MOLECULAR AND CELLULAR BIOLOGY, Feb. 1993, p. 1290-12950270-7306/93/021290-06$02.00/0Copyright X 1993, American Society for Microbiology

Inhibition of Protein Kinase C 4 Subspecies Blocksthe Activation of an NF-KB-Like Activity in

Xenopus laevis OocytesISABEL DOMINGUEZ,' LAURA SANZ,1 FERNANDO ARENZANA-SEISDEDOS,2MARIA T. DIAZ-MECO,1 JEAN-LOUIS VIRELIZIER,2 AND JORGE MOSCATV*Centro de Biologia Molecular UAM-CSIC, Canto Blanco, 28049 Madrid, Spain, 1 and

Laboratoire d'Immunologie Virale, Institut Pasteur, Panis, France2

Received 27 July 1992/Returned for modification 28 August 1992/Accepted 28 October 1992

Nuclear factor KB (NF-KB) plays a critical role in the regulation of a large variety of cellular genes. However,the mechanism whereby this nuclear factor is activated remains to be determined. In this report, we presentevidence that in oocytes from Xenopus laevis, (i) ras p21- and phospholipase C (PLC)-mediated phosphatidyl-choline (PC) hydrolysis activates NF-KB and (ii) protein kinase C C subspecies is involved in the activation ofNF-cB in response to insulin/ras p21/PC-PLC. Thus, the microinjection of either ras p21 or PC-PLC, or theexposure of oocytes to insulin, promotes a significant translocation to the nucleus of an NF-KB-like activity.This effect is not observed when oocytes are incubated with phorbol myristate acetate or progesterone, both ofwhich utilize a ras p21-independent pathway for oocyte activation. These data strongly suggest a critical roleof the insulin/ras p21/PC-PLC/protein kinase C C pathway in the control of NF-KB activation.

Although nuclear factor KB (NF-KB) was originally iden-tified as a complex binding to the KB enhancer element of thegene coding for the immunoglobulin light chain (35), it nowseems apparent that it is involved in the regulation of arelatively large variety of cellular genes (for reviews, seereferences 16 and 22). An important example is the bindingof NF-KB to the enhancer of human immunodeficiency virus(HIV), which appears to be critical in viral activation (26).NF-KB is located in the cytoplasm of resting cells as an

inactive form complexed to an inhibitory protein termed IKB(3, 4). Upon cell activation, the dissociation of the NF-KB/IKB complex takes place and NF-KB is translocated to thenucleus, where it carries out its transactivating function (3,4). NF-KB is composed of two different subunits: p50, a50-kDa DNA-binding protein, and an associated 65-kDaprotein, p65, with apparently weak DNA-binding properties(5, 25). The latter is the subunit that interacts with IKB (28,31). The genes coding for both NF-KB components haverecently been cloned and sequenced. Both show a highhomology to c-rel and v-rel and to the Drosophila maternalmorphogen dorsal (28, 31).The mechanisms that regulate the dissociation of the

NF-KB/IKdB complex and the resulting activation of NF-KBare unclear. Thus, although from a series of in vitro exper-iments, activation of protein kinase A (PKA) and PKC andthe subsequent phosphorylation of IKB have been proposedto be a major stimulatory pathway for NF-KB (15, 36),additional studies suggest that other signaling mechanismsoperate in vivo (18, 24). In this regard, it is noteworthy thatactivation of NF-KB translocation in response to tumornecrosis factor alpha (TNF-ao) is not sensitive to stauro-sporin, a broadly specific inhibitor of protein kinases, includ-ing PKC (18, 24). Therefore, even though a PKC artificialstimulant like phorbol myristate acetate (PMA) may be ableto activate NF-KB translocation, the role of this kinase inresponse to a natural agonist in vivo is not as apparent.

* Corresponding author.

