The ins and outs of IgE-dependent mast-cell exocytosis

The ins and outs of IgE-dependentmast-cell exocytosisUlrich Blank1 and Juan Rivera2

1INSERM E 0225, Bichat Medical School, 16 rue Henri Huchard, BP 416, 75870 Cedex 18, France2Molecular Inflammation Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and

Musculoskeletal and Skin Diseases, National Institutes of Health, Building 10, Room 9N228, Bethesda, MD 20892-1820, USA

Mast cells (MCs) are able to secrete the contents of

preformed cytoplasmic secretory granules (SGs) on

encountering certain stimulants. For MCs, this process

is fundamental to their role in innate and acquired

immunity. Immunological adaptation through the pro-

duction of IgE antibodies, to normally innocuous sub-

stances, has kidnapped the MC exocytotic response to

cause allergic disease. Thus, understanding the molecu-

lar events in IgE-dependent MC exocytosis holds prom-

ise for therapeutic intervention. Recent advances, in

deciphering the coupling of the high affinity IgE recep-

tor (Fc1RI) to the secretory machinery, promote a new

paradigm of the molecular coordination of MC exocyto-

sis. A clearer picture of the link between Fc1RI stimu-

lation and SG exocytosis is emerging.

Mast cells (MCs) can mediate adaptive immune responsesthrough the high affinity IgE receptor (Fc1RI) (reviewed inRef. [1]). They store histamine and serotonin in preformedcytoplasmic secretory granules (SGs) as well as a variety ofproteases that become active only on their release. Unlikemany other secretory cells, they can release substantialamounts of vesicular content in a single stimulatory eventthrough a process in which vesicle membranes fuse withthe plasma membrane (PM) and with each other to formchannels (compound exocytosis), which ensures a maximalbiological effect. This massive exocytosis causes many ofthe symptoms associated with acute and chronic allergicdisease. Chronic allergic disease is also coupled to secre-tion of SG-stored cytokines [e.g. tumor necrosis factor(TNF)] and to the MCs ability to generate a so-called latephase reaction, by producing prostaglandins, leukotrienes

and inflammatory and immunoregulatory cytokines(reviewed in Ref. [2]).

The function of MCs in immediate hypersensitivity isevolutionary conserved from amphibians and birds [3,4].Similarly, MC exocytosis relies on the basic machinery ofmembrane trafficking conserved from yeast to humans [5],although differences in the regulation and execution of theprocess are apparent (Table 1). Although various triggerscan elicit the MC exocytotic response [6], new informationon how the IgE-triggered process is driven has emerged.Here, we attempt to integrate this information towards amore cohesive view of the requirements in IgE-dependentMC exocytosis.

Regulation of SG exocytosis by Fc1RI

The Fc1RI is a multisubunit receptor with a ligand-binding a subunit, a signal-amplifying membrane-tetra-spanning b subunit and a homodimeric disulfide-linkedg subunit that provides the signaling ability of this recep-tor (reviewed in Ref. [7]) (Figure 1). Initiation of signalingis mediated through the immunoreceptor tyrosine-basedactivation motif (ITAM) encoded in the cytoplasmic tails ofthe b and g subunits. Monomeric IgE binding to Fc1RI, inthe absence of antigen, increases expression of the receptorand causes the generation of survival signals (reviewed inRef. [8]) (Figure 2). These signals result from autologous orcross-reactive aggregation of IgE once bound to MCs [9].However, at the minimal concentration sufficient to occupyall receptors on the cell surface, monomeric IgE fails toelicit considerable MC exocytosis.

By contrast, antigen-driven aggregation of IgE anti-body-occupied Fc1RI elicits intracellular signals that

Table 1. Comparison of exocytosis in MCsa and neurons

Parameter MC Neuron

Granule type Secretory lysosome Specialized secretory granule

Granule size 300–1000 nm 50 nm

Granule number #1000 per cell 200–500 per synaptic terminal

Calcium requirement ,1 mM . 20 mM

PKC requirement PKCb and/or PKCd No

Kinetics Minutes Milliseconds

Recycling (granule) Long (hours to days) Very short (minutes)

Release (type) One single release of #100% (compound or

multigranular mode)

Multiple release, one granule per fusion event

aAbbreviations: MC, mast cell; PKC, protein kinase C.

