INTRODUCTION

A growing body of evidence suggests that the induction of the mitogen-activated protein kinase (MAPK)¹ pathway leading to activation of extracellular signal-regulated kinase (ERK)-1 and -2 is essential for mitogenic signal transduction (1-4). ERK1 and -2 are rapidly activated after growth factor stimulation or overexpression of constitutively active Ras, Raf, or MAPK/ERK kinase (MEK) (2, 4). Following activation the ERKs translocate to the nucleus to phosphorylate their nuclear substrates (5-8). Results obtained by the use of dominant interfering mutants and antisense RNA suggest that activation of the ERKs may be required for proliferation of fibroblasts (9). The only direct activators of the ERKs identified so far are MEK1 and -2 (2, 4, 10). These dual specificity kinases activate ERK1 and -2 by phosphorylating both the threonine and the tyrosine residue in the sequence motif TEY (3). MEK1, but not MEK2, forms a ternary complex with Ras and Raf-1 (11). The Raf-1 kinase has been regarded as the major MEK1 activator in most cell systems (2, 12). However, it is now evident that several proteins may contribute to MEK1 activation (10, 13-16). Of particular relevance to this study is the finding that the atypical protein kinase C subtype  (PKC) is activated by tyrosine kinase receptors (17) and is shown to phosphorylate and activate MEK1 in vitro as well as in vivo (6, 18, 19). We (20, 21) and others (22, 23) have previously used the notion PKC for this PKC subspecies cloned from Xenopus laevis. However, the more recent description of /PKC (24, 25) identified this atypical PKC subspecies originating from Xenopus as PKC (19).

Polypeptide-derived growth factors and activation of Ras or Src cause an increase in the hydrolysis of phosphatidylcholine (PC) resulting in a sustained elevation in intracellular levels of PC-derived diacylglycerol (DAG) (26-32). Chronic stimulation of PC hydrolysis by stable expression of the gene (plc) encoding Bacillus cereus phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) causes severe growth deregulation and morphological transformation of NIH 3T3 fibroblasts without activating Ras (20, 21). Moreover, expression of B. cereus PC-PLC was able to release NIH 3T3 cells from a block to proliferation imposed by expression of dominant negative N-17 Ras but not so when the block was due to expression of dominant negative Raf-1 (33, 34). Consistently, expression of dominant negative Raf-1 reverted the transformed phenotype induced by plc expression (20). Furthermore, addition of purified bacterial PC-PLC to quiescent NIH 3T3 cell cultures induced Raf-1 kinase activity, whereas an inhibitor of endogenous PC-PLC activity blocked Raf-1 activation in response to serum (33-35). These findings suggest that the generation of DAG by PC hydrolysis is located downstream of Ras but upstream of Raf in the mitogenic signal transduction pathway. Thus, PC-derived DAG may directly or indirectly be involved in the poorly defined activation of the Raf kinase family members (12).

Several proteins, including PKC, bind to Ras in a GTP-dependent manner suggesting that several pathways are involved in relaying Ras-mediated signals (20, 36-42). PKC (the human homolog is named PKC) constitutes together with PKC, the atypical PKCs. These PKC subtypes contain only one cysteine-rich zinc finger and is not activated by phorbol esters or Ca²⁺ ions (43). A requirement for functional PKC in the Ras-mediated insulin-induced maturation pathway in Xenopus oocytes and for serum-activated DNA synthesis in murine fibroblasts has been demonstrated (22, 23). Consistent with the notion of PKC as a downstream target of activated Ras, a dominant negative mutant of PKC, as well as dominant negative Raf-1, was found to revert both v-ras- and plc-induced transformation (20). The atypical PKCs have been reported to be involved in several signaling pathways activating different downstream components. PKC is important for NFB activation in different cell lines, including NIH 3T3, probably through activation of an IB kinase (18, 19). Furthermore, PKC plays a critical role during stromelysin promoter activation by PDGF in fibroblasts (44). Recent experiments, using antisense oligonucleotides against different PKC subtypes, have shown that PKC acts as a mediator of PDGF-induced ₂-integrin gene expression in human dermal fibroblasts (45). Both PKC and PKC have been suggested as downstream components for PI 3-kinase-mediated signaling (17, 46). Thus, atypical PKCs clearly play critical roles in several signaling pathways.

