ipmtvwkwTiPpragaog

p

omtobpr1tgtPsaictc

2

Experimental Cell Research 253, 15–24 (1999)Article ID excr.1999.4671, available online at http://www.idealibrary.com on

Phospholipase C-g as a Signal-Transducing ElementGraham Carpenter1 and Qun-sheng Ji

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

wpoidvgfkc

cndg[astatlhc

YmfSsmrahhoat“irSt1pp

A ubiquitous signaling event in hormonal responsess the phospholipase C (PLC)-catalyzed hydrolysis ofhosphatidylinositol 4,5-bisphosphate to produce theetabolite second messenger molecules inositol 1,4,5-

risphosphate and diacylglycerol. The former pro-okes a transient increase in intracellular free Ca21,hile the latter serves as a direct activator of proteininase C. In tyrosine kinase-dependent signaling path-ays this reaction is mediated by the PLC-g isozymes.hese are direct substrates of many tyrosine kinases

n a wide variety of cell types. The mechanism ofLC-g activation involves its association with andhosphorylation by receptor and non-receptor ty-osine kinases, as well as interaction with specializeddaptor molecules and, perhaps, other second messen-er molecules. However, the biochemistry of PLC-g ist a more advanced state than a clear understandingf exactly how this signaling element functions in theeneration of a mitogenic response. © 1999 Academic Press

Key Words: phospholipase C; growth factors;hosphotyrosine.

INTRODUCTION

The signal transduction pathways for a wide varietyf hormone-dependent responses include the transientobilization of intracellular-free Ca21 and the activa-

ion of protein kinase C isozymes [1, 2]. The activationf these two second messenger pathways is controlledy the phospholipase C (PLC)-dependent hydrolysis ofhosphatidylinositol 4,5-bisphosphate (PIP2), whicheleases the second messengers molecules inositol,4,5-trisphosphate (IP3) and diacylglycerol. To datehere are three subfamilies of PLC isozymes and 10 plcenes in mammalian cells [3]. Many agonists that ac-ivate G protein-coupled receptors provoke increasedLC activation by a mechanism that involves the as-ociation of heterotrimeric G protein-derived activatedsubunits or b:g subunit complexes with PLC-b family

sozymes, of which there are four distinct members. Inontrast, signaling pathways employed by agonistshat depend on tyrosine kinases as crucial mediatorsommunicate with members of the PLC-g family, of

1 To whom reprint request should be addressed. Fax: (615) 322-931. E-mail: Graham.Carpenter@mcmail.vanderbilt.edu.

15

hich there are two members. Less understood are thehysiological roles played by any of the four membersf the PLC-d family. Hence, of the known PLCsozymes specific for the hydrolysis of phosphoinositi-es, the two g isozymes (PLC-g1 and PLC-g2) are de-oted to tyrosine kinase-dependent signaling for mito-enic responses to diverse agents, such as growthactor activation of transmembrane receptor tyrosineinases, antigenic stimulation of multichain immuneell receptors, and fertilization of eggs.The structures of PLC-g1 and PLC-g2, based on

DNA sequences, are depicted in Fig. 1A. All phosphoi-ositide-specific PLC isozymes share the catalytic sub-omains designated as X and Y [3]. The crystallo-raphic structure of a PLC-d isoform has been reported4] and one would expect the general configuration of Xnd Y domains in other PLCs to be similar. The dtructure shows that these two domains fold togethero compose the catalytic site. Within the catalytic sitend catalytic ridge are several residues known by mu-agenesis and X-ray structure to be essential for cata-ytic activity [56]. Two of these, His 356 and His 335,ave been mutated in PLC-g1, with a reduction inatalytic activity of 90–95% in vitro [7].In the PLC-g isozymes, the region between the X anddomains is enlarged to contain SH2 and SH3 do-

ains unique to the g isozymes. Both of these motifsacilitate PLC-g association with other proteins. TheH2 domains do so by recognizing phosphotyrosineequences in other proteins, while the SH3 domainediates interaction with proteins containing proline-

ich sequences. Several proteins are known to associ-te with PLC-g by SH2–phosphotyrosine interactions;owever, a physiological partner for the SH3 domainas not been identified. The central SH domain regionf PLC-g may also have a role in regulating the enzymectivity of the enzyme. It has been proposed [8] thathis region may function as an intramolecular “cap” orlid” to occlude the active site in the absence of activat-ng events (Fig. 1B). Modulation of this “lid” by ty-osine phosphorylation and/or protein binding to theH domains may alter the relationship of the “lid” tohe active site. This model is based on experimental [9,0] and the crystal of SHP-2 [11], a phosphotyrosinehosphatase in which an SH2 domain occupies thehosphatase-active site in the absence of a phosphoty-

0014-4827/99 $30.00Copyright © 1999 by Academic Press

All rights of reproduction in any form reserved.

ri

dsbmmd

Pamis

iioe

bnofgav

Atfm[i

iLfsvbp

dXe

16 CARPENTER AND JI

osine ligand for the SH2 domain and represses activ-ty.

In addition to SH domains, PLC-g also contains a C2omain and two putative PH domains, one of which isplit (Fig. 1A). These domains are features shared with

and d family members and likely serve a generalechanism in the catalysis of PIP2 [12]. C2 domainsediate interaction with Ca21/phospholipids, while PH

omains recognize polyphosphoinositides.In recent years, several reviews have focused on

LC-g or the families of PLC isozymes [3, 8, 13]. In thisrticle we attempt to focus on more recent develop-ents, especially within the function of PLC-g

sozymes as signal transducers in various biologicalystems.

GENETIC MODULATION OF PLC-g1

Recently, genetic approaches have started to yieldnformation regarding the function of PLC-g isozymesn organisms and cells. In mice, the targeted disruptionf the Plcg1 gene leads to early embryonic lethality atmbryonic day 9.0 (E9.0) for nullizygous (Plcg12/2) em-

FIG. 1. Organization of domains in PLC-g isozymes. (A) Based oomains (X and Y) and regulatory domains (SH2, SH3, PH, and C2) aand Y domains must fold together to create the catalytic site in

nzymatic activity, a model for these regions in the inactive or basa

ryos [14], while heterozygous (Plcg11/2) mice appearormal. It is not an uncommon feature for the “knock-ut” of signal transducing components that embryosail at approximately this point or slightly later inestation. While exact causes of the lethal phenotypere not always clear, often the failure of embryonicasculature and/or blood cell development is involved.To date, the knockout of Plcg2 has not been reported.s the expression of this isozyme is more restricted

hat of PLC-g1, the phenotype may not be as severe. Allour Plcb genes have been disrupted in mice and, while

ost produce restricted mild phenotypes in adult mice15–17], the b-3 mutation is embryonic lethal at E2.5,.e., prior to implantation [18].

In lower organisms, a Plcgg gene has been identifiedn Drosophila, as identical to the gene small wing [19].oss of function mutations in this gene result in the

ormation of extra R7 photoreceptors in the eye. Themall wing phenotype results from attenuated ectopicein formation. The observed phenotypes also haveeen recorded for gain of function mutations in the Rasathway and the authors concluded that, in Drosoph-

rimary sequence information, the linear arrangements of catalyticdepicted for PLC-g1 and PLC-g2. (B) Based on the evidence that thenative protein and that the SH region may act to prepress basalzyme state and the activated state of the enzyme is depicted.

n prethel en

ii

ogfwtpPfiipTspcIav

aDuasdc

Cfiyptpmsl

ps(pbs3gssEde

mcdPt[trpmsreS

dbsmliffolcPtPmajbacft[

PdtppfNopueNfwbptE

17PLC-g AS A SIGNAL-TRANSDUCING ELEMENT

la, PLC-g functions to suppress Ras-dependent signal-ng.

