Positioning Atypical Protein Kinase C Isoforms in the UV-Induced ...

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1997, p. 4346–4354 0270-7306/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 17, No. 8

Positioning Atypical Protein Kinase C Isoforms in the UV-Induced Apoptotic Signaling Cascade EDURNE BERRA, MARIÁ M. MUNICIO, LAURA SANZ, SONIA FRUTOS, MARIÁ T. DIAZ-MECO, AND JORGE MOSCAT* Laboratorio Glaxo Wellcome-CSIC de Biologıá Molecular y Cellular, Centro de Biologıá Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Cientı´ficas-Universidad Auto ńoma de Madrid), Universidad Auto ńoma, Canto Blanco, 28049 Madrid, Spain Received 13 February 1997/Returned for modification 26 March 1997/Accepted 5 May 1997

Recent studies have documented the involvement of the atypical protein kinase C (aPKC) isoforms in important cellular functions such as cell proliferation and survival. Exposure of cells to a genotoxic stimulus that induces apoptosis, such as UV irradiation, leads to a profound inhibition of the atypical PKC activity in vivo. In this study, we addressed the relationship between this phenomenon and different proteins involved in the apoptotic response. We show that (i) the inhibition of the aPKC activity precedes UV-induced apoptosis; (ii) UV-induced aPKC inhibition and apoptosis are independent of p53; (iii) Bcl-2 proteins are potent modulators of aPKC activity; and (iv) the aPKCs are located upstream of the interleukin-converting enzymelike protease system, which is required for the induction of apoptosis by both Par-4 (a selective aPKC inhibitor) and UV irradiation. We also demonstrate here that inhibition of aPKC activity leads to a decrease in mitogen-activated protein (MAP) kinase activity and simultaneously an increase in p38 activity. Both effects are critical for the induction of apoptosis in response to Par-4 expression and UV irradiation. Collectively, these results clarify the position of the aPKCs in the UV-induced apoptotic pathway and strongly suggest that MAP kinases play a role in this signaling cascade. The atypical protein kinase C (aPKC) isoforms zPKC and l/iPKC constitute a special subfamily of PKCs with specific characteristics (1, 42, 43, 49). Thus, their regulatory domain is significantly different from that of the other PKCs in that it does not contain a Ca21-binding motif and has only a zinc finger lipid binding region (43). Consequently, and unlike other PKC isoforms, the aPKCs cannot be activated by Ca21, phorbol esters, or diacylglycerol (1, 43, 49) but are regulated by other important lipid cofactors such as phosphatidylinositol (PI) 3,4,5-P3 and ceramide (32, 35a, 37, 40, 41), which are generated following cell activation by inflammatory cytokines and growth factors (19, 24, 29, 33). Consistent with this finding, the aPKCs appear to be involved in a number of important cellular functions, including maturation of (16) and NF-kB activation in (17) Xenopus oocytes, as well as the reinitiation of DNA synthesis in quiescent mouse fibroblasts (5), mitogenactivated protein kinase (MAPK) activation (6, 7, 18), kB- and AP-1-dependent promoter activity (12, 14, 17, 21, 26), interleukin-1b-stimulated prostaglandin E2 release (47), a2 integrin gene expression (56, 57), and the differentiation of PC12 cells in response to nerve growth factor (NGF) (53, 54). Of potential relevance, it has also recently been demonstrated in vivo that l/iPKC and most probably also zPKC are decisive downstream effectors of PI 3-kinase (2, 4). This is particularly relevant because PI 3-kinase has been shown to be critical for cell survival. Thus, inhibition of PI 3-kinase with wortmannin inhibited the ability of NGF to prevent apoptosis in PC12 cells, whereas platelet-derived growth factor (PDGF) blocked apoptosis in NGF-deprived PC12 cells expressing the wild-type

PDGF receptor but not a receptor mutant that failed to activate PI 3-kinase (59). In an effort to identify potentially novel modulators of the aPKC isoforms, we have recently used the yeast two-hybrid system with the regulatory domain of zPKC as the bait (15). Interestingly, we found that the product of the par-4 gene, which is induced in cells undergoing apoptosis (50), specifically interacts with the zinc fingers of zPKC and of l/iPKC in vitro and in vivo, which leads to the blockade of their enzyme activity (15). Therefore, it seems that inhibition of the aPKCs may be necessary for the induction of apoptosis. It was found that overexpression of l/iPKC and zPKC inhibited UV-induced cell death (15), whereas exposure of cells to UV irradiation leads to a dramatic reduction of zPKC activity. Collectively, these results suggest a role for these PKC isotypes in cell survival and apoptosis. The identification of molecules that modulate these two antagonistic processes has progressed dramatically in recent years. The Bcl-2 family of proteins have extensively been demonstrated to play decisive roles as either antiapoptotic (Bcl-2 itself, A1, Mcl-1, and Bcl-xL) or proapoptotic (Bax, Bak, Bad, and Bcl-xs) agents (20, 44). On the other hand, different genotoxic stresses, including UV light (34), induce p53 that, depending on the cell system, results in growth arrest or apoptosis (23, 28). The ability of p53 to induce cell cycle arrest seems to be mediated by (among other proteins) p21, whereas the induction of cell death could be mediated, at least in part, through the expression of the Bax protein (38). Bax dimerizes with Bcl-2 and prevents its ability to inhibit cell death (28). Genetic studies of Caenorhabditis elegans identify Ced-3, an interleukin-converting enzyme (ICE)-like gene, as a downstream target of Ced-9, which is the worm homolog of Bcl-2 (see references 22 and 25 for recent reviews). Interestingly, ICE-like proteases have also been shown to be critically implicated during apoptosis in mammalian cells (22, 25). In this study, we have addressed the potential involvement of the ICE-like proteases as well as that of p53 and Bcl-2 in the

* Corresponding author. Mailing address: Centro de Biologıá Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Cientı´ficas-Universidad Auto ńoma de Madrid), Universidad Auto ńoma, Canto Blanco, 28049 Madrid, Spain. Phone: 34-1-397 8039. Fax: 34-1397 8344. E-mail: [email protected]. 4346