In the investigation presented here, oocytes fromXenopuslaevis were used as a model system. Oocytes are recognizedas suitable for investigating the involvement of differentenzymatic activities in relevant signal transduction pathways(6, 20). Xenopus oocytes undergo, for example, a maturationprogram following stimulation with either insulin or proges-

terone, and several lines of evidence indicate the specificinvolvement of ras p21 in the signaling cascades activated byinsulin but not in those activated by progesterone (20). Also,because of the large size of Xenopus oocytes, distinctbiochemical parameters of signal transduction pathways canbe quantitated along with evaluation of the functional capa-bilities of different stimuli. This analysis can be carried outfollowing the microinjection of relatively large molecules,like the products of oncogenes, or potentially inhibitorypeptides. This approach allows a direct correlation betweenboth types of measurements (i.e., biochemical and func-tional parameters of signaling pathways).

In this report, we present evidence for the involvement ofPKC t subspecies (t-PKC) in the activation of an NF-K<B-likeactivity by insulin/ras p21/phosphatidylcholine (PC)-phos-pholipase C (PLC) in Xenopus oocytes.

MATERIALS AND METHODS

Oocyte culture. Oocytes were prepared in accordance withstandard procedures (14). Briefly, ovaries from X. laevisfrogs (Blades Biologicals, Oxford, United Kingdom) wereincubated with collagenase (2 mg/ml; Boehringer, Mann-heim, Germany) for 45 min in modified Barth solutionwithout Ca2" (110 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1mM CaCl2, 2 mM NaHCO3, 10 mM N-2-hydroxyethylpiper-azine-N'-2-ethanesulfonic acid [HEPES] [pH 7.5]). Afterextensive washing, stage VI oocytes were selected andincubated overnight at 20°C.

Isolation of PC-PLC from BaciUus cereus. PC-PLC wasisolated from cultures of B. cereus SE-1 essentially asdescribed previously (21). The enzyme preparation was thenpurified to complete homogeneity, as confirmed by sodium

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dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The specific activity of thepurified enzyme was 1.5 U/,ug.

Preparation of v-H-ras p21 proteins. Transforming andnormal v-H-ras p21 proteins were expressed in bacteria aspreviously described (14). A final step of purification con-sisted of gel filtration chromatography through a SephadexG-100 column (2.5 by 90 cm); fractions containing thepurified protein were pooled and dialyzed extensivelyagainst 20 mM Tris-HCI (pH 7.5) to remove urea and kept at-70°C until used.Analysis of oocyte maturation. Groups of 20 oocytes were

cultured at 20°C in modified Barth solution, and germinalvesicle (nuclear) breakdown was assessed by the appearanceof a white spot in the animal pole. In some cases, nuclearbreakdown was confirmed by dissection of trichloroaceticacid (10%)-fixed oocytes (14).

Maturation-promoting factor Hi kinase assay. Twentyoocytes were homogenized in a buffer containing 20 mMHEPES (pH 7.0), 10 mM ,-glycerophosphate, 5 mM EGTA[ethylene glycol-bis(3-aminoethyl ether)-N,N,N',N'-tetra-acetic acid], 5 mM MgCl2, 50 mM NaF, 2 mM dithiothreitol,100 ,ug of leupeptin per ml, and 100 p,M phenylmethylsulfo-nyl fluoride. Following centrifugation at 13,000 x g for 15min, extracts (1 to 2 mg per assay) were assayed for 10 minat 30°C in a final reaction volume of 50 ,ul containing 20 mMHEPES (pH 7.0), 5 mM ,B-mercaptoethanol, 10 mM MgCI2,100 ,uM [-y-32P]ATP (2 to 5 dpm/fmol), 0.2 p,g of heat-stableinhibitor of cyclic AMP-dependent protein kinase, and 0.6mg of Sigma type III-S calf thymus histone per ml. Reactionswere terminated, spotted onto Whatman P81 phosphocellu-lose paper, washed, and quantitated as described previously(14). In some experiments, extracts were incubated withpl3sucl linked to agarose beads, and histone 1 (H1) kinaseactivity was determined in the precipitates and then sepa-rated by SDS-PAGE (9).

Preparation of nuclear extracts and GMSAs. Oocyte nu-clear extracts were prepared as described previously (32).Gel mobility shift assays (GSMAs) were performed by usingin each assay 15,000 cpm of a 32P-end-labeled fragment ofthe HIV enhancer containing the NF-KB binding site or amutated sequence (2).

