Requirement of Phospholipase C-catalyzed Hydrolysis of ...

Val. 266, No . 11, Issue of April 15. pp. 6825-6829.1991 Printed In U.S.A.

THEJOURNALOF BIOLOCICAL CHEMISTRY 1991 by The American Society for Biochemistry and Molecular Biology, Inc. (K.

Requirement of PhospholipaseC-catalyzed Hydrolysis of Phosphatidylcholine for Maturation of Xenopus laevis Oocytes in Response to Insulin andras p21* (Received for publication, October 9, 1990)

Antonio Garcia de HerrerosS, Isabel DorninguezSg, Maria T. Diaz-Meco$Tl, Grazia GrazianiII, Maria E. Cornet$**,Per Henrik Guddal$$$§,Terje Johansen$$llll, andJorge Moscat$II 11 Prom the SMedicina y Cirugia Experimental, Hospital General “GregorioMaranon, Dr. Esquerdo 46, 28007 Madrid, Spain, the 11 Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutesof Health, Bethesda, Maryland 20892, and the $$Institute of Medical Biology, University of Tromso, 9001 Tromso, Norway I’

Recent studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine both by growth factors and by the product of ras oncogene, ras p21. Also, evidence has been presented indicating that the stimulation of this phospholipid-degradative pathway is sufficient to activate mitogenesis in fibroblasts. In Xenopus laevis oocytes, microinjection of transforming ras p21is a potent inducerof maturation, whereas microinjection of a neutralizing anti-ras p21antibody specifically inhibits maturation induced byinsulin but not by progesterone. The results presented here demonstrate that microinjection of phosphatidylcholine-hydrolyzing phospholipase C is sufficient to induce maturation of Xenopus laevis oocytes. Furthermore, microinjection of a neutralizing anti-phosphatidylcholine-hydrolyzing phospholipase C specifically blocks the maturation program induced by ras p2 l/insulin but not byprogesterone.

by growth factors (3-5). Recently,evidence has been presented showing that the activation of phosphatidylcholinehydrolyzing PLC (PC-PLC)by platelet-derived growth factor isanimportantstepinthe mitogenicsignaling pathways activated by this agonist ( 5 ) . PLC-mediated PC hydrolysis has also been shown to be stimulated by the product of ras oncogene, rasp21 (6-8), whose involvementinmitogenic cascades has been demonstrated (9, 10).Therefore,these results permit one to suggest that PC-PLC activation could be critically involved in pathways controllingcell growth and tumour transformation. Oocytes from Xenopus laeuis are recognized as a suitable systemfor investigating theinvolvement of different enzymatic activities in relevant signal transductionpathways (11, 12). Thus, Xenopus oocytesundergoa maturationprogram following thestimulation with either insulin or progesterone, and several lines of evidence indicate the specific involvement of ras p21 in the maturation signaling cascades activated by insulin: 1) microinjection of ras p21 activates maturation in oocytes (13); 2) microinjection of a neutralizing anti-rus p21 antibody(Y13-259) blocks the matPhospholipase C (PLC)’-mediated degradationof phospho- uration program induced by insulin but not by progesterone lipids has been implicatedin thecontrol of a number of (11).Also, because of the large size of Xenopus oocytes, the parameters of signal cellular functions (1).Although most of the studieshave been quantitation of severalbiochemical transduction pathways can be determined alongwith the focused on the phosphoinositide (PI) turnover, a number of recent reports suggest the existenceof PI-independent signal evaluation of the maturating capabilitiesof different stimuli. transduction pathways involving the phosphodiesterase-me- This allows a direct correlation between both types of measdiated hydrolysis of phosphatidylcholine (PC)(2). Both phos- urements. The aim of this study was to establish theinvolvepholipid-degradative routes have been shown to be activated ment and importance of PC-PLC in the maturationprogram activated by insulin and ras p21. We demonstrate here that PLC-mediated hydrolysis of PC is both necessary and suffi*This work was supported in part by Grant SAL90-0070from Comision Interministerial deCiencia y Tecnologia. Thecosts of cient for activation of maturation of X . laeuis oocytes by publication of this article were defrayed in part by the payment of insulinlras p21.