Corresponding author: Juan Rivera ( [email protected]).

Review TRENDS in Immunology Vol.25 No.5 May 2004

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result in MC exocytosis. Signals generated by Fc1RIaggregation are subject to kinetic proofreading [10].Aggregation must persist for sufficient time to enablethe execution of the molecular events required for aproductive response (Figure 2). Because of its persistentinteraction with IgE (Figure 2), a high rather than alow affinity antigen is more likely to fulfill the kineticproofreading requirements. This might avoid spuriousexocytosis with a low affinity cross-reactive antigen.Nonetheless, some responses escape kinetic proofreadingbecause monomeric IgE, low affinity antigens and lowoccupancy of Fc1RI can all cause responses, such assurvival and lymphokine production, in the absence of SGexocytosis (reviewed in Ref. [8]) [11,12] (Figure 2). Thisuncoupling of a signal-generating Fc1RI from exocytosisis an apparent consequence of preferential engagementof certain intracellular signals [12]. The physiologicaladvantage is unclear, yet one might predict that a limitedresponse to a weak challenge might be advantageous by

priming the host immune system and initially avoidingpossible injurious effects of a full inflammatory response.

Protein tyrosine kinases and their relative importance to

SG exocytosis

Several Src family protein tyrosine kinases (PTKs) areexpressed in MCs (Table 2). These and other PTKs, such asSyk and Btk, rapidly respond to Fc1RI engagement bylocalizing to the appropriate site of function and/or byincreasing their activity (reviewed in Ref. [13]). The SrcPTK, Lyn, which is weakly associated with Fc1RI [14],phosphorylates the canonical ITAM-tyrosines of the b andg subunits (reviewed in Ref. [7]) (Figure 1). Although allthe steps required to initiate phosphorylation are still notcompletely defined, there is growing acceptance of a rolefor specialized lipid environments in receptor phosphoryl-ation [15]. It was recently recognized that the Src PTKFyn is also activated as a Fc1RI-proximal event that isindependent of Lyn activation [16] (Figure 1). Fyn does not

Figure 1. A model of structure and functional coupling of the high affinity receptor for immunoglobulin E (Fc1RI) to early signaling events. Fc1RI is comprised of an

IgE-binding a chain, a tretraspanning b chain and two disulfide linked g chains. Antigen-aggregation of IgE-occupied Fc1RI results in phosphorylation of the immuno-

receptor tyrosine-based activation motifs (ITAMs) by Lyn kinase (Lyn), activation of Syk kinase (Syk) through ITAM binding, and inclusion of the Fc1RI in lipid rafts. Aggre-

gation of Fc1RI also rapidly activates Fyn kinase (Fyn), whose activity is important for phosphorylation of the adaptor known as Grb-2 associated binder-like protein 2

(Gab2) and activation of phosphoinositide 3-kinase (PI3-K) activity in mast cells (MCs). Both Fyn and Lyn co-immunoprecipitate with Fc1RI but although Lyn is known to

directly interact with the tretraspanning b chain, it is not known whether Fyn directly or indirectly interacts with Fc1RI. Moreover, the requirements for Fc1RI-dependent Fyn

activation are unclear (þ?). Lyn kinase negatively regulates the activity of Fyn but positively regulates the activation of Syk and phosphorylation of another adaptor protein,

linker for activation of T cells (LAT), which scaffolds a complex required for normal MC calcium responses. Fyn kinase positively regulates Gab2 and its association with

PI3-K and is essential for initiation of the degranulation response. Signals generated by the two distinct adaptor complexes (LAT and Gab2) synergize to support novel PKC

activation (Fyn–Gab2–PI3-K) and calcium signals (Lyn–Syk–LAT–SLP-76–Vav1). This synergy provides competence for MC degranulation. Abbreviations: Ag, antigen;

PLCg, phospholipase Cg; PKC, protein kinase C; SHP-2, Src homology 2 domain-containing protein tyrosine phosphatase-2; SLP-76, Src homology 2 domain-containing

leukocyte phosphoprotein of 76 kDa.