Activity of the HIV LTR-CAT plasmid. Reporter plasmidHIV LTR (long terminal repeat)-CAT (chloramphenicolacetyltransferase) or HIV LTRmut-CAT, harboring eitherthe wild type or a mutated form of the NF-K.B enhancerregion, respectively, was microinjected (5 ng) into the nucleiof oocytes in accordance with standard procedures. Afterovernight incubation at 21°C, oocytes were stimulated withdifferent agents, and CAT activity was determined in ex-tracts as described previously (7). Both plasmids have beendescribed previously (12).

RESULTS

Presence of NF-KB in cytosol extracts fromX. laevis oocytes.NF-KcB is found in the cytoplasm of many uninduced cellsbound to IKB, which renders it incapable of binding DNA(28). This cytoplasmic form of NF-KB can be released fromIKB by treating cytosolic extracts with sodium deoxycholate(DOC), making it competent for DNA binding. To determinethe presence of NF-KB activity in oocytes, we initiallycarried out a series of GSMAs using a double-strandedoligonucleotide with the KB sequence present in the HIVLTR enhancer (2). GSMA of oocyte cytosolic extractstreated with DOC displayed retarded bands that were not

FIG. 1. Detection of NF-KB activity in oocyte extracts. Cytosolextracts from oocytes (Cyt; 5 ,ug) were incubated with a 32P-labeleddouble-stranded oligonucleotide containing the wild-type bindingsite for NF-KB of the HIV LTR (2 ng/15,000 cpm) either in theabsence or in the presence of 0.8% DOC. Some experiments werealso carried out in the presence of a 100-fold molar excess of coldoligonucleotides, either wild type (WT) or mutated (MUT). NF-KBactivity was determined by GSMA as described in Materials andMethods. The position of the retarded band which runs in parallelwith the nuclear NF-KB activity induced by ras p21 (not shown) ismarked with an arrowhead. Essentially identical results were ob-tained in three independent experiments.

observed in untreated extracts (Fig. 1). These bands werespecifically competed for by an excess of wild-type unla-beled oligonucleotide but not by point mutations that abolishNF-KB binding. These results suggest the presence of KB-binding activities in the cytosol of Xenopus oocytes.

Activation of NF-KB in Xenopus oocytes stimulated withdifferent agents. To determine whether the NF-KB activitydetected in cytosolic extracts of oocytes is susceptible ofactivation, the translocation of KB-binding activity to thenuclei of stimulated oocytes was determined. To this end,oocytes were treated with insulin or were microinjected withtransforming H-ras p21 (both potent stimulants of oocytematuration). Afterwards, oocyte nuclear extracts were pre-pared and GSMA was carried out. Nuclear extracts fromresting oocytes displayed an undetectable basal KB-bindingactivity which was significantly induced in a time coursefollowing stimulation of oocytes with insulin (Fig. 2). ras p21also potently induced this NF-KB-like activity (Fig. 3). Tofurther characterize the NF-KB activity stimulated in treatedoocytes, the following experiments were carried out. Nu-clear extracts from ras p21-microinjected oocytes wereincubated with a 100-fold molar excess of unlabeled oligo-nucleotide prior to the addition of the labeled oligonucle-otide. The KB-binding activity induced by v-H-ras p21 wasspecific, since it was completely competed for by the unla-beled oligonucleotide (Fig. 4A). This retarded band comi-grated with the upper band observed in Fig. 1, which isreleased in cytosolic extracts treated with DOC (not shown).The fast-migrating band shown in Fig. 1 migrated in aposition between the NF-KB activity and the nonspecificband in GSMAs of nuclear extracts of induced oocytes. Thenature of that cytosolic KB-binding activity remains unclear,but it is not induced in nuclei. Incubation of nuclear extractsfrom ras p21-microinjected oocytes with recombinant IKB(MAD-3) completely abolished the retarded band induced by