page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate MATERIALSANDMETHODS this fact. $ Fellow from the Gobierno Vasco. Oocyte CultureandLabeling-Oocytes were prepared following !I Fellow from the Ministerio de Educacion. standard procedures (14).Briefly, ovaries from X . laeuis frogs (Blades ** Fellow of the Comunidad de Madrid. Biologicals, United Kingdom) were incubated with 2 mg/ml collagen$0 Research Fellow of the Norwegian Research Council for Science ase (Boehringer Mannheim) for 45 rnin in modified Barth solution and the Humanities. without ca’+ (110 mM NaCI, 2 mM KCI, 1 mM MgCI,, 1 mM CaC12, 2 lln Postdoctoral Fellow of the NorwegianResearchCouncilfor mM NaHCO:,, 10 mM HEPES, pH7.8).After extensive washing, stage Science and the Humanities. VI oocytes were selected and incubated overnight at 20 “C. Selected 11 )I To whom correspondence and reprint requests should be ad- oocytes were labeled according to the experiments either with 1 2 pCi/ dressed. Tel. (1) 586 8312; Fax: (1) 586 8018. ml [rnethyl-14C]choline(Amersham International;specific radioactiv1 Theabbreviations used are:PLC,phospholipase C; PC-PLC, ity 55 mCi/mmol) or with 24 pCi/ml [U-’4C]glycerol (Amersham phosphatidylcholine-hydrolyzing phospholipase C; PLD, phospholiInternational; specific radioactivity 141 mCi/mmol) for 24 h in modpase D; GVBD,general vesicle breakdown; PA, phosphatidic acid; ified Barth solution, after whichmedium was removed and fresh, PC, phosphatidylcholine; PCho, phosphocholine; PI, phosphoinosilabel-free medium was added, and experimentswere carried out after tides;PEt,phosphatidylethanol; DAG, diacylglycerol; PBS,phosa 30-min equilibrationperiod. For determination of levels of different phate-buffered saline; HEPES, 4-(2-hydroxyethyl)-l-piperazineeth- phospholipids, cells were labeled as follows: for PC and sphingomyeanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic lin, as above; for phosphatidylethanolamine, with 10 pCi/ml [2-14C] acid. ethan-1-olamine (specific radioactivity 55 mCi/mmol); for phospha-

