A Novel Bifunctional Phospholipase C That Is Regulated by Ga12and Stimulates the Ras/Mitogen-activated Protein Kinase Pathway*

Received for publication, September 5, 2000, and in revised form, October 3, 2000Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M008119200

Isabel Lopez‡, Eric C. Mak‡, Jirong Ding‡, Heidi E. Hamm§, and Jon W. Lomasney‡§¶i

From the ¶Veterans Affairs Chicago Health Care System-Lakeside Division and the ‡Department of Pathology andFeinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois 60611 and the§Institute of Neuroscience and Department of Molecular Pharmacology and Biological Chemistry, Northwestern UniversityMedical School, Chicago, Illinois 60611

Three families of phospholipase C (PI-PLCb, g, and d)are known to catalyze the hydrolysis of polyphosphoi-nositides such as phosphatidylinositol 4,5-bisphosphate(PIP2) to generate the second messengers inositol 1,4,5trisphosphate and diacylglycerol, leading to a cascadeof intracellular responses that result in cell growth, celldifferentiation, and gene expression. Here we describethe founding member of a novel, structurally distinctfourth family of PI-PLC. PLCe not only contains con-served catalytic (X and Y) and regulatory domains (C2)common to other eukaryotic PLCs, but also contains twoRas-associating (RA) domains and a Ras guanine nucle-otide exchange factor (RasGEF) motif. PLCe hydrolyzesPIP2, and this activity is stimulated selectively by a con-stitutively active form of the heterotrimeric G proteinGa12. PLCe and a mutant (H1144L) incapable of hydro-lyzing phosphoinositides promote formation of GTP-Ras. Thus PLCe is a RasGEF. PLCe, the mutant H1144L,and the isolated GEF domain activate the mitogen-acti-vated protein kinase pathway in a manner dependent onRas but independent of PIP2 hydrolysis. Our findingsdemonstrate that PLCe is a novel bifunctional enzymethat is regulated by the heterotrimeric G protein Ga12and activates the small G protein Ras/mitogen-activatedprotein kinase signaling pathway.

Many hormones, neurotransmitters, and growth factorselicit intracellular responses by activating a family of inositolphospholipid-specific phospholipase C (PLC)1 isozymes (1, 2).

There are three established families of PLC termed b (;150kDa), g (;145 kDa), and d (; 85 kDa) (3). All three families ofthe PI-PLC family are able to recognize phosphatidylinositol(PI), phosphatidylinositol 4-phosphate, and phosphatidylinosi-tol 4,5-bisphosphate (PIP2) and to carry out the Ca21-depend-ent hydrolysis of these inositol phospholipids. It is presumedthat the primary substrate for hydrolysis is PIP2, which yieldsthe second messengers inositol 1,4,5-trisphosphate (IP3) anddiacylglycerol (DAG). IP3 releases intracellular Ca21 from theendoplasmic reticulum via interaction with a specific receptorlocated on the surface of the endoplasmic reticulum. DAG, aswell as increased intracellular Ca21, activate protein kinase Cleading to a cascade of intracellular events including regulationof cellular growth, smooth muscle contraction, and cardiachypertrophy (2).

The mode of regulation differs considerably for members ofthe different isoform families. The b isoforms are regulated bylarge heterotrimeric G proteins. After activation by agonistssuch as epinephrine, a1 adrenergic receptors are able to coupleto Ga subunits from the Gq/G11 family and stimulate hydrolysisof phosphatidyl inositol lipids via PLC b isoforms. For some bisoforms, Gq alone is sufficient for activation, whereas for oth-ers the coordinated action of both Gq and bg is necessary. Theg isoforms of PLC contain SH2 and SH3 domains; hence, theyare activated by both receptor and nonreceptor tyrosine ki-nases. Until recently, the mode of regulation of the d class wasunknown. Work from our laboratory and that of others hasdetermined that this class is regulated in vitro by lipid ligandsand ionized free calcium (regulation by calcium via C2 domainis described by Lomasney et al.)2 (4–7).

Activation of G protein-coupled receptors modulates variousaspects of cellular growth and proliferation, processes that areprimarily controlled by small Ras-related G proteins and theirdownstream effector, the mitogen-activated protein (MAP) ki-nases (8, 9). There appear to be multiple mechanisms involvingheterotrimeric Ga and bg subunits by which G protein-coupledreceptors regulate small monomeric G protein function (10).Gbg subunits can induce phosphorylation of the Shc adapterprotein leading to association with the Grb2 docking proteinand eventual stimulation of Ras guanine nucleotide exchangeactivity (11). Gai and Gao subunits can lead to activation ofMAP kinase via a protein kinase C-dependent pathway (12).Recently, a novel molecule, p115 RhoGEF, has been identifiedthat serves as a direct link between the heterotrimeric Gasubunit Ga13 and the small G protein Rho (13, 14). Ga13 stim-ulates the nucleotide exchange activity of p115 RhoGEF forRho. p115 RhoGEF also serves as a GTPase-activating proteinfor Ga13 and Ga12. This is the first example of a protein that is

* This work was supported by a Merit Review Grant from the De-partment of Veterans Affairs (to J. W. L.), by National Institute ofHealth Grants HL03836 (to I. L.) and HL55591 and HL03961 (toJ. W. L.), by the Robert H. Lurie Comprehensive Cancer Center’sAmerican Cancer Society Institutional Research Grant IRG-93-037-06(to I. L.), and by American Heart Association Grant-in-Aid 9951330Z (toI. L.). The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF170071.

i To whom correspondence should be addressed: Dept. of Pathologyand Feinberg Cardiovascular Research Institute, Tarry Bldg. 12-703,Northwestern University Medical School, 303 E. Chicago Ave., Chicago,IL 60611. Tel.: 312-503-0450; Fax: 312-503-0137; E-mail: j-lomasney@northwestern.edu.

