INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Although activation of mitogen-activated protein (MAP) kinases (extracellular signal-related kinase 1 [ERK1] and -2) by growth factors can lead to a variety of cellular fates, including growth and differentiation, the mechanism by which specificity is determined is not known. One explanation for the observed differences involves the duration of ERK activation (32). For example, epidermal growth factor (EGF) induces transient activation of ERKs and stimulates proliferation of pheochromocytoma (PC12) cells, while fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) stimulate prolonged ERK activation and induce cellular differentiation (46). Another potential explanation is that there are differences in the intracellular signals that couple growth factors to ERKs. Several molecules exclusive of the linear Ras, c-Raf, MAP kinase kinase (MEK), and MAP kinase pathways have been shown to mediate ERK activation (4, 13, 41, 47) and among these are members of the protein kinase C (PKC) family.

To date, 11 members of the PKC superfamily have been identified (reviewed in references 9, 22, and 36). The PKCs have been classified into three groups based upon their ability to be activated by Ca²⁺ and diacylglycerol (DAG). The classical PKCs (cPKCs) are activated by both Ca²⁺ and DAG and include the , I, II, and  isoforms. The Ca²⁺-independent but DAG-dependent isoforms (, , , and ) comprise the novel PKCs (nPKCs). Finally, the atypical PKCs (aPKCs), , /, and µ, are both Ca²⁺ and DAG independent. Various PKCs have been shown to mediate or modulate the activation of ERKs by growth factors, hormones, and phorbol esters (3, 31, 41, 45). Many of these PKCs act as potentiators of cell cycle progression.

Interestingly, PKC differs from other closely related PKCs, such as PKC. PKC has a distinct subcellular localization (15) and mediates tetradecanoyl phorbol acetate-induced differentiation of murine myeloid progenitor cells into macrophages (34) as well as the secretory response of antigen-stimulated rat basophilic RBL-2H3 cells (34, 44). Overexpression of PKC leads to growth arrest in vascular smooth muscle, capillary endothelial, NIH 3T3, and CHO cells (14, 20, 33, 48), a function often associated with differentiation. Furthermore, N-myc-induced transformation in a rat neuroblastoma cell line results in a decrease in PKC expression (2). Consistent with these studies, PKC blocks cellular transformation by Src and has been postulated to be a tumor suppressor (29). Finally, PKC, but not PKC, is translocated to the membrane in response to NGF in PC12 cells (38). Thus, PKC appears to have a growth-inhibiting and differentiating function in a variety of cell types.

In the present study, we have investigated the role of PKC in the activation of MAP kinase and the induction of neurite outgrowth in PC12 and H19-7 cells. The conditionally immortalized H19-7 cell line was generated by transducing rat E17 hippocampal cells with a retroviral vector expressing a temperature-sensitive simian virus 40 large T antigen (10). At the nonpermissive temperature, when T is inactivated, H19-7 cells differentiate upon stimulation by FGF, but not upon exposure to EGF (10, 25, 26). In contrast to PC12 cells, H19-7 cells lack the Trk receptor and therefore are nonresponsive to NGF (11).