Several investigations have reported the propertiesf cell lines in which PLC-g expression has been abro-ated. Immortalized mouse embryo fibroblasts, derivedrom Plcg12/2 embryos, show no change compared toild-type cells in epidermal growth factor (EGF) induc-

ion of MAP kinase, expression of c-fos, entry into Shase, or migration into a wounded area [20]. Clearly,LC-g1 is not essential for the growth of immortalizedbroblasts. The only recorded deficits in these cells are

n growth factor activation of phospholipase D [21] andhosphorylation of Tiam1, a Rac1 exchange factor [22].wo reports have employed antisense approaches touppress PLC-g1 levels. In one case, this maneuverrevented or decreased the differentiation of keratino-ytes provoked by high levels of extracellular Ca21 [23].n hepatocytes, antisense vectors to PLC-g1 attenu-ted growth factor-induced Ras and MAP kinase acti-ation as well as entry into DNA synthesis [24].B cells express PLC-g2 and relatively little PLC-g1,

nd the gene for the former has been disrupted in theT40 cell line [25], which normally responds to stim-lation of surface IgM receptors by growth inhibitionnd apoptosis. However, in cells deficient in PLC-g2,ignaling for apoptosis was significantly attenuated,emonstrating an essential role for PLC-g2 in thatellular response to activation by antigen.

ACTIVATION BY RECEPTOR TYROSINE KINASES

PLC-g1 tyrosine phosphorylation, IP3 formation, anda21 mobilization are provoked by nearly all growth

actor receptors. Three tyrosine residues have beendentified as sites of receptor tyrosine kinase phosphor-lation [26, 27] and mutagenesis indicates that thehosphorylation at Tyr 783 is essential for IP3 forma-ion, phosphorylation of Tyr 771 is dispensable, andhosphorylation of Tyr 1254 is necessary to achieveaximal IP3 formation [28]. The phosphorylation

eems direct as the purified EGF receptor phosphory-ates and activates the enzyme in vitro [29].

The capacity of tyrosine kinase receptors to phos-horylate PLC-g1 depends on the formation of an as-ociation complex between PLC-g1 and the receptorFig. 2, step 1), which is mediated by recognition ofhosphotyrosine-containing sequences in the receptory the SH2 domains of PLC-g1 [30]. In some receptors,uch as the platelet-derived growth factor (PDGF) [31–3], fibroblast growth factor (FGF) [34, 35], and nerverowth factor (NGF) [36, 37] receptors, there is onepecific autophosphorylated tyrosine that specifies as-ociation with PLC-g1. In other receptors, such as theGF receptor, no single autophosphorylation site me-iates this association [38], but rather preferred sitesxist [39, 40].The two SH2 domains of PLC-g1 are dissimilar and

ay therefore mediate recognition with different re-eptor autophosphorylation sites. However, both SH2omains of PLC-g1 have been shown to be required forDGF-induced receptor association and Ca21 mobiliza-

ion [41] and also for association with the EGF receptor40]. In PLC-g1 association with PDGF or EGF recep-ors, the N-SH2 has a more predominant or primaryole compared to the C-SH2 domain. There are twolausible mechanisms to explain why both SH2 do-ains are necessary. First, there may be a primary and

econdary autophosphorylation docking site on eacheceptor monomer. Second, since activated receptorsxist as dimers, the PLC-g1 molecule may use bothH2 domains to bridge monomers within the dimer.While tyrosine phosphorylation seems to have pre-

ominant role in PLC-g1 activation, other factors maye physiological contributors to the full activationtate. Addition of growth factors to cells not only pro-otes receptor association, but also stimulates trans-

ocation from the cytosol to the membrane, as depictedn Fig. 2, step 3 [42]. Given the location of the substrateor this enzyme, translocation is likely to be a criticaleature of PLC-g1 activation and is a common featuref receptor proximal molecules in signal transductionogistics. To a limited extent, receptor association couldontribute to the increased pool of membrane-associateLC-g1. However, the amount of PLC-g1 present in

hese complexes is less than 0.5% of the total cellLC-g1 [38] and the amount of enzyme translocateday be as much as 20% [42]. It is possible that receptor

ssociation contributes to the activation of PLC-g1,ust by binding the SH2 domains. Indirect evidence haseen cited to this effect [43, 44]. However, attempts toctivate PLC-g1 in vitro with various phosphotyrosine-ontaining peptides have not been particularly success-ul [45]. This is in contrast to the capacity of phospho-yrosine peptides to activate SHP-1 more than 10-fold46].

PLC-gs have, by sequence homology, an N-terminalH domain plus a split PH domain that bounds the SHomains (Fig. 1A). Whether either of these is func-ional is unclear. However, PH domain recognition ofhosphatidylinositol 3,4,5-trisphosphate (PIP3) mightrovide a means to localize PLC-g to the cytoplasmicace of the plasma membrane (Fig. 2). Clearly, the-terminal PH domain of PLC-d is required for activity

f the enzyme [12]. One report has investigated theotential role of the N-terminal PH domain of PLC-g1,sing a competitive inhibitor approach [47]. In thesexperiments, a GST (or GFP) fusion protein with the-terminus of PLC-g1 was employed. This PH domain

usion protein was localized to the plasma membranehen cells were stimulated by growth factor and did soy interacting with PIP3 or other PI-3 kinase lipidroducts. Importantly, this overexpressed fusion pro-ein also reduced by about 60% the formation of IP3 inGF-treated cells, implying that it interfered with

mtdPb3

mctvtt

PeatPnbsii

ar

mattbtswmeslnt

Cn

kdpfhm

18 CARPENTER AND JI

embrane localization of endogenous PLC-g1. Inhibi-ion of PI-3 kinase activity by various means also re-uced IP3 formation. In vitro studies show that theLC-g1 PH domain, as an isolated protein fragment,inds 39 phosphorylated phosphoinositides in the PI-P . PI 3,4-P2 . PI 3,4,5-P3 [48].However, the influence of PIP3 on PLC-g activityay have a second locus of action. It is know that PIP3

an recognize SH2 domains by a mechanism unrelatedo phosphotyrosine recognition of SH2 domains [49]. Initro evidence has shown that PIP3 can interact withhe C-SH2 domain of PLC-g1 and increase activity ofhe enzyme in vitro and in vivo [50, 51].

In regard to the role of PI-3 kinase and PIP3 inLC-g1 activation, a few cautionary comments are nec-ssary. In some instances inhibition of PI-3 kinasectivity has not resulted in decreased Ca21 mobiliza-ion in growth factor-treated cells [41]. Also, while theH domain of PLC-g1 can be mutated so that it doesot bind PIP3, this mutation and its effect have noteen analyzed in the intact PLC-g1 molecule. The re-ults cited above depend on the specific action of dom-nant-negative inhibitors or chemical inhibitors. Thenfluence of PIP3 and other lipid modulators of PLC-g

FIG. 2. Activation cycle for PLC-g isozymes in the intact cells. Shinases. Following growth factor binding to the receptor tyrosineocking sites that interact with PLC-g SH2 domains to produce a rhosphorylation of PLC-g1 and its dissociation from the receptor (stace of the plasma membrane, perhaps by PH domain recognition of Pydrolysis and the production of the second messengers IP3 and dembrane and be dephosphorylated to return to its basal state (ste

ctivity, such as phosphatidic acid, have recently beeneviewed elsewhere [52].The influence of PI-3 kinase on PLC-g1 activationay be complex and involve issues such as cell type

nd strength of the primary activating events, i.e.,yrosine phosphorylation. In the growth factor activa-ion of MAP kinase [53] and Ras [54], PI-3 kinase haseen shown to have such a secondary role that is de-ermined by the strength of the primary activatingignal. It is plausible, based on these reports, thathen tyrosine phosphorylation of PLC-g1 is maxi-ized, by saturating concentration of growth factor, for

xample, then PI-3 kinase activity may have no mea-urable influence on PLC-g1 activation. However, atower levels of tyrosine phosphorylation the PI-3 ki-ase dependence of PLC-g1 activation may be substan-ial.