1 The abbreviations used are: PLC, phospholipase C; PI-PLC, PI-PLC; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; MAP, mitogen-activated protein;RasGEF, Ras guanine nucleotide exchange factor; RA, Ras-associating;RBD, Ras-binding domain; PI, phosphatidylinositol; EST, expressedsequence tag; PCR, polymerase chain reaction; HA, hemagglutinin;GST, glutathione S-transferase. 2 J. W. Lomasney and K. King, submitted for publication.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 4, Issue of January 26, pp. 2758–2765, 2001Printed in U.S.A.

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able to directly link large and small G protein pathways. In thisreport we identify a novel fourth class of PI-PLC that wedesignate PLCe and demonstrate that PLCe interacts withlarge and small G proteins, although it is very different fromp115 RhoGEF.

EXPERIMENTAL PROCEDURES

Cloning of hPLCe cDNA—A computer search of the human Gen-BankTM expressed sequence tag (EST) data base was conducted usingthree relatively short amino acid sequences from the conserved X and Ydomains of the mammalian PLCs. An EST clone, zb59f12.s1, showed ahigh degree of homology and contained a putative open reading frame.Screening of a human placental cDNA library with the EST cDNA probeyielded a larger (3.0 kilobases) but still incomplete cDNA fragment. Thefull-length PLCe cDNA was generated by 59 rapid amplification ofcDNA ends utilizing the Marathon cDNA amplification kit (CLON-TECH). The cDNA was reverse transcribed from poly(A)1 mRNA ob-tained from human heart (CLONTECH), using the cDNA synthesisprimer provided with the kit. The PCR amplification was carried outusing adaptor primer 1 (59-CCATCCTAATACGACTCACTATAGGGC-39) and a gene specific reverse primer, 59-TCCACCGTCTGCCACCAA-ACAACTCCACA-39. The first round PCR was performed according tothe recommendations of the manufacturer of 94 °C for 1 min, five cyclesat 94 °C for 5 s and 72 °C for 4 min, another five cycles at 94 °C for 5 sand 70 °C for 4 min, followed by 25 cycles at 94 °C for 5 s and 68 °C for4 min. The second round of PCR was performed under the same con-ditions using a 1:200 dilution of the first round PCR amplificationmixture as the template, adaptor primer 2 (59-ACTCACTATAGGGCT-CGAGCGGC-39) and a nested gene-specific primer (59-AGCAGCGGG-CAGAGAGGTGTGTGTCC-39). Southern blot analysis using anend-labeled internal oligonucleotide (59-TGTCAACAGCATCTTTCAG-GTCATCC-39) 59 from the PCR primers confirmed that the PCR productcontained the expected sequence. The PCR product was subcloned intothe PCR2.1 TA cloning vector (InVitrogen) according to the recommen-dations of the manufacturer. Positive clones were identified by hybrid-izing filter lifts with the same labeled oligonucleotide that was de-scribed for the Southern blots. Clones containing the expected size DNAfragment were then sequenced along both strands using the Taq dyeterminator method at the University of Georgia Molecular GeneticsFacility, Department of Genetics, using Applied Biosystems 373 and377 automatic sequencers. The entire hPLCe cDNA was obtained fromheart cDNA by PCR using the following primers: 59-ATGGTTTCAGA-AGGAAGTGCAGCAGGAA-39 (sense) and 59- TCACTGTCGGTAATCC-ATTGTGTCACTGG-39 (antisense). The PLCe cDNA was subcloned in-frame into pBluescript KS1. The sequence of the inserted DNA wasdetermined as previously mentioned.

Creation of the Phosphodiesterase-deficient Mutant hPLCe H1144L—The phosphodiesterase-deficient mutant of hPLCe, PLCe H1144L, wasgenerated by changing the histidine residue at position 1144 to leucineusing the QuickChange Kit (Stratagene). Briefly, the fragment of PLCecontaining the mutation was amplified with Pfu Turbo Polymerase(Stratagene) using sense (59-GCTTGACGGCGCCTCCGG-39) and an-tisense (59-CTACCCTACGGGTAGTAAATAGAACCTGTATGCGACTG-TTGGTTC-39) primers containing the mutation changing the histidine(CAT) to a leucine (CTT). The PCR was performed according to therecommendations of the manufacturer of 95 °C for 30 s, 12 cycles at95 °C for 30 s, 1 min at 55 °C and 4 min at 68 °C. A second fragmentoverlapping the first was amplified using the sense primer, 59-GATG-GGATGCCCATCATTTATCTTGGACATACGCTGACAACCAAG-39 andan antisense primer (59-AACGGGGAGGGGGCACGG-39) correspond-ing to pcDNA3 vector, 39 from the multiple cloning site of the vector.The PCR was also performed according to the recommendations of themanufacturer of 95 °C for 30 s, 12 cycles at 95 °C for 30 s, 1 min at55 °C, and 6 min at 68 °C. The overlapping products were then used toamplify the entire PLCe cDNA from nucleotide1278–6507 using thesense primer 59-GCTTGACGGCGCCTCCGG-39 and the antisenseprimer corresponding to the vector just 39 of the multiple cloning site.The PCR conditions were 95 °C for 30 s, 12 cycles at 95 °C for 30 s, 55 °Cfor 1 min, and 10 min at 68 °C. After ligation and plasmid preparation,the mutant insert was digested with SacII and XbaI. The mutatedfragment was gel purified using the QIAquick gel extraction kit (Qia-gen) and ligated into pcDNA3 in place of the wild type fragment. Thesequence was then verified for the presence of the H1144L mutationand the absence of any additional mutations.