VOL. 17, 1997

ATYPICAL PKCs IN UV-INDUCED APOPTOTIC PATHWAY

4347

mechanisms of zPKC inhibition produced by UV irradiation. We also investigated the mechanisms whereby the inhibition of zPKC triggers apoptosis. MATERIALS AND METHODS Plasmids. pCDNA3-HA-zPKC, pCDNA3-HA-l/iPKC, and pRcCMVzPKCDEL have previously been described (6, 14). The Bcl-2 expression vector pSFFV-Bcl-2, based on the long terminal repeat of the splenic focus-forming virus, was provided by C. Martinez (Centro Nacional de Biotecnologia [CNB], Madrid, Spain). pRK-CrmA, containing the cowpox virus CrmA-encoding gene, was provided by D. Goeddel (Tularik, Inc.). The expression vector for Bax was a gift from S. Farrow (Glaxo Wellcome). Epitope-tagged MAPK and stress-activated protein kinase (SAPK) have been described previously (6) and were provided by J. Pouyssegur and J. R. Woodgett, respectively. The pcDNA3-HA-p38 construct was obtained by PCR using a human placenta cDNA as a template and the following primers: 59-ATACCCGGGATGTCTCAGGAGAGGCCCACG-39 and 59-GCCTCTAGATCAGGAACTCCATCTCTTCTTG-39. The amplified product was digested with SmaI and XbaI and subcloned into EcoRV/XbaI sites of pcDNA3-HA. The dominant negative mutant of p38 (pCMV5-Flag-p38AGF) was a generous gift from R. Davis (55). An EcoRV/XbaI fragment encompassing amino acids 69 to 332 of Par-4 (15) was subcloned into pcDNA3-HA or pcDNA3-myc to obtain epitope-tagged Par4DNLS. Cell culture and transfections. p53-deficient (p53-/-) mouse embryo fibroblasts (MEFs) and control cells were kindly provided by T. Jacks and have been described previously (3). NIH 3T3 and Cos cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, penicillin G (100 mg/ml), and streptomycin (100 mg/ml) (Flow). Subconfluent cells were transfected by the calcium phosphate method (Clontech, Inc.). Some experiments were performed in the presence of the ICE inhibitor YVAD-fmk or z-VAD-fmk (Bachem). aPKC activity. NIH 3T3 cells incubated at different times after UV light exposure were extracted with lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM EGTA, protease inhibitors) and immunoprecipitated with an affinity-purified rabbit polyclonal antibody generated against the last 16 amino acids of zPKC, which are conserved also in l/iPKC (2 mg of antibody). Immunoprecipitates were washed seven times with lysis buffer containing 0.5 M NaCl. For in vitro kinase assay, immunocomplexes were incubated with 1 mg of myelin basic protein (MBP) and 5 to 10 mCi (100 mM) of [g-32P]ATP in kinase buffer (35 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 0.5 mM EGTA, 0.1 mM CaCl2, 1 mM phenylphosphate) for 30 min at 30°C in a final volume of 20 ml. Reactions were stopped by the addition of concentrated sample buffer. Samples were boiled for 3 min and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis followed by exposure and quantitation in an InstantImager (Packard). The activity of transfected hemagglutinin (HA)-zPKC was determined in immunoprecipitations with an anti-HA antibody (12CA5; Boehringer, Mannheim, Germany) as described above. MAPK, SAPK, and p38 activities. Extracts of NIH 3T3 or Cos cells transfected with pCDNA-HA-zPKC, pCDNA-HA-MAPK, pCDNA-HA-SAPK, or pCDNAHA-p38 were immunoprecipitated with anti-HA antibody (2 mg/mg of protein extract) as described above. For the MAPK assay, phosphorylation of 3 mg of MBP was determined in buffer containing 35 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.5 mM EGTA, 0.1 mM CaCl2, and 1 mM phenylphosphate. For the SAPK assay, phosphorylation of 3 mg of glutathione S-transferase–c-Jun-(5-89) was determined in buffer containing 20 mM morpholine propane sulfonic acid (pH 7.2), 2 mM EGTA, 10 mM MgCl2, 1 mM dithiothreitol, and 0.1% Triton X-100. For the p38 assay, Cos cells transfected with pCDNA3-HA-p38 were lysed with buffer A (20 mM Tris [pH 7.5], 10% glycerol, 1% Triton X-100, 0.137 M NaCl, 25 mM b-glycerophosphate, 2 mM EDTA, 0.5 mM dithiothreitol, 1 mM orthovanadate, 2 mM PPi, 10 mg of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride). HA-p38 was immunoprecipitated with an anti-HA antibody. The activity was determined by phosphorylation of 1 mg of glutathione S-transferase– ATF2 in 25 mM HEPES–25 mM b-glycerophosphate–25 mM MgCl2–0.5 mM dithiothreitol–0.1 mM sodium orthovanadate. All kinase reactions were terminated after 30 min at 30°C by addition of Laemmli sample buffer. The phosphorylation of the substrate proteins was examined after sodium dodecyl sulfatepolyacrylamide gel electrophoresis followed by exposure and quantitation in an InstantImager (Packard). Apoptosis assays. b-Galactosidase cotransfection assays and immunofluorescence analysis for determination of cell death were performed as described by Diaz-Meco et al. (15). For UV treatment, culture medium was removed, dishes were washed once with phosphate-buffered saline and UVC irradiated (180 J/m2), and fresh medium was added to the cells. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) analysis was performed by using an in situ cell death detection kit (Boehringer Mannheim).

RESULTS Bcl-2 delays the inhibition of the aPKCs in UV-treated NIH 3T3 fibroblasts. We initially determined the effect of UVC

FIG. 1. Inhibition of aPKC activity in response to UV irradiation. NIH 3T3 cells were induced (bars and circles) or not (squares) to undergo apoptosis by UV irradiation, and cell extracts were prepared at different times; then the aPKCs were immunoprecipitated in the absence (empty bars) or presence (striped bars) of competing peptide, and enzyme activity was determined as described in Materials and Methods (bars). In parallel cultures, the induction of apoptosis was determined by TUNEL (circles and squares). Results are the means 6 standard deviations of three independent experiments with incubations in duplicate (A). The upper panel is a representative experiment showing MBP phosphorylation. A fraction of the immunoprecipitates of the aPKCs in the absence of competing peptide was analyzed by immunoblotting with the same antibody to determine the amount of aPKC recovered (B).

irradiation on the activity of the aPKCs in NIH 3T3 cells. Extracts obtained from cells incubated at different times after UV light exposure were immunoprecipitated with an antibody generated against the last 16 amino acids of zPKC, which are conserved also in l/iPKC. This antibody has been affinity purified in our laboratory and was shown to react with both aPKCs but not with any other PKC isoform (not shown). From Fig. 1, it seems clear that the activity of the aPKCs was dramatically diminished by 1 h after UV irradiation and completely abrogated by 4 to 6 h. TUNEL analysis of parallel cultures indicated that DNA fragmentation becomes detectable around 4 to 6 h after irradiation (Fig. 1). Morphological changes indicative of apoptosis, including membrane blebbing and shrunken cytoplasm, were not detected until 2 h after UV irradiation (not shown). Immunoblot analysis of extracts from UV-irradiated cells demonstrated that aPKC protein levels remained constant during the time course of the experiment (Fig. 1). Therefore, the inhibition of the aPKCs precedes the induction of apoptosis in NIH 3T3 cells. To address the potential role of Bcl-2 molecules in the inhibition of the aPKC activity, NIH 3T3 cells were transfected with an HA-tagged version of zPKC (14) along with either a plasmid control or expression vectors for Bcl-2 or Bax. Transfected HA-zPKC was then immunoprecipitated with the monoclonal anti-HA antibody 12CA5, and zPKC activity was determined. Expression of Bcl-2 significantly enhances the basal activity of zPKC (Fig. 2), with no effect on the amount of the expressed HA-zPKC (Fig. 2). Interestingly, expression of Bcl-2 dramatically delays the inhibition of zPKC produced by UV irradiation (Fig. 3). Thus, whereas zPKC activity is re-

4348

BERRA ET AL.