In this paper we report that ERK1 and -2 are constitutively activated and localized to the nucleus in both v-ras- and plc-transformed cells. Expression of a dominant negative mutant of PKC abolished ERK activation in response to v-ras or plc expression. Consistently, v-ras- and plc-induced GAL-ElkC-transactivation was found to be dependent on functional PKC but did not involve activation of JNK. Moreover, the dominant negative mutant of PKC blocked v-ras but not activated MEK induction of ERK kinase activity. We also show that v-ras or plc transformation lead to PDGF receptor down-regulation leading to abolished superinduction of DNA synthesis in response to PDGF. This was also the case for a cell line stably overexpressing an activated mutant of MEK1. Reversion of the v-ras- and plc-transformed phenotype by expression of a dominant negative mutant of PKC re-established the receptor level and PDGF-induced receptor autophosphorylation, ERK kinase activation, and induction of DNA synthesis.

MATERIALS AND METHODS

NIH 3T3 fibroblasts (passage 123) were purchased from the American Type Culture Collection (ATCC CRL 1658) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum (HyClone, Logan, UT), penicillin (100 units/ml), and 100 µg/ml streptomycin (Life Technologies, Inc.) in a CO₂ incubator (5% CO₂) at 37 °C. NIH 3T3 cells transformed with the v-Ha-ras or v-src oncogenes (26, 28) were grown in the same medium. A well characterized PC-PLC expressing clone (clone P18)(20) was grown in the presence of hygromycin B (Calbiochem) at 300 µg/ml, whereas the doubly transfected cell lines, v-ras-dnPKC and plc-dnPKC (20), were grown in the presence of both hygromycin B and 400 µg/ml G418 (Life Technologies, Inc.).

Measurements of the induction of DNA synthesis were performed as described previously (21). The MEK inhibitor PD 098059 (New England Biolabs) was dissolved in dimethyl sulfoxide and added to the the cell cultures to a final concentration of 0.5% (v/v) of vehicle.

For preparation of cytosolic extracts, serum-starved or PDGF-stimulated cell cultures were rinsed twice with ice-cold phosphate-buffered saline and lysed in the dishes by adding ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄) for 30 min on ice. The lysates were collected with a rubber policeman, and insoluble material was removed by centrifugation in a microcentrifuge for 5 min at 13,000 rpm. The supernatant was aliquoted and stored at 70 °C. The protein concentrations in the cytosolic extracts were determined using a detergent-compatible protein assay kit (Bio-Rad DC Protein Assay) with bovine serum albumin as the standard.

Whole cell and nuclear extracts were prepared as described by Westwick and Brenner (47) and by Sjøttem et al. (48), respectively, in the presence of the following protease/phosphatase inhibitors: 2 µg/ml aprotinin, 40 µg/ml bestatin, 0.5 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 0.7 µg/ml pepstatin A, 20 mM -glycerophosphate, 50 µM Na₃VO₄, and 10 mM p-nitrophenyl phosphate (all from Sigma).

Activation of ERK1/2 in situ was determined using a rabbit polyclonal antibody raised against a synthetic phosphotyrosine peptide corresponding to residues 196-209 of human ERK1/p44 MAP kinase (9101, New England Biolabs, Inc., Beverly, MA). This antibody specifically recognizes activated ERK1 and -2. The different cell lines were seeded into 8-well chamber slides (Nunc, Inc., Naperville, IL) at subconfluent cell densities (less than 30% confluence) in DMEM supplemented with 10% calf serum and left for 24 h. The cultures were subsequently serum-starved for 20 h in DMEM supplemented with 0.1% calf serum and either stimulated with 10 ng/ml PDGF (BB homodimer, Sigma) for 15 min or left untreated. The immunostaining was performed essentially as described in the protocol obtained from the supplier (New England Biolabs). Briefly, the cells were fixed in 3% paraformaldehyde, permeabilized using a buffer containing 0.1% Triton X-100, and preincubated for 1 h in 5.5% normal horse serum. The primary antibody was added for 48 h at 4 °C in a humidified chamber, and the staining was developed using biotinylated secondary antibodies and preformed streptavidin-peroxidase complexes in a Ni(II)-enhanced DAB reaction (Vectastain, Vector Laboratories).