Northern Blot Analysis—Human multiple-tissue Northern blot I andblot IV membranes (CLONTECH) containing approximately 2 mg ofpoly(A)1 RNA/lane were probed with full-length cDNA of hPLCe or

b-actin according to the manufacturer’s instructions. Briefly, 30 ng ofhPLCe or b-actin were random-primed with [a-32P]dCTP (,6000 Ci/mmol; PerkinElmer Life Sciences) using the Prime-It II random primerlabeling kit (Stratagene). Unincorporated radionucleotide was removedfrom the probe by using Bio-Spin 30 chromatography columns (Bio-Rad). The radiolabeled probes were then denatured by boiling for 5 minand placed on ice. Membranes were prehybridized in 15 ml of Expresshyb solution (CLONTECH) with continuous shaking at 68 °C for 1 h.The prehybridization buffer was replaced with 10 ml of fresh Ex-pressHyb solution containing the radiolabeled probe. After a 1-h incu-bation at 68 °C with continuous shaking, the blots were quickly rinsedseveral times with 23 SSC, 0.05% SDS at room temperature and thenwashed twice for 10 min at room temperature in the same buffer. Thiswas followed by two 20-min washes with fresh 0.13 SSC, 0.1% SDS at50 °C and continuous shaking. The blots were then wrapped in plasticwrap and exposed to HyperFilm MP x-ray film (Amersham PharmaciaBiotech) at 280 °C with two intensifying screens for 4–24 h beforedeveloping the film. Blots exposed to multiple probes were stripped ofthe first probe as suggested by the manufacturer’s protocols.

Transient Transfection of TSA201 Cells—The hPLCe cDNA was sub-cloned in-frame into the unique HindIII and NotI sites of the mamma-lian expression vector pcDNA3 (InVitrogen) downstream of the cyto-megalovirus promotor. A Kozak consensus sequence (TAAT) and a Myctag (59-ATGGAGCAGAAGCTGATCAGCGAGGAGGACCTG-39) wereincorporated in frame before the start codon. Plasmids were purified fortransfection using Qiagen kits. TSA201 cells were cultured in completeDulbecco’s modified Eagle’s medium (Life Technologies, Inc.) containingglutamine, high glucose, 10% fetal bovine serum (Life Technologies,Inc.), and 50 mg/ml gentamicin (Life Technologies, Inc.) at 37 °C in ahumidified 5% CO2 incubator. The cells were transfected with Myc-hPLCe or pcDNA3 empty vector using LipofectAMINE (Life Technolo-gies, Inc.) according to manufacturer’s instructions with slight modifi-cations. Cells were harvested 48 h after transfection.

Immunoblotting—Samples were subjected to 6% or 15% SDS-poly-acrylamide gel electrophoresis and were electrophoretically transferredto nitrocellulose as described previously (15). hPLCe was detected byWestern blot analysis using a primary anti-Myc tag antibody (InVitro-gen). Ga* subunits were detected using specific anti-Ga antibodies.Alkaline phosphatase or horseradish peroxidase-conjugated secondaryantibody (IgG) was used for detection.

PLC Activity in TSA201 Cells—TSA201 cells were transfected witheither pcDNA3 vector (control), hPLCe, hPLCe H1144L, Gas*, Gai*,Ga12*, Ga13* cDNA, or Ras (2 mg/35-mm plate) alone or in combinationusing LipofectAMINE (Life Technologies, Inc). To maintain uniformamount of transfected DNA, empty vector was added to the transfectionmixture when necessary. The cells were labeled with [3H]myo-inositol(PerkinElmer Life Sciences) for 24 h and harvested 48 h after transfec-tion. The amount of inositol phosphates (inositol 1-phosphate, inositol1,4-bisphosphate, and IP3) was determined using anion exchange chro-matography (16). The percentage of PI hydrolyzed is expressed as thetotal inositol phosphates formed relative to the amount of [3H]myo-inositol incorporated into the phospholipid pool.

Cell Free Assays—TSA201 cells were transfected with either vector(control) or hPLCe cDNA (12 mg DNA/100-mm plate). Cells were har-vested 48 h after transfection and homogenized in ice-cold lysis buffer(50 mM Tris, pH 7.5, 30 mM NaCl, 1 mM EDTA, 1 mM phenylmethyl-sulfonyl fluoride, 2 mM leupeptin, 1 mM aprotinin, and 1 mM pepstatin).Cytosolic and particulate fractions were isolated as described previ-ously (15). Cytosolic and membrane fractions were then assayed forPLC activity using a vesicle assay previously described with slightmodification (17). Substrate was provided as mixed phospholipid vesi-cles containing phosphotidylinositol-4,5-bis phosphate, PIP2 (28 mM),and phosphatidylethanolamine (280 mM) in a ratio of 1:10 with 25,000-35,000 cpm of [3H]PIP2/assay. PLC assays were performed at 37 °C forvarying time periods in a mixture (70 ml) containing 50 mM HEPES, pH7.3, 3 mM EGTA, 0.2 mM EDTA, 1 mM MgCl2, 20 mM NaCl, 30 mM KCl,4 mM dithiothreitol, 0.1 mg/ml acetylated bovine albumin, 1.6 mM

sodium deoxycholate, and 1 mM CaCl2 The reactions were started byaddition of the transfected cell membranes and terminated by adding350 ml of chloroform/methanol/concentrated HCl (500:500:3, v/v/v). Thesamples were vortexed, and 100 ml of 1 M HCl containing 5 mM EGTAwas added. Phases were separated by centrifugation and assayed forradioactivity by liquid scintillation counting.