MOL. CELL. BIOL.

z-VAD, an inhibitor with a reported broader specificity than YVAD (not shown). Lack of a role of p53 in UV-inhibited aPKC activity. The protein p53 has been shown to be induced by a number of genotoxic stresses, including UV irradiation (34). To address if p53 plays any role in the inhibition of the aPKC activity induced by UV light, early-passage p53-/- MEFs were exposed to UV light, and the activity of each of the aPKCs was determined as described above. Interestingly, the absence of p53 did not prevent the inhibition of the aPKC activity or the induction of apoptosis (Fig. 5). Although the kinetics of the aPKC inhibition was slower in p53-/- MEFs than in NIH 3T3 cells, it was identical to that of control normal MEFs (Fig. 5). Role of Bcl-2 proteins, ICE-like proteases, and p53 in the induction of apoptosis by Par-4. The foregoing results suggest that Bcl-2 proteins are upstream regulators of the aPKC activity in the UV-induced apoptotic pathway, whereas ICE-like proteases and p53 are not. However, it is possible that the ICE-like proteases and p53 are located downstream of the inhibition of the aPKCs. To address this possibility, we explored the effects of both kind of modulators during the induction of apoptosis by inhibition of the aPKCs. In this regard, we have recently reported that expression of Par-4, a selective inhibitor of the aPKCs, induces apoptosis that is abrogated by

FIG. 2. Effect of Bcl-2 proteins on aPKC activity. NIH 3T3 cells were transfected with 10 mg of HA-tagged zPKC along with different concentrations of expression vectors for Bcl-2 (squares) or Bax (circles) and enough empty vector to give 30 mg of total DNA. Cells were harvested 24 h after transfection, and HA-zPKC activity and expression were determined. (A) Results are the means 6 standard deviations of three independent experiments with incubations in duplicate. (B and C) Representative experiments for Bcl-2 and Bax, respectively, showing MBP phosphorylation and HA-zPKC expression levels.

duced to 20% at 2 h, a comparable reduction of that activity in the Bcl-2 transfectants was detected as late as 8 h (Fig. 3). Consistent with the role of Bcl-2 in the control of the aPKC activity are the results of Fig. 2, demonstrating that expression of Bax dramatically reduces, whereas Bcl-2 stimulates, the activity of the cotransfected zPKC in NIH 3T3 cells, with no effect on the amount of expressed or recovered enzyme (Fig. 2). Together these results are in keeping with Bcl-2 proteins having a role in the regulation of the aPKCs in response to apoptotic stimuli such as UV irradiation. To rule out the possibility that the observed effects of Bax expression on zPKC activity could be ascribed to secondary responses due to the actions of Bcl-2 proteins on the cell apoptotic machinery, the experiment shown in Fig. 2 was repeated in the presence of YVAD (a potent inhibitor of apoptosis [see below]); identical results were obtained (not shown). These observations further emphasize the notion that inhibition of zPKC activity by apoptotic stimuli precedes and most probably is important for the induction of cell death. Inhibition of the aPKC activity by UV irradiation is not affected by YVAD. The role of the ICE-like proteases on the inhibition of the aPKCs by UV irradiation was explored in NIH 3T3 cells that were either untreated or incubated with YVAD, an inhibitor of the ICE-like protease activity. Interestingly, the ability of UV light to inhibit the aPKC activity was not affected by YVAD (Fig. 4), whereas apoptosis was greatly diminished (Fig. 4). This result indicates that ICE proteases do not appear to be upstream modulators of the aPKCs but play a critical role in UV-induced cell death. Similar results were obtained with

FIG. 3. Bcl-2 delays the inhibition of aPKC activity by UV irradiation. NIH 3T3 cells were transfected with 10 mg of HA-zPKC along with 10 mg of either plasmid control (circles) or the Bcl-2 expression vector (squares). After 24 h, cells were UV irradiated, and the enzyme activity of zPKC was determined at different times thereafter. (A) Results are the means 6 standard deviations of three independent experiments with incubations in duplicate. (B and C) Representative experiments showing MBP phosphorylation and HA-zPKC expression control of cells transfected with an empty plasmid and Bcl-2 expression vector, respectively.

VOL. 17, 1997


4349

FIG. 4. Lack of a role of the ICE-like proteases in the UV-induced inhibition of the aPKCs. NIH 3T3 cells were incubated either with buffer control (circles) or with 100 mM YVAD (squares) 30 min prior to UV irradiation. Cell extracts were prepared at different times, after which the aPKCs were immunoprecipitated and enzyme activity and expression were determined as described in Materials and Methods (filled symbols). In parallel cultures, the induction of apoptosis was determined by TUNEL (empty symbols). (A) Results are the means 6 standard deviations of three independent experiments with incubations in duplicate. (B) Representative experiment showing MBP phosphorylation and zPKC expression levels.

expression of catalytically active zPKC or l/iPKC (15). Par-4 was recently reported to bind the nuclear protein WT1 (27). If true, this observation implies that Par-4 can modulate not only the aPKCs, which are located in the cytosol (15a), but also a nuclear activity. To rule out any hypothetical nuclear function of Par-4 in apoptosis, the theoretically predicted nuclear localization signal located at amino acids 24 to 29 of the human par-4 gene product was deleted to generate Par-4DNLS. A Myc-tagged version of this Par-4 mutant was transfected into

FIG. 6. Morphological changes induced by Par-4 expression. NIH 3T3 cells were transfected with 5 mg of pCDNA3-myc-Par-4DNLS plus 15 mg of pRcCMV (A to D), pRcCMV-zPKCCAT (E and F), pCDNA-HA-MAPK (G and H), or pRcCMV5-HA-p38AGF (I and J). Cells were changed 24 h posttransfection to 1% serum-containing medium for 12 h and then immunostained with anti-Myc antibody (C, E, and G) or Hoechst 3358 (D, F, and H). Some cultures were analyzed 4 h posttransfection (A [immunostaining] and B [staining with Hoechst 3358]). Essentially identical results were obtained in another three experiments.

FIG. 5. Lack of a role of p53 in the UV-induced inhibition of the aPKCs. Control (empty bars and squares) or p53-/- (striped bars and circles) MEFs were UV irradiated, and cell extracts were prepared at different times thereafter; then the aPKCs were immunoprecipitated and the enzyme activity was determined (bars). In parallel cultures, the induction of apoptosis was determined by TUNEL (circles and squares). Results are the means 6 standard deviations of three independent experiments with incubations in duplicate.