We now show that PKC is required for ERK activation by FGF and other differentiating factors in both H19-7 and PC12 cells. Preincubation of cells with rottlerin, a PKC inhibitor with specificity for the  isoform (17), blocks activation of MEK but not Raf, suggesting that PKC acts either downstream or in parallel with Raf. Finally, rottlerin inhibits neurite outgrowth in response to activated Raf in H19-7 cells and FGF or NGF in PC12 cells. The results presented here suggest that PKC is required for the activation of MEK by FGF or NGF in neuronal cells, and this pathway may account for some of the selective effects of differentiating versus growth-promoting factors.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. Receptor-grade EGF was purchased from Biomedical Technologies, Inc. (Stoughton, Mass.). Basic FGF was purchased from Research Diagnostics, Inc. (Flanders, N.J.). Phorbol 12, 13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate, phosphatidylserine, wortmannin, myelin basic protein (MBP), peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG), and peroxidase-conjugated goat anti-mouse IgG were purchased from Sigma Chemicals (St. Louis, Mo.). Chelerythrine chloride, Gö 6976, and rottlerin were purchased from Calbiochem (La Jolla, Calif.). The MEK inhibitor PD098059 was a gift from Alan Saltiel (Parke-Davis). Anti-MAP kinase antiserum (Ab283) was developed as previously described (25). Monoclonal antibodies 12CA5 and 9E10 against the hemagglutinin (HA) and myc epitopes, respectively, were purchased from BabCo (Emeryville, Calif.). High-affinity rat anti-HA monoclonal antibody (3F10), grade II NGF, and peroxidase-conjugated, affinity-purified sheep anti-rat Fab Ig were purchased from Boehringer Mannheim (Indianapolis, Ind.). Anti-active MAP kinase polyclonal antibody was purchased from Promega (Madison, Wis.). Phospho-specific MEK 1/2 (Ser 217/221) polyclonal antibody was purchased from New England BioLabs (Beverly, Mass.). Monoclonal antibody M5 against the FLAG epitope and X-Omat film were purchased from Eastman Kodak Co. (New Haven, Conn.). Polyclonal MEK antibody (C-18) was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). The PKC monoclonal antibody sampler kit was purchased from Transduction Labs (Lexington, Ky.). Polyclonal rabbit anti-PKC was from Zymed (South San Francisco, Calif.). Protein G-Sepharose (4 Fast Flow) was purchased from Pharmacia Biotech AB (Uppsala, Sweden). Protein A-Sepharose was supplied by Jeffrey Bluestone (University of Chicago). Enhanced chemiluminescence reagents and [-³²P]ATP (6,000 Ci/mmol) were purchased from DuPont/NEN Research Products (Boston, Mass.). The purified, kinase-dead MEK (MEK K97A) was a gift from Angus MacNichol (University of Chicago). The activated MEK-2E and HA-tagged mouse ERK2 constructs were described previously (25). The FLAG-Raf construct was a gift from Andrey Shaw (Washington University). The myc-PKC plasmid was a gift from Peter Parker (ICRF, London, United Kingdom). Plasmid DNAs were prepared by CsCl-ethidium bromide gradient centrifugation as previously described (25) or by purification through columns according to the manufacturer's instructions (Qiagen, Chatsworth, Calif.).

Cell culture. The immortalized H19-7 cells were generated from embryonic rat hippocampal cells as previously described (10). A subclone of H19-7 stably expressing an oncogenic Raf-estrogen receptor (ER) fusion protein, Raf-1:ER, was previously described (25). PC12 cells were grown on tissue culture plates coated with poly-L-lysine in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 5% horse serum and supplemented with antibiotics (50 U of penicillin per ml, 50 µg of streptomycin per ml) at 37°C in a 95% air-5% CO₂ atmosphere. Quiescent cells were obtained by starving with 2% FBS and 1% horse serum overnight. NIH 3T3 cells were grown on tissue culture plates in DMEM containing 10% fetal calf serum (FCS) supplemented with antibiotics (50 U of penicillin per ml, 50 µg of streptomycin per ml) at 37°C in a 95% air-5% CO₂ atmosphere. Quiescent cells were obtained by starving with 0.5% FCS overnight.

Transient transfections. Approximately 10⁶ cells were seeded on 100-mm-diameter plates and incubated overnight. The medium was changed to serum-free OptiMem (Gibco/BRL), and cells were transfected with a total of 10 µg of plasmid DNA and 40 µl of TransIt LT-1 according to the manufacturer's protocol (Pan Vera Corp., Madison, Wis.). Ten percent of the total plasmid DNA consisted of pGreen Lantern-1 (Gibco/BRL), and the percentage of green fluorescent protein-expressing cells was scored to normalize transfection efficiency between groups. Cells were made quiescent for 24 h prior to treatment and harvesting. PC12 cells were transfected as described above, except that Effectene transfection reagent (Qiagen) was used as per the manufacturer's protocol.