MAP Kinase Assay—TSA201 cells were co-transfected (12 mg DNA/100 mm plate) with vector, hPLCe, hPLCe H1144L, or RasGRF2 incombination with HA-tagged MAP kinase (2 mg). 24 h after transfectionthe cells were serum starved over night. Cells were lysed 48 h aftertransfection as described previously with slight modifications (18).

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Briefly, cells were washed twice with ice-cold phosphate-buffered saline(Life Technologies, Inc.) and lysed by addition of one volume 103 MAPkinase lysis buffer (5.5% Triton X-100, 0.2 mM phenylmethanesulpho-nyl fluoride, 0.7 mg/ml pepstatin A, 10 mg/ml leupeptin, 2 mg/mlaprotinin, 20 mM sodium orthovanadate, and 20 mM sodium pyrophos-phate) to 10 volumes phosphate-buffered saline. The cells were incu-bated for 10 min at 4 °C and centrifuged at 4 °C for 15 min at 10,000 3g. Activated MAP kinase was immunoprecipitated by incubating thecells for 2 h with anti-HA antibodies (Upstate Biotechnology Inc., 0.4mg/ml). At the end of the incubation period precleared protein A beads(50% slurry) were added, and the samples were rotated 2 h at 4 °C. Theability of MAP kinase to phosphorylate myelin basic protein was meas-ured following the manufacturer’s protocol (Upstate BiotechnologyInc.). Protein samples were separated on 6% (PLCe and H1144L) and15% SDS-polyacrylamide gel and subsequently transferred to nitrocel-lulose at room temperature for 1 or 2 h at 5 or 15 V, respectively, usinga semidry transfer cell (Bio-Rad). The HA monoclonal antibody (clone12CA5; Roche Molecular Biochemicals) was used to detect HA-MAPkinase, anti-FLAG M2 antibody (Sigma) was used to detect FLAG-tagged RasGRF2, and anti-Myc antibody (InVitrogen) was used to de-tect hPLCe and the H1144L mutant. The horseradish peroxidase-cou-pled goat anti-mouse antibody (1:2,000; Sigma) was used as thesecondary antibody. The blots were developed by ECL (AmershamPharmacia Biotech).

Ras Pull-down Assays—TSA201 cells were transiently co-transfectedas previously mentioned with either control plasmid (pcDNA3), hPLCe,hPLCe H1144L, or RasGRF2 and Ha-Ras. The cells were serum-starvedfor 24 h. 48 h after transfection the cells from a 10-cm dish were lysedand scraped in 1 ml of RIPA buffer containing 50 mM Tris, pH 8.0, 150mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 0.1 mM

aprotinin, 1 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride.Lysates were centrifuged at 14,000 rpm for 8 min at 4 °C to removenuclei. The desired amount of bacterial lysate containing the expressedGST-Ras-binding domain (GST-RBD) of Raf1 (prepared as described byde Rooij and Bos (19)) was thawed on ice and incubated with glutathi-one-agarose beads at 4 °C for 1 h. The beads were isolated by centrif-ugation and washed three times with RIPA buffer containing 50 mM

Tris, pH 8.0, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1%SDS, 0.1 mM aprotinin, 1 mM leupeptin, 1 mM pepstatin, and 1 mM

phenylmethylsulfonyl fluoride at 4 °C. Cell lysates were added to GST-RBD precoupled to glutathione-agarose beads and incubated at 4 °C for1 h. Beads were collected by centrifugation, washed three times withRIPA buffer, and resuspended in SDS sample buffer. GTP bound Raswas identified by precipitation with GST-Raf RBD followed by immu-noblotting as described previously by de Rooij and Bos (19). The proteinsamples were separated on 6% (PLCe and H1144L) and 15% SDS-polyacrylamide gel and subsequently transferred to nitrocellulose atroom temperature for 1 h at 5 V using a semidry transfer cell (Bio-Rad).The monoclonal antibody Y13–259 (1:500, Transduction Labs) was usedto detect Ras and anti-Myc tag antibody was used to detect hPLCe andthe H1144L mutant. The horseradish peroxidase-coupled goat anti-mouse antibody (1:2,000, Sigma) was used as the secondary antibody.The blot was developed by ECL (Amersham Pharmacia Biotech).

RESULTS AND DISCUSSION

Isolation of the cDNA Encoding Human PLCe, Determina-tion of Expression in Human Tissues, and Identification ofStructural Features—X and Y domains are regions of ;170 and;260 amino acids, respectively, which share 60% to 40% aminoacid identity among all PLC isoforms. The X and Y domains arenecessary for phosphodiesterase activity of PLC. We selectedthree relatively short amino acid sequences from the conservedX and Y domains to use in a BLAST search. The Basic LocalAlignment Search Tool (BLAST) is an extremely powerful toolthat allows one to simultaneously search multiple nucleotidesequence data bases. Using the BLAST server at the NationalCenter for Biotechnology Information, we were able to identifyan EST clone that partially encoded for a novel PLC isoform.Using the EST cDNA as a probe, a human placental cDNAlibrary was screened yielding a larger but still incompletecDNA. A full-length cDNA clone was ultimately generated with59 rapid amplification of cDNA ends PCR using human heartmRNA as template that we termed PLCe. The full-lengthcDNA (Fig. 1A) possesses an open reading frame of 6.05 kilo-

bases encoding a 1994-amino acid protein with a calculatedmolecular mass of 230,000, making this the largest PLC iso-lated to date. The next largest would be a member of the b

family at ;1300 amino acids and ;150 kDa. Hybridization ofhuman multiple tissue Northern blots with PLCe cDNA re-vealed a ;7.5-kilobase message corresponding to PLCe ex-pressed in a wide variety of tissues including brain, lung,kidney, testis, and colon with highest expression detected inthe heart (Fig. 2). An additional transcript of larger size (;9.5kilobases) could be observed in most tissues, suggesting thepossibility of an alternatively spliced form of PLCe or differen-tial polyadenylation. Results from Southern blotting indicatethat isoforms likely do not exist (data not shown).