NIH 3T3 cells, and expression of the protein was monitored by immunofluorescence with an anti-Myc antibody. Shortly after transfection (4 h), Par-4DNLS was located in the cytosol, and about 50% of the cells displayed a completely normal morphology with normal nuclei (Fig. 6A and B). When cells were visualized at longer times (12 to 16 h), most displayed an apoptotic morphology with no staining of Par-4 in the nucleus

4350

BERRA ET AL.

MOL. CELL. BIOL.

TABLE 1. Roles of Bcl-2 and ICE-like proteases in cell death induced by Par-4 expression and UV irradiationa No. of blue cells/well

Plasmid or treatment

Control

Bcl-2

CrmA

YVAD

Control Par-4DNLS Par-4 zPKCMUT UV irradiation

4,520 6 410 280 6 35 400 6 50 425 6 50 380 6 250

4,825 6 375 4,605 6 420 4,900 6 550 820 6 50 3,800 6 350

4,800 6 520 4,500 6 350 4,600 6 420 4,200 6 400 3,750 6 300

4,720 6 550 4,580 6 419 4,700 6 500 4,550 6 560 3,550 6 420

a NIH 3T3 cells were transfected with pCMV-bgal (2.5 mg) and 5 mg of either plasmid pCDNA3 (control) or an expression vector for Par-4DNLS (Par4DNLS), Par-4, zPKCMUT, Bcl-2, or CrmA. Twenty-four hours posttransfection, cells were changed to 0.5% serum-containing medium for 24 h, after which they were fixed and stained with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal). In other experiments, cells were induced to undergo apoptosis by UV irradiation and 12 h later were fixed and stained as described above. In some experiments, cells were incubated with 100 mM YVAD 30 min prior to the transfection or stimulation with UV irradiation. Results are the means 6 standard deviations of three independent experiments with incubations in duplicate.

but with clearly apoptotic nuclei (Fig. 6C and D). Collectively, these findings indicate that Par-4 does not have to be in the nucleus to induce apoptosis. Transfection of a constitutively active form of zPKC completely abrogated the induction of apoptosis by Par-4DNLS (Fig. 6E and F), in good agreement with previous data for the full-length protein (15). We next transfected either a Par-4DNLS or Par-4 expression vector along with a control plasmid or expression vector for Bcl-2 or CrmA into NIH 3T3 cells either untreated or incubated with 100 mM YVAD. Results in Table 1 show a series of b-galactosidase cell viability assays. Interestingly, transfection of Bcl-2 inhibited the ability of Par-4 and of Par-4DNLS to induce apoptosis as did transfection of CrmA or incubation with YVAD (Table 1). The same kind of response was obtained when cells were exposed to UV light (Table 1). This observation is consistent with a model whereby ICE-like proteases may be located downstream of the inhibition of the aPKCs in the apoptotic cascade. These results also indicate that the negative signals induced by Par-4 that lead to inhibition of the aPKCs are counteracted by positive signals produced by expression of Bcl-2. To more firmly support this notion, NIH 3T3 cells (Fig. 7) were transfected with HAtagged zPKC along with Myc-tagged Par-4 with either a control plasmid or an expression vector for Bcl-2. Interestingly, the ability of Par-4 to inhibit zPKC was dramatically reduced by expression of Bcl-2, which also increased the basal activity of the transfected zPKC (Fig. 7). Consistently, transfection of Bcl-2 did not inhibit the induction of apoptosis by dominant negative zPKC (Table 1), suggesting that Bcl-2 proteins are actually located upstream of zPKC. Also, transfection of a permanently active mutant of zPKC reproducibly reduced the induction of apoptosis by Bax in NIH 3T3 cells (not shown). Together, these results indicate that the aPKC activity is modulated by Par-4 and Bcl-2 in opposite manners. Regarding the potential role of p53 as a downstream event in the induction of apoptosis by Par-4, the results in Table 2 demonstrate that the induction of apoptosis by Par-4 or UV irradiation was completely independent of p53. This finding, together with the data in Fig. 5, indicates that p53 is neither upstream nor downstream of the aPKCs in the UV-induced apoptotic pathways and probably is not involved in the induction of cell death by this genotoxic stimulus. Effects of Par-4 expression on the activities of different MAPKs. We (6) and others (7, 18) have recently shown that the aPKCs may be critically involved in the regulation of

FIG. 7. Effect of Bcl-2 on the inhibition of zPKC by Par-4 expression. NIH 3T3 cells were transfected with 10 mg of HA-tagged zPKC along with different concentrations of an expression vector for Par-4DNLS together with 10 mg of either a control plasmid (circles) or an expression vector for Bcl-2 (squares). Empty vector was added to give 40 mg of total DNA. After 24 h, transfected zPKC activity and expression were determined. (A) Results are the means 6 standard deviations of three independent experiments with incubations in duplicate. (B and C) Representative experiments showing MBP phosphorylation and HA-zPKC expression control of cells transfected an empty plasmid and Bcl-2 expression vector, respectively.

MAPK but not SAPK (6). This could be of potential relevance because the balance of MAPK activity to that of SAPK and p38 can sometimes be critical for the induction of apoptosis (51, 55). Thus, we decided to determine the effect of the expression of Par-4 on the activity of MAPK, SAPK, and p38. Therefore, HA-tagged versions of zPKC or MAPK were transfected in Cos (Fig. 8) or NIH 3T3 (not shown) cells along with different concentrations of Myc-tagged Par-4DNLS expression vector, after which zPKC and MAPK activities were determined in cells that were either untreated or stimulated with PDGF.

TABLE 2. Role of p53 in cell death induced by Par-4 expression and UV irradiationa No. of blue cells/well


Control

p53-/-

Control Par-4DNLS UV irradiation

4,650 6 440 290 6 40 370 6 28

4,825 6 375 260 6 42 380 6 50

a Early-passage normal or p53-/- MEFs were transfected with pCMV-bgal (2.5 mg) and 5 mg of either plasmid pCDNA3 (control) or an expression vector for Par-4DNLS (Par-4DNLS). Twenty-four hours posttransfection, cells were changed to 0.5% serum-containing medium for 24 h, after which they were fixed and stained with X-Gal. In other experiments, cells were induced to undergo apoptosis by UV irradiation and 12 h later were fixed and stained as described above. Results are the means 6 standard deviations of three independent experiments with incubations in duplicate.