Treatment of cells with ODNs. H19-7 or PC12 cells were seeded in six-well poly-L-lysine-coated plates to near confluency. H19-7 cells were switched to N2 medium at 39°C, and PC12 cells were switched to 1% FCS-0.5% horse serum before oligonucleotide (ODN) addition. The antisense sequences used were 5' GAAGGAGATGCGCTGGAA 3' for PKC and 5' GCCATTGAACACTACCAT 3' for PKC (12). The antisense sequence for PKC is based on nucleotides 10 to 27 of the murine coding sequence, while the sequence for PKC is based on the start codon plus the next 15 downstream nucleotides. The appropriate sense sequence was used as a control. ODNs were added daily to a final concentration of 30 µM, and then the cells were incubated for 3 days (H19-7) or 7 days (PC12) prior to growth factor stimulation.

In vitro Raf and HA-ERK2 kinase assays. FLAG-Raf or HA-ERK2 was overexpressed in H19-7 cells and pretreated with rottlerin or vehicle. Cells were then treated with or without FGF followed by two washings with phosphate-buffered saline (PBS). Cells were then lysed with 1% Triton-based lysis buffer (TLB) containing 1% Triton X-100, 50 mM Tris-HCl [pH 7.5], 40 mM -glycerophosphate, 100 mM NaCl, 50 mM NaF, 2 mM EDTA, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg of aprotinin per ml, 1 µg of leupeptin per ml, and 20 mM -nitrophenyl phosphate. The cell debris was removed by centrifugation (14,000 rpm for 10 min [Biofuge Fresco; Heraeus Instruments] at 4°C), and protein concentrations were determined by a Bio-Rad protein assay (Bio-Rad, Hercules, Calif.) with bovine serum albumin as a standard. Lysates were precleared with 50 µl of protein G-Sepharose for 30 min at 4°C. The M5 and 12CA5 monoclonal antibodies against the FLAG and HA epitopes, respectively, were coupled to protein G-Sepharose beads by adding 20 µg of M5 or 12CA5 to 1 ml of a 50:50 slurry of protein G-Sepharose in TLB overnight at 4°C. One hundred microliters of the antibody-protein G-Sepharose complex was added to 500 µg of cellular lysate protein, and this mixture was incubated for 3 h at 4°C. Immune complexes were then washed three times with TLB and two times in kinase buffer (1× kinase buffer is 25 mM HEPES [pH 7.4], 10 mM MgCl₂, 1 mM MnCl₂, 1 mM dithiothreitol, and 0.2 mM sodium vanadate). The final pellet was resuspended with 1× (vol/vol) kinase buffer, and reactions were started by addition of 2 µM ATP, 5 µCi of [-³²P]ATP, and 100 ng of purified MEK K97A or MBP and carried out for 30 min at 30°C. Reactions were stopped by addition of 6× concentrated sample buffer and boiling for 5 min at 100°C. Beads were pelleted by centrifugation (14,000 rpm for 5 min), and supernatants were loaded onto a 10% acrylamide separation gel. Proteins were transferred to nitrocellulose and subjected to autoradiography.