Phosphorylated/activated ERK1 and -2 were specifically detected using the phospho-ERK-specific antibody described above. The total level of ERK2 was determined using a monoclonal anti-ERK2 antibody (clone B9, Upstate Biotechnology Inc.) recognizing both phosphorylated and non-phosphorylated forms. The cell cultures were treated as indicated in the respective figure legends. Thrombin (-thrombin) was purchased from Hoffman-La Roche. The cell cultures were harvested directly into 1 × SDS-PAGE gel load buffer and immediately heated to 95 °C for 5 min and sonicated briefly on ice. The extracts (20 µl, corresponding to 2 × 10⁵ cells per lane) were separated by SDS-PAGE, electrotransferred onto polyvinylidene difluoride membranes (Millipore), and developed following protocols obtained from the respective suppliers of antibodies. Tyrosine phosphorylation of the PDGF receptor was determined using a phosphotyrosine-specific monoclonal antibody following the protocol obtained from the supplier (clone 4G10, Upstate Biotechnology Inc.). To verify protein loading, some of the immunoblots were stripped for 2 h in 0.2 M glycine, pH 2.4, 1% SDS at 65 °C, blocked, and reprobed with the anti-ERK2 antibody. The level of PDGF receptor was determined as described by Vaziri and Faller (49) using a polyclonal PDGF receptor antibody (PDGFR--specific, Santa Cruz Biotechnology). All immunoblots were developed using alkaline phosphatase-conjugated secondary antibodies and the chemiluminescent substrate CDP-Star (New England Biolabs or Boehringer Mannheim).

ERK activities in cytosolic or nuclear extracts were determined as described by Sale et al. (50). The activity of hemagglutinin (HA)-tagged ERK1 in extracts from transient transfected cell lines was measured by an immune complex kinase assay using MBP as the substrate. Subconfluent cell cultures were transfected using lipofectamine (Life Technologies, Inc.) according to the instructions of the manufacturer. Following 4 h of incubation with DNA, the cells were incubated for 24 h in the presence of 10% serum and then starved in 0.1% serum for another 24 h. Preparation of cell extracts and immunoprecipitation of HA-ERK1 using a monoclonal antibody recognizing the HA epitope (12CA5, Boehringer Mannheim) was performed as described (6). The immune complexes were washed three times with cell lysis buffer containing 0.5 M NaCl and twice with MBP kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl₂, 0.1 M Na₃VO₄). The complexes were resuspended in kinase buffer containing 1 mM dithiothreitol and 0.3 mg/ml MBP, and kinase reactions were initiated by adding [-³²P]ATP (0.1 mM, 5 cpm/fmol final). Following incubation for 15 min at 30 °C, the kinase reactions were terminated by adding 5 × SDS-PAGE gel load buffer and boiled immediately for 5 min. The phosphorylated proteins were separated on a 12.5% polyacrylamide gel and electrotransferred to a polyvinylidene difluoride membrane (Millipore). The phosphorylated proteins were detected by autoradiography and quantitated using a PhosphorImager (Molecular Dynamics). The activation status of the c-Jun N-terminal kinase (JNK) in the different cell lines was measured by a solid-phase kinase assay. To make GST-Jun_5-115 a sequence encoding amino acids 5-115 of c-Jun was amplified by polymerase chain reaction (5-GAATTGGATCCATGGAAACGACCTTCTATGAC-3 and 5-GAATTCTCGAGTGCTCATCTGTCACGTTCTTG-3) and inserted into the BamHI and XhoI sites of pGEX-4T-3 (Pharmacia Biotech Inc.) producing an in-frame fusion with glutathione S-transferase (GST). The fusion protein (GST-Jun_5-115) was expressed in Escherichia coli LE392 (51). GST-Jun_5-115 was coupled to glutathione-agarose beads and stored as a 20% suspension in NETN buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 0.5% (v/v) Nonidet P-40, 2 mM dithiothreitol, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 0.7 µg/ml pepstatin A). The kinase assays were performed as described by Westwick and Brenner (47). Phosphorylated proteins were resolved on 10% SDS-polyacrylamide gels that were dried and subjected to autoradiography at 70 °C with intensifying screens. Equal protein loading was verified by immunoblotting with an antibody against JNK1 (C-17; Santa Cruz Biotechnology).