Structural analysis reveals that PLCe contains the con-served catalytic X and Y domains, thus identifying this isoformconclusively as a Pl-PLC (Fig. 1B). Like members of the threeother PLC families, PLCe also contains the regulatory C2 do-main. Unlike other eukaryotic PLCs, PLCe appears not tocontain a pleckstrin homology domain. A phylogenetic compar-ison of all known mammalian PLC isoforms (Fig. 1C) demon-strates that PLCe shares little homology with other PLC fam-ilies (b, g, and d) and therefore constitutes a distinct family ofPLCs. PLCe is most similar to PLC210, a largely uncharacter-ized isoform from the nematode Caenorhabditis elegans (20).PLC210 was first identified by open reading frame prediction ofgenomic sequences generated from the C. elegans genome se-quencing project. Although there are significant similarities,human PLCe also differs considerably from PLC210. PLCe isconsiderably larger than PLC210 (;200 amino acids) and dif-fers extensively in the primary structure of the C terminus andportions of the N terminus. We predict that the C terminus ofPLCe will mediate protein-protein interactions that are com-pletely different from those of PLC210. Interestingly, bothPLCe and PLC210 share structural domains that are not pres-ent in any other PLC, further suggesting the existence of anovel PLC family. Both PLCs contain a Ras binding motifdenoted as the RA domain (21). Although the functional role ofRA domains is presently unknown, a recent report has demon-strated that PLC210 binds to Ha-Ras via this domain, suggest-ing a possible role of this isoform in Ras signaling (20). Anumber of other RA domain-containing proteins such as Ral-GDS and RGL are also known to bind to Ras (21). PLCe has twoRA domains at the C terminus (amino acids 1688–1792 and1813–1916), suggesting that it might interact with the effectorregion of Ras (Fig. 1B). PLCe also contains domains in the Nterminus, which suggests that it may interact with small Gproteins of the Ras superfamily. Very significant homology (p ,0.00001) is found with aimless RasGEF from Dictyostelium,CDC25 RasGEF from yeast (both Candida albicans and Sac-charomyces cerevisiae), human RasGEF homolog Sos1, humanRasGEF H-GRF55, and son-of-sevenless RasGEF from mouse.The area of homology is encompassed by the RasGEF catalyticdomain signature (G/A/P)CVP(F/Y)X4(L/I/M/F/Y)X(D/N)(L/I/V/M) PROSITE121 (22). No other eukaryotic PLC containsthese Ras binding motifs. This strongly suggests that PLCe

activates Ras signal transduction pathways.Expression of PLCe in TSA201 Cells and Characterization of

Polyphosphoinositide Hydrolysis—Western blot analysis of celllysates from TSA201 cells (a clone of human embryonic kidney293 cells stably expressing simian virus 40 large T antigen)revealed the presence of a single protein of approximately 230kDa from cells transfected with PLCe cDNA inserted into themammalian expression vector pcDNA3 but not from controlcells (Fig. 3A) (23). The observed apparent molecular mass of230 kDa is consistent with the predicted molecular mass ofPLCe. Extensive PLCe immunoreactivity resides in the partic-

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ulate fraction of transfected TSA201 cells (data not shown)similar to eukaryotic PLCb isoforms but unlike PLCg andPLCd isozymes that are primarily localized in cytosolic frac-tions (24, 25).

PLCe has Pl-PLC activity, measured by its ability to hydro-lyze exogenous PIP2, a selective substrate of PI-PLCs. As canbe seen in Fig. 3B, 10 mg of plasma membranes obtained fromPLCe transfected TSA201 cells had a 2–3-fold (61–114 pmol

PIP2 hydrolyzed) greater PLC activity over a 15-min incubationperiod than membranes of cells transfected with control vector(20–46 pmol of PIP2 hydrolyzed).

The Heterotrimeric G Protein Ga12 Selectively StimulatesPLCe-mediated Hydrolysis of Polyphosphinositides—As de-picted in the phylogenetic tree (Fig. 1C), the closest mamma-lian homolog of PLCe is PLCb, an effector for heterotrimeric Gprotein Ga and bg subunits. Currently, there are no identifia-

FIG. 1. Structure and comparison ofPLCe. A, Full-length amino acid se-quence of human PLCe. Highlighted res-idues indicate the identified protein do-mains. X and Y, X and Y domains,catalytic core regions of PI-PLCs; PH,pleckstrin homology domains; C2, calci-um-regulated lipid binding domain; RA,Ras-associating domain. B, schematiccomparison drawn to scale of the PLCfamily functional domains. X and Y, Xand Y domains, catalytic core regions;PH, pleckstrin homology domains; C2,calcium-regulated lipid binding domain;SH, Src homology domain; RA, Ras-asso-ciating domain. C, evolutionary compari-son depicting the relationships among allknown isoforms of eukaryotic PLC andPLCe. Clustal X was used to generatealignments of the primary structureswhich were then displayed with Tree-View. The scale bar is used to quantitatebranch lengths. A scale of “0.1” means 0.1amino acid substitutions/residue.