PLC-g SIGNALING AND RESPONSES THROUGHRECEPTOR TYROSINE KINASES

The PLC-g signaling pathways downstream froma21 mobilization and protein kinase C activation areot well defined in cells such as fibroblasts and epithe-

n are various steps in the activation of PLC-g1 by receptor tyrosinease, autophosphorylation of the receptor creates phosphotyrosineptor: PLC-g1 complex (step 1). This is rapidly followed by tyrosine2). The phosphorylated PLC-g1 then interacts with the cytoplasmicor other mechanisms (step 3). Membrane association facilitates PIP2

ylglycerol (step 4). Finally, the enzyme must dissociate from the).

owkineceepIP3

iacp 5

lttidg

PssaPwbncpciDtPmfiPnc

srtPswgDrwcat[ktrs(Dt[tt

trmm

oRnmdhttafcn

csitprvfaPcAoc

amgnmkwPtkpqtbpPsfapkdk[Ip

19PLC-g AS A SIGNAL-TRANSDUCING ELEMENT

ial cells where the primary signaling event is activa-ion of a receptor tyrosine kinase. In part this is due tohe pleitrophic effects of Ca21 and protein kinase C, butt also is due to the fact that the definition of PLC-g-ependent signaling response points, such as specificene expression, are unclear in these cell systems.Receptor tyrosine kinase signaling downstream of

LC-g1 has been studied in one of two contexts. First,everal approaches have been used to abrogate PLC-gignaling while all other signaling pathways remainctive. In this context the experiments would revealLC-g1 signaling responses that are not redundantith other signaling pathways. The experiments haveeen performed using selective inhibitors (dominant-egative inhibitors, antibodies), receptor mutations, orells in which the Plcg1 gene has been disrupted. Ex-eriments with the inhibitor approaches have indi-ated that PLC-g1 is necessary for expression of themmediate early gene c-fos and for the induction ofNA synthesis in quiescent cells [7, 55, 56]. However,

he opposite conclusion has been reached by examiningDGF and FGF receptors mutated to specifically re-ove the PLC-g1 association site [31, 32, 34, 35] and inbroblasts genetically compromised at the level of thelcg1 gene [20]. Other than cell type differences, it isot possible to reconcile these divergent results andonclusions.The second approach to PLC-g signaling has been to

electively activate PLC-g, but not other receptor ty-osine kinase-dependent signaling pathways. Twoypes of experiments have been reported. PurifiedLC-g1 has been microinjected into cells and found totimulate DNA synthesis [7]. In those experiments itas also reported that catalytically compromised PLC-1, by mutation of active site residues, also provokedNA synthesis. However, the mutated enzyme still

etained 5–10% of its lipase activity and this combinedith amount of enzyme injected may have been suffi-

ient to initiate DNA synthesis. A more sophisticatedpproach to selectively activate PLC-g1 has involvedhe use of “add back” mutants of the PDGF receptor57]. The receptor was first mutated at each of fivenown autophosphorylation sites to produce an F5 mu-ant that is unable to stimulate DNA synthesis inesponse to the ligand. Subsequently, the PLC-g1 as-ociation site (Tyr 1021) is mutated back to tyrosineF1021Y). This “add back” mutant is able to stimulateNA synthesis and activate Ras [57], as well as induce

he expression of immediate early genes such as c-fos58]. However, these systems in which PLC-g1 is selec-ively activated have not yielded mechanistic informa-ion.

The role of PLC-g1 has also been investigated inhree other biological systems generally activated byeceptor tyrosine kinase: transformation, cell move-ent, and differentiation. Often when signaling ele-ents downstream of receptor tyrosine kinases are

verexpressed, oncogenic transformation is produced.ecently, several papers have reported this phenome-on for PLC-g1 [59–63]. Cell movement, in the form ofigration or chemotaxis has also been reported to be

ependent on PLC-g1 [6369]. However, other reportsave concluded that PLC-g1 is not essential for theseypes of cell movement [20, 70–72]. Frequently, recep-or tyrosine kinase-dependent signaling elements aressayed for their capacity to stimulate or inhibit dif-erentiation signaling pathways. Several reports indi-ate a positive role for PLC-g1 in the induction ofeurite outgrowth in neuronal cell cultures [73–77].

ACTIVATION BY IMMUNE CELL RECEPTORS

In T and B cells antigens bind cell surface receptoromplexes that are composed of multiple membrane-panning polypeptides and thereby activate PLC-gsozymes. The resultant Ca21 mobilization activateshe transcription factor NF-AT and induces gene ex-ression [79]. In T cells, expression of the IL-2 geneequires NF-AT as well as other transcriptional acti-ators and expression of IL-2, a secreted growth factoror T cells, is necessary for clonal expansion of thentigen-activated cell. The role of Ca21 mobilization byLC-g in this process is to activate the phosphatasealcineurin, which dephosphorylates and activates NF-T. Hence, in T cells there is a clear model for the rolef PLC-g in gene expression and the mechanism ofellular proliferation.The activation of PLC-g isozymes in immune cells

lso involves several interesting variations on theechanism described above for cells treated with

rowth factors and activated by receptor tyrosine ki-ases. In T and B cell receptor complexes none of theultiple constituent polypeptide chains are tyrosine

inases. Rather cytosolic tyrosine kinases associateith antigen-bound receptor complexes and provokeLC-g tyrosine phosphorylation [80]. During T cell ac-

ivation, members of three different cytosolic tyrosineinase families are involved in PLC-g tyrosine phos-horylation. Src family members are indirectly re-uired as these kinases serve to phosphorylate specificyrosine residues within immunoreceptor tyrosine-ased activation motifs (ITAMs) present on the cyto-lasmic domains of T cell receptor polypeptide chains.hosphorylated ITAM motifs then serve as dockingites for members of the ZAP-70/Syk tyrosine kinaseamily, which contain SH2 domains. When ZAP-70/Sykre associated with receptor ITAMs, they are tyrosinehosphorylated at specific sites and activated. Theseinases then are able to form a complex with the SH2omains of PLC-g1 and are likely candidates for theinases that directly phosphorylate PLC-g1 in T cells81]. However, the presence of the Tec family kinasestk and Rlk is also required for maximal PLC-g phos-horylation and IP3 formation [82, 83]. ZAP-70/Syk

kslflkP

iLipkZmis[PZyuPy

mptLemwmtEcltbptd

taSpPaigtppHc

aicor1BaaadtPm

S[BtnLpTbfC

eNBtSmwPot

PbktfWwgttprted[

20 CARPENTER AND JI

inases are membrane localized by virtue of their as-ociation by SH2 domains with tyrosine phosphory-ated receptor polypeptides. Itk has a PH domain toacilitate membrane association, while Rlk is palmitoy-ated. Hence, there are at least two distinct tyrosineinase families that may be directly involved inLC-g1 phosphorylation in T cells.Another novel feature of PLC-g1 activation in T cells

s the requirement for the specific adaptor proteinsAT and SLP-76 [80, 84, 85]. LAT is a 36- to 38-kDa

ntegral membrane protein that is highly tyrosinehosphorylated following T cell activation [86]. Theinase that phosphorylates LAT is thought to beAP-70 and once phosphorylated, LAT associates withultiple signaling proteins that contain SH2 domains,