To measure the activity of the C-terminal transactivation domain of Elk-1 in the different cell lines, pGAL4-ElkC was made by inserting a BglII-XbaI fragment from pBS-Elk-1 into the BamHI and XbaI sites of pSG424 (52). Subconfluent cultures of the different cell lines in 100-mm diameter Petri dishes were transfected with 2 µg of pGAL4-ElkC or pSG424 (vector control), 2 µg of the reporter vector pG₅E1bTATA-CAT (53), and 2 µg of salmon sperm carrier DNA using the calcium phosphate coprecipitation method. The precipitates were left on the cells for 4 h in medium containing 10% serum, after which the cells were glycerol-shocked for 90 s in 15% (v/v) glycerol in HBS buffer (137 mM NaCl, 5 mM D-glucose, 0.9 mM NaH₂PO₄, 21 mM HEPES, pH 7.08). The cells were washed three times with DMEM and serum-starved for 48 h in 0.1% serum. The cells were harvested and chloramphenicol acetyltransferase assays were performed as described previously (20).

RESULTS

ERK1 and -2 Are Constitutively Activated and Translocated to the Nucleus in v-ras- and plc-transformed Cells in a PKC-dependent Manner

We have previously shown that chronic stimulation of PC hydrolysis by expression of B. cereus PC-PLC caused induction of DNA synthesis, enhanced proliferation in the absence of added growth factors, and led to transformation of NIH 3T3 cells (21). Expression of a dominant negative mutant of PKC (dnPKC) led to reversion to a non-transformed, normal phenotype of both plc- and v-ras-expressing cells (20). Here, we wanted to further evaluate the signal mediators downstream of PC-derived DAG. Recently, PKC was found to activate MEK1 both in vitro and in vivo (6). To begin elucidating if the MEK/ERK pathway is involved in transduction of the mitogenic signal generated by PC-derived DAG, we used a novel inhibitor, PD098059, that specifically interferes with the activation of MEK1 and MEK2 (54). The ability of plc- and v-ras-expressing cells to induce DNA synthesis in the absence of added growth factors was completely lost in the presence of PD098059 (Fig. 1A). Also, prolonged treatment of the plc- and v-ras transformed cell lines with this inhibitor caused complete reversion to a normal, non-transformed phenotype (data not shown), indicating that MEK is a necessary downstream component for mitogenic signaling and transformation mediated by PC-derived DAG and v-ras. However, we found no significant increase in cytosolic ERK activity in serum-starved cell cultures of v-ras- or plc-transformed cells (Fig. 1B). Furthermore, treatment of these cell lines with PDGF did not induce increased ERK activity. Consistently, immunoblotting analyses of whole cell extracts using an antibody specifically recognizing phosphorylated and activated ERK1 and -2 did not reveal any potent ERK activation in response to stable expression of either v-ras or plc (Fig. 1C). Interestingly, unlike their parental transformed cells, the cell lines reverted by expression of dnPKC-induced phosphorylation of myelin basic protein in response to PDGF (Fig. 1B). The reverted cell lines also consistently showed a pronounced PDGF-induced phosphorylation of ERK1 and -2 (Fig. 1C). The activation of ERK1 and -2 in response to serum was indistinguishable between the different cell lines, suggesting that the differential activation of ERK in transformed and reverted cell lines was specific for PDGF-induced signaling.

Activation of the MAPK pathway is accompanied by translocation of ERK1 and -2 to the nucleus (4-8). Thus, we next analyzed the ERK activity in nuclear extracts from cells expressing v-ras or plc alone or together with dnPKC (Fig. 2). The basal nuclear ERK activity was increased in both v-ras- and plc-expressing cells compared with quiescent NIH 3T3 cells. However, the increased basal ERK activity displayed by the transformed cells was inhibited by expression of dnPKC. To further analyze this, serum-starved or PDGF-stimulated cell cultures were immunostained with an antibody specifically recognizing phosphorylated ERK1 and -2 (Fig. 3). These experiments confirmed that ERK1 and -2 were constitutively activated both in v-ras- and in plc-transformed cell lines. Moreover, phosphorylated ERKs were exclusively localized to the nuclei. Treating the transformed cell lines with PDGF for 15 min did not further increase the intensity of the nuclear staining (data not shown). Reversion of the v-ras- or plc-induced transformation by expression of dnPKC abolished the chronic activation of the nuclear ERKs. However, the phosphorylation of ERK1 and -2 in the reverted cell lines could be induced by PDGF. These data suggest that expression of v-ras as well as increased intracellular levels of PC-derived DAG caused by expression of B. cereus PC-PLC both result in activation and nuclear translocation of ERK1 and -2. Furthermore, the activation of ERKs in response to Ras and PC-PLC is dependent on functional PKC.