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ble motifs for proteins that bind to heterotrimeric G proteins.However, truncation of the terminal 112 amino acids of PLCb1(Gln1030–Leu1142) has been shown to totally abolish regulationof this isoform by Gaq, suggesting that the C terminus isnecessary for Ga subunit interaction (26). Like PLCb, PLCehas a long C terminus, suggesting the possibility for regulationby heterotrimeric G proteins. The ability of eight differentconstitutively active (GTPase-deficient) mutants of G protein asubunits (Ga*) to stimulate PLCe activity in TSA201 cells wasdetermined. In the absence of any extracellular activators,co-transfection of Ga12* with PLCe augmented PLC activitynearly 3-fold greater than control (Fig. 4A). Cells transfectedwith Ga12* alone did not have increased PLC activity. Co-transfection with Ga13* also led to an increase in PLC activity;however, Ga13 alone increased endogenous PLC activity, sug-gesting that the effects obtained with Ga13* co-transfected withPLCe were in fact equal in magnitude to the sum of the indi-vidual effects of PLCe and Ga13* alone. Because co-expressionof Ga13* with PLCe tended to decrease PLCe expression some-what, we cannot be sure that Ga13* has no effect. In manysystems Ga12 and Ga13 are interchangable, that is they bothregulate the same effectors. A clear exception has been de-scribed for p115RhoGEF. Although both Ga12 and Ga13 canbind to this novel RhoGEF (p115RhoGEF serves as a GAP forboth Ga subunits), only Ga13 can activate the GEF activitytoward Rho (14). Gas* and Gai* had no effect on PLCe activity.The ability of other Ga subunits: Gai2*, Gaz*, and Gao* toregulate PLCe were also determined; however, none of theseGa subunits increased PLC activity when co-transfected withPLCe (data not shown). Gaq* expressed in cells greatly in-

creased basal PLC activity most likely by activation of endog-enous PLCb found in these cells. Co-transfection of PLCe withGaq* did not increase PLC activity above Gaq* transfectedcells, suggesting that this Ga subunit does not activate PLCe.PLCe activity was obtained under conditions where similaramounts of PLCe and Ga subunits were expressed (Fig. 4, Band C).

PLCe is one of very few known effectors for Ga12, because theGa12/Ga13 family of heterotrimeric G proteins is currentlypoorly characterized (27). Ga12 and Ga13 appear to play animportant role in regulating cellular and cytoskeletal changesand may themselves be regulated by the receptors for thrombinand lysophosphatidic acid and other mitogenic agonists (9).Ga12 is in fact a highly oncogenic Ga subunit. A GTPase defi-cient mutant Ga12 Q229L fully transforms NIH 3T3 cells. Thetransformed cells form foci, grow in semisolid medium, andform tumors in nude mice (28). Ga12 has also been found toregulate extracellular signal-regulated kinase and c-Jun ki-nase pathways (29, 30). It appears that Ga12 stimulates c-Junvia activation of Ras; however, the mechanism by which Ga12

activates Ras is unknown (31). Perhaps PLCe can act as adirect link between Ga12 and Ras.

PLCe Activates Ras and a Downstream Serine/ThreonineKinase MAP Kinase—The presence of a RasGEF motif in the Nterminus of PLCe suggests that PLCe can activate Ras byacting as an exchange factor by promoting the exchange of GTPfor bound GDP. Ras mediates its effects on cellular growth andtransformation mainly by activating a cascade of serine/threo-nine kinases including Raf, MEK, and MAP kinase (32). Be-cause MAP kinase (ERK1/2) is a downstream effector of Ras

FIG. 2. Tissue distribution of hPLCemRNA. Multiple human tissue Northernblots were analyzed as described under“Experimental Procedures.” Each lanecontains approximately 2 mg of poly(A)1

RNA. Blots were hybridized with the full-length cDNA or b-actin probe. The hy-bridized membrane was exposed to x-rayfilm for 24 h. kb, kilobases.

FIG. 3. Expression of PLCe in TSA201 cells reveals a PI-PLC. A, Western blot analysis reveals a band of approximately 230 kDacorresponding to Myc-tagged PLCe (arrow) only in cells lysates (50 mg) obtained from TSA201 cells transfected with PLCe, whereas none is foundin lysates (50 mg) obtained from TSA201 cells transfected with control (vector). B, membranes (10 mg) isolated from TSA201 cells expressing PLCe(filled circles) exhibit time dependent increases in InsP formation when reconstituted with mixed phospholipid vesicles containing [3H]PIP2 asdescribed under “Experimental Procedures.” Data represent the averages 6 S.E. of duplicate samples. Similar results were obtained in threeindependent experiments. Where error bars are not shown, the range was less than the size of the point. MWt., molecular mass.

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signaling, the ability of PLCe to activate Ras was determinedby measuring phosphorylation of MAP kinase. To determinewhether PLCe could activate Ras and the MAP kinase pathwayindependent of PI hydrolysis, the ability of an X domain phos-phodiesterase deficient mutant of PLCe was examined. X and Ydomains are regions of ;170 and ;260 amino acids, respec-tively, which share 60% to 40% amino acid identity among alleukaryotic PI-PLCs. The X and Y domains are necessary forphosphodiesterase activity and make up the catalytic core ofthe enzyme. Bacterial PLCs contain only the X domain. Wehave previously demonstrated through extensive site-directedmutagenesis of PLC d1 that the X domain is responsible for thecatalytic hydrolysis of polyphosphoinositides, whereas the Ydomain is responsible for substrate binding (33). Mutation ofamino acid residues Arg338, Glu341, and His356 in the X domainof PLC d1 lead to cleavage defective enzymes (33). These resi-dues are absolutely conserved in all eukaryotic PLCs includingPLCe. A cleavage-defective PLCe was created by mutating theconserved histidine at position 1144 to leucine. This residue isanalogous to the histidine at position 356 of PLC d1. PLCe

H1144L is expressed normally as determined by Western blotusing anti-Myc antibodies (Fig. 5B) but does not hydrolyzesubstrate (data not shown). Thus, any effects on Ras mediatedby PLCe H1144L would be due to the Ras binding domains ofPLCe rather than due to indirect effects of PI hydrolysis andthe production of the second messengers DAG and IP3.