6825

6826

Phosphatidylcholine Hydrolysis

i n Oocyte Maturation

tidylserine, with 10 pCi of ~-[U-'~C]serine (specific radioactivity 55 Analysis of Oocyte Maturation-Groups of 20 oocytes were cultured mCi/mmol); for polyphosphoinositides,with 10 pCi of myo-[2-'HH] at 20 "C in modified Barth solution; and germinal vesicle (nuclear) inositol (specific radioactivity 16.3 Ci/mmol). breakdown (GVBD) was assessed by the appearance of a white spot Analysis of Products of Phospholipid Metabolism-Labeled oocytes in the animalpole. In some cases, nuclear breakdown was confirmed were treated or not with the corresponding agonists or were microinby dissection of trichloroacetic acid (10%)-fixed oocytes (24). jectedwith Bacillus cereus PC-PLC or with rus p21. At different Histone 1 Kinase Assay-Twenty oocytes were homogenized in a times, reactions were stopped by adding ice-cold methanol. Methabuffer containing20 mM HEPES, pH 7.0,lO mM @-glycerophosphate, nolic cell extracts were fractionated into chloroform and aqueous 5 mM EGTA, 5 mMMgC12, 50 mM NaF, 2 mM dithiothreitol, 100 pg phases aspreviously described (15). Thepresence and levels of water- of leupeptin/ml, and100 p~ phenylmethylsulfonyl fluoride. Following soluble choline metabolites were evaluated in the aqueous phases by centrifugation at 13,000 X g for 15 min, extracts(1-2 mg/assay) were thin layer chromatography (16),followed by autoradiography ofplates assayedfor 10 min at 30 "C in a finalreaction volume of 50 wl in which standards corresponding to the different water-soluble cho- containing 20 mM HEPES, pH 7.0,5mM P-mercaptoethanol, 10 mM line metaboliteswereincluded.Organic phases from methanolic MgCI?, 100 p M [T-"'P]ATP (2-5 dpm/fmol), 0.2 pg of heat-stable extracts were dried down under N,, and lipids were fractionated by inhibitor of CAMP-dependent protein kinase, and 0.6 mg/ml Sigma thin layer chromatography using the following solvent systems. For type 111-S calf thymus histone. Reactions were terminated, spotted theseparation ofDAG: hexane:diethylether:aceticacid (60:40:1; onto WhatmanP-81phosphocellulose paper, washed, and quantitated v/v).Fortheseparation of different phospholipids,chloro-form: as described (25). methano1:ammonia (65:25:4) (v/v) was used in the first dimension, and ch1oroform:acetone:methanol:acetic acid:water (30:40:10:10:5) RESULTS (v/v) was used in the second dimension. For determination of PA Activation of PLC-mediated Hydrolysis of Phosphatidylchoand phosphatidylethanol production,lipid extracts from [U-'4C]glycerol-labeled oocytes were fractionated with the upper phase of the line in Xenopus Oocytes-We initially determined whether following solvent system: ethyl acetate:trimethylpentane:acetic addition of insulin or progesterone or microinjection of transacid:water (90:50:20:100) (v/v). Different lipids were visualized after forming ras p21 activated the phosphodiesterase degradation autoradiography of plates where the corresponding standards were of PC. Thus, the release of phosphocholine (PCho), which included. along with DAG arises after the activation of PC-PLC, was Isolation of PC-PLC fromBacillus cereus and Preparationof Affinity-purified Antibody-PC-PLC was isolated from cultures of B. cer- measured a t different times following the addition of either 1 eus SE-1essentially as described by Myrnes and Little (17). Following p M insulin or 1 p M progesterone to [rnethyl-14C]choline-laJohansen et al. (18)protocol, the enzyme preparation was purified to beled X . laevis oocytes. Results from Fig. 1 indicatethat complete homogeneity as confirmed by sodium dodecyl sulfate-polyinsulin promotesa potent release of PCho which is detectable acrylamide gel electrophoresis followed by silverstaining. The specific by 1.5 h and maximal by 4 h after agonist addition. Progesactivity of the purified enzyme was 1.5 units/pg (19). A rabbit antiterone produced little or no effect on this parameter (Fig. 1). serum was raised against this B. cereus PC-PLC by multiple intradermal injections with 75 pg of this enzyme. Serum was diluted 1:3 Microinjection of transforming (Fig. 1)but not normal (not in phosphate-buffered saline (PBS) and applied to an Affi-Gel 10 shown) ras p21 promotes a rapid and dramatic increase in (Bio-Rad) column containing immobilized B. cereus PC-PLC. The PCho levels which is detectable by 5 to 10 min and maximal column was washed with PBS, with PBS withincreasing salt (up to by 1 h. Therefore,theseresults suggest thatPC-PLCis 1 M NaCI), and with PBS containing 3 M urea before elution with 4 activated by insulinand ras p21andareconsistent with M urea, 0.5 M NaCl adjusted to pH3.0 with acetic acid. The affinityprevious reports demonstrating thatras p21 is an important purified antibody was eluteddirectly into 1 M glycine-NaOH, pH 10.5, and dialyzed extensively against a suitable buffer. The sole intermediary in the maturation cascade induced by insulin presence of heavy and light antibody chains in the final preparation but not by progesterone (11, 13). Of note is that no evidence was confirmed by sodium dodecyl sulfate-polyacrylamide gel electro- of stimulation of PI turnover was found in [3H]inositolphoresis followed by silver staining. labeled oocytes either stimulated with insulin (1 p M ) or proPreparation of ras p21 Proteins-Transforming and normal ras gesterone (1 w ~ or) microinjected with 4 ng of transforming p21 proteins were expressed in bacteria as previously described (20). ras p21 (data not shown). A final step of purification consisted in a gel filtration chromatograA potentially alternativesource of PCho inras p21-microinphythrough a Sephadex G-100column(2.5 X 90cm);fractions containing the purified protein were pooled and dialyzed extensively jected oocytes could be the activation of a choline kinase. In against 20 mM Tris-HCI, pH 7.5, to remove urea and kept a t -70 "C order torule out thispossibility, we measured PCho synthesis until utilized. in X . laevis oocytes that were either untreated or microinjected Determination of PI-specific PLC and PC-PLD ainCell-free Assay for different times with 4 ng of transforming ras p21. These System-Oocytes were extracted by10 strokesin a glass/Teflon Potter homogenizer in 0.25 M sucrose, in the following buffer 0.1 mM ' l A dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM EGTA, m and 50 mM Tris-HCI, pH 7.4. The homogenate was centrifuged twice 1.2 r 1.21 a t 20,000 X g for 15 min a t 4 "C; supernatants were collected and r centrifuged at 100,000 X g for 60 min a t 4 "C. High speedsupernatants were decanted and stored at-70 "C. x 1 PI-PLC was routinely assayed either with ~-3-phosphatidyl[2-''H] E Q inositol (Amersham; specific radioactivity 10-15 Ci/mmol) or phos0 0.8 phatidyl[l,2-:'H]inositol4,5-bisphosphate(DuPont-NewEngland Nuclear; specific radioactivity 3.6 Ci/mmol), as described previously 0.6 (21, 22) without a final Ca" concentration of 80 p M . For the measV urement of phosphatidylinositolandphosphatidylinositol 4,5-bisa phosphate activities, the release of inositol phosphate and inositol 0.4; ' ' ' ' ' ' ' 1 2 3 4 5 6 7 8 trisphosphates was measured as described (21). To measure PC-PLD in oocyte extracts, apreviouslydescribed Time (hr) protocol was followed (23), by using 1,2-dipalmitoyl-sn-glycerol-3FIG. 1. Time course of PCho release in response to the adphosphoryl[N-methyl-"H]choline (Amersham International; specific radioactivity 40-80 Ci/mmol). The amount of [N-methyl-"Hlcholine dition of insulin, progesterone,or to the microinjection of ras X.laevis oocytes were either incureleased which was separated from phosphorylcholine as described p21. [rnethyl-14C]Choline-labeled (16) representsthe hydrolytic activity of PLD. To obliterate [N- batedwith 1 p~ insulin (B)or 1 p~ progesterone ( X ) or were methyl-:'H]choline from hydrolysis of phosphoryl[N-methyl-"Hlcho- microinjected with 4 ng of transforming ras p21 ( O ) ,for different line originat.ed by PC-PLC, 10 mM unlabeled phosphorylcholine, and times, after which PCho levels were measured. Results, expressed in disintegrations/min/5 oocytes, are representative of three other ex50 p~ p-nitrophenyl phosphate,a phosphatase inhibitor,were present periments with essentially identical results. in the incubation media.