Transformation by v-ras or plc leads to constitutive activation of nuclear ERK1 and -2, whereas expression of dnPKC abolishes v-ras- or plc-induced ERK activation.

Expression of dnPKC Blocks Both v-ras- and plc-induced Activation of Elk-1

We have previously shown that dnPKC abolished the Ras- or PC-PLC-induced activation of both NF-B and AP-1 (20). However, since we found constitutive activation and nuclear translocation of ERKs in Ras- and PC-PLC-transformed cell lines, the activation status of the transcription factor Elk-1 was of interest. For this purpose, a chimeric transcription factor composed of the DNA binding domain of yeast GAL4 and the transactivating domain of Elk-1 (GAL4-ElkC) was constructed. The transactivation potential of this nuclear fusion protein is strongly enhanced by specific MAPK phosphorylation within the transactivation domain of Elk-1 (55). Consistent with the nuclear localization of activated ERKs in the transformed cell lines, we found that the transactivating potential of GAL4-ElkC was strongly induced in response to transformation by v-ras or plc (Fig. 4A). Reversion of the transformed cell lines by expression of dnPKC coincided with a large decrease in the transactivation potential of GAL4-ElkC down to the background level seen in quiescent NIH 3T3 cells. Thus, both v-ras and plc induced the transactivating potential of Elk-1 in a PKC-dependent manner. Recently, it was reported that another subclass of the MAPK family, termed the c-Jun N-terminal kinases (JNKs) or stress-activated protein kinases, phosphorylates and activates the transactivation domain of Elk-1 (56-58). To determine the activity of the JNKs in the transformed and reverted cell lines, whole cell extracts were used in a solid phase kinase assay using GST-Jun_5-115 as a substrate (Fig. 4B). GST-Jun_5-115 was extensively phosphorylated when treated with extract from UV-stimulated NIH 3T3 cells. However, we found no increase in JNK activity in ras-transformed cells and only a slight increase in plc-transformed cells. The same results were obtained by carrying out immune complex kinase assays using an antibody specifically recognizing JNK1 (data not shown). Prolonged treatment of ras- and plc-transformed cells with the specific MEK inhibitor PD 098059 totally abolished the GAL4-ElkC activity (data not shown). Altogether, these results clearly suggest that the increased transactivation potential observed for GAL4-ElkC is due to activation by nuclear ERK1 and -2 with very little, if any, contribution by JNKs.

Activation and nuclear translocation of ERKs in response to v-ras and plc is accompanied by PKC-dependent induction of Elk-1 transactivation.

Expression of dnPKC Does Not Affect MEK-induced ERK Activation

The expression of dnPKC blocked ras- and plc-induced activation of ERK as well as Elk-1 transactivation suggesting that PKC acts downstream of Ras and PC-PLC but upstream of ERK. To more firmly localize PKC with respect to MEK, we performed transient transfections measuring the activation of HA-tagged ERK1 (59) in response to Ha-Ras V-12 (22) or an activated mutant of MEK1 (aMEK) (60), in the presence or absence of dnPKC (19). The activation of ERK1 induced by Ras was blocked by cotransfection with the dnPKC expression vector. However, dnPKC did not affect MEK1-induced activation of ERK1 in NIH 3T3 cells (Fig. 5). Immunoblot analysis of cell extracts from the different transfected cell cultures showed that the differences in ERK activity were not due to differences in the expression of HA-tagged ERK1 (data not shown). Collectively, these results suggest that PKC is located downstream of Ras but upstream of MEK in the MEK/ERK pathway. To further evaluate the effect of dnPKC on MEK-mediated signaling in NIH 3T3 cells, we first made a cell line that stably overexpressed aMEK. The aMEK expressing cells displayed a transformed phenotype, induced DNA synthesis in the absence of added growth factors, showed a constitutively increased nuclear ERK activity and an increased GAL-ElkC transactivation potential (data not shown). However, several attempts to establish clones that stably coexpressed dnPKC and aMEK failed. Of more than 50 clones analyzed from two experiments performed with two different expression vectors for dnPKC, none expressed dnPKC. All of them had retained the transformed phenotype (data not shown). This indicates that dnPKC interferes with the survival of aMEK-transformed cells without affecting the activation of ERK. Thus, in addition to the MEK/ERK pathway, PKC also acts in other signaling pathways (17-20, 22, 44-46, 61), some of which may be critical for cell survival.