ncluding PLC-g1. Also, association with LAT is neces-ary for PLC-g tyrosine phosphorylation and activation87]. It has been proposed that the two SH2 domains ofLC-g1 are employed simultaneously to associate withAP-70 and LAT to maximize both tyrosine phosphor-lation and membrane localization [80]. This is notnlike the previously described SH2 requirement forLC-g1 to maximally associate with and be phosphor-lated by PDGF receptors [41].LAT is palmitoylated within its transmembrane do-ain and this modification is required for its tyrosine

hosphorylation, association with PLC-g1, and activa-ion of PLC-g1 in T cells [88, 89]. The palmitoylation ofAT localizes it to plasma membrane microdomainsnriched in cholesterol, known as detergent-resistantembrane rafts [88–91]. It remains to be determinedhether PLC-g1 is activated within these microdo-ains, though tyrosine phosphorylated PLC-g1 is de-

ected within these specialized membrane regions [92].vidence for the targeting of PLC-g to membrane mi-

rodomains has also been reported for antigen-stimu-ated basophilic leukemia cells [93]. In the case of PIP2

urnover by receptor tyrosine kinases, evidence haseen presented to support the notion that PLC-g1 isresent in membrane microdomains [94] and that EGFreatment provokes a decrease of PIP2 within theseomains [95].In activated T cells the cytosolic adaptor SLP-76 is

yrosine phosphorylated by ZAP-70/Syk kinases andlso associates with PLC-g1 [96, 97]. T cells deficient inLP-76 showed impaired capacity to tyrosine phos-horylate and activate PLC-g1. Hence, models forLC-g1 activation in T cells require inclusion of thisdaptor molecule. SLP-76 multiple potential means tonteract with other proteins: a central proline-rich re-ion that may associate with SH3 domains, a carboxy-erminal SH2 domain that could recognize tyrosinehosphorylated proteins, and amino-terminal tyrosinehosphorylation sites that may bind SH2 domains.ow SLP-76 actually associates with PLC-g1 is un-

lear.In B cells the predominant PLC-g isoform is PLC-g2

nd, in general, its activation following antigen bind-ng to the B cell receptor parallels that of PLC-g1 in Tells [99–101]. The Syk tyrosine kinase functions anal-gously to ZAP-70 in T cells and associates with and isequired for PLC-g2 tyrosine phosphorylation [102–04]. In addition, it is clear that the Tec family kinasetk is also necessary for PLC-g2 phosphorylation inctivated B cells [105, 106]. Btk has a PH domain andn SH2 domain both of which are necessary for PLC-g2ctivation [105]. PIP3 has been shown to bind to the PHomain of Btk and to participate in Btk phosphoryla-ion of PLC-g2 [107, 108]. PIP3-dependent activation ofLC-g1 has also been observed in antigen-stimulatedast cells [109, 110] and platelets [111].In B cells the cytosolic adaptor BLNK (analogous to

LP-76 in T cells) is involved in PLC-g2 activation112–114]. BLNK is tyrosine phosphorylated by Syk in

cells and interacts with the SH2 domains of PLC-g2o facilitate PLC-g2 phosphorylation by Syk. B cells doot seem to have a membrane adaptor analogous toAT in T cells; however, BLNK is translocated to thelasma membrane following B cell activation [113].hat this is relevant to PLC-g2 activation is evidencedy the fact that when a membrane-targeted chimericorm of PLC-g2 is expressed in BLNK-deficient cells,a21 mobilization is restored [114].Interestingly, both SH2 domains of PLC-g2 are nec-

ssary for IP3 formation in activated B cells, though the-SH2 domain seems to provide association withLNK [115]. In this case the C-SH2 domain may func-

ion to associate with a tyrosine kinase, as proposed foryk [104] or perhaps to associate with a second BLNKolecule. Hence, in PDGF-treated fibroblasts [41] asell as activated T [80] and B cells [115], activation ofLC-g1 requires both SH2 domains. However, in nonef the systems described to date has a clear function forhe SH3 domain been presented.

EGG FERTILIZATION

A unique biological system that seems to involveLC-g isoforms is the fertilization of eggs, which haseen studied in several experimental systems. It isnown that Ca21 oscillations are produced in eggs fer-ilized by sperm and that PLC-g and other PLC iso-orms are present in oocytes [116] and eggs [117].

hen a receptor tyrosine kinase (PDGF/FGF chimera)as expressed in starfish eggs, the addition of therowth factor produced Ca21oscillations similar tohose by fertilization with sperm [118]. The capacity ofhis model system to produce Ca21 oscillations de-ended on a functional docking site for PLC-g in theeceptor cytoplasmic domain. Similar results were ob-ained when a truncated form of the c-kit receptor wasmployed and GST fusion proteins with PLC-g SHomain used to specifically block the egg response119]. Studies using similar PLC-g1 SH domains as

ca[dndt

sa

C-g1 in mammalian growth and development. Proc. Natl.

21PLC-g AS A SIGNAL-TRANSDUCING ELEMENT

ompetitive inhibitors also implicate PLC-g1 functions required for sperm induced Ca21 oscillations in eggs120–122]. In contrast to the other biological systemsescribed above, it is not yet clear what tyrosine ki-ase(s) is involved in the egg system, although evi-ence has been presented that implicates a Src familyyrosine kinase upstream of PLC-g1 (123).

The authors appreciate the assistance of Sue Carpenter in manu-cript and figure preparation. Support from NIH Grant CA75195 iscknowledged.

REFERENCES

1. Nishizuka, Y. (1984). The role of protein kinase C in cellsurface signal transduction and tumor promotion. Nature 308,693–697.

2. Berridge, M. J., and Irvine, R. F. (1984). Inositol trisphos-phate, a novel second messenger in cellular signal transduc-tion. Nature 312, 315–321.

3. Rhee, S. G., and Bae, Y. S. (1997). Regulation of phosphoinosit-ide-specific phospholipase C isozymes. J. Biol. Chem. 272,15045–15048.

4. Essen, L.-O., Perisic, O., Cheung R., Katan, M., and Williams,R. L. (1996). Crystal structure of a mammalian phosphoinosit-ide-specific phospholipase Cd. Nature 380, 595–602.

5. Ellis, M. V., James, S. R., Perisic, O., Downes, C. P., Williams,R. L., and Katan, M. (1998). Catalytic domain of phosphoi-nositide-specific phospholipase C (PLC). Mutational analysisof residues within the active site and hydrophobic ridge ofPLC-d1. J. Biol. Chem. 273, 11650–11659.

6. Heinz, D. W., Essen, L.-O., and Williams, R. L. (1998). Struc-tural and mechanistic comparison of prokaryotic and eukary-otic phosphoinositide-specific phospholipases C. J. Mol. Biol.275, 635–650.

7. Smith, M. R., Liu, Y.-L., Matthews, N. T., Rhee, S. G., Sung,W. K., and Kung, H.-F. (1994). Phospholipase C-g1 can induceDNA synthesis by a mechanism independent of its lipase ac-tivity. Proc. Natl. Acad. Sci. USA 91, 6554–6558.

8. Kamat, A.m and Carpenter, G. (1997). Phospholipase C-g1:Regulation of enzyme function and role in growth factor-de-pendent signal transduction. Cytokine Growth Factor Rev, 8,109–117.

9. Horstman, D. A., DeStefano, K., and Carpenter, G. (1996).Enhanced phospholipase C-g1 activity produced by associationof independently expressed X and Y domain polypeptides.Proc. Nat. Acad. Sci. USA 93, 7518–7521.

10. Zhou, C., Horstman, D., Carpenter, G.m and Roberts, M. F.(1999). Action of phosphatidylinositol-specific phospholipaseCg1 on soluble and micellar substrates: Separating effects oncatalysis from modulation of the surface. J. Biol. Chem. 274,2786–2793.