A

r"

I

'

Phosphatidylcholine Hydrolysis in Oocyte Maturation

6827

absence or in the presence of 0.5% oocytes were microinjected with 1 pCi of [methyl-'4C]choline ras p21 either in the for 15min prior to terminationof the reactions. By using this ethanol. It has been demonstrated that the presence of this experimental approach, the choline-containing phospholipid alcohol in cell systems where a PLD is activated leads to the pool was not significantly labeled. Importantly, little or no accumulation of the metabolically stable phosphatidylethanol change was detected in PCholabeling in oocytes microinjected (PEt) (8, 27). If PA was actually being originated as a consequence of ras microinjection, P E t should be detected in those with ras p21 as compared to controls (data not shown). These oocytes microinjected in the presenceof ethanol. Our results results indicate that ras p21 produces no changes in PCho unless phospholipids are labeled, which strongly supports the indicate that PEtlevels in oocytes microinjected with ras p21 presence of notion that ras p21 activatesa PC-specific phosphodiesterase (235 f 15 dpm/oocyte) do notchangeinthe ethanol (258 f 32 dpm/oocyte). PEt levels in control oocytes degradative pathway. On the other hand, although we did not detect any changes in the presenceof ethanol were 255 f 25 dpm/oocyte and 238 in cholinelevels, ras-induced PChorelease couldtheoretically f 15 dpm/oocyte in its absence. Again, as a control, microinalso be accounted for by the rapid action of a phospholipase jection of 50 microunits of PLD leads to the release of PA by D (PLD). Thus, PLD-mediated hydrolysis of PC would give 5 min (Table I). The presence of 0.5% ethanol under these conditions promotes the appearance of PEt (405 f 32 dpm/ rise to choline which could be rapidly converted to PCho. This PLD pathway would also originate phosphatidic acid oocyte uersus 255 f 25 in control oocytes). These data are in (PA) which upon the actionof a PA-phosphohydrolase would good agreement with recently published results in fibroblasts produce diacylglycerol (DAG). Therelease of DAG is potently (8). PLC-mediated Hydrolysis of Phosphatidylcholine Promotes activatedin X . laevis oocytesmicroinjected with ras p21 (Table I) in accordance with previously publishedresults (12); Maturation of Xenopus Oocytes-To evaluate the possible these changes in DAG were not accompanied by alterations importance of PC-PLC activation in the maturating capabilin PA levels (Table I). This strongly suggests that ras-acti- ities of ras p21 and insulin, we examined in a time course vated PC hydrolysis takes place through the activation of a whether or not PC-PLC was sufficient to trigger the matuPLC instead of a PLD. However, from these results, a rapid ration response in oocytes. For this purpose,we used a highly and therefore undetectable stimulation of PLD cannot be purified, permanently activated PC-PLC from E . cereus that completely ruled out. This together with a very active PA- has been characterized extensively (18). Thus, oocytes were phosphohydrolase could theoretically account for the release either stimulatedwith 1 p~ insulin or they were microinjected of DAG observed in ras p21-microinjected oocytes. If this with4ng of transforming ras p21 or 25 microunits of B. vesicle breakdown (GVBD), were the case, treatment of X . laeuis oocytes withpropranolol, cereus PC-PLC, and the germinal a very well known inhibitor of PA-phosphohydrolase (26), a good parameter of oocyte maturation, was determined at should inhibit the productionof DAG by ras p21 and should differenttimesthereafter.Results from Fig. 2 show that favor the accumulation of PA. Results from Table I clearly microinjection of ras p21 induces maturation in oocytes to an produced by insulin, inaccordance with oocytes were microin- extent similar to that show thatwhen [U-'4CC]glycerol-labeled previously published results (13).Also from Fig. 2, it is clear jected with transforming ras p21 in the presence of 200 p~ propranolol, no changes were detected either in the production that ras p21 promotes oocyte maturation more rapidly than GVBD induced by insulin is detectable of DAG or in the levels of PA. As a control, microinjection of insulin. Thus, whereas 50 microunits of PLD from peanut into [U-14C]glycerol-la- by 5.5 h and gives a maximal value by 6.5 h after agonist beled oocytes lead to the release of PA by 5 min and to the addition, the microinjection of ras p21 promotes significant production of DAG by 15 min (Table I). The presence of maturation by 3 h, being this parametermaximal by 5 h after propranolol inhibited the productionof DAG and favored the microinjection.More importantly,maturation of X . laevis accumulation of PA released as a consequence of PLD mi- oocytes following microinjection of 25 microunits of B. cereus PC-PLC, which promotes PC hydrolysis to an extent similar croinjection (Table I). Takentogether,alltheseresults strongly support a model whereby ras p21 activates directly to that produced by insulin or ras p21 (Fig. 3A), slightly or indirectly a PLC specific for PC in X . laeuis oocytes. In precedes GVBD induced by ras p21 (Fig. 2 ) . Thus, significant ordertofurthersubstantiatethisnotion, [U-'4C]glycerol- maturation is detected as early as2 h after microinjection of labeled oocytes were microinjected with 4 ng of transforming B. cereus PC-PLC, being maximal GVBD values attained by 4 h. Of noteisthat microinjection of B. cereus PC-PLC TABLEI Effect of propranolol on diacylglycerol and phosphatidic acid levels of X . laevis oocytes microinjected either with transformingras p21 or with phospholipase D [U-'"C]Glycerol-labeledX.laevis oocytes were either uninjected or microinjected with 4 ng of ras p21 or with 50 microunits of phospholipase D (PLD, Sigma), either in the absence or in the presence of 200 p M propranolol (Prop). Reactions were stopped, and DAG and PA levels were determined as described under "Materials and Methods." Results are meank S.D. of three independent experiments with incubations in duplicate. 5 min