Coexpression of dnPKC blocks the activation of ERK induced by Ha-Ras^V12 but not the activation caused by an activated mutant of MEK1.

Expression of dnPKC Prevented the Constitutive Down-regulation of the PDGF Receptor Observed in Both v-ras- and plc-transformed Cell Lines

While determining ERK activities in whole cell extracts, we noted that the reverted cell lines responded more potently to PDGF than their parental transformed cell lines did (see Fig. 1). Expression of v-ras has previously been shown to cause suppression of PDGF receptor autophosphorylation (49, 62-64). Several mechanisms may be involved in this desensitization to PDGF stimulation including Ras-induced activation of protein-tyrosine phosphatase activity (64), induction of a membrane-bound inhibitor of receptor phosphorylation (63), and reduced transcription of the gene encoding the receptor (49). Thus, it was of interest to determine whether plc transformation inhibited PDGF receptor autophosphorylation as found for v-ras and if reversion of the transformed phenotype by dnPKC also restored receptor autophosphorylation. We therefore performed immunoblot analyses of tyrosine-phosphorylated proteins in extracts prepared from cells that were either untreated or stimulated with PDGF using thrombin stimulation as a negative control (Fig. 6A). Interestingly, a phosphotyrosine-containing protein with a migration corresponding to the PDGF receptor was only detected after PDGF stimulation of NIH 3T3 cells and the reverted cell lines. This tyrosine-phosphorylated protein could not be detected after PDGF stimulation of the transformed cell lines. Immunoblotting with an antibody against the PDGF receptor revealed complete down-regulation of the receptor in both v-ras and plc-transformed cells, whereas reversion of the transformed phenotype by expression of dnPKC coincided with restored receptor levels (Fig. 6B). PDGF treatment of the v-ras- and plc-transformed cell lines was not able to further increase the induction of DNA synthesis, whereas addition of 10% serum caused a superinduction of DNA synthesis in v-ras- and plc-transformed cells (Fig. 6C). This suggests that the insensitivity to PDGF was not due to a maximum stimulation of DNA synthesis by Ras or PC-PLC. On the other hand, PDGF did induce DNA synthesis in the reverted cell lines. Thus, reversion of the transformed cells by dnPKC expression prevented the constitutive down-regulation of the PDGF receptor allowing receptor autophosphorylation and ERK activation in response to PDGF. Since both v-ras and plc activate other signaling pathways in addition to the MEK/ERK pathway, we looked at PDGF receptor expression in cells stably transfected with aMEK. Western blot analyses revealed down-regulation of the PDGF- receptor showing that constitutive ERK activation is alone able to lead to receptor down-regulation (data not shown).

The constitutive down-regulation of the PDGF- receptor observed in both v-ras and plc-transformed cells does not occur in the dnPKC-reverted cell lines.