11. Hof, P., Pluskey, S., Dhe-Paganon, S., Eck, M. J., and Shoel-son, S. E. (1998). Crystal structure of the tyrosine phosphataseSHP-2. Cell 92, 441–450.

12. Katan, M., and Allen, V. L. (1999). Modular PH and C2 do-mains in membrane attachment and other functions. FEBSLett. 452, 36–40.

13. Katan, M. (1998). Families of phosphoinositide-specific phos-pholipase C: Structure and function. Biochim. Biophys. Acta1436, 5–17.

14. Ji, Q.-s., Winnier, G. E., Niswender, K. D., Horstman, D.,Wisdom, R., Magnuson, M. A., and Carpenter, G. (1997). Es-sential role of the tyrosine kinase substrate phospholipase

Acad. Sci. USA 94, 2999–3003.15. Kim, D., Jun, K. S., Lee, S. B., Kang, N.-G., Min, D. S., Kim,

Y.-H., Ryu, S. H., Suh, P.-G., and Shin, H.-S. (1997). Phospho-lipase C isozymes selectively couple to specific neurotransmit-ter receptors. Nature 389, 290–293.

16. Jiang,H., Lyubarsky, A., Dodd, R., Vardi, N., Pugh, E., Baylor,D., Simon, M. I., and Wu, D. (1996). Phospholipase C b4 isinvolved in modulating the visual response in mice. Proc. Natl.Acad. Sci. USA 93, 14598–14601.

17. Jiang, H., Kuang, Y., Wu, Y., Xie, W., Simon, M. I., and Wu, D.(1997). Roles of phospholipase Cb2 in chemoattractant-elicitedresponses. Proc. Natl. Acad. Sci. USA 94, 7971–7975.

18. Wang, S., Gebre-Medhin, S., Betsholtz, C., Stålberg, P., Zhou,Y., Larsson, C., Weber, G., Feinstein, R., Oberg, K., Gobl, A.,and Skogseid, B. (1998). Targeted disruption of the mousephospholipase C b3 gene results in early embryonic lethality.FEBS Lett. 441, 261–265.

19. Thackeray, J. R., Gaines, P. C. W., Ebert, P., and Carlson, J. R.(1998). small wing encodes a phospholipase C-g that acts as anegative regulator of R7 development in Drosophila. Develop-ment 125, 5033–5042.

20. Ji, Q.-s., Ermini, S., Baulida, J., Sun, F.-l., and Carpenter, G.(1998). Epidermal growth factor signaling and mitogenesis inPlcg1 null mouse embryonic fibroblasts. Mol. Biol. Cell 9,749–757.

21. Hess, J. A., Ji, Q.-s., Carpenter, G., and Exton J. H. (1998).Analysis of platelet-derived growth factor-induced phospho-lipase D activation in mouse embryo fibroblasts lacking phos-pholipase C-g1. J. Biol. Chem. 273, 20517–20524.

22. Fleming, I. N., Elliott, C. M., and Exton, J. H. (1998). Phos-pholipase C-g, protein kinase C and Ca21/calmodulin-depen-dent protein kinase II are involved in platelet-derived growthfactor-induced phosphorylation of Tiam1. FEBS Lett. 429,229–233.

23. Xie, Z., and Bikle, D. D. (1999). Phospholipase C-g1 is requiredfor calcium-induced keratinocyte differentiation. J. Biol.Chem. 274, 20421–20424.

24. Nebigil, C. G. (1997). Suppression of phospholipase C b, g, andd families alters cell growth and phosphatidylinositol 4,5-bisphosphate levels. Biochemistry 36, 15949–15958.

25. Takata, M., Homma, Y., and Kurosaki, T. (1995). Requirementof phospholipase C-g2 activation in surface immunoglobulinM-induced B cell apoptosis. J. Exp. Med. 182, 907–914.

26. Kim, J. W., Sim, S. S., Kim, Y.-H., Nishibe, S., Wahl, M. I.,Carpenter, G., and Rhee, S. G. (1990). Tyrosine residues inbovine phospholipase C-g phosphorylated by the epidermalgrowth factor receptor in vitro. J. Biol. Chem. 265, 3940–3943.

27. Wahl, M. I., Nishibe, S. Kim, J. W., Kim, H., Rhee, S. G., andCarpenter, G. (1990). Identification of two epidermal growthfactor-sensitive tyrosine phosphorylation sites of phospho-lipase C-g in intact HSC-1 cells. J. Biol. Chem. 265, 3944–3948.

28. Kim, H. K., Kim, J. W., Zilberstein, A., Margolis, B., Kim, J. G.,Schlessinger, J., and Rhee, S. G. (1991). PDGF stimulation ofinositol phospholipid hydrolysis requires PLC-g1 phosphory-lation on tyrosine residues 783 and 1254. Cell 65, 435–441.

29. Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T.,Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990). Increaseof the catalytic activity of phospholipase C-g1 by tyrosinephosphorylation. Science 250, 1253–1256.

30. Anderson, D., Koch, C. A., Grey, L., Ellis, C., Moran, M. F., andPawson, T. (1990). Binding of SH2 domains of phospholipaseCg1, GAP, and Src to activated growth factor receptors. Sci-ence 250, 979–982.

31. Ronnstrand, L., Mori, S., Arridsson, A.-K., Eriksson, A., Wern- specific phospholipase Cg1 structure and activity. Biochem. J.

22 CARPENTER AND JI

stedt, C., Hellman, U., Claesson-Welsh, L., and Heldin, C.-H.(1992). Identification of two C-terminal autophosphorylationsites in the PDGF b-receptor: Involvement in the interactionwith phospholipase C-g. EMBO J. 11, 3911–3919.

32. Valius, M., Bazenet, C., and Kazlauskas, A. (1993). Tyrosines1021 and 1009 are phosphorylation sites in the carboxy termi-nus of the platelet-derived growth factor receptor b subunitand are required for binding of phospholipase Cg and a 64-Kilodalton protein, respectively. Mol. Cell. Biol. 13, 133–143.

33. Larose, L., Gish, G., Shoelson, S., and Pawson, T. (1993).Identification of residues in the b platelet-derived growth fac-tor receptor that confer specificity for binding to phospholipaseC-g1. Oncogene 8, 2493–2499.

34. Peters, K. G., Marie, J., Wilson, E., Ives, H. E., Escobedo, J.,Del Rosario, M., Mirda, D., and Williams, L. T. (1992). Pointmutation of an FGF receptor abolishes phosphatidylinositolturnover and Ca21 flux but not mitogenesis. Nature 358, 678–681.

35. Mohammadi, M., Dionne, C. A., Li, W., Li, N. Spivak, T.,Honegger, A. M., Jaye, M., and Schlessinger, J. (1992). Pointmutation in FGF receptor eliminates phosphatidylinositol hy-drolysis without affecting mitogenesis. Nature 358, 681–684.

36. Obermeier, A., Halfter, H., Wiesmuller, K.-H., Jung, G.,Schlessinger, J., and Ullrich, A. (1993). Tyrosine 785 is a majordeterminant of Trk–substrate interaction. EMBO J. 12, 933–941.

37. Middlemas, D. S., Meisenhelder, J., and Hunter, T. (1994).Identification of TrkB autophosphorylation sites and evidencethat phospholipase C-g1 is a substrate of the TrkB receptor.J. Biol. Chem. 269, 5458–5466.

38. Soler, C., Beguinot, L., and Carpenter, G. (1994). Individualepidermal growth actor receptor autophosphorylation sites donot stringently define association motifs for several SH2-con-taining proteins. J. Biol. Chem. 269, 12320–12324.