PI3-K

TRENDS in Immunology

α

Syk

βγ

Lipid Raft

Grb2

+

+

+

+

Cbp

Csk–

+?

IgE

α

γ β

Ag

SHP-2

Gab2

Fyn–

PKC

Ras

Sos

Degranulationcytokine production

=(pY)ITAM

FcεRI

PI3-K Rac Vav

SLP-76

Gads

PLCγ2PLCγ1

Ca2+

LAT

Lyn

Review TRENDS in Immunology Vol.25 No.5 May 2004 267

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appear to effectively phosphorylate b and g chains [17],suggesting that its function is distinct from Lyn. Acti-vation of Btk also occurs rapidly on stimulation, seeminglypreceding protein kinase C (PKC) activation [18]. Btkactivation in MCs requires both Lyn and Syk activity,although the exact mechanism of activation remainsunclear.

Recent studies with gene-inactivated mice addressedthe question of the relative importance of the receptor-proximal PTKs to SG exocytosis. Studies of Lyn-deficientMCs demonstrate that Lyn is dispensable [16,18,19]. Infact, Lyn-deficient MCs are hyper-responsive because theSG contents are extruded at antigen concentrations thatdo not elicit a response from wild-type cells and the totalamount released is enhanced. By contrast, Fyn is essentialbecause MCs deficient in this kinase show a significant

defect (80–90% inhibition) in SG exocytosis [16]. Syk isalso essential because exocytosis is abrogated in Syk-deficient MCs. Btk-deficient MCs also show a defect indegranulation, albeit less pronounced than that of Fyn orSyk-deficient MCs, with higher antigen concentrationseliciting as much as 70% of the response of wild-type cells(reviewed in Ref. [13]). However, it should be noted that inthe background of Lyn-deficiency, the absence of Btkcaused dramatic inhibition (80–90%) of exocytosis [18].Nonetheless, it appears that a deficiency in Fyn or Syk hasmore severe inhibitory consequences on MC exocytosisthan the absence of either Btk or Lyn.

The importance of adaptor molecules in SG exocytosis

MCs express numerous adaptor proteins that coordinateand assemble the molecular apparatus of cell signaling

Figure 2. Stimulus strength and mast cell (MC) effector responses. Binding of monomeric IgE to its high affinity receptor (Fc1RI) can cause cellular responses, including

increased Fc1RI cell surface expression and enhanced MC survival, probably through a paracrine or autocrine lymphokine effect. Monomeric IgE does not effectively induce

secretory granule (SG) exocytosis. The interaction of a low affinity allergen with cell surface bound IgE antibody, or the limited occupancy of Fc1RI with IgE and allergen,

results in preferential induction of cell signals and in limited responses (i.e. lymphokine production but not SG exocytosis), which resemble some of the effects of mono-

meric IgE binding. By contrast, a high affinity allergen or aggregation of large numbers of Fc1RI induces robust cellular signals that can effectively induce MC effector

responses inclusive of SG exocytosis. Abbreviation: Fab, antigen-binding fragment.

lymphokines

Low affinity allergen, Low IgE occupancy

Small numbers of receptor aggregates

induction of preferential signals

(c)

Monomeric IgE binding

Receptor aggregates weak signals?

increased receptor numbers/lymphokines/increased cell survival.

(a) (b)

IgE

FabFab

FabFab

FcεRIα


High affinity allergen

Large numbers of receptor aggregates robust signaling

degranulation, lymphokines, other effectors.