DISCUSSION

In this study we present evidence for chronic activation and nuclear translocation of ERK1 and -2 in NIH 3T3 cells transformed with v-ras or plc. Expression of a dominant negative mutant of PKC blocked the activation of ERK1 and -2. A sustained activation of ERK1 and -2 has been suggested to be a prerequisite for proliferation of fibroblasts and differentiation of PC12 cells (4). Interestingly, the ERKs located in the cytosol were not phosphorylated/activated in the transformed cells. Previously, Gardner et al. (65) reported no significant activation of MEK in v-ras-transformed NIH 3T3 cells. These workers found only modest constitutive activation of ERKs in Ras-transformed NIH 3T3 cells and no persistent ERK activity in similarly transformed Rat 1a fibroblasts (66). Their conclusion was based on the use of cytosolic extracts. These previous results are therefore entirely consistent with our present findings showing no constitutive activation of ERKs in the cytosol of the transformed cells. However, we show here that both v-ras- and plc-transformed cells contain constitutively elevated nuclear ERK activity. This may suggest that the mechanisms for terminating ERK activation by dephosphorylation mediated by protein phosphatases are probably operating normally in the cytosol, whereas the regulation of nuclear ERK activity is subverted in the transformed cells. Our results together with the recent demonstration that Ras transformation is inhibited by coexpression of kinase-defective mutants of ERK1 and -2 (38) indicate that constitutive nuclear ERK activity may be a prerequisite for transformation by v-ras or plc. Thus, it seems logical to assume that the transformed phenotype is dependent on persistent ERK-mediated phosphorylation of transcription factors. Consistent with this notion we found a potent activation of the chimeric transcription factor GAL4-ElkC, which contains the DNA binding domain and nuclear localization signal from yeast GAL4 and the transactivation domain of Elk-1, in the transformed cells. Intriguingly, the ERKs do not contain any known nuclear localization signals, and mutants that lack the TEY motif (T192A, Y194F) can be translocated to the nucleus (5, 7, 67). Therefore, although sustained ERK activation always seems to be associated with nuclear translocation, transient activation does not lead to nuclear translocation (4), and activation is not required for nuclear localization.

The activation of ERKs in response to Ras and PC-PLC was blocked by expression of a dominant interfering mutant of PKC. Thus, PKC may be critical for the Ras- and PC-PLC-mediated activation of MEK1. Although Raf-1 has been regarded as the major MEK1 activator in most cell systems, it is now evident that several protein kinases may serve as MEK1 activators (10, 13-15, 18, 68). In fact, Raf-1 may not be an important activator of MEK1 upon stimulation with serum or insulin (15). Also, a MEK1 mutant unable to bind to Raf-1 and B-Raf was still potently activated in response to serum, thrombin, and v-Ras (10). Of direct relevance to our present results is the finding that PKC was able to phosphorylate and activate a MEK1 preparation from COS-1 cells in vitro (18). Furthermore, by the use of transient transfections, it was recently demonstrated that both MEK and ERK were activated in vivo by expressing activated PKC and that the dominant negative mutant of PKC severely impaired the activation of both kinases following stimulation with serum or tumor necrosis factor- (6). The N-terminal regulatory domain of PKC has been found to bind to Ras in a GTP-dependent manner in vitro and PKC co-immunoprecipitated with Ras-GTP in vivo (37). Therefore, the full-length construct of a dominant interfering mutant of PKC used in this and other studies (6, 20, 22, 37) could simply function to sequester Ras from interacting productively with other downstream targets. However, cotransfection studies using a mutant construct expressing only a catalytically inactive C-terminal kinase domain of PKC, unable to interact with Ras-GTP, inhibited MEK1 activation as potently as the full-length dominant negative mutant (6). Consistent with these observations we found that the dominant negative PKC mutant blocked Ras- but not MEK1-induced ERK activation when assayed by transient overexpression. Together, these results place PKC upstream of MEK in Ras- and PC-PLC-mediated signaling. However, several attempts to establish clones that coexpressed dnPKC with aMEK failed. This is not due a blockade of the MEK/ERK pathway since transient transfections of aMEK cells with a dnPKC expression plasmid did not abolish the GAL-ElkC activity nor did it affect the activation of HA-tagged ERK. This suggests that dnPKC interferes with the survival of aMEK-transformed cells without affecting the activation of ERK. Interestingly, Moscat and co-workers (61) have recently reported that the atypical PKCs are clearly involved in cell survival. The zinc finger domain of both PKC and PKC was shown to interact with the product of the par4 gene that is involved in growth inhibition and induction of apoptosis. The interaction of Par4 with PKC/PKC reduced the activity of the kinases dramatically. Furthermore, overexpression of Par4 as well as dnPKC/dnPKC in NIH 3T3 cells induced apoptosis, whereas the cells survived when Par4 was coexpressed with the wild-type kinases. Altogether, these findings suggest that atypical PKCs have a role in mediating cell survival signals. It has been reported that PI 3-kinase is required for generating survival signals in PC12 cells (69). Interestingly, the kinase activity of PKC, which is closely related to PKC, is stimulated in vitro by phosphatidylinositol 3,4,5-triphosphate (46). Furthermore, PI 3-kinase was recently suggested as an in vivo activator of PKC based on measurements of AP-1 transactivation (17). We found that reversion of Ras- or PC-PLC-transformed cells by stable expression of dnPKC was accompanied by loss of growth factor-independent AP-1- and NFB-mediated transactivation (20). Constitutively active mutants of Ras relay signals through several parallel pathways (37, 38, 40, 70). Hydrolysis of phosphatidylcholine has also been reported to be involved in several signaling pathways (20, 27, 71, 72). However, the ERKs are the only known substrates for MEK. Since we were unable to establish cell lines where activated MEK1 and dnPKC were stably coexpressed, activation of the ERKs is by itself evidently not sufficient to overcome the apoptotic effect of overexpressing dnPKC. We found that the ERK activation in response to PDGF or serum was not blocked in cells coexpressing Ras or PC-PLC and dnPKC. Expression levels of dominant interfering mutants that completely block Ras-dependent signaling would clearly be lethal to the cells. Therefore, clones that stably express cytotoxic levels of dnPKC cannot be established as stably transfected cell lines. Our previous findings that expression of dnPKC reverted v-ras and plc-induced transformation and our present results, demonstrating a block in the activation (and nuclear translocation?) of ERK1 and -2, implicate PKC as an important mediator of ERK activation in response to Ras and PC-PLC. However, although PKC acts upstream of MEK1 in the MEK/ERK pathway, it is also required in other pathways where one or more of these may be critical for cell survival.