15 min

Treatment

DAG

DAG

PA

PA

dpmloocyte

None Prop ras p2 1 ras p21 + Prop PLD PLD + Prop

2 2 0 2 30 225 4 25 3 4 0 % 20 350 2 25 240 f 24 235 2 21

320 f SO 3 3 0 f 45 3 1 8 k 30 330 f 25 640 f 50 800 -c 72

2 4 0 f 25 222 f 20 450 f 25 480 f 41 380f 24 225 f 15

335 f 39 3 5 0 f 25 330 f 40 325 f 29 800 f 70 1200 180

1 Ime ( r i r )

FIG. 2. Time course of induction of X. laevis oocyte maturation by different agents. Selected oocyteswere either incubated in the presence of 1 pM insulin (W) or were microinjected with 4 ng of ras p21 (0)or with 25 microunits of B. cereus PC-PLC (A),and the breakdown of the germinal vesicle was determined at different times thereafter. Essentially identical results were obtained in three other experiments.

Phosphatidylcholine Hydrolysis in Oocyte Maturation

6828 .

TABLE111 Activation of HI kinase by B. cereus PC-PLC and ras p21 Oocytes were microinjectedeither with 10 ng of ras p21 or with 25 microunits of B. cereus PC-PLC for different times, afterwhich they werehomogenized andH1kinaseactivity was determined as described under “Materials and Methods.” Results are meanf S.D. of three independent experiments with incubations in duplicate. ND, not determined.