(d)

Table 2. Src family protein tyrosine kinases (PTKs) expressed in mast cells (MCs)a

Src PTKb Bone marrow-derived

MCs (BMMCs)

RBL-2H3 PT-18 HMC-1 HuMCc

Fgr þþ þþ þ /2 þ Not determined

Fyn þþþ þ þþþþ þþþ þþþ

Hck þþ þþ þ þþ þþ

Lyn þþþþ þþþþ þþþ þþþ þþþþ

c-Src þþ þþþ þþ þþ þþþ

Yes þ /2 þ /2 þ /2 þ þ

aAbbreviations: HMC-1, human immature MC line-1; HuMC, CD34þ cultured human MCs; PT-18, murine MC line; RBL-2H3, rat basophilic leukemia-clone 2H3.bSrc family kinases were detected by RT-PCR and Western blotting for cultured BMMCs or the MC lines RBL-2H3 and PT-18. Src PTKs in the human MC line HMC-1 was

determined by Western blot. Relative levels are indicated by þ , results where detection was variable either by RT-PCR, Western blot or both is indicated by þ /2 . Results for

BMMCs and limited results for RBL-2H3 and PT-18 were previously published [16].cWestern blot data from human MC (CD34þ cultured MC) was kindly provided by Alasdair Gilfillan (pers. commun.).




(reviewed in Ref. [20]). However, only a few of theseadapters, such as Grb2-associated binder-like protein 2(Gab2), SLP-76 (Src homology 2 domain-containingleukocyte phosphoprotein of 76 kDa) and the linker foractivation of T cells (LAT), showed considerable involve-ment in SG exocytosis. Some (SLP-76 and LAT) appear toinfluence SG exocytosis through their essential role incalcium mobilization and potentially their role in theactivation of small GTPases, such as Rac and Cdc42. Some(SLP-76 and LAT) appear to influence SG exocytosisthrough their essential role in calcium mobilization andpotentially through their role in the activation of smallGTPases, such as Rac and Cdc42, which regulate IgE-dependent SG exocytosis (reviewed in Ref. [21]). Bycontrast, Gab2 appears to affect degranulation primarilythrough its effect on phosphoinositide 3-kinase (PI3-K)activity and PKC activation [16,22]. Mice deficient forGab2, LAT and SLP-76 were resistant to an anaphylacticchallenge.

Of particular interest is the ‘current hypothesis’ thatLAT and SLP-76 function together in a pathway distinctfrom that in which Gab2 functions (reviewed in Ref. [20])(Figure 1). Whereas LAT and SLP-76 require Lyn activity,Lyn and LAT seem dispensable for Gab2 phosphorylation,which instead requires Fyn activity leading to subsequentactivation of PI3-K [16]. In agreement with this, LAT andGab2 localize to different regions of the PM [23]. Theexistence of distinct early signaling pathways is alsosupported by studies on both murine and human MCsdemonstrating a PI3-K-independent activation of phos-pholipase Cg (PLCg) and calcium responses (Box 1).Whereas calcium responses are defective in LAT-deficientMCs (they fail to effectively activate PLCg), only a modestreduction in calcium was observed in Gab2-deficient MCs(in which PI3-K activity is dramatically reduced) [22,24].The perception that these pathways function indepen-dently of each other, however, must be regarded cautiouslybecause both pathways have crucial importance inexocytosis and it appears that LAT and Gab2 complementat the level of calcium mobilization and PKC activation,respectively [16,24].

MC apparatus for secretion

MCs contain the molecular machinery that drives mem-brane fusion during SG exocytosis. Essential to thisprocess are SNARE [soluble NSF (N-ethyl-maleimide-sensitive factor) attachment protein receptor] proteinsthat lie on opposing cellular membranes to form a stablemultimeric complex that catalyzes fusion (reviewed inRef. [25]). A typical SNARE complex includes a vesicularSNARE (R-SNARE), such as the vesicle-associated mem-brane protein (VAMP), which pairs with two PM-localizedSNAREs (Q-SNAREs), such as SNAP-23 (ubiquitous) orSNAP-25 (neuronal), and syntaxin (reviewed in Ref. [25]).MCs express several PM- and SG-localized SNAREs thatcould be part of the exocytotic machinery. They include, forexample, SNAP-23, syntaxin 2, 3 and 4 Q-SNAREs andseveral VAMP R-SNAREs (VAMP 2, 3, 7, 8) (reviewed inRef. [26]). Some, such as SNAP-23 [27] and syntaxin 4,have been implicated in IgE-dependent fusion [28](Figure 3). Yet, given the multiple fusion events during

compound exocytosis, it is likely that several otherSNAREs participate in this process.