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FIG. 2. Time course of the induction of NF-KB in response toinsulin in X. laevis oocytes. Oocytes were incubated with insulin (1,uM), nuclear extracts were prepared at different times, an aliquot(20 ,ug) was incubated with the labeled probe described in the legendto Fig. 1, and NF-KB activity was determined by GSMA. Autorad-iograms from three experiments were scanned, and the meanamount of the NF-KB complex formed ± standard deviation isrepresented by arbitrary units. The insert is a representative exper-iment of another three with similar results.

v-H-ras p21 (Fig. 4A). This result may suggest the presenceof p65 in the NF-KB activity stimulated by ras p21, whichwould be consistent with recent data demonstrating a highdegree of homology between Xenopus and human p65 (19,33). However, p65 has weak or no DNA-binding activity (28,31), and NF-KB requires the simultaneous presence of p50 tobe able to bind DNA. To examine the presence of such aprotein in ras p21-induced NF-KB, nuclear extracts wereincubated with two different antibodies prior to addition ofthe labeled probe. The antibodies used were pSOAB1, whichwas generated against recombinant p50, and p5OAB2, whichis an antipeptide antibody raised against a small sequencelocated at the C terminus of the protein (generously providedby A. Israel). Both antibodies abolished the NF-KB activityinduced in nuclear extracts from ras p21-microinjectedoocytes (Fig. 4A), indicative of the possible presence of p50in this NF-KB-like activity. When this experiment wasperformed with an irrelevant antibody, no effect on the

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FIG. 3. NF-KB activation in X. laevis oocytes. Oocytes weremicroinjected with either 20 ng of v-H-ras p21 or buffer control orwere incubated in the presence of 1 ,uM insulin (Ins) for 6 h. Thennuclear extracts were prepared, and NF-KB activity was determinedin GSMA by using a double-stranded oligonucleotide correspondingto the wild-type NF-KB sequence of the HIV LTR enhancer asdescribed in the legend to Fig. 2. Essentially identical results wereobtained in three independent experiments.

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FIG. 4. Characterization of the NF-KB activity induced in rasp21-stimulated oocytes. Nuclear extracts from oocytes, either con-trol or microinjected with 20 ng of v-H-ras p21 (A) or 25 ,uU of B.cereus PC-PLC (B), were analyzed by GSMA as described in thelegend to Fig. 2. Some incubations with extracts from stimulatedoocytes were carried out in the presence of a 100-fold molar excessof a cold double-stranded oligonucleotide corresponding to thewild-type NF-KB sequence of the HIV LTR enhancer (WIT). An-other set of incubations was performed in the presence of either 20ng of recombinant I-KB or 10 ng of anti-p5O antibody p50AB1 orp5OAB2 (described in Results). Essentially identical results wereobtained in three independent experiments.

appearance of the NF-KB-like retarded band was observed(data not shown).

It has previously been demonstrated that v-H-ras p21promotes a potent induction of PC-PLC activity in bothfibroblasts and Xenopus oocytes which is critical for ras p21mitogenic function (14, 23). Our earlier published results areconsistent with the notion that microinjection of a perma-nently activated PC-PLC from B. cereus mimics the signal-ing pathways activated by v-H-ras p21 (14). Therefore, todemonstrate a role of PC-PLC in the induction of NF-KB inXenopus oocytes by ras p21, B. cereus PC-PLC was micro-injected and the NF-KB activity in nuclear extracts wasdetermined. Microinjection of B. cereus PC-PLC promoted apotent induction of NF-KB activity in oocytes (Fig. 4B). Thisactivity was competed for by incubation of nuclear extractswith either recombinant IKB or pSOAB1 (Fig. 4B). Interest-ingly, progesterone, a potent inducer of oocyte maturation(20), does not regulate NF-KB activity; also, PMA, a potenteffector of some PKC isotypes, does not stimulate translo-cation of NF-KB to the nucleus in Xenopus oocytes (data notshown). This observation is consistent with our recentfinding that PMA is a poor stimulant of oocyte maturation (8)and suggests that at least in the oocyte system, classicalPMA-sensitive PKC isotypes do not appear to be involved inthe regulation of NF-KB.

