcium into mast cells in the presence ofPtdIns(3,4,5)P3 [34].

Targets of phosphoinositides

Phospholipase D (PLD)Described above are mechanisms whereby PtdIns(4,5)P2

is modified by hydrolysis and reversible phosphorylationto regulate mast cell degranulation. It is important tonote however that PtdIns(4,5)P2 can also influence mastcell signalling by virtue of its modulating enzyme activ-ities. PLD 1 and 2 (Figure 3), respectively localised to secre-tory granules and the plasma membrane of mast cells,convert PtdCho to phosphatidic acid (PtdOH) and choline[35], and are stimulated catalytically by PtdIns(4,5)P2 [36].PtdIns(3,4,5)P3 has also been suggested to regulate PLDactivity [37], but the very low levels of this lipid presentunder conditions where PLD is active indicate thatPtdIns(3,4,5)P3 is unlikely to be a major regulator of PLD[38]. PLD has N-terminal PH and Phox domains, both ofwhich have the ability to bind PI lipids [39]. N-terminal



mutants of PLD2 which lack these domains remain cata-lytically active, and retain responsiveness to the presenceof the Arf GTPase [40]. However, the PH domain, althoughnot critical for PLD catalytic activity is required for thecorrect localisation of the enzyme; in its absence PLD2mislocalises to endosomes [41].

How PLD activity modulates the exocytosis of immuno-genic mediators is becoming clear. Current hypothesessuggest that after FcRI activation, the ordered activationof Arf GTPase by ArfGEFs (both capable of binding eitherPtdIns(4,5)P2 or PtdIns(3,4,5)P3), in the presence ofPtdIns(4,5)P2 stimulates PLD activity, and thus the genera-tion of PtdOH [42]. This lipid in turn stimulates PtdIns(4)Pkinase Ia (PIPKIa), which generates the production offurther PtdIns(4,5)P2 from PtdIns(4)P [43]. Such a genera-tion of PtdOH and PtdIns(4,5)P2 is subsequently thoughtto facilitate membrane fusion events between secretoryvesicles and the plasma membrane (see below). The gen-eration of PtdIns(4,5)P2 in this scheme may also be facili-tated by the PITPs, probably by virtue of PITP supplyingsubstrate for PtdIns(4,5)P2 synthesis. In permeabilisedand stimulated RBL 2H3 cells, PITPa increases the levelsof PtdIns(4)P and PtdIns(4,5)P2, and it prevents the loss ofthese lipids during ,run-down’ (the progressive decreasein mast cell ability to exocytose) after permeabilisation[44, 45]. However, contrary to these reports, PITPa –/–mast cell lineages from embryonic stem cells manufac-ture secretory granules correctly, and exocytose as wellas PITPa +/+ cells [46]. Moreover, the PITPa +/+ and –/– cells

,run-down’ at the same rate [46]. Thus the role of PITPs inmast cell signalling remains unclear.

In accordance with the role for PLD in exocytosis, pri-mary alcohols which inhibit PLDs also inhibit PtdOHaccumulation and degranulation [47, 48]. In this scheme,PtdOH is described primarily as a signalling second mes-senger. One must consider however a second mechanismby which this lipid may act. In converting PtdCho toPtdOH PLD will, over a localised area modify both thecharge and size of phospholipid headgroups. Such a

change may promote negative curvature of the mem-brane, another feature which induces membrane fusion[49]. PLD is also a calcium dependent enzyme. Thus, onecan see how the activation of the PI3K and PLC familymembers comes together to facilitate the release ofimmunogenic mediators from mast cells.

BtkThe activation of the PI3Ks after mast cell activation willlead to the generation of PtdIns(3,4,5)P3 on the plasmamembrane. This lipid will serve to recruit PH domain-containing proteins to the plasma membrane in a con-certed fashion. One of these proteins is Btk (Figure 2). Btkis a tyrosine kinase, and has among its targets PLCc [28].The tyrosine phosphorylation of PLCc serves to enhanceits catalytic activity and thus its ability to generate DAGand InsP3. The calcium rises generated from intracellularstores such as the endoplasmic reticulum are essentialfor the release of pre-formed mediators. In this manner,btk–/– murine BMMCs display a reduction in histaminerelease, InsP3 generation and calcium transients after sti-mulation [28, 50]. Long term responses such as cytokineproduction are also reduced [28, 50, 51]. Thus one canconsider Btk to be an essential PI effector in mast cell exo-cytosis, linking upstream PI phosphorylation events todownstream PtdIns(4,5)P2 hydrolysis.