SNAREs in MCs function with accessory proteins, suchas the NSF ATPase, which dissassembles SNARE com-plexes [29], and Rab3 GTPases (Rab3A, Rab3D), which aregenerally involved in regulating exocytotic traffickingsteps (reviewed in Refs [25,26]). Recently, the role ofRab3D in MC exocytosis has been challenged becauseperitonal MCs from Rab3D-deficient mice showed normalexocytosis in patch clamp studies [30]. However, whetherfunctional redundany by other Rab isoforms could bepresent or whether other compensatory mechanisms couldexist under these circumstances was not addressed by thisstudy. Accessory molecules also include syntaxin-bindingSec1 or Munc18 [Sec–Munc (SM)] family members, suchas Munc18–2 and Munc18–3, the former having beenimplicated in SG exocytosis [31] (Figure 3). SM proteinsare thought to have both essential and inhibitory functionsin exocytosis (reviewed in Ref. [32]). Although the essen-tial role remains unclear, inhibition results from Munc18-binding to syntaxin, which fixes the latter in a closedconformation unable to form SNARE complexes. Takentogether, the role of accessory proteins is to increase fusionefficiency by regulating the fusion-competent state ofSNAREs (reviewed in Ref. [25]). The fusion efficiency couldalso depend on the interaction of the fusion machinery

Box 1. Does phosphoinositide 3-kinase (PI3-K) regulate

secretory granule (SG) exocytosis independent of

phospholipase Cg (PLCg) and calcium?

There is growing evidence that mast-cell (MC) calcium responses,

but not SG exocytosis, are relatively insensitive to major reductions

in PI3-K activity. Thus, Grb2-associated binder-like protein 2 (Gab2)-

deficient MCs, which show a dramatic loss of total PI3-K activity, have

relatively normal calcium responses but defective SG exocytosis

[22]. This is mirrored by the Src protein tyrosine kinase (PTK) Fyn-

deficient MCs, which are also defective in activation of total PI3-K,

PI3-K-dependent kinase 1 (PDK1) and survival kinase Akt, as well as

SG exocytosis, yet show normal calcium responses [16]. Recent

studies demonstrate that PLCg activation is dependent on the

adaptor protein, linker for activation of T cells (LAT) because the

absence of LAT or expression of a LAT Y136 mutant (the binding site

for PLCg) inhibited its activation, whereas PI3-K-dependent Akt

phosphorylation appeared normal [24,65]. This evolving view of

PI3-K-independent activation of PLCg is strongly supported by

several recent studies. In human MCs, inhibitors of PI3-K activity

had little effect on PLCg activation and the calcium response [66].

These authors also showed that PLCg activation occurred in MCs

deficient in the regulatory p85a and b subunit of PI3-K. Others have

shown, by microinjection of inhibitory antibodies, that the PI3-K

catalytic subunits p110b and p110d are important in regulating

IgE-dependent calcium influx through the enhancement of inositol

1, 4, 5-trisphosphate [Ins(1,4,5)P3], which binds to receptors in the

endoplasmic reticulum eliciting calcium release [67]. However, the

p110a isoform showed no apparent role in the rise of intracellular

calcium. Nonetheless, inhibitory antibodies to the p110a isoform

were more recently demonstrated to ablate IgE-mediated MC exo-

cytosis [68]. This disconnection between calcium and SG exocytosis

is further shown in the SrcPTK Lyn-deficient MC, which has a

dramatic loss and prolonged delay of calcium responses, but an

enhanced SG exocytosis that is abrogated by PI3-K inhibitors

[16,18,19]. Regardless, although intracellular chelators of calcium

demonstrate the essential nature of calcium for SG exocytosis, the

collective studies point to a greater role for PI3-K in SG exocytosis

than was previously appreciated.




with the cytoskeleton, which has been recognized asanother important regulatory step [33]. MC exocytosis isindeed accompanied by the extensive reorganisation ofthe actino-myosin cortex [34] and the controlled movementof SGs along microtubular structures [35]. All of thesemolecular participants must coordinately function inresponse to Fc1RI stimulation.