Involvement ofC-PKC in the activation of NF-KB inXenopusoocytes. Our earlier work (8) demonstrated the critical roleplayed by the 4 isotype but not the a, 1, -y, 8, or e isotype ofPKC in the ras p21/PC-PLC signal transduction pathway.Since PMA is unable to promote NF-idB activation inoocytes, it is very unlikely that PMA-sensitive PKC isotypea, 1, or -y would be involved in the regulation of thisparameter. Therefore, if any PKC isotype plays a role in theregulation of NF-KB activation in Xenopus oocytes, the (isotype appears to be a good candidate. Following a strategysimilar to that described previously (8), peptides A and Zwere used. Peptide A has a sequence conserved in thepseudosubstrate region of PKC isotypes at, 1, and -y andtherefore should be a good candidate inhibitor of theseisotypes (29, 30). Peptide Z has a sequence identical to that

MOL. CELL. BIOL.

NF-KB ACTIVATION 1293

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FIG. 5. Involvement of t-PKC in the activation of NF-KB inXenopus oocytes. Oocytes, either untreated or microinjected withpeptide A (PSA) or peptide Z (PSZ) (final concentration, 5 I1M),were subsequently microinjected with 20 ng of transforming v-H-rasp21 and incubated for 6 h. Then NF-KB activity was determined inGSMA of nuclear extracts. The amino acid sequences of peptides Aand Z (one-letter code) are RKGALRQKN and RRGARRWRK,respectively. Essentially identical results were obtained in threeindependent experiments.

of the t-PKC pseudosubstrate region, which differs signifi-cantly from that of isotype at, 1, or -y (30). Therefore, Xenopusoocytes were microinjected with either peptide A or Z, afterwhich they were microinjected with transforming v-H-ras p21or control buffer. The presence of 5 ,uM peptide Z completelyinhibited NF-KB activation by v-H-ras p21, whereas themicroinjection of peptide A had little or no effect on thisparameter (Fig. 5). Activation of NF-KB by incubation ofoocytes with insulin or by microinjection of B. cereus PC-PLC was likewise inhibited by peptide Z but not by peptide A(data not shown). A control experiment demonstrating thatpeptide A effectively inhibited PMA-sensitive PKCs, al-though they are not involved in NF-KB activation, is shown in

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FIG. 6. Control of specificity of peptides A and Z. Stage VIoocytes, either untreated or microinjected with 1 ng of purifiedbovine brain PKC (isotypes a, 1, and -y), were microinjected eitherwith distilled water (open bars) or with 5 ,uM (final concentration)peptide A (cross-hatched bars) or Z (black bars). Subsequently,oocytes were either untreated or incubated in the presence of PMA(100 ng/ml) or progesterone (1 ,uM). Hi kinase activity in theextracts was determined as described in Materials and Methodswhen oocytes microinjected with v-H-ras p21 without peptideinhibitors displayed a 50% induction of germinal vesicle breakdown.Results are means standard deviations of three independentexperiments with incubations in duplicate.

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Five nanograms of reporter plasmid HIV LTR-CAT or HIV LTR-mut-CAT (see Materials and Methods) was microinjected into thenuclei of oocytes. Following overnight incubation at 21'C, oocyteswere microinjected with water (open bars) or peptide A (cross-hatched bars) or Z (black bars) (final concentration, 5 ,uM), afterwhich they were microinjected with either a buffer control or 20 ngof v-H-ras p21. Reactions were stopped 6 h after microinjection ofv-H-ras p21, and CAT activity in the extracts was determined asdescribed in Materials and Methods. Results are means + standarddeviations of three independent experiments with incubations induplicate.

Fig. 6. In that experiment, we took advantage of the fact thatPMA can induce maturation-promoting factor Hi kinaseactivity in oocytes overloaded with a mixture of PKC isotypesa, 1B, and fy (8). Thus, it is clear from Fig. 6 that peptide Ainhibits PMA-induced maturation in PKC (isotypes a, 1, andy)-microinjected oocytes, whereas peptide Z is unable to doso, in agreement with previously published results (8). Fur-thermore, the activating potential of progesterone, whichinduces oocyte maturation through a ras p21- and PC-PLC-independent pathway (8, 20), is not affected by the microin-jection of either peptide. This property represents a goodcontrol for the specificity and lack of toxicity of these mole-cules.