PKCThe activation of PLCc and its hydrolysis of PtdIns(4,5)P2

have been described above. Its products, calcium andDAG are likely to have a number of roles in the degranu-lation process. Calcium itself is thought to have a num-ber of distinct roles, one of which will certainly be theregulation of machinery triggering the release of inflam-matory mediators (see below) [52]. Another role for cal-cium is believed to be the activation of some PKC iso-forms present in RBL2H3 cells. On FceRI activation, ormast cell activation by compound 48/80, PKCs translo-cate to the plasma membrane and are activated [53, 54].For the calcium-dependent PKCa and PKCb isoforms, thisprocess is dependent upon the PLCc products, DAG andcalcium. DAG binds to a module within PKC enzymescalled a C1 domain, recruiting them to the plasma mem-brane. Additionally, mast cells express other PKC iso-forms, including PKCd, PKCe and PKCf [54]. These iso-forms are calcium-independent, their activation requir-ing DAG and phosphorylation by the upstream serine-threonine kinase phosphoinositide-dependent kinase 1[55–57]. Activated PKC enzymes have several targets,including components of the exocytotic machinerywhich regulate the release of histamine and b-hexos-aminidase [58, 59]. In addition, PKC will phosphorylate


Figure 3. Metabolism of phospholipids by phospholipases. Theconnected metabolism of phospholipids is illustrated. Lipids areindicated in bold, modifying enzymes in normal type.


and activate phospholipase A2 (PLA2) (Figure 2 & 3) [60].PLA2 will generate arachidonic acid (AA) and the corre-sponding lysophospholipid from phospholipids such asPtdCho and PtdEtn with an arachidonate (20:4) fatty acidchain at the sn-2 position [61]. AA will subsequently bemetabolised to other inflammatory mediators includingthe leukotrienes, prostaglandins, thromboxanes and pro-tacyclins. It should be noted that due to the interconver-sion of the products of all the phospholipases, AA maynot only be generated by PLA2, but also by the concertedaction of PLC and a DAG lipase, a pathway utilising PtdInsas the initial substrate (Figure 3).

In addition to PKC being a major target of DAG, andthe possible conversion of DAG to AA, it is possible thatother DAG targets exist in mast cells. It is now widelyaccepted that there are several DAG effectors, which maybe responsible for effects previously attributed to PKC[62]. Mast cell degranulation is also regulated in part byVav, a Rac GEF and the small GTPases Ras and Rac [1, 4].Both Ras GAPs and Vav contain C1 domains. Thus DAGmay in part regulate all of these proteins, and thusimpinge on multiple areas of mast cell signalling.

Negative regulation of phosphoinositide signallingin mast cellsGiven the dependence of degranulation on both proteinand lipid phosphorylation events, it is natural that nega-tive regulation of mast cell exocytosis will be under thecontrol, at least in part of protein and lipid phosphatases.The protein tyrosine phosphatases negatively regulatingmast cell secretion have been reviewed elsewhere [1, 4].For every PI kinase that exists, a complementary phos-phatase activity has been described (Figure 1). Classically,the tumour suppressor phosphatase and tensin homolo-gue (PTEN) is the antagonist of the class I PI3Ks, convert-ing PtdIns(3,4,5)P3 to PtdIns(4,5)P2 [63, 64]. To date little isknown about the function of PTEN in mast cell signal-ling. This awaits the generation of PTEN –/– BMMCs orthe application of novel PTEN inhibitors to this system[65].