VOL. 17, 1997


4351

ability of Par-4 to induce apoptosis, as illustrated in Table 3. It seems clear that the overexpression of wild-type MAPK severely inhibits cell death by Par-4 or UV irradiation, as determined in b-galactosidase cell viability assays. Conversely, expression of dominant negative MAPK, which by itself induces a moderate reduction in the number of viable cells, sensitizes cells to the proapoptotic actions of Par-4 and UV-irradiation (Table 3). Of great interest, transfection of dominant negative p38 severely abrogates apoptosis induced by Par-4 or UV irradiation, whereas wild-type p38 behaved similarly to dominant negative MAPK, making cells more sensitive to Par-4 and UV effects (Table 3). Experiments carried out in parallel, using different reporter genes for MAPK and p38 (45), demonstrate the actual dominant negative abilities of the MAPKMUT and

FIG. 8. Par-4 expression inhibits MAPK activity. Cos cells were transfected with 10 mg of either HA-zPKC (upper panel) or HA-MAPK (lower panel) along with different concentrations of a Myc-tagged Par-4DNLS expression vector and enough empty vector to give 35 mg of total DNA. After 24 h, cells were made quiescent by serum starvation and either not stimulated or stimulated with 100 ng of PDGF per ml. Epitope (HA)-tagged zPKC and MAPK were immunoprecipitated, and their activities were determined by using MBP as the substrate. Extracts were analyzed by immunoblotting with anti-HA for expression of zPKC and MAPK and with anti-Myc for Par-4. Essentially identical results were obtained in another three experiments.

Both the basal and the PDGF-stimulated activities of zPKC and MAPK were severely abrogated by expression of Par4DNLS (Fig. 8), with no effect on SAPK or p70S6K (used as a negative control) activity (not shown), in agreement with previous results (6). Of potential great relevance, inhibition of the aPKCs by expression of Par-4 dramatically activates cotransfected p38, to an extent comparable to that produced by UV irradiation both in Cos cells (Fig. 9A) and NIH 3T3 fibroblasts (not shown). Transfection of a constitutively active zPKC mutant severely impaired both Par-4- and UV-induced p38 activation (Fig. 9B and C), suggesting that the inhibition of zPKC is a prerequisite for the activation of p38 by both apoptotic stimuli. Also, the ability of Par-4 to induce p38 was not affected by the presence of YVAD, which completely abrogates apoptosis. Together, these data indicate that inhibition of the aPKC activity leads to a decrease in the MAPK-p38 balance of activities that may be decisive for the induction of apoptosis in response to UV irradiation. The results in Fig. 10 demonstrate that exposure of NIH 3T3 cells to UVC light leads to a significant decrease of MAPK activity that is concomitant with a dramatic and sustained increase in p38 activity. This timedependent imbalance of MAPK to p38 begins as early as 1 h after UV irradiation (Fig. 10) and precedes the induction of apoptosis (see above), suggesting a critical role for these kinases in the control of the programmed cell death pathways. Role of the MAPKs in Par-4- and UV-induced effects. The alteration of the MAP kinase-p38 balance is decisive for the

FIG. 9. Par-4 expression activates p38 activity. (A) Cos cells were transfected with 10 mg of HA-p38 along with different concentrations of myc-Par-4DNLS expression vector, after which cells were extracted, p38 was immunoprecipitated, and its activity was determined by using recombinant ATF2 as the substrate. Cultures transfected with HA-p38 plus control vector were exposed to UV irradiation, and the activity of p38 was determined. Expression levels of HA-p38 and myc-Par-4DNLS were determined by immunoblotting with anti-HA and anti-Myc antibodies, respectively. Essentially identical results were obtained in another three experiments. (B) Cos cells were transfected with 10 mg of HA-p38 along with either control vector (lanes 1 and 4) or myc-Par-4DNLS expression plasmid (15 mg [lanes 2 and 5] or 25 mg [lanes 3 and 6]) either with control vector or an expression plasmid for zPKCCAT. The activity of p38 was determined as described above. Expression levels of HA-p38, myc-Par-4DNLS, and zPKCCAT were determined by immunoblotting with the corresponding antibodies. Essentially identical results were obtained in another three experiments. (C) Cos cells transfected with 10 mg of HA-p38 and either a control plasmid or an expression vector for zPKCCAT were exposed or not exposed to UV light, and p38 activity was determined 120 min thereafter as described above. Expression levels of HA-p38 and zPKCCAT were determined by immunoblotting with the corresponding antibodies. Essentially identical results were obtained in another three experiments.

4352

BERRA ET AL.

MOL. CELL. BIOL.

FIG. 10. Effect of UV irradiation on MAPK and p38 activities. NIH 3T3 cells were left intact or irradiated with UVC at different times as described in Materials and Methods. Cell lysates were analyzed by immunoblotting with antibodies to phospho-p38 or phospho-MAPK. Essentially identical results were obtained in another three experiments.

p38AGF constructs (not shown). Therefore, inhibition of MAPK and activation of p38 appear to be important events in the mechanism of action of Par-4. To further confirm this notion, Myc-tagged Par-4DNLS was transfected into NIH-3T3 cells along with either a control vector or an expression plasmid for dominant negative p38 or wild-type MAPK, after which transfectants were visualized by immunofluorescence with an anti-Myc antibody. We found that transfection of dominant negative p38 (Fig. 6I and J) or wild-type MAPK (Fig. 6G and H) abrogated apoptosis induced by Par-4. DISCUSSION Understanding the signaling events that control proliferation, survival, and programmed cell death is of paramount importance in the identification of potentially novel targets for better and novel therapeutic approaches in cancer and inflammation. The aPKCs have recently been proposed to play critical roles in these pathways. Thus, inhibition of the aPKCs by a variety of strategies blocks the ability of cells to grow (5) and even to survive, as determined by cell colony assays (13). More recently, the identification of a selective protein modulator, named Par-4 (50), that binds and inhibits both aPKCs and is induced during apoptosis (15) suggests a novel role for these kinases in the control of cell death. Actually, inhibition of both aPKCs by the forced expression of Par-4 or with dominant negative mutants promotes apoptosis as scored by changes in the morphology of the transfectants and by the appearance of the characteristic DNA laddering, indicative of oligonucleosomal DNA fragmentation (15). Consistent with the role of the aPKCs in cell survival is the fact that exposure of cells to UV irradiation, a potent genotoxic stimulus linked to the induction of apoptosis, provokes a severe reduction in the activity of the aPKCs prior to the beginning of cell death (reference 15 and Fig. 1). Apoptosis is regulated by a number of different molecules ranging from Bcl-2 proteins to ICE-like proteases and p53 (9,