Role of C-PKC in transactivation of the HIV enhancer. Todetermine whether the activation of NF-KB by the rasp21/PC-PLC/t-PKC pathway is critical for the functionaltransactivation of KB-containing enhancer elements, thefollowing series of experiments was carried out. A vectorthat permits expression of the CAT gene under the control ofthe regulatory region of the HIV-1 LTR was constructed (1).As a control, a plasmid harboring this regulatory region withan inactivating mutation in the KB enhancer was also pre-pared. Either plasmid was microinjected into the nuclei ofoocytes, and following overnight incubation, the oocyteswere microinjected with v-H-ras p21 either in the absence orin the presence of peptide A or Z. v-H-ras p21 potentlyinduced a 12-fold enhancement of CAT activity comparedwith oocytes injected with control buffer (Fig. 7). Thepresence of 5 pM peptide A did not inhibit ras p21-inducedCAT activity, but it potentiated that parameter in compari-son with peptide A-microinjected controls. Remarkably, thepresence of peptide Z inhibited the ability of ras-p21 toinduce HIV transactivation. Thus, microinjection of peptideZ reduces the enhancement of the CAT activity in rasp21-microinjected oocytes from 12-fold to only 3-fold com-pared with their respective controls. The sole presence ofpeptide A or Z significantly reduced or augmented, respec-tively, the HIV LTR-regulated CAT activity in resting

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1294 DOMINGUEZ ET AL.

oocytes. As a control, v-H-ras p21 was unable to stimulateCAT activity in oocytes microinjected with the plasmidharboring the inactivating mutation in the KB enhancerregion (Fig. 7). Interestingly, similar results were obtainedwith oocytes treated with insulin or microinjected with B.cereus PC-PLC (data not shown).

DISCUSSION

NF-KB and Rel-related proteins regulate an increasingnumber of genes through binding to a specific sequence motifin their enhancers (16, 22, 35). A particularly importantexample is the HIV LTR (26). A critical aspect of thepathogenesis of AIDS is an understanding of the mechanismof viral reactivation that takes place in infected quiescentcells when mitogenically induced (13).

Stimulation of the T-cell receptor promotes NF-KB acti-vation (17). Lymphocyte activation through the CD3/CD2pathway leads to significant changes in the GTP/GDP ratio ofras p21, indicative of the likely involvement of this oncogeneproduct in T-cell receptor signaling pathways (10). Interest-ingly, previous data demonstrated that ras p21 inducespotent transactivation of the HIV LTR through the cBregion of its enhancer element (1). This finding stronglysuggests an important role for ras p21 in the pathwayscontrolling NF-KB activation. H-ras p21 promotes the PLC-mediated breakdown of PC, with no effect on the classicalphosphoinositide pathway, in both fibroblasts (23) and X.laevis oocytes (14). The activation of PC hydrolysis has beenshown to be critical in mitogenic signal transduction by rasp21 (14). Conceivably, stimulation of NF-KB translocation inresponse to this oncogene will involve the PC-PLC-mediatedsignaling pathway. The downstream mechanisms activatedby ras p21/PC-PLC remain to be elucidated. In this regard,since PLC-mediated PC hydrolysis generates diacylglycerol,which is an important activator of PKC (27), the involvementof this kinase in the signaling cascades activated by PC-PLChas been considered an intriguing possibility. Previous datademonstrate that down-regulation of PKC by chronic expo-sure of cells to phorbol esters such as PMA does not affectthe ability of PC-PLC to promote mitogenesis in fibroblasts(21). Furthermore, expression of the stromelysin gene inresponse to platelet-derived growth factor/ras p21/PC-PLCis not affected by down-regulation of PKC (7). Interestingly,transient expression experiments with plasmids harboringdifferent deletions and mutations in the stromelysin pro-moter region linked to a reporter gene demonstrate that thePMA-responsive element located in that promoter is notrequired to transmit signals generated by PC-PLC (7). Takentogether, these results strongly suggest that a classical PKCsensitive to PMA does not appear to be involved in rasp21/PC-PLC signaling. This suggestion opens the possibilityfor the participation of novel PKC isotypes in mitogenicsignaling cascades. It is noteworthy that recent evidencefrom this laboratory strongly suggests an important role of(-PKC in ras p21/PC-PLC-triggered mitogenic pathways (8).The mechanisms whereby NF-KB is activated remain