The SH2 domain containing inositol 5-phosphatase(SHIP) converts PtdIns(3,4,5)P3 to PtdIns(3,4)P2 [66]. SHIP isapproximately 145 kDa in size, having a 5-phosphataseconserved catalytic core, an N-terminal SH2 domain anda C-terminal proline rich domain [66]. This latter domain,along with the catalytic core is essential to SHIP functionin BMMCs, probably by regulating its interaction withSH3 domain-containing proteins [67]. Unlike PTEN, SHIPhas been studied extensively in mast cells, and appears tobe a strong negative regulator of mast cell function. Thisnegative regulation takes place through the low-affinityIgG FccRIIB receptor [68]. Antigen, as well as binding IgE

will also bind simultaneously to IgG. Thus, upon antigenpresentation to a mast cell, IgE will bind to FceRI, and theIgG to FccRIIB, bringing the activatory FceRI and inhibi-tory FccRIIB receptors into proximity. Lyn, as well ascreating ITAMs on FceRI will phosphorylate FccRIIB onspecific tyrosine residues to generate immunoreceptortyrosine-based inhibitory motifs (ITIMs). SHIP will bindITIM motifs through its SH2 domain, bringing it fromthe cytosol into the proximity of its substrate PI lipids atthe plasma membrane. Through the conversion of itssubstrates SHIP inhibits mast cell activation. For exam-ple, SHIP –/– BMMCs virally infected with catalyticallyactive SHIP display reduced levels of PtdIns(3,4,5)P3 gen-eration and b-hexoaminidase release in response to steelfactor treatment, compared to their uninfected counter-parts [67]. Moreover, SHIP–/– BMMCs degranulate morereadily, have higher PtdIns(3,4,5)P3 levels, increased PKCactivation and exhibit enhanced calcium influx inresponse to IgE-antigen or steel factor, compared toSHIP+/+ BMMCs [69, 70]. Thus pre-formed mediator exocy-tosis is negatively regulated by SHIP. In addition, the denovo synthesis and release of cytokines from mast cells isalso under the control of SHIP. PKB is activated upon IgEtreatment of mast cells in a PtdIns(3,4,5)P3 dependentmanner [71], and subsequently phosphorylates the IjBportion of the IjB/NF-jB complex, causing them to dis-sociate. Under such dissociated conditions, the freeNF-jB transcription factor positively regulates Il-6 tran-scription. Hence, in SHIP–/– BMMCs, where PtdIns(3,4,5)P3 is more abundant PKB is more readily activated,Il-6 production is enhanced [72]. In a SHIP+/+ background,PtdIns(3,4,5)P3 levels are diminished, PKB as a populationis less active, hence Il-6 production is reduced [72].

PtdIns(4,5)P2, whose accumulation is described aboveas being essential for degranulation to take place, mayalso have an inhibitory role in exocytosis. One of theenzymes responsible for PtdIns(4,5)P2 synthesis, PIPKIahas been identified as a negative regulator of BMMCs [11].PIPKIa–/– BMMCs (expressing normal levels of PIPIb and c

isoforms) exhibit larger calcium fluxes and cytokinelevels in comparison to PIPKIa +/+ BMMCs. Moreover, theinitial signalling events of the exocytotic pathway arealso altered in PIPKIa–/– cells, with PLCc and PKB beingmore readily phosphorylated on tyrosine and serine resi-dues respectively. It was also observed that the FceRIreceptor was more abundant in lipid rafts in the PIP-KIa–/– BMMCs after mast cell stimulation, indicating anegative role for PtdIns(4,5)P2 in FceRI receptor aggrega-tion. This effect would explain the enhanced signallingphenotype seen in the PIPKIa –/– cells. Independent ofthis effect, but also PtdIns(4,5)P2 mediated, PIPKIa waspostulated to modulate the cytoskeleton, leading to an



enhanced b-hexoaminidase release in PIPKIa–/– BMMCs.Thus one can see that mast cells are not only negativelyregulated by the removal of certain PI lipids from theplasma membrane, but also by PI generation in specificpools by specialised enzymes.