I

Treatment

l h

4h

fmollminloocyte

None

Fln . “I

x

I

B. cereus PC-PLC ras p21

72 f 6 176 f 15 ND

76 ? 5 550 k 40 325 f 21

60

It has extensively been characterized that oocyte maturation is activated by a protein kinase complex called maturationpromotingfactor whichincludes p34CDC28/cdc2+ (for a > 0 review, see Ref. 28). The activity of this complex can easily 20 be followed by its ability, when activated, to phosphorylate histone H1 (28). Therefore, in order to further establish that r: 0 PC-PLC actually controls thebiochemical mechanisms activating oocyte maturation, H1 kinase activity was determined FIG. 3. Effect of anti-B. cerehs PC-PLC antibody on PCho at different times following microinjection of 25 microunits release and maturation of X . laevis oocytes. A, [methyl-I4C] of B. cereus PC-PLC or 10 ng of ras p21. Results from Table choline-labeledoocyteswere either untreated (dotted) or microinI11 show that microinjection of B. cereus PC-PLC actually jected with 80 ng of anti-B. cereus PC-PLC antibody (striped), after activates oocyte’s H1 kinasetoanextentsimilartothat which they were either untreated or microinjectedeitherwith 25 produced by ras p21. microunits of B. cereus PC-PLC or with4 ng of transforming ras p21, PC-PLC Activity Is Required for Maturation Induced by ras orthey wereincubatedin the presence of 1 p M insulin or 1 p M progesterone. Reactions were stopped 1 h after microinjection of B. p21 and Insulin-The results presented so far demonstrate cereus PC-PLC or ras p21,or 4 h after the addition of hormones. that PC-PLC is sufficient to trigger the maturation cascade PCho levels were determined as described in the legend to Fig. 1. B, of oocytes in a time course compatible with the activationof selected oocytes were treated as described above, and the breakdown PLC-mediated PC hydrolysis by ras p21 and by insulin. To of the germinal vesicle was determined 3 h after microinjection of B. demonstrate that PC-PLC stimulationby these two agents is cereus PC-PLC or ras p21, or 7 h following the addition of hormones. actually required for the controlof maturation, itis necessary Essentially identical results were obtained inthree other experiments. to establish that the blockade of PC-PLC abolishes the ability of insulin and ras p21 but not of progesterone to induce TABLEI1 maturation in oocytes. For this purpose,we used a neutralizSpecificity of B. cereus PC-PLC ing affinity-purified anti-B. cereus PC-PLC polyclonal antiX . laeuis oocytes were labeled with different precursors as described body ( 5 ) . It has previously been shown by Clark et al. (29) under “Materials and Methods”after which they were microinjected to B. cereus PC-PLC cross-react with 25 microunits of B. cereus PC-PLC for 30 min. Reactions were that antibodies prepared with a PC-PLC in mammaliancells. We have confirmed and stoppedandlipids were extractedandfractionatedasdescribed. Results are expressed as percent of control values and are mean f extended these findings.’ Thus, preliminary results with this S.D. of three independent experiments performed in duplicate. Con- antibody indicate that it inhibits and immunoprecipitates a trol values for different phospholipids were as follows: PC, 128,000 mammalian PC-PLC activity detected in lysatesfrom Swiss dpm/oocyte; sphingomyelin, 8,800 dpm/oocyte; phosphatidylethanol-3T3 fibroblasts’; also, it recognizes a single band in immuamine, 104,000 dpm/oocyte; phosphatidylserine, 19,300 dpm/oocyte; noblots from lysates of Xenopus laeuis oocyteswhich are polyphosphoinositides, 35,000 dpm/oocyte. competed by preincubation of the antibody with B. cereus Phospholipid levels” (% control) PC-PLC.2 Therefore,we initially examined whethermicroinAddition PC PE PS SM PIPs X. jection of this antibody into [nethyl-’4C]choline-labeled lueuis oocytes prevented PChorelease in responseto B. cereus 100 100 100 100 100 None H. cereusPC-PLC 75-C 5 91 + 8 97 f 3 1 O O f 3 l o o + 5 PC-PLC, ras p21, or insulin. Results from Fig. 3A show that (25 microunits) the ability of B. cereus PC-PLC,insulin,andrasp21 to ” The abbreviations used are: SM, sphingomyelin; PE, phospbati- activate the phosphodiesterase hydrolysis of PC was comdylethanolamine; PS, phosphatodylserine; PIPs, polyphosphoinosi- pletely abolished in oocytes that have previously been mitides. croinjected with anti-B. cereus PC-PLC antibody. NO effects on the release of PCho in response to microinjection of B. or no cereus PC-PLC or rus p21 or to the addition of insulin were promotestheprompt hydrolysis of PCwithlittle changes in the levels of other lipids (Table 11), in good observed in oocytesmicroinjectedwith nonrelevant rabbit agreement with previously published data inSwiss 3T3 fibro- IgG (data not shown). These results indicate that the anti-B. blasts (5). Taken together, all these results indicate that the cereus PC-PLC antibody actually inhibits not only B. cereus simple microinjection of a permanently activated PC-PLC PC-PLC but also the oocyte’s enzyme that is activatable by from B. cereus is capable of triggeringafull maturation insulin and ras p21. As a further control of the specificity of response in X. laeuis oocytes. Furthermore, the time course this antibody, we sought to determine whether it inhibited shown in Fig. 2 is in good agreement with the idea that ras PC-PLD or PI-PLC activities in a Xenopus oocyte cell-free p21 and PC-PLC activation are proximal events(Fig. 1; Ref. 8),and that both appear tobe late intermediary steps in the ’ I. Dominguez, M. E. Cornet, A. Garcia de Herreros, andJ . Moscat, unpublished observations. maturation-signaling cascade activated by insulin. 40