Regulation of the secretory apparatus by calcium

MC exocytosis and SNARE complex formation in regu-lated-secretion competent cells requires calcium (reviewedin Refs [21,36]). During the last decade, a family of calciumsensors, the synaptotagmins (syt), emerged as moleculartargets of the calcium fusion machinery (Figure 3). Syt aremembrane-anchored proteins (vesicular- or PM-bound)that bind calcium by conserved tandem C2 domains (C2Aand C2B), albeit the affinity differs among the variousfamily members. Calcium binding promotes oligomeriz-ation of syt and interaction of their C2 domains withnegatively charged membrane phospholipids, similar to

what has been observed in other C2-containing proteins(such as PKC and phospholipase A2) (reviewed inRef. [37]). It also promotes the interaction of syt withmembrane-localized syntaxin and SNAP-25. It is thoughtthat the calcium-induced oligomerization of syt and theircombined binding to SNAREs and phospholipids functionto promote calcium-dependent SNARE assembly andmembrane fusion (reviewed in Ref. [37]) (Figure 3). MCsexpress several syt isoforms, such as II, III, V and IX [38].Some of them function to regulate intracellular traffickingsteps [39]. However, which syt might serve as the physio-logic sensor involved in the regulation of SG exocytosisremains at present unclear.

Another calcium sensor is calmodulin (CaM) (reviewedin Ref. [40]). In yeast, an organism that does not expresssyt, CaM regulates the calcium-dependent homotypicfusion of vacuoles through its association with proteinphosphatase 1 (PP1) (see later) and the vacuolar Hþ

ATPase [41,42]. In cells from higher organisms, the role ofCaM is more complex because it targets multiple proteins

Figure 3. The molecular events in secretory granule (SG) fusion and exocytosis. Mast cells (MCs) contain molecular machinery involved in the regulation of the final steps

of membrane fusion of SGs between themselves and with the plasma membrane (PM). This machinery includes soluble NSF (N-ethyl-maleimide-sensitive factor) attach-

ment protein receptor (SNARE) proteins, such as the vesicle-associated membrane protein (VAMP), and the plasma membrane-associated syntaxin 4 and SNAP-23 that are

able to form a stable multimolecular complex that energetically favors the merger of lipid membranes. SNARE complex formation is thought to be regulated by multiple

accessory proteins, such as the SNARE-priming NSF, small Rab3 GTPases with associated kinases, such as the Rab3D-associated kinase (Rak3D), Munc18 proteins and

phosphatases. For some of these proteins the precise mechanism of action remains unclear (?). In the resting state, some of the accessory proteins restrain primed and

docked SG from fusion. On stimulation, the cellular secondary messenger calcium and protein kinase C (PKC) activity act to reverse these inhibitory mechanisms but also

cause activation of proteins and cellular events that promote fusion. These include the recruitment of phosphatases to the membrane, the activation of a calcium sensor(s)

and cytoskeletal changes. Abbreviations: Ag, antigen; SNAK, SNARE kinase.


PP1/2

L

SG

Munc18-2/18-3

SNAP-23

Resting cell

Syntaxin 3?

SG

Munc18-2

Ca2+ PKC

Ag

?

?

Munc18-3

PM PM

FcεRI

IgE

?

Ca2+ sensor

Rab3D

Rak3D

ADP

Actin

NSF

SNAK

Microtubules

VAMP?

Priming

ATP

ADP

Rak3D–Rab3D

ATP

Syntaxin 4

Activated cell

P

P




with a potential role in exocytosis [40]. In MC exocytosis,sudies with CaM antagonists show conflicting resultsranging from no effect to an inhibitory effect [43,44].Potential CaM targets in MCs include: myosin light chainkinase (MLCK) [45]; CaM kinase II [46]; syntaxin 3 [47]and phosphlipase D (PLD) [48], a lipid-modifying enzymeimplicated in regulating SG exocytosis (Box 2). Newtargets also include VAMP proteins. VAMP-2 binds CaMand membrane phospholipids in a mutually exclusivemanner within a region adjacent to its transmembraneanchor. CaM binding activates VAMP-2 for fusion byliberating it from its lipid-bound state (reviewed inRef. [49]). In MCs, VAMP-2 incorporates into the PM ofdegranulating MCs (reviewed in Ref. [26]). Also, calciuminteracts directly with several other signaling molecules(see next section) and thus has a central role in coordinat-ing the molecular processes of cell signaling and SG fusion.Nonetheless, there is considerable evidence that calcium isbut one of many activators in SG exocytosis [21] (Box 1).