20, 22, 25). To better understand how the aPKCs participate in cell proliferation and survival, the connection between these apoptosis-related molecules and the regulation of the aPKCs needed to be clarified. In this regard, we show here that p53 is not involved in the inhibition of aPKC activity by UV irradiation. In addition, according to our results, p53 does not appear to mediate apoptosis by UV light or by inhibition of the aPKCs. It seems, however, that UV irradiation, and other genotoxic stresses, signal to p53, promoting its accumulation (34). The available data indicate that the kinetics of p53 induction in response to UV irradiation is slower than that of, for example, g irradiation. Thus, cell exposure to UV light promotes p53 accumulation by 14 h in MEFs whereas g irradiation maximally induces p53 by 1 h (58). Therefore, the results shown here indicate the existence of alternative p53-independent routes during UV-induced apoptosis that appears to be mediated by inhibition of the aPKCs. This aPKC-regulated pathway is abrogated by two different inhibitors of the ICE-like protease system, YVAD and CrmA. This finding, together with the fact that UV-induced aPKC inhibition is not affected by the blockade of the ICE-like protease system, locates the aPKCs as potential upstream modulators of the protease cascade. In some situations, such as in the Fas signaling route, the pathway appears to be unexpectedly direct from the membrane receptor to the protease cascade, leaving little room for further modulation. Thus, Fas binds to the death domain-containing protein FADD that binds and directly signals to FADD-like ICE (FLICE), which itself is an ICE-like protease (7, 39). However, in other cellular situations, such as induction of apoptosis by doxorubicin, vincristine, or interleukin-3 withdrawal of 32D cells, apoptosis is completely resistant to protease inhibitors, suggesting the existence of ICE-like proteaseindependent pathways to cell death (36). In the UV response, like in other apoptotic pathways, the protease cascade is modulated by an upstream step that determines whether cells survive or die (25). This decision step may be modulated by Bcl-2 (11, 25). Interestingly, Bcl-2 is shown here to be a potent in vivo activator of the aPKCs, whereas the proapoptotic Bax is an inhibitor. The mechanism whereby Bcl-2 proteins modulate the aPKCs is unclear, and the existence of a completely indirect interaction between Bcl-2 and the aPKCs cannot be ruled out at this moment (52). Bcl-2 has been proposed to act as a docking system that positions Raf-1 to the mitochondria which serves to trigger an apparently MAPK-independent, as yet undefined survival signaling pathway (52). We do not know if the aPKCs could be similarly translocated to the mitochondria, and there is no evidence for a direct binding of Bcl-2 to the aPKCs as has been shown for Raf-1 (52). Nevertheless, we show here that, contrary to Raf-1 (52), the MAPK cascades appear to be decisively involved in the mechanism of action of the aPKCs in signaling survival. Thus, inhibition of MAPK and the parallel

TABLE 3. Roles of MAPK and p38 in cell death induced by Par-4 expression and UV irradiationa No. of blue cells/well


Control

MAPK

MAPKMUT

p38

p38AGF

Control Par-4 UV irradiation

4,720 6 410 270 6 40 380 6 45

5,225 6 375 5,020 6 540 4,580 6 450

1,825 6 250 60 6 50 180 6 70

3,825 6 275 160 6 60 156 6 50

4,825 6 375 4,760 6 420 4,580 6 550

a NIH 3T3 cells were transfected with pCMV-bgal (2.5 mg) and 5 mg of either plasmid pCDNA3 (control) or an expression vector for Par-4DNLS (Par-4), MAPK, or p38 (wild type or dominant negative). Twenty-four hours posttransfection, cells were changed to 0.5% serum-containing medium for 24 h, after which they were fixed and stained with X-Gal. In other experiments, cells were induced to undergo apoptosis by UV irradiation and 12 h later were fixed and stained as described above. Results are the means 6 standard deviations of three independent experiments with incubations in duplicate.

VOL. 17, 1997


activation of p38 were observed as consequences of the blockade of the aPKCs by Par-4 and in UV-irradiated cells. This mechanism is more similar to that of other systems where the balance of MAPK to SAPK and p38 controls cell survival (51, 55). It is noteworthy that the simple activation of SAPK and p38 may not be critical in some circumstances to induce apoptosis (35) and that the inhibition of MAPK is a prerequisite for SAPK and p38 to be able to promote cell death (9). Actually, our own unpublished data indicate that transfection of p38 along with activated MKK6 potently induces apoptosis in NIH 3T3 cells only when cells are incubated with limiting amounts of serum. In the presence of high (10%) serum concentrations, p38 plus activated MKK6 induced apoptosis only when cells were cotransfected with dominant negative MAPK or with Par-4 (which reduces MAPK by inhibiting zPKC). Therefore, downregulation of MAPK appears to be necessary for SAPK and p38 to induce programmed cell death. Also the continuance of the activation of p38 and SAPK seems critical. Thus, in marked contrast to MAPK, the activation of p38 by UV light is dramatic and sustained (Fig. 10). Interestingly, at early times (1 to 15 min) after UV irradiation, not only MAPK but also zPKC and AP-1 are significantly activated (15a, 46), whereas at later times, the three activities dramatically diminish (Fig. 1 and 10). The early increase in prosurvival signals can be interpreted as an attempt of the cell to survive and may be accounted for by the induction of a potent cross talk between different cytokine receptors at the plasma membrane (46). However, despite this early UV-induced “pseudomitogenic” response, cells ultimately die. The exact trigger of the apoptotic signal that reverses the early positive activation of mitogenic signaling molecules is not fully clear, but we showed previously that Par-4 is induced by UV irradiation, which leads to the inhibition of the aPKCs (15). This inhibition of aPKC by Par-4 contributes significantly to the subsequent decrease in the MAPK-p38 balance of activities. The fact that genotoxic stimuli activate the p38 and SAPK pathways in a sustained manner whereas mitogens do it transiently has been shown to be critical to determine whether the stimulated cells proliferate or enter apoptosis (10). In this regard, when the transient stimulation of SAPK and p38 induced by a mitogenic stimulus is sustained by incubation with a phosphatase inhibitor (10), the biological response produced by the mitogen is apoptosis instead of proliferation (10). Therefore, both the inhibition of MAPK and the duration of the p38 activation response emerge as two critical parameters that may determine the ability of p38 and SAPK to induce apoptosis. The ability of MAPK to serve as a survival signal may be related to its role in the activation of the c-Fos transcription factor through the phosphorylation of Elk-1 (31, 48). It is also possible that the alteration in the MAPK-p38 balance may affect molecules critical for the apoptotic response other than transcription factors. Consistent with this concept, it seems clear that the MAPKs phosphorylate a number of potentially important different signaling proteins (31). The fact that the ICE-like proteases are downstream of the inhibition of the aPKCs, together with the observation that the inhibition of these proteases reduces at least in part the induction of apoptosis by p38 plus activated MKK6 (15a), suggests that the ICE proteases may be among the important targets of the MAPK-p38 signaling cascade. However, it should be noted that other pathways may critically participate in the protection of cells from apoptosis by UV irradiation. For example, recent results demonstrate that overexpression of active mutants of PI 3-kinase or c-AKT significantly improves cell survival in UV-irradiated cells (30, 44a). All of these routes may constitute alternative or redundant mechanisms of cell survival. The different contribution of one pathway relative to