To determine whether PLCe and PLCe H1144L could indeedactivate the MAP kinase pathway, TSA201 cells were co-trans-fected with either pcDNA3 vector (control), PLCe, PLCeH1144L, or RasGRF2 (as a control for activation of the Ras/MAP kinase pathway) and HA-tagged MAP kinase. Immuno-precipitates obtained from cells expressing PLCe show a 3-fold(454 pmol/min/mg total protein) increase in phosphorylation ofMAP kinase relative to control immunoprecipitates (139 pmol/min/mg) obtained from cells transfected with vector alone (Fig.5A). Expression of PLCe H1144L also stimulates phosphoryla-tion of MAP kinase, indicating that PI hydrolysis is not neces-sary for activation of the Ras effector MAP kinase. In fact, PIhydrolysis seems to inhibit the activation of MAP kinase, be-cause H1144L-stimulated MAP kinase approximately 2-foldgreater than wild type PLCe (907 and 454 pmol/min/mg, re-spectively). The expression of wild type PLCe and H1144L wasnearly identical (31.9 versus 29.3 relative units, respectively) inTSA201 cells, as was the level of HA-MAP kinase. Therefore,levels of expression did not account for the differences in activ-ity (Fig. 5B). The products of PI hydrolysis IP3 and DAG couldact as indirect negative regulators of the RasGEF activity andthereby act as a negative feedback loop. For example, stimula-tion of protein kinase C by DAG might lead to phosphorylation

FIG. 4. Overexpression of GTPase-deficient heterotrimeric Gprotein a subunits stimulates PLCe enzymatic activity. A, PIhydrolysis (InsP formation) is stimulated by co-expression of Ga12 withPLCe in TSA201 cells. Each value represents the mean 6 S.E. of fourexperiments each carried out in duplicate. B, protein (100 mg) obtainedfrom cells transfected with either control (vector), Ga subunits, andPLCa alone or in combination with Ga subunits were separated bySDS-polyacrylamide gel electrophoresis on an 6% gel and transferredunto nitrocellulose. Levels of expression of Ga subunits were analyzedby Western blotting using antisera directed against Gas, Gai, Ga12, andGa13 as described under “Experimental Procedures.” This figure is arepresentative of four different experiments with similar results. C,protein (100 mg) obtained from cells were separated on a 6% gel andtransferred unto nitrocellulose. Levels of expression of PLCe were an-alyzed using anti-Myc antibody as described under “Experimental Pro-cedures.” This figure is a representative of four different experimentswith similar results.

FIG. 5. PLCe activates the MAP kinase pathway. A, cell lysates(;3–5 mg of total protein) were acquired from control, PLCe, PLCeH1144L, and RasGRF2 transfected cells, and MAP kinase activity wasassayed as described under “Experimental Procedures.” The data arethe means 6 S.E. of two to six experiments using separate transfec-tions. B, Western blot analysis of cell lysates (100 mg), using HAantibody 12CA5 for MAP kinase, anti-Myc for PLCe, or anti-FLAGantibody for RasGF2 as described under “Experimental Procedures.”Antibodies were used at a dilution of 1:1000. This figure is a represent-ative of three to five different experiments repeated with similarresults.

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of PLCe and inhibition of the RasGEF activity. The knownRasGEF, RasGRF2, gave the most robust stimulation (1231pmol/min/mg) yet was comparable with the stimulation byH1144L and PLCe, demonstrating that PLCe is a fairly robustactivator of MAP kinase.

The role of the Ras-associating or RA domains is presentlyunknown. Proteins such as RalGDS that contain both Ras-GEFand RA domains are assumed to serve as links between differ-ent Ras family members. Although RA domains are known tobind to the effector loop of activated members of the Ras su-perfamily, the RA domain seems to not be necessary for regu-lation of GDP/GTP exchange by some proteins. For example,activated Ras stimulates RGL (for RalGDS-like), which ex-changes GDP for GTP on Ral in vivo. In vitro, however, the RAdomain of RGL is not necessary for GDP/GTP exchange on Ral(34). It is presumed that the role of the RA domain in RGLactivation of Ral is to mediate redistribution of RGL to themembranes where Ral is located. To differentiate the role of theRasGEF domain from that of the RA domain of PLCe in stim-ulating MAP kinase, a Myc-tagged construct PLCe-GEF wasmade of the N-terminal 600 amino acids of PLCe. This con-struct contains the entire RasGEF domain but lacks the X andY phosphodiesterase domains and both of the C-terminal RAdomains. PLCe-GEF was able to stimulate MAP kinase to alevel comparable with the wild type holo enzyme (Fig. 6), sug-gesting that the RasGEF domain is sufficient for activation ofthe Ras/MAP kinase pathway by PLCe and that the C-terminalRA domains are not necessary. This result is consistent withthe previously described findings for RGL (34).

It is difficult to predict the function of the RA domains in

FIG. 7. Activated Ras inhibits PLCe phosphodiesterase activ-ity in vivo. A, PI hydrolysis (InsP formation) is assayed in whole cellsafter transfection in TSA201 cells. Each value represents the means 6S.E. of two experiments each carried out in duplicate. B, Western blotdemonstrating expression of v-Ras and PLCe. This figure is a repre-sentative of two different experiments with similar results.