PKC and other Ser–Thr kinases in the regulation of

fusion and exocytosis

PKC activation has been established as a prerequisite forMC exocytosis (reviewed in Ref. [50]). PKC is partlycontrolled by the PI3-K-dependent kinase 1 (PDK1) [51],whose activity requires both Fyn and Gab2 [16,22]. Both acalcium-dependent, PKCb, and a calcium-independent,PKCd, PKC were implicated in degranulation usingdepletion–reconstitution or mutational studies thataddressed the calcium–PKC relationship or the conse-quences of PKC phosphorylation of cellular substrates(reviewed in Ref. [50]). Nonetheless, the recent findingthat PKCd-deficient MCs showed enhanced SG exocytosis[52] brings into question the essential nature of thisisozyme, although this apparent controversy might beexplained by direct or indirect crosstalk between Srcfamily kinases and PKCd [53].

Regardless, it is clear that PKC regulates late stepsin exocytosis. In vitro, PKC directly phosphorylates

recombinant SNAP-25 and syntaxin 4 fusion proteins,thereby inhibiting the interaction with other SNAREpartners. However, the functional role of this event in vivoremains to be established [54,55] (Figure 3). Patch clampstudies on eosinophils [56] and chromaffin cells [57]demonstrated that fusion pore expansion is sensitive toPKC inhibitors and is enhanced by the addition of PMA(phorbol 12-myristate 13-acetate). Whether this is due tothe phosphorylation of SNAREs, syt or other physiologicsubstrates is presently unclear. However, PKC phosphory-lates the neuronal SM family protein Munc18–1 [58]. Thisinhibits its ability to bind syntaxin 1 in vitro [58] and couldthus regulate the amount of syntaxin able to engage withother SNARE partners within the cell. In MCs, the PKCphosphorylation sites of Munc18–2 are conserved [31](Figure 3) and our preliminary evidence indicates thatMunc18–2 is phosphorylated in these cells.

Other Ser–Thr kinases, such as SNARE kinase(SNAK), might also regulate MC exocytosis. SNAK phos-phorylates SNAP-23 [59] (Figure 3), which renders itincapable of forming a SNARE complex but increases itscellular lifespan. Therefore, SNAK-dependent phosphoryl-ation could indirectly promote fusion by increasing theintracellular concentration of SNAP-23. Fusion could alsobe regulated by the putative kinase Rak3D, whose activityco-immunoprecipitates with granule-localized Rab3D [60].Rak3D phosphorylates syntaxin 4, but not syntaxin 2 and3, thereby inhibiting its binding to SNAP-23 in vitro(Figure 3). Rak3D activity decreases on Fc1RI stimulationin a calcium-dependent manner and thus this loss ofactivity could enable SNAP-23 to interact with syntaxin 4promoting fusion [60]. This provides another possiblemechanism for maintaining SNARE proteins in a fusion-incompetent state until intracellular calcium concen-trations increase.

Role of phosphatases in the regulation of fusion

Dephosphorylation of cellular proteins by phosphatasesalso has a central role in the activated state. For example,the protein tyrosine phosphatases, Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) andSHP-2, and the lipid phosphatase Src homology 2-contain-ing 50-inositol phosphatase (SHIP) (reviewed in Ref. [61]),function in early Fc1RI-dependent events that initiate andregulate exocytosis. Recently, a role for Ser–Thr phospha-tase PP1 in the homotypic fusion of yeast vacuoles hasbeen demonstrated. PP1 was detected in a macromolecularcomplex with CaM, and was required at multiple intra-cellular trafficking steps in yeast [41]. In MCs, the phos-phatase PP2A is recruited to the PM in a manner thatcorrelates with the kinetics of exocytosis [62]. PP1 and PP2phosphatase inhibitors affect SG release to agents thatbypass early-receptor-mediated signals indicating thatthese phosphatases act at a late step in exocytosis [62].Immunofluorescent imaging demonstrated that both PP1and PP2A transiently associated with cortical myosin II,suggesting a role in the rearrangement of the actino-myosin cortex [63]. A crucial target for phosphataseactivity could be a PKC-dependent phosphorylation sitein myosin light and heavy chains [63]. However, given thedefined role in yeast vacuole membrane bilayer mixing, as

Box 2. Phospholipase D (PLD), an integrator of mast-cell

signaling and function?