39. Rotin, D., Margolis, B., Mohammadi, M., Daly, R. J., Daum, G.,Li, N., Fischer, E. H., Burgess, W. H., Ullrich, A., and Schless-inger, J. (1992). SH2 domains prevent tyrosine dephosphory-lation of the EGF receptor: Identification of Tyr992 as thehigh-affinity binding site for SH2 domains of phospholipaseCg. EMBO J. 11, 559–567.

40. Chattopadhyay, A., Vecchi, M., Ji, Q.-S., Mernaugh, R., andCarpenter, G. (1999). The role of individual SH2 domains inmediating association of phospholipase C-g1 with the acti-vated EGF receptor. J. Biol. Chem. 274, 26091–26097.

41. Ji, Q.-s., Chattopadhyay, A., Vecchi, M., and Carpenter, G.(1999). Physiological requirement for both SH2 domains forphospholipase C-g1 function and interaction with platelet-derived growth factor receptors. Mol. Cell. Biol. 19, 4961–4970.

42. Todderud, G., Wahl, M. I., Rhee, S. G., and Carpenter, G.(1990). Stimulation of phospholipase C-g1 membrane associa-tion by epidermal growth factor. Science 249, 296–298.

43. Kumjian, D. A., Barnstein, A., Rhee, S. G., and Daniel, T. O.(1991). Phospholipase Cg complexes with ligand-activatedplatelet-derived growth factor receptors. An intermediate im-plicated in phospholipase activation. J. Biol. Chem. 266, 3973–3980.

44. Vega, Q. C., Cochet, C., Filhol, O., Chang, C.-P., Rhee, S. G.,and Gill, G. N. (1992). A site of tyrosine phosphorylation in theC terminus of the epidermal growth factor receptor is requiredto activated phospholipase C. Mol. Cell. Biol. 12, 128–135.

45. Koblan, K. S., Schaber, M. D., Edwards, G., Gibbs, J. B., andPompliano, D. L. (1995). src-homology 2 (SH2) domain ligationas an allosteric regulator: Modulation of phosphoinositide-

305, 745–751.

46. Barford, D., and Neel, B. G. (1998). Revealing mechanisms forSH2 domain mediated regulation of the protein tyrosine phos-phatase SHP-2. Structure 6, 249–254.

47. Falasca, M., Logan, S. K., Lehto, V. P., Baccante, G., Lemmon,M. A., and Schlessinger, J. (1998). Activation of phospholipaseCg by PI 3-kinase-induced PH domain-mediated membranetargeting. EMBO J. 17, 414–422.

48. Kavran, J. M., Klein, D. E., Lee, A., Falasea, M., Isakoff S. J.,Skolnik, E. Y., and Lemmon, M. A. (1998). Specificity andpromiscuity in phosphoinositide binding by pleckstrin homol-ogy domains. J. Biol. Chem. 273, 30497–30508.

49. Rameh, L. E., Chen, C.-S., and Cantley, L. C. (1995). Phospha-tidylinositol (3,4,5)P3 interacts with SH2 domains and modu-lates PI 3-kinase association with tyrosine-phosphorylatedproteins. Cell 83, 821–830.

50. Bae, Y. S., Cantley, L. G., Chen, C.-S., Kim, S.-R., Kwon, K.-S.,and Rhee, S. G. (1998). Activation of phospholipase C-g byphosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273,4465–4469.

51. Rameh, L. E., Rhee, S. G., Spokes, K., Kazlauskas, A., Cantley,L. C., and Cantley, L. G. (1998). Phosphoinositide 3-kinaseregulates phospholipase Cg-mediated calcium signaling.J. Biol. Chem. 273, 23750–23757.

52. Sekiya, F., Bae, Y. S., and Rhee, S. G. (1999). Regulation ofphospholipase C isozymes: Activation of phospholipase Cg inthe absence of tyrosine-phosphorylation. Chem. Phys. Lipids98, 3–11.

53. Duckworth, B. C., and Cantley, L. C. (1997). Conditional inhi-bition of the mitogen-activated protein kinase cascade byWortmannin: Dependence on signal strength. J. Biol. Chem.272, 27665–27670.

54. Wennstrom, S., and Downward, J. (1999). Role of phosphoino-sitide 3-kinase in activation of Ras and mitogen-activatedprotein kinase by epidermal growth factor. Mol. Cell. Biol. 19,4279–4288.

55. Roche, S., McGlade, J., Jones, M., Gish, G. D., Pawson, T., andCourtneidge, S. A. (1996). Requirement of phospholipase Cg,the tyrosine phosphatase Syp and the adaptor proteins Shcand Nck for PDGF-induced DNA synthesis: evidence for Ras-independent pathways. EMBO J. 15, 4940–4948.

56. Wang, Z., Gluck, S., Zhang, L., and Moran, M. F. (1998).Requirement for phospholipase C-g1 enzymatic activity ingrowth factor-induced mitogenesis. Mol. Cell. Biol. 18, 590–597.

57. Valius, M., and Kazlauskas, A. (1993). Phospholipase C-g1and phosphatidylinositol 3 kinase are the downstream medi-ators of the PDGF receptor’s mitogenic signal. Cell 73, 321–334.

58. Montmayeur, J.-P, Valius, M., Vandenheede,J., and Kazlaus-kas, A. (1997). The platelet-derived growth factor b receptortriggers multiple cytoplasmic signaling cascades that arrive atthe nucleus as distinguishable inputs. J. Biol. Chem. 272,32670–32678.

59. Borrello, M. G., Alberti, L., Arighi, E., Bongarzone, I., Battis-tini, C., Bardelli, A., Pasini, B., Piutti, C., Rizzetti, M. G.,Mondellini, P., Radice, T., and Pierotti, M. A. (1996). The fulloncogenic activity of Ret/ptc2 depends on tyrosine 539, a dock-ing site for phospholipase Cg. Mol. Cell. Biol. 16, 2151–2163.

60. DeMali, K. A., Whiteford, C. C., Ulug, E. T., and Kazlauskas,A. (1997). Platelet-derived growth factor-dependent cellulartransformation requires either phospholipase Cg or phospha-tidylinositol 3 kinase. J. Biol. Chem. 272, 9011–9018.

61. Chang, J.-S., Noh, D. Y., Park, I. A., Kim, M. J., Song, H., Ryu, tion signals are controlled by nerve growth factor receptor/Trk

23PLC-g AS A SIGNAL-TRANSDUCING ELEMENT

S. H., and Suh, P.-G. (1997). Overexpression of phospholipaseC-g1 in rat 3Y1 fibroblast cells leads to malignant transforma-tion. Cancer Res. 57, 5465–5468.

62. Smith, M. R., Court, D. W., Kim, H.-k., Park, J. B., Rhee, S. G.,Rhim, J. S., and Kung, H.-f. (1998). Overexpression of phos-phoinositide-specific phospholipase Cg in NIH 3T3 cells pro-motes transformation and tumorigenicity. Carcinogenesis 19,177–185.

63. Yu, J.-C., Li, W., Wang, L. M., Uren, A., Pierce, J. H., andHeidaran, M. A. (1995). Differential requirement of a motifwithin the carboxyl-terminal domain of a-platelet-derivedgrowth factor (aPDGF) receptor for PDGF focus forming ac-tivity, chemotaxis, or growth factor. J. Biol. Chem. 270, 7033–7036.

64. Khoshyomn, S., Penar, P. L., Rossi, J., Wells, A., Abramson,D. L., and Bhushan, A. (1999). Inhibition of phospholipaseC-g1 activation blocks glioma cell motility and invasion of fetalrat brain aggregates. Neurosurgery 44, 568–578.

65. Ronnstrand, L., Siegbahn, A., Rorsman, C., Johnell, M., Han-sen, K., and Heldin, C.-H. (1999). Overactivation of phospho-lipase C-g1 renders platelet-derived growth factor b-receptor-expressing cells independent of the phosphatidylinositol3-kinase pathway for chemotaxis. J. Biol. Chem. 274, 22089–22094.