Cellular fractionations. H19-7 cells were starved overnight prior to treatment. Cells were lysed by addition of a hypotonic buffer (40 mM HEPES, 4 mM EDTA, 2 mM EGTA, 10 mM dithiothreitol, 1 mM sodium vanadate, 1 mM PMSF, 1 µg of aprotinin per ml, 1 µg of leupeptin per ml, 20 mM -nitrophenyl phosphate) and incubated for 30 min at 4°C. The cells were then sonicated twice with a Sonifier cell disruptor model W140 (Heat Systems-Ultrasonics, Inc., Plainview, N.Y.) at setting 5. Membranes were pelleted by centrifugation (100,000 rpm for 1 h at 4°C) in an Optima TLX ultracentrifuge by using the TLA 120.1 rotor and polycarbonate centrifuge tubes (8 by 34 mm) (Beckman, Palo Alto, Calif.). The supernatant was collected as the cytosolic fraction, and the pellets were washed twice with PBS. The final pellet was resuspended in TLB and incubated with agitation for 1 h at 4°C. Membranes were collected as the supernatant following centrifugation (14,000 rpm for 5 min at 4°C). Protein concentrations in each fraction were quantitated by Bio-Rad protein assay, and equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Western analysis. Cell extracts (10 to 20 µg) were resolved on a 10% acrylamide separation gel by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane. Membrane blocking, washing, antibody incubation, and detection by enhanced chemiluminescence were performed as previously described (26). When antibodies against phospho-specific peptides were used, blots were stripped by washing six times for 5 min each with Tris-buffered saline (TBS)-Tween (0.1%) at room temperature, 30 min at 55°C with stripping buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS, 100 mM 2-mercaptoethanol), and finally six times for 5 min each with TBS-Tween at room temperature. The stripped blots were then reprobed with the corresponding pan-ERK or pan-MEK antibody to ensure equal protein loading.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

PKC is activated by FGF but not EGF in H19-7 cells. Since PKC is activated in response to differentiating agents in PC12 cells, as shown by membrane translocation (38), we initially determined whether PKC is similarly expressed and activated in a conditionally immortalized rat hippocampal cell line, H19-7. As shown in Fig. 1A, PKC was detected in H19-7 cells by immunoblotting with an anti-PKC antibody. As in other cells, chronic pretreatment of H19-7 cells with PDBu led to proteolytic degradation of PKC. However, no change in PKC expression was observed in response to rottlerin, a bisindolylmaleimide kinase inhibitor with selectivity for PKC (30). The 50% inhibitory concentrations (IC₅₀s) of rottlerin were 3 to 6 µM for PKC; 30 to 42 µM for PKC, -, and -; and 80 to 100 µM for PKC, -, and - (17). Upon treatment of H19-7 cells with the differentiating agent FGF, PKC was activated, as monitored by translocation from the cytosol to the membrane. In contrast, no membrane translocation occurred in response to EGF or when cells were pretreated with the kinase inhibitor rottlerin prior to FGF treatment (Fig. 1B). Consistent with these results, FGF but not EGF stimulated transiently transfected PKC kinase activity (7). These results indicate that PKC is activated in response to differentiating signals in H19-7 cells.

View larger version (35K): [in this window] [in a new window]   FIG. 1.   PKC is expressed in H19-7 cells and is selectively activated by neurogenic factors in H19-7 and PC12 cells. (A) H19-7 cells in N2 medium at 39°C were either untreated (CTRL), stimulated with 10 ng of FGF per ml for 10 min, pretreated with 400 nM PDBu for 24 h, or pretreated with 5 µM rottlerin (Rott) for 5 h. Cells were lysed, and equal protein aliquots were resolved by SDS-PAGE (10% polyacrylamide) and then immunoblotted with anti-PKC antibody. (B) H19-7 cells were either untreated (CTRL), stimulated with 10 ng of EGF per ml, stimulated with 10 ng of FGF per ml, or pretreated with 5 µM rottlerin for 6 h prior to stimulation with 10 ng of FGF per ml for 10 min. The cells were lysed and fractionated into cytosolic (C) and membrane (M) fractions and immunoblotted with anti-PKC antibody as described in Materials and Methods.

PKC inhibitors block ERK activation by FGF, NGF, and Raf in neuronal cells. The inhibition of FGF-induced PKC activation by rottlerin is associated with an inhibition of MAP kinase (ERK1 and ERK2) activity. Pretreatment of H19-7 cells with 5 µM rottlerin completely suppressed FGF-induced phosphorylation of the conserved TEY motif within the activation loop in ERK (6), as shown by immunoblotting with an anti-phospho-ERK antibody (Fig. 2A). This dose of rottlerin is within the range of concentrations that inhibit PKC, but not other known PKC isozymes, and the effect was observed for at least 2 h following FGF treatment (7). In contrast, no inhibition of EGF-induced