It was recently reported that murine fibroblasts transformed by either the v-ras or v-src oncogenes or following prolonged growth factor stimulation of normal cells expressed low levels of PDGF receptors compared with quiescent nontransformed cells (49). The reduced receptor levels coincided with a lower transcription of the PDGF- receptor gene and reduced receptor mRNA levels. We found that the down-regulation of the PDGF- receptor in response to v-ras was mimicked by transformation induced by elevated levels of PC-derived DAG. Furthermore, overexpression of aMEK also led to a reduced PDGF- receptor level. Reversion of both v-ras and plc-transformed cells by expression of dnPKC restored the receptor levels. Taken together, our results suggest that constitutive nuclear ERK activity leads to down-regulation of the PDGF- receptor perhaps via effects on transcription factors regulating the transcription of the PDGF- receptor gene.

Together with previous findings (6, 20), the results reported here strongly suggest that PC-derived DAG acts via the ERK1 and -2 MAPK pathway with PKC as a downstream mediator of MEK activation. PC-derived DAG acts late in the G₁ phase of the cell cycle as suggested from the kinetics of induction of DNA synthesis in quiescent fibroblasts treated with B. cereus PC-PLC (73). Furthermore, in keratinocytes the production of PC-derived DAG is inhibited by transforming growth factor- (74) which induces G₁ arrest by inhibiting various cyclin-Cdk kinases through the cooperative action of at least two Cdk inhibitors (75). Interestingly, a recent report demonstrated that in the yeast cell cycle the only PKC found in this organism, PKC1, functions downstream of the activation of the CDC28 kinase at the late G₁ restriction point START (76). Strikingly, the activation of PKC1, which acts via a MAPK module involving a MEK kinase (BCK1), two MEKs (MKK1 and -2), and a MAPK (MPK1) (77), was linked to a CDC28-dependent production of PC-derived DAG most probably due to activation of a PC-PLC. These findings strongly support our notion outlined above concerning the location of Ras and PC-derived DAG in the mitogenic MAPK pathway and provide evidence that a signaling pathway generally thought of as solely stimulated by membrane-bound receptors is also employed by a cell cycle regulatory kinase in the absence of extracellular stimuli. In light of the conserved nature of MAPK modules and the cell cycle regulatory machinery, future work aimed at elucidating the functions of PC-derived DAG will clearly profit from parallel experimental approaches involving both mammalian and yeast cell systems.