66. Kundra, V., Escobedo, J. A., Kazlauskas, A., Kim, H. K., Rhee,S. G., Williams, L. T., and Zetter, B. R. (1994). Regulation ofchemotaxis by the platelet-derived growth factor receptor-b.Nature 367, 474–476.

67. Ming, G.-l., Song, H.-j., Berninger, B., Inagaki, N., Tessier-Lavigne, M., and Poo, M.-m. (1999). Phospholipase C-g andphosphoinositide 3-kinase mediate cytoplasmic signaling innerve growth cone guidance. Neuron 23, 139–148.

68. Derman, M. P., Chen, J. Y., Spokes, K. C., Songyang, Z., andCantley, L. G. (1996). An 11-amino acid sequence from c-metinitiates epithelial chemotaxis via phosphatidylinositol 3-ki-nase and phospholipase C. J. Biol. Chem. 271, 4251–4255.

69. Wells, A., Gupta, K., Chang, P., Swindle, S., Glading, A., andShiraha, H. (1998). Epidermal growth factor receptor-medi-ated motility in fibroblasts. Microsc. Res. Technol. 43, 395–411.

70. Clyman, R. I., Peters, K. G., Chen, Y. Q., Escobedo, J., Wil-liams, L. T., Ives, H. E., and Wilson, E. (1994). PhospholipaseCg activation, phosphatidylinositol hydrolysis, and calciummobilization are not required for FGF receptor-mediated che-motaxis. Cell Adhes. Commun. 1, 333–342.

71. Landgren, E., Klint, P., Yokote, K., and Claesson-Welsh, L.(1998). Fibroblast growth factor receptor-1 mediates chemo-taxis independently of direct SH2-domain protein binding.Oncogene 17, 283–291.

72. Yokote, K., Mori, S., Siegbahn, A., Ronnstrand, L., Wernstedt,C., Heldin, C.-H., and Claesson-Welsh, L. (1996). Structuraldeterminants in the platelet-derived growth factor a-receptorimplicated in modulation of chemotaxis. J. Biol. Chem. 271,5101–5111.

73. Williams, E. Y., Farness, J., Walsh, Y. S., and Doherty, P.(1994). Characterisation of the second messenger pathwayunderlying neurite outgrowth stimulated by FGF. Develop-ment 120, 1685–1693.

74. Stephens, R. M., Loeb, D. M., Copeland, T. D., Pawson, T.,Greene, L. A., and Kaplan, D. R. (1994). Trk receptors useredundant signal transduction pathways involving SHC andPLC-g1 to mediate NGF responses. Neuron 12, 691–705.

75. Obermeier, A., Bradshaw, R. A., Seedorf, K., Choidas, A.,Schlessinger, J., and Ulrich, A. (1994). Neuronal differentia-

binding sites for SHC and PLC-g. EMBO J. 13, 1585–1590.

76. Hall, H., Williams, E. Y., Moore, S. E., Walsh, Y. S., Prochi-antz, A., and Doherty, P. (1996). Inhibition of FGF-stimulatedphosphatidylinositol hydrolysis and neurite outgrowth by acell-membrane permeable phosphopeptide. Curr. Biol. 6, 580–587.

77. Saffrell, J. L., Williams, E. Y., Mason, I. J., Walsh, Y. S., andDoherty, P. (1997). Expression of a dominant negative FGFreceptor inhibits axonal growth and FGF receptor phosphory-lation stimulated by CAMs. Neuron 18, 231–242.

78. Bae, S. K., Lee, Y. H., Chang, J.-S., Galadari, S. H., Kim, Y. S.,Ryu, S. H., and SH, P.-G. (1998). Src homology domains ofphospholipase C g1 inhibit nerve growth factor-induced differ-entiation of PC12 cells. J. Cell. Neurochem. 71, 178–185.

79. Rao, A., Luo, C., and Hogan, P. G. (1997). Transcription factorsof the NFAT family: Regulation and function. Annu. Rev.Immunol. 15, 707–747.

80. van Leeuwen, J. E. M., and Samelson, L. E. (1999). T cellantigen-receptor signal transduction. Curr. Opin. Immunol.11, 242–248.

81. Williams, B. L, Irvin, B. J., Sutor, S. L., Chini, C. C. S.,Yacyshyn, E., Wardenburg, J. B., Dalton, M., Chan, A. C., andAbraham, R. T. (1999). Phosphorylation of Tyr319 in ZAP-70 isrequired for T-cell antigen receptor-dependent phospholipaseC-g1 and Ras activation. EMBO J. 18, 1832–1844.

82. Liu, K. B., Bunnell, S. C., Gurniak, C. G., and Berg, L. J.(1998). T cell receptor-initiated calcium release is uncoupledfrom capacitative calcium entry in itk-deficient T cells. J. Exp.Med. 187, 1721–1727.

83. Schaeffer, E. M., Debnath, J., Yap, G., McVicar, D., Liao, X. C.,Littman, D. R., Sher, A., Varmus, H. E., Lenardo, M. J., andSchwartzberg, P. L. (1999). Requirement for Tec kinases Rlkand Itk in T cell receptor signaling and immunity. Science 284,638–641.

84. Rudd, C. E. (1999). Adaptors and molecular scaffolds in im-mune cell signaling. Cell 96, 5–8.

85. Clement, J. L., Boerth, N. J., Lee, J. R., and Koretzky, G. A.(1999). Integration of T cell receptor-dependent signalingpathways by adapter proteins. Annu. Rev. Immunol. 17, 89–108.

86. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P., andSamelson, L. E. (1998). LAT: The ZAP-70 tyrosine kinasesubstrate that links T cell receptor to cellular activation. Cell92, 83–92.

87. Finco, T. S., Kadlecek, T., Zhang, W., Samelson, L. E., andWeiss, A. (1998). LAT is required for TCR-mediated activationof PLCg1 and the Ras pathway. Immunity 9, 617–626.

88. Zhang, W., Irvin, B. J., Trible, R. P., Abraham, R. T., andSamelson, L. E. (1999). Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkatcell line. Int. Immunol. 11, 943–950.

89. Zhang, W., Trible, R. P., and Samelson, L. E. (1998). LATpalmitoylation: Its essential role in membrane microdomaintargeting and tyrosine phosphorylation during T cell activa-tion. Immunity 9, 239–246.

90. Brdicka, T., Cerny, J., Horejsı, V. (1998). T cell receptor sig-nalling results in rapid tyrosine phosphorylation of the linkerprotein LAT present in detergent-resistant membrane mi-crodomains. Biochem. Biophys. Res. Commun. 248, 356–360.

91. Xavier, R., and Seed, B. (1999). Membrane compartmentationand the response to antigen. Curr. Opin. Immunol. 11, 265–269.

92. Xavier, R., Brennan, T., Li, Q., McCormack, C., and Seed, B.

1

1

1

1

1

1

1

1

1

109. Barker, S. A., Caldwell, K. K., Pfeiffer, J. R., and Wilson, B. S.

1

1

1

1

1

1

1

1

1

1

1

1

1

1

R

24 CARPENTER AND JI

(1998). Membrane compartmentation is required for efficientT cell activation. Immunity 8, 723–732.

93. Stauffer, T. P., and Meyer, T. (1997). Compartmentalized IgEreceptor-mediated signal transduction in living cells. J. Cell.Biol. 139, 1447–1454.

94. Hope, H. R., and Pike, L. J. (1996). Phosphoinositides andphosphoinositide-utilizing enzymes in detergent-insolublelipid domains. Mol. Biol. Cell 7, 843–851.