4353

the others may depend on the cell type or other environmental constraints. ACKNOWLEDGMENTS This work was supported by grants SAF96-0216 from CICYT and PB93-180 from DGICYT. M.M.M. and S.F. are Fellows from GlaxoCSIC and Ministerio de Educacio ń, respectively. This work was funded in part by Glaxo Wellcome Spain and has benefited from an institutional grant from Fundacio ń Ramo ń Areces to the CBM. We are indebted to Esther Garcia, Carmen Iban ˜ez, and Beatriz Ranera for technical assistance. We thank Gonzalo Paris and Isabel Perez for help and enthusiasm. E.B., M.M.M., and L.S. contributed equally to this study. REFERENCES 1. Akimoto, K., K. Mizuno, S. Osada, S. Hirai, S. Tanuma, K. Suzuki, and S. Ohno. 1994. A new member of the third class in the protein kinase C family, PKCl, expressed dominantly in an undifferentiated mouse embryonal carcinoma cell line and also in many tissues and cells. J. Biol. Chem. 269:12677– 12683. 2. Akimoto, K., R. Takahashi, S. Moriya, N. Nishioka, J. Takayanagi, K. Kimura, Y. Fuqua, S.-I. Osada, K. Mizuno, S.-I. Hirai, A. Kazlauskas, and S. Ohno. 1996. EGF or PDGF receptors activate atypical PKCl through phosphatidylinositol 3-kinase. EMBO J. 15:788–798. 3. Attardi, L. D., S. W. Lowe, J. Brugarolas, and T. Jacks. 1996. Transcriptional activation by p53, but not induction of the p21 gene, is essential for oncogene-mediated apoptosis. EMBO J. 15:3693–3701. 4. Bandyopadhyay, G., M. L. Standaert, L. Zhao, Y. Binzhi, A. Avignon, L. Galloway, P. Karnam, J. Moscat, and R. V. Farese. 1996. Activation of protein kinase C (a, b, z) by insulin in 3T3/L1 cells. Transfection studies suggest a role for PKC-z in glucose transport. J. Biol. Chem. 272:2551–2558. 5. Berra, E., M. T. Diaz-Meco, I. Dominguez, M. M. Municio, L. Sanz, J. Lozano, R. S. Chapkin, and J. Moscat. 1993. Protein kinase C z isoform is critical for mitogenic signal transduction. Cell 74:555–563. 6. Berra, E., M. T. Diaz-Meco, J. Lozano, S. Frutos, M. M. Municio, P. Sanchez, L. Sanz, and J. Moscat. 1995. Evidence for a role of MEK and MAPK during signal transduction by protein kinase C z. EMBO J. 14:6157– 6163. 7. Bjorkoy, G., M. Perander, A. Overvatn, and T. Johansen. 1997. Reversion of Ras and phosphatidylcholine-hydrolyzing phospholipase C-mediated transformation of NIH 3T3 cells by a dominant interfering mutant of protein kinase Cl is accompanied by the loss of constitutive nuclear MAPK/ERK activity. J. Biol. Chem. 272:11557–11565. 8. Boldin, M. P., T. M. Goncharov, Y. V. Goltsev, and D. Wallach. 1996. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85:803–815. 9. Canman, C. E., and M. B. Kastan. 1996. Three paths to stress relief. Nature 384:213–214. 10. Chen, Y., X. Wang, D. Templeton, R. J. Davis, and T.-H. Tan. 1996. The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and g radiation. J. Biol. Chem. 271:31929–31936. 11. Chinnaiyan, A. M., K. Orth, K. O’Rourke, H. Duan, G. G. Poirier, and V. M. Dixit. 1996. Molecular ordering of the cell death pathway. J. Biol. Chem. 271:4573–4576. 12. Diaz-Meco, M. T., E. Berra, M. M. Municio, L. Sanz, J. Lozano, I. Dominguez, V. Diaz-Golpe, M. T. Lain de Lera, J. Alcami, C. V. Paya, F. Arenzana-Seisdedos, J. L. Virelizier, and J. Moscat. 1993. A dominant negative protein kinase C z subspecies blocks NF-kB activation. Mol. Cell. Biol. 13:4770–4775. 13. Diaz-Meco, M. T., J. Lozano, M. M. Municio, E. Berra, S. Frutos, L. Sanz, and J. Moscat. 1994. Evidence for the in vitro and in vivo interaction of Ras with protein kinase C z. J. Biol. Chem. 269:31706–31710. 14. Diaz-Meco, M. T., M. M. Municio, P. Sanchez, J. Lozano, and J. Moscat. 1996. Lambda-interacting protein, a novel protein that specifically interacts with the zinc finger domain of the atypical protein kinase C isotype l/i and stimulates its kinase activity in vitro and in vivo. Mol. Cell. Biol. 16:105–114. 15. Diaz-Meco, M. T., M. M. Municio, S. Frutos, P. Sanchez, J. Lozano, L. Sanz, and J. Moscat. 1996. The product of par-4, a gene induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C. Cell 86:777–786. 15a.Diaz-Meco, M. T., et al. Unpublished data. 16. Dominguez, I., M. T. Diaz-Meco, M. M. Municio, E. Berra, A. Garcia de Herreros, M. E. Cornet, L. Sanz, and J. Moscat. 1992. Evidence for a role of protein kinase C z subspecies in maturation of Xenopus laevis oocytes. Mol. Cell. Biol. 12:3776–3783. 17. Dominguez, I., L. Sanz, F. Arenzana-Seisdedos, M. T. Diaz-Meco, J. L. Virelizier, and J. Moscat. 1993. Inhibition of protein kinase z subspecies blocks the activation of a NF-kB-like activity in Xenopus laevis oocytes. Mol. Cell. Biol. 13:1290–1295.

4354

BERRA ET AL.