FIG. 8. Effect of PLCe and H1144L on Ras activation. A, cellswere transfected with either vector (control), PLCe, PLCe H1144L, orRasGRF2 in combination with Ha-Ras. Cell lysates were prepared, andthe amount of protein was determined. GTP-Ras was isolated by bind-ing to GST-RBD and visualized by Western blot Analysis as describedunder “Experimental Procedures.” Approximately equal amounts ofprotein were loaded per lane. This figure is a representative of three tofive different experiments with similar results. B, Western blot analysisof cells lysates (100 mg), using anti-Myc, anti-HA, anti-FLAG, or anti-Ras antibodies. Antibodies were used in dilutions of 1:1000.

FIG. 6. The GEF domain of PLCe is sufficient to activate MAPkinase. A, cell lysates (;3–5 mg of total protein) were acquired fromcontrol, PLCe, PLCe-GEF transfected cells, and MAP kinase activityassayed as described under “Experimental Procedures.” The data arethe means 6 S.E. of three experiments using separate transfections. B,Western blot analysis of cell lysates (100 mg), using HA antibody 12CA5for MAP kinase, and anti-Myc for PLCe and PLCe-GEF as describedunder “Experimental Procedures.” Antibodies were used at a dilution of1:1000. This figure is a representative of three different experimentswith similar results.

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PLCe from these studies. They do not seem to be involved withmembrane association, because the PLCe-GEF construct ishighly targeted to membranes (data not shown). The domainsmight be closely associated with the phosphodiesterase cata-lytic domains (X and Y) because when one or more of the RAdomains are truncated, the resulting enzyme has very littlephosphodiesterase activity, suggesting that these domains arenecessary for proper folding of the enzyme (data not shown).This is similar to truncation of the d1 isoform of PLC, wheredeletion of even a few C-terminal residues leads to inactivationof phosphodiesterase activity.3 Because of the potential inter-action of the RA domains with the X and Y domains, the abilityof activated Ras to regulate the phosphodiesterase activity ofPLCe was assessed by co-transfection. As can be seen from Fig.7A, v-Ras does not stimulate but may inhibit PLCe in vivo.Transfection of cells with PLCe alone increased PI hydrolysis3.5-fold, whereas co-transfection with PLCe and v-Ras lead toan increase of only 2-fold. Expression levels of v-Ras and PLCedid not vary substantially among different conditions. Actually,PLCe expression was slightly greater when co-transfected withv-Ras. Overall, these results suggest a potential role for PLCein regulating Ras activation and thus activation of the MAPkinase pathway in a manner dependent upon the RasGEFdomain and independent of PI hydrolysis.

PLCe Activates Ras—More direct evidence of the ability ofPLCe to activate Ras comes from experiments in which GTP-Ras is trapped using the Ras effector Raf1. The minimal RBDof Raf1 (amino acids 51–131) binds very tightly and specificallyto the GTP-bound form of Ras (Kd 20 nM), whereas the affinityfor RasGDP is 3 orders of magnitude lower (35). To determinewhether PLCe could indeed act as an exchange factor for Ras,TSA201 cells were transiently co-transfected with eitherpcDNA3 (control), PLCe, PLCe H1144L, or RasGRF2 (as a GEFcontrol for activation of Ras) and Ha-Ras. The cells were har-vested after 48 h and RasGTP was identified by precipitationwith GST-RBD and immunoblotting using anti-Ras antibody.Fig. 8A demonstrates that GST-RBD bound RasGTP was in-creased significantly in cells transfected with PLCe, PLCeH1144L, and the known RasGEF, RasGRF2. The fold increasein RasGTP for PLCe, H1144L, and RasGRF was 4.2-, 9.3-, and4.1-fold, respectively. Although levels of expression for PLCeand the phosphodiesterase deficient mutant H1144L were verysimilar (Fig. 8B), H1144L was 2.2-fold more potent for activa-tion of Ras. As discussed in the previous section, H1144L wasalso more potent in activating MAP kinase. The increase inH1144L activity suggests that the phosphodiesterase activitymay serve as a negative feedback regulator of the RasGEFactivity. Levels of expression for Ha-Ras were similar for eachof the experimental conditions (Fig. 8B). There was no appre-ciable RasGTP detected in control cells. Basal levels of RasGTPwere reduced significantly by preincubation of the cells inserum-free medium for 12 h. Taken together, these resultsprovide more direct evidence that PLCe indeed activates Ras byacting as a RasGEF.

This work demonstrates that PLCe is a widely expressedunique PLC enzyme that constitutes a new PLC family pos-sessing bifunctional activity, both PLC and RasGEF activity.

As such, PLCe may mediate the effects of G protein-coupledreceptors, especially those coupled with Ga12/Ga13 through twodivergent pathways involving phosphatidylinositol hydrolysisas well as direct activation of the Ras/MAP kinase pathway.Thus, this new member of the PLC family may play a vital rolein transducing signals from the plasma membrane to the nu-cleus through multiple pathways to modulate cytoskeletalchanges, cell growth, and mitogenesis.

Acknowledgments—We thank Tatyana A. Voyno-Yasenetskaya forkindly providing the constitutively active Gas*, Gai*, Ga12*, Ga13*,Gaz*, Gao*, and Ga16* and David R. Manning for providing antibodiesagainst Gas, Ga12, and Ga13. We also thank Dr. Alan Wolfman andJohannes L. Bos for kindly providing GST-Raf1 minimal Ras-BindingDomain (GST-RBD), Mike Moran for providing RasGRF2, and MarkMarshall for v-Ras G12V and Ha-Ras. We also thank John Campion fortechnical assistance.

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Isabel Lopez, Eric C. Mak, Jirong Ding, Heidi E. Hamm and Jon W. Lomasneythe Ras/Mitogen-activated Protein Kinase Pathway

and Stimulates12αA Novel Bifunctional Phospholipase C That Is Regulated by G

doi: 10.1074/jbc.M008119200 originally published online October 5, 20002001, 276:2758-2765.J. Biol. Chem. 

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