unknown, although it is clear that this factor belongs to theclass of transcription factors whose nuclear translocation isstimulated by external signals. In this study, we presentresults for X. laevis oocytes that confirm and extend the roleof ras p21 in NF-KB activation. This system was chosenbecause the large size of the oocyte permits microinjectionof the product of ras genes and evaluation kinetically of thetriggered signaling cascades. We showed earlier that thisapproach allowed us to establish the importance of PC-PLC

in ras p21-induced mitogenic signaling (14). In this report,we demonstrate that PC degradation is able by itself toactivate NF-KB and transactivate a CAT reporter plasmidharboring the regulatory region of the HIV LTR. Therefore,bearing in mind the functional link existing between insulin,ras p21, and PC-PLC (14, 20), these results identify PChydrolysis as a further downstream step responsible forNF-KB induction and HIV transactivation.At least one of the critical targets of PC-PLC signals

leading to NF-KB activation has also been identified in thisstudy. Thus, we demonstrated that a specific peptide inhib-itor of (-PKC inhibits NF-KcB translocation and HIV trans-activation in oocytes. However, a specific inhibitor peptidefor isotypes ao, P, and -y does not affect the ability of v-H-rasp21 or B. cereus PC-PLC to activate NF-KB-dependentpathways. The specificity and lack of toxicity of thesepeptides have been validated in the results shown in Fig. 6and in experiments recently published (8). Interestingly,progesterone, which potently stimulates oocyte maturationthrough a pathway completely independent of ras p21,PC-PLC, or (-PKC, does not activate NF-KB. This findingindicates that NF-KB is a transcription factor specificallylocated in the pathway controlled by insulin. This informa-tion is important because it not only helps to clarify themechanisms controlling NF-KB stimulation and HIV trans-activation but also serves to identify transcription factors assignaling molecules that transmit information of specificpathways from the membrane to the nucleus.The fact that PMA does not induce NF-KB activation is

consistent with the notion that phorbol esters do not activatematuration in oocytes unless they are overloaded with apartially purified mixture of brain PKC isotypes a, ,B, and -y(8). PMA has been shown to activate NF-KB in other cellsystems; however, the significance of this fact in vivo is notclear. For example, TNF-ao is able to promote NF-KBactivation in the absence of functional PMA-sensitive PKCs(24). The signaling pathways activated by this cytokine arepresently unclear. It has recently been shown that TNF-otactivates a novel phospholipid-degradative pathway involv-ing the activation of a sphingomyelinase (11). It would be ofinterest to determine whether sphingomyelin degradationplays any role in the activation of NF-KB. Alternatively, thePC-PLC/t-PKC pathway defined here could also be impor-tant in the TNF-a signaling mechanisms. In this regard, it isnoteworthy that Schutze et al. have recently presentedevidence that TNF-a is a potent inducer of PC phosphodi-esterase hydrolysis (34). The functional role potentiallyplayed by PC degradation in NF-KB activation in response toTNF-a warrants further clarification.

ACKNOWLEDGMENTS

This work was supported in part by grants SAL90-0070 andPTR91-0022 from CICYT, PB90-0074 from DGICYT, and C256-91from Comunidad de Madrid. I.D. is a Fellow from Gobierno Vasco.M.T.D-M. and L.S. are Fellows from Ministerio de Educaci6n. Wethank Glaxo Spain for partially funding this work.The two first authors equally contributed to this study.

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VOL. 13, 1993