Phosphoinositides in exocytosisAside from the hydrolysis or reversible phosphorylationof PIs described above which are upstream events in thedegranulation process, there are potential roles for PIs atother stages of the secretory pathway. The emptying ofsecretory granules in response to stimuli is a compoundexocytotic event. In other words, it entails multiple mem-brane fusion events. First, heterotypic fusion events,whereby the secretory granules fuse with the plasmamembrane. Second, homotypic fusion events in whichsecretory vesicles fuse with each other forming channels,ensuring that maximal release of pre-stored mediatorstakes place. These multiple fusion events will requiremany proteins to regulate them. Numerous proteinshave now been identified as key regulators of membranefusion events, including the SNARE family of proteins,which are themselves regulated by additional proteinsincluding N-ethyl-maleimide sensitive factor, the cal-cium-dependent synaptotagmins, Rab3 and the Munc18family members. These proteins have been reviewedextensively elsewhere [52, 73–76]. The role of lipids inmembrane fusion events is less well documented, but itis clear that the lipids themselves, or the pathways theyregulate are vital for membrane fusion events [77–82].For example, the role of the PIs in calcium mobilisationwas outlined above. Calcium will play a number of criti-cal roles in exocytosis; it regulates calcium-dependentPKCs which phosphorylate fusion machinery includingsyntaxin 4 and Munc18 [58, 59]. It also binds to the C2domain of synaptotagmins [83], inducing their oligomer-isation and enhancing the interaction of the C2 domainwith acidic phospholipids, in turn aiding the formationof SNARE complexes [84, 85].

The fusion of secretory granules may be under the con-trol of phospholipids and their effectors, but the PI lipidsand their derivatives also regulate other aspects of fusion.The fusion of two membranous compartments and theirsubsequent content mixing is characterised by their com-ing together to form a ,stalk’, a process which requiresthe two compartments to remodel their membranes,enhancing membrane curvature at their points of con-tact [86]. The negative membrane curvature required canbe attained by remodelling the outer leaflet of the plasmamembrane, replacing phospholipids with bulky head-groups with those with smaller ones. This was exempli-fied above by the conversion of PtdCho to PtdOH by PLD

after mast cell activation. It may be similarly achieved bythe hydrolysis of PtdIns(4,5)P2 to DAG by PLC, anotherdownstream effector of FceRI. DAG and PtdOH have longbeen identified as fusogenic lipids [49, 78], and mightwell regulate exocytosis by facilitating homo- and hetero-typic fusion events at the mast cell plasma membrane.

Finally, the vesicle trafficking bringing together secre-tory granules and the plasma membrane will requirecomponents of the cytoskeleton that may be under thecontrol of PI signalling pathways [87]. PKC, activated inpart by DAG has myosin light and heavy chains amongits targets, whose phosphorylation has been shown tocorrelate with exocytosis [88]. Rac regulates the F-actinnetwork, and it has been demonstrated in mast cells thatthe V12Rac1 mutant, which is constitutively activeincreases mast cell secretion [89]. Conversely, inhibitionof Rac with pharmacological inhibitors decreases exocy-tosis [89]. Rac is activated by the Rac-GEF Vav1, which hasa DAG binding C1 domain and a PtdIns(3,4,5)P3 bindingPH domain within its structure. Finally, the actin cytoske-leton is postulated to regulate degranulation, since itsdisruption with latrunculin B inhibits secretion inducedby the secretagogue compound 48/80 [90]. The actincytoskeleton is known to be strongly influenced by PIlipids, including PtdIns(4,5)P2 and PtdIns(3,4,5)P3.PtdIns(4,5)P2 is in part responsible for promoting theassembly of the actin cytoskeleton at defined sites withina cell. Both positive and negative regulators of the actincytoskeleton are modulators of, or modulated byPtdIns(4,5)P2 levels. For example, gelsolin, CapZ, Arp2/3,Wikcott-Aldrich syndrome protein (WASP), and poten-tially WASP-related proteins are all modified byPtdIns(4,5)P2 to promote actin polymerisation. Conver-sely, ADF/cofilin, a promoter of actin depolymerisation isinhibited by PtdIns(4,5)P2, and the PtdIns(4,5)P2 hydrolys-ing 5-phosphatase synaptojanin reduces the number ofactin stress fibres. For a more detailed description of PIregulation of the cytoskeleton the reader is directed torecent excellent reviews on the subject [87, 91]. Together,it is clear that PI lipids regulate the final stages of thedegranulation process, through alterations in membranecharge and hence curvature, fusion machinery and cyto-skeletal control.

Summary

With asthma and other mast cell-influenced diseases soprevalent in society, this area of research will continue tobe the focus of much attention. While our knowledge ofproteins and mast cell function is substantial, lipid sig-nalling in this area is less well understood. However, it is



clear that phospholipids, and in particular the PI lipidsare critical regulators of mast cell function, impingingon the degranulation process from start to finish, andthat alterations in lipid signalling events may contributeto mast cell related disease states.

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The role of phosphoinositides in mast cell signalling

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