18. Duang-Fang, L., M. Brett, D. Nicholas, and C. B. Bradford. 1997. Protein kinase C-z mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. J. Biol. Chem. 279:8146–8160. 19. Exton, J. H. 1994. Phosphatidylcholine breakdown and signal transduction. Biochim. Biophys. Acta 1212:26–42. 20. Farrow, S. N., and R. Brown. 1996. New members of the Bcl-2 family and their protein partners. Curr. Opin. Genet. Dev. 6:45–49. 21. Folgueira, L., J. A. McElhinny, G. D. Bren, W. S. MacMorran, M. T. DiazMeco, J. Moscat, and C. V. Paya ´. 1996. Protein kinase C-z mediates NF-kB activation in human immunodeficiency virus-infected monocytes. J. Virol. 70:223–231. 22. Fraser, A., and G. Evan. 1996. A license to kill. Cell 85:781–784. 23. Gottlieb, M. T., and M. Oren. 1996. p53 in growth control and neoplasia. Biochim. Biophys. Acta 1287:77–102. 24. Hannun, Y. A. 1994. The sphingomyelin cycle and the second messenger function of ceramide. J. Biol. Chem. 269:3125–3128. 25. Henkart, P. A. 1996. ICE family proteases: mediators of all apoptotic cell death? Immunity 4:195–201. 26. Huang, C., W. Ma, G. T. Bowden, and Z. Dong. 1996. Ultraviolet B-induced activated protein-1 activation does not require epidermal growth factor receptor but it is blocked by a dominant negative PKCl/i. J. Biol. Chem. 271:31262–31268. 27. Johnstone, R. W., R. H. See, S. F. Sells, J. Wang, S. Muthukkumar, C. Englert, D. A. Haber, J. D. Licht, S. P. Sugrue, T. Roberts, V. M. Rangnekar, and Y. Shi. 1996. A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms’ tumor suppressor WT1. Mol. Cell. Biol. 16:6945–6956. 28. Ko, L. J., and C. Prives. 1996. p53: puzzle and paradigm. Genes Dev. 10:1054–1072. 29. Kolesnick, R., and D. W. Golde. 1994. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 77:325–328. 30. Kulik, G., A. Klippel, and M. J. Weber. 1997. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol. Cell. Biol. 17:1595–1606. 31. Kyriakis, J. M., and J. Avruch. 1996. Sounding the alarm: protein kinase cascades activated by stress and inflammation. J. Biol. Chem. 271:24313– 24316. 32. Limatola, C., B. Barabino, A. Nista, and A. Santoni. 1997. Interleukin 1-binduced protein kinase C-z activation is mimicked by exogenous phospholipase D. Biochem. J. 321:497–501. 33. Liscovitch, M., and L. C. Cantley. 1994. Lipid second messengers. Cell 77:329–334. 34. Liu, Z.-G., R. Baskaran, E. T. Lea-Chou, L. D. Wood, Y. Chen, M. Karin, and J. Y. J. Wang. 1996. Three distinct signalling responses by murine fibroblasts to genotoxic stresses. Nature 384:273–276. 35. Liu, Z-G, H. Hsu, D. V. Goeddel, and M. Karin. 1996. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kB activation prevents cell death. Cell 87:565–576. 35a.Long, S. D., and P. H. Pekala. 1996. Lipid mediators of insulin resistance: ceramide signalling down-regulates Glut4 gene transcription in 3T3/L1 adipocytes. Biochem. J. 319:179–184. 36. Lotem, J., and L. Sachs. 1996. Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents. Proc. Natl. Acad. Sci. USA 93:12507–12512. 37. Lozano, J., E. Berra, M. M. Municio, M. T. Diaz-Meco, I. Dominguez, L. Sanz, and J. Moscat. 1994. Protein kinase C z isoform is critical for kBdependent promoter activation by sphingomyelinase. J. Biol. Chem. 269: 19200–19202. 38. Miyashita, T., and J. C. Reed. 1995. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80:293–299. 39. Muzio, M., A. M. Chinnaiyan, F. C. Kischkel, K. O’Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J. D. Bretz, M. Zhang, R. Gentz, M. Mann, P. H. Krammer, M. E. Peter, and V. M. Dixit. 1996. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-

MOL. CELL. BIOL. inducing signaling complex. Cell 85:817–827. 40. Nakanishi, H., and J. H. Exton. 1992. Purification and characterization of the zeta isoform of protein kinase C from bovine kidney. J. Biol. Chem. 267:16347–16354. 41. Nakanishi, H., K. A. Brewer, and J. H. Exton. 1993. Activation of the z isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 268:13–16. 42. Nishizuka, Y. 1992. Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C. Science 258:607–614. 43. Ohno, Y., T. Fuji, K. Ogita, U. Kikkawa, K. Igarashi, and Y. Nishizuka. 1989. Protein kinase C z subspecies from rat brain: its structure, expression, and properties. Proc. Natl. Acad. Sci. USA 86:3099–3103. 44. Oltavi, Z. N., and S. J. Korsmeyer. 1994. Checkpoints of dueling dimers foil death wishes. Cell 79:189–192. 44a.Parrizas, M., A. R. Saltiel, and D. LeRoith. 1997. Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 39-kinase and mitogenactivated protein kinase pathways. J. Biol. Chem. 272:154–161. 45. Raingeaud, J., A. J. Whitmarsh, T. Barrett, B. Derijard, and R. Davis. 1996. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol. Cell. Biol. 16:1247–1255. 46. Rosette, C., and M. Karin. 1996. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science 274:1194–1197. 47. Rzymkiewicz, D. M., T. Tetsuka, D. Daphna-Iken, S. Srivastava, and A. R. Morrison. 1996. Interleukin-1b activates protein kinase Cz in renal mesangial cells. J. Biol. Chem. 271:17241–17246. 48. Schreiber, M., B. Baumannn, M. Cotten, P. Angel, and E. F. Wagner. 1995. Fos is an essential component of the mammalian UV response. EMBO J. 14:5338–5349. 49. Selbie, L. A., C. Schmitz-Peiffer, Y. Sheng, and T. J. Biden. 1993. Molecular cloning and characterization of PKCi, an atypical isoform of protein kinase C derived from insulin-secreting cells. J. Biol. Chem. 268:24296–24302. 50. Sells, S. F., D. P. Wood, S. S. Joshi-Barve, S. Muthukumar, R. J. Jacob, S. A. Crist, S. Humphreys, and V. M. Rangnekar. 1994. Commonality of the gene programs induced by effectors of apoptosis in androgen-dependent and -independent prostate cell. Cell Growth Differ. 5:457–466. 51. Verheij, M., R. Bose, X. H. Lin, B. Yao, W. D. Jarvis, S. Grant, M. J. Birrer, E. Szabo, L. I. Zon, J. M. Kyriakis, A. Haimovitz-Friedman, Z. Fuks, and R. N. Kolesnick. 1996. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380:75–79. 52. Wang, H.-G., U. R. Rapp, and J. C. Reed. 1996. BCl-2 targets the protein kinase Raf-1 to mitochondria. Cell 87:629–638. 53. Wooten, M. W., G. Zhou, M. L. Seibenhener, and E. S. Coleman. 1994. A role for z protein kinase C in nerve growth factor induced differentiation of PC12 cells. Cell Growth Differ. 5:395–403. 54. Wooten, M. W., M. L. Seibenhener, L. H. Matthews, G. Zhou, and E. S. Coleman. 1996. Modulation of z-protein kinase C by cyclic AMP in PC12 cells occurs through phosphorylation by protein kinase A. J. Neurochem. 67:1023–1031. 55. Xia, Z., M. Dickens, J. Raingeaud, R. J. Davis, M. E. Greenberg. 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326–1332. 56. Xu, J., M. M. Zutter, S. A. Santoro, and R. Clark. 1996. PDGF-induction of a2 integrin gene expression is mediated by protein kinase C z. J. Cell Biol. 134:1301–1311. 57. Xu, J., and R. Clark. 1997. A three-dimensional collagen lattice induces protein kinase C-z activity. Role in a2 integrin and collagenase mRNA expression. J. Cell Biol. 136:473–483. 58. Xu, Y., and D. Baltimore. 1996. Dual roles of ATM in the cellular response to radiation and in cell growth control. Genes Dev. 10:2401–2410. 59. Yao, R., and G. M. Cooper. 1995. Requirement of phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267: 2003–2006.