Calcium has an important role in the activation of PLD family

members whose activities have been implicated in exocytosis [69]

and cytoskeletal rearrangement [70]. Interestingly, protein kinase C

(PKC) is also a modulator of PLD activity because both the secretory

granule (SG)-localized PLD1 and plasma membrane (PM)-localized

PLD2 are activated by PKCa in Sf9 cells [71]. Although the specifics

are still unclear, PLD activity might have multiple downstream

targets, including PKC and sphingosine kinase (reviewed in Ref. [72]),

and via the latter might modulate calcium [73]. Another recent study

in human A431 epidermoid carcinoma cells elucidates a possible link

between PM-localized PLD2 and early signals activated by tyrosine

phosphorylation [74]. It showed that PLD2 is a substrate of Src

protein tyrosine kinase (PTK), c-Src, and to a lesser extent of the

SrcPTK, Fyn, whereas PLD1 did not appear to be a substrate for Src

PTK activity. This tyrosine phosphorylation is promoted by protein–

protein interaction between the c-Src catalytic domain and the PH

domain of PLD2. Because both c-Src and Fyn are activated on

stimulation of the high affinity receptor for immunoglobulin E (Fc1RI)

[16], the findings suggest that the signaling axis of Src PTKs–PLD2–

PKC–sphingosine kinase–calcium could be important for mast-cell

exocytosis.




yet unidentified targets in the MC vesicular fusionmachinery might exist. Other phosphatases in the latesteps of MC exocytosis could include the recently dis-covered and SG-localized protein tyrosine phosphatase(PTP)-megakaryocyte cytosolic protein tyrosine phospha-tase 2 (MEG2) [64]. Overexpression of this phosphatase inrat basophilic leukemia (RBL) cells resulted in formationof large SGs, suggestive of a role in granule fusion.However, the relative importance of these and otherphosphatases on MC exocytosis, for the most part, remainsto be explored.

Concluding remarks

As becomes evident from this brief Review, MC exocytosisis a complex process that requires a signal input and asophisticated molecular machinery for output. A molecu-lar architecture is also required for coordination of bothearly and late signaling events. Not only must the exo-cytotic machinery be mobilized but signals must coordi-nately promote the activation of multiple enzymes,generate second messengers, activate specific moleculartargets, cause cytoskeletal changes and promote move-ment and fusion of SG. This coordination is provided byspecific protein–protein interactions regulated by theaction of kinases, phosphatases, calcium and lipidsignals. It is not surprising that this complex andenergy-demanding process requires fine-tuning by bothpositive and negative regulators that control the occur-rence and extent of the response. Future efforts are likelyto focus on a molecular understanding of how theexocytotic fusion machinery merges with the myriad ofearly and intermediate IgE-dependent signals. This willrequire identification of the key MC exocytotic moleculesthat respond to these signals and can initiate vesiclefusion. Nonetheless, the recent progress provides a betterunderstanding of the hierarchy of this exquisitely coordi-nated biological process.

Acknowledgements

We thank members of our respective laboratories, collaborators andcolleagues who contributed to the work described herein. We also wish toacknowledge the important contribution of colleagues whose work was notincluded due to space constraints. The work of J.R. was supported by theDepartment of Health and Human Services, National Institutes of Health,National Institute of Arthritis and Musculoskelelatal and Skin Diseases,and Grant No. 2000016 from the United States-Israel Binational ScienceFoundation. The work of U.B. was supported by a grant from theAssociation de la Recherche sur le Cancer (ARC N84389).

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The ins and outs of IgE-dependent mast-cell exocytosis

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