PhosphatidylcholineHydrolysis in Oocyte Maturation

6829

system. Our results demonstrate that the presence of either paper) further supports the idea that PC-PLC is a specific nonrelevant IgG or anti-B. cereus PC-PLC antibody (100 pg/ critical step in the maturation cascade activated by insulin/ ml) did not affect the activities of either PC-PLD (6.2 & 0.3 ras p21. On the other hand, the time course of the effect of nmol of choline/h/mg of protein) or PI-PLCs (phosphatidyl- B. cereus PC-PLC and ras p21-induced maturation differs inositol hydrolytic activity: 6.5 0.7 nmol of inositol phos- significantly from that produced by insulin (Fig. 2). In addiphate/min/mg of protein; phosphatidylinositol 4,5-bisphos- tion, the kinetics of induction of PC-PLC by ras p21 also phate hydrolytic activity: 3.4 k 0.2 nmol of inositol trisphos- differs from that activated by insulin (Fig. 1).From all this, concluded that ras p21 and PC-PLC, whose activities phate/min/mg of protein). Therefore, this antibody is a useful it can be crucial tool for investigating therole of PLC-mediated PChydrolysis appear to be proximal to each other, are both late in the controlof maturation inoocytes. Consequently, oocytes events in signal transduction cascades. A similar conclusion with a were either untreated or microinjected with anti-B. cereus was drawn from experiments carried out in fibroblasts PC-PLCantibodyafter which they were stimulated with mutant of ras oncogene which is temperature-sensitive for either 1 p~ insulin or 1 p M progesterone, or they were mi- transformation (8).Data presentedin that papershowed that croinjected either with25 microunits of B. cereus PC-PLC or activation of ras p21 leads to a more rapid stimulation both with 4 ng of ras p21. Results from Fig. 3B show that oocytes of PC hydrolysis and mitogenesis than in control cells trigwhereby microinjectedwith anti-B. cereus PC-PLCantibody were gered with serum(8).Although the exact mechanism unable to maturate in response B. to cereus PC-PLC, ras p21, ras p21 activates PLC-mediated hydrolysis of PC remains to or insulin. However, microinjection of anti-B. cereus PC-PLC be clarified, the results presented here indicate that PC-PLC antibody did not block the induction of GVBD by progester- should clearly be considered one of the important targets of one (Fig. 3B).Microinjection of a nonrelevantrabbit IgG ras action. produced little or no effect on the maturationinduced by any of these stimulants. Taken together, these results demonstrateAcknowledgments-We are grateful toStuart A. Aaronson for suggestions and critical reading of this manuscript. The technical a specific criticalstepinthe thatPC-PLCactivationis assistance of M" Jeslis Sanchez isgreatly appreciated. maturation program induced by insulinlrus p21 but not by progesterone. REFERENCES

*

1. Berridge, M. J. (1987) Annu. Reu. Biochem. 5 6 , 159-194 2. Exton, J. H. (1990) J. Biol. Chem. 2 6 5 , 1-4 3. Besterman, J. M., Duronio, V. & Cuatrecasas, P. (1986) Proc. Natl. Acad. Sei. U. S. A. 8 3 , 6785-6789 A number of recent studies suggest that PLCs could be 4. Pessin, M. S., Baldassare, J. J. & Raben, D. M. (1990) J. Eiol. Chem. 2 6 5 , importantstepsinthecontrol of cell growth andtumor 7959-7966 5. Larrodera, P., Cornet, M. E., Diaz-Meco, M. T., Lopez-Barahona, M., Diaztransformation (1-8). This is based, in part, on the fact that Laviada, I., Guddal, P. H., Johansen, T. & Moscat, J. (1990) Cell 6 1 , several growth factors as well as certain oncogene products 1113-1120 6. Lacal, J. C., Moscat, J. & Aaronson, S.A. (1987) Nature 3 3 0 , 267-272 potentlyactivatethese phospholipid-degradative pathways 7. Price, B. D., Morris, J. D. H., Marshall, C. J. & Hall, A. (1989) J. Eiol. (3-8). In fibroblasts, microinjection of PI-PLC or the exogeChem. 2 6 4 , 16638-16643 8. Lopez-Barahona, M., Kaplan, P. L., Cornet,M. E., Diaz-Meco, M. T., nous addition of PC-PLC both promote DNA synthesiswith Larrodera, P., Diaz-Laviada, I., Municio, A. M. & Moscat, J. (1990) J . a kinetic consistent with a model whereby PI-PLC may be Biol. Chem. 265,9022-9026 9. Mulcahy, L. S., Smith, M. R.& Stacey, D. W. (1985) Nature 313,241-243 considered an early step whereas PC-PLC activationa islate 10. Smith, M.R., DeGudicihus, S. J. & Stacey, D. W. (1986) Nature 320,540event in the mitogenic cascade ( 5 , 30). Neither insulin, pro541 gesterone (31), norras p21 (this paper)trigger P I turnover in 11. Korn, L. J., Siebel, C. W., McCormick, F. & Roth, R. A. (1987) Science 236,840-842 X . luevis oocytes at the concentrations used in the experi12. Lacal, J. C., Pefia,P.,Moscat, J., Barreno, P. G., Anderson,P. S. & ments shown here (4-10 ng/oocyte), despite the fact that they Aaronson, S.A. (1987) Science 238,533-536 13. Birchmeier, C., Broek, D. & Wigler, M. (1985) Cerl43, 615-621 are able to induce a full maturating response. Although mi14. Colman, A. (1984) in Tramcription and Translatlon: A PracttcalApproach (Hames, B. D. & Higgins, S. J., eds) pp.271-300, IRL Press, Washington, croinjection of 20-fold higher concentrations (80-150 ng/