95. Pike, L., and Casey, L. (1996). Localization and turnover ofphosphatidylinositol 4,5-bisphosphate in caveolin-enrichedmembrane domains. J. Biol. Chem. 271, 26453–26456.

96. Jackman, J. K., Motto, D. G., Sun, Q., Tanemoto, M., Turck,C. W., Peltz, G. A., Koretzky, G. A., and Findell, P. R. (1995).Molecular cloning of SLP-76, a 76-kDa tyrosine phosphopro-tein associated with Grb2 in T cells. J. Biol. Chem. 270, 7029–7032.

97. Wardenburg, J. B., Fu, C., Jackman, J. K., Flotow, H., Wilkin-son, S. E., Williams, D. H., Johnson, R., Kong, G., Chan, A. C.,and Findell, P. R. (1996). Phosphorylation of SLP-76 by theZAP-70 protein-tyrosine kinase is required for T-cell receptorfunction. J. Biol. Chem. 271, 19641–19644.

98. Yablonski, D., Kuhne, M. R., Kadlecek, T., and Weiss, A.(1998). Uncoupling of nonreceptor tyrosine kinases fromPLC-g1 in a SLP-76-deficient T cell. Science 281, 413–415.

99. Kurosaki, T. (1997). Molecular mechanisms in B cell antigenreceptor signaling. Curr. Opin. Immunol. 9, 309–318.

00. Tamir, I., and Cambier, J. C. (1998). Antigen receptor signal-ing: integration of protein tyrosine kinase functions. Oncogene17, 1353–1364.

01. Campbell, K. S. (1999). Signal transduction from the B cellantigen-receptor. Curr. Opin. Immunol. 11, 256–264.

02. Takata, M., Sabe, H., Hata, A., Inazu, T., Homma, Y., Nukada,T., Yamamura, H., and Kurosaki, T. (1994). Tyrosine kinasesLyn and Syk regulate B cell receptor-coupled Ca21 mobiliza-tion through distinct pathways. EMBO J. 13, 1341–1349.

03. Kurosaki, T., Johnson, Sara A., Pao, L., Sada, K., Yamamura,H., and Cambier, J. C. (1995). Role of the Syk autophosphor-ylation site and SH2 domains in B cell antigen receptor sig-naling. J. Exp. Med. 182, 1815–1823.

04. Law, C.-L., Chandran, K. A., Sidorenko, S. P., and Clark, E. A.(1996). Phospholipase C-g1 interact with conserved phospho-tyrosyl residues in the linker region of Syk and is a substratefor Syk. Mol. Cell. Biol. 16, 1305–1315.

05. Takata, M., and Kurosaki, T. (1996). A role for Bruton’s ty-rosine kinase in B cell antigen receptor-mediated activation ofphospholipase C-g2. J. Exp. Med. 184, 31–40.

06. Fluckiger, A.-C., Li, Zuomei, Kato, R. M., Wahl, M. I., Ochs,H. D., Longnecker, R., Kinet, J.-P., Witte, O. N., Scharenberg,A. M., and Rawlings, D. J. (1998). Btk/Tec kinases regulatesustained increases in intracellular Ca21 following B-cell re-ceptor activation. EMBO J. 17, 1973–1985.

07. Scharenberg, A. M., El-Hillal, O., Fruman, D. A., Beitz, L. O.,Li, Z., Lin, S., Gout, I., Cantley, L. C., Rawlings, D. J., andKinet, J.-P. (1998). Phosphatidylinositol-3,4,5-trisphosphate(PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signalingpathway: a target for SHIP-mediated inhibitory signals.EMBO J. 17, 1961–1972.

08. Scharenberg, A. M., and Kinet, J.-P. (1998). PtdIns-3–4–5-P3:A regulatory nexus between tyrosine kinases and sustainedcalcium signals. Cell 94, 5–8.

(1998). Wortmannin-sensitive phosphorylation, translocation,and activation of PLCg1, but not PLCg2, in antigen-stimu-lated RBL-2H3 mast cells. Mol. Biol. Cell. 9, 483–496.

10. Barker, S. A., Lujan, D., and Wilson, B. S. (1999). Multipleroles for PI 3-kinase in the regulation of PLCg activity andCa21 mobilization in antigen-stimulated mast cells. J. Leuko-cyte Biol. 65, 321–329.

11. Gratacap, M.-P., Payrastre, B., Viala, C., Mauco, G., Planta-vid, M., and Chap, H. (1998). Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C-g2 isan early key event in FcgRIIA-mediated activation of humanplatelets. J. Biol. Chem. 273, 24314–24321.

12. Fu, C., and Chan, A. C. (1997). Identification of two tyrosinephosphoproteins, pp70 and pp68, which interact with phos-pholipase Cg, Grb2, and Vav after B cell antigen receptoractivation. J. Biol. Chem. 272, 27362–27368.

13. Fu, C., Turck, C. W., Kurosaki, T., and Chan, A. C. (1998).BLNK: A central linker protein in B cell activation. Immunity9, 93–103.

14. Ishial, M., Kurosaki, M., Pappu, R., Okawa, K., Ronko, I., Fu,C., Shibata, M., Iwamatau, A., Chan, A. C., and Kurosaki, T.(1999). BLNK required for coupling Syk to PLCg2 and Rac1-JNK in B cells. Immunity 10, 117–125.

15. Ishiai, M., Sugawara, H., Kurosaki, M., and Kurosaki, T.(1999). Cutting edge: Association of phospholipase C-g2 Srchomology 2 domains with BLNK is critical for B cell antigenreceptor signaling. J. Immunol. 163, 1746–1749.

16. Dupont, G., McGuinness, O. M., Johnson, M. H., Berridge,M. J., and Borgese, F. (1996). Phospholipase C in mouse oo-cytes: characterization of b and g isoforms and their possibleinvolvement in sperm-induced Ca21 spiking. Biochem J. 316,583–591.

17. De Nadai, C., Cailliau, K., Epel, D., and Ciapa, B. (1998).Detection of phospholipase Cg in sea urchin eggs. Dev. GrowthDiffer. 40, 669–676.

18. Shilling, F. M., Carroll, D. J., Muslin, A. J., Escobedo, J. A.,Williams, L. T., and Jaffe, L. A. (1994). Evidence for bothtyrosine kinase and G-protein-coupled pathways leading tostarfish egg activation. Dev. Biol. 162, 590–599.

19. Sette, C., Bevilacqua, A., Geremia, R., and Rossi, P. (1998).Involvement of phospholipase Cg1 in mouse egg activationinduced by a truncated form of the c-kit tyrosine kinasepresent in spermatozoa. J. Cell Biol. 142, 1063–1074.

20. Carroll, D. J., Ramarao, C. S., Mehlmann, L. M., Roche, S.,Terasaki, M., and Jaffe, L. A. (1997). Calcium release at fer-tilization in starfish eggs is mediated by phospholipase Cg.J. Cell Biol. 138, 1303–1311.

21. Carroll, D. J., Albay, D. T., Terasaki, M., Jaffe, L. A., andFoltz, K. R. (1999). Identification of PLCg-dependent and -in-dependent events during fertilization of sea urchin eggs. Dev.Biol. 206, 232–247.

22. Shearer, J., De Nadai, C., Emily-Fenouil, F., Gache, C., Whi-taker, M., and Ciapa, B. (1999). Role of phospholipase Cg atfertilization and during mitosis in sea urchin eggs and em-bryos. Development 126, 2273–2284.

23. Giusti, A. F., Carroll, D. J., Abassi, Y. A., and Foltz, K. R.(1999). Evidence that a starfish egg Src family tyrosine kinaseactivates with PLC-g1 SH2 domains at fertilization. Dev. Biol.208, 189–199.

eceived September 1, 1999