DISCUSSION

oocyte) of transforming ras p21 promotes a slight increase ( 1 5 2 0 % ) in PI turnover metabolites (data not shown and Ref. 12), itdoes not appear to be significant from a functional point of view. All these results strongly suggest that at least in this particular system, P I hydrolysis does not appear to play a meaningful role in mitogenic signal transduction. The lack of effect of ras-mediated transformation onP I turnover has previously been reported in other cellular systems (6-8). It has been reported that besides PLC, a PLD specific for PC can also be involved in the activation of PC turnover in several cell systems (for recent reviews, see Refs. 2 and 32). Although from our results a PLD does not appear to play a role in the signaling mechanisms activated by ras p21 (8, and this paper), its importance in the control of other cellular functions is an exciting possibility. The datashown here also demonstrate that PC-PLC activity is not only sufficient to induce maturation in X . laeuis oocytes, but also we show that activation of PC-PLC by ras p21 and insulin is required to induce maturation by these agents. The evidence that progesterone-activated GVBD is not affected by the blockade of ras p21 (11) or PC-PLC (this

D.C.

15. Bligh, E. & Dyer, W. (1959) Can. J . Eiochem. Physiol. 37,911-917 16. Diaz-Meco, M. T., Larrodera,P.,Lopez-Barahona, M., Cornet,M. E., Barreno, P. G. & Moscat, J. (1989) Eiochem. J. 2 6 3 , 115-120 17. Myrnes, B. J. & Little, C. (1980) Acta Chem. Scand. Scr. E Org. Chem. Biochem. 32,375-377 18. Johansen, T., Holm, T., Guddal, P. H., Sletten, K., Haugli, F. B. & Little, C. (1988) Gene (Amst.)65,293-304 19. Little, C. (1988) Eiochem. SOC.Trans. 17,271-273 20. Lacal, J. C., Santos, E., Notario, V., Barbacid, M., Yamakazi,S., Kong, H., Seamans, C., McAndrew, S. & Crowl, R. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1 , 5305-5309 21. Moscat, J., Herrero, C., Garcia-Barreno, P. & Municio, A. (1987) Eiochem. J. 242,441-445 22. Herrero, C., Cornet, M. E., Lopez, C., Garcia-Berreno, P. & Municio, A. M. (1988) Eiochem. J. 255,807-812 23. Quian, Z. & Drewes, L. R. (1989) J. Eiol. Chem. 2 6 4 , 21720-21724 24. Wasserman, W. J., Houle, J. G. & Samuel, D. (1984) Deu. Eiol. 105,315324 25. Glass, D. B., Masarachia, R. A,, Feramisco, F. R. & Kemp, B. E. (1978) Anal. Eiochem. 87.5704-5708 26. Billah, M. M., Eckei, S.,Mullmann, T. J., Egan, R. W. & Siegel, M. I. (1989) J. Eiol. Chem. 2 6 4 , 17069-17077 27. Billah, M. M., Pai, J., Mullmann, T. J., Egan, R. W. & Siegel, M. I. (1989) J. B i d . Chem. 264,9069-9076 28. Murray, A. W. & Kirschner, M. W. (1989) Nature 3 3 9 , 275-286 29. Clark, M. A., Shorr, R. G . L. & Bomalaski, J. S. (1986) Eiochem. Eiophys. Res. Commun. 1 4 0 , 114-119 30. Smith, M. R., Ryu, S.,Suh, P., Rhee, S. & Kung, H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3659-3663 31. Pan, B. T. & Cooper, G . M. (1990) Mol. Cell. Bid. 1 0 , 923-929 32. Billah, M. M. & Anthes, J. C. (1990) Eiochem. J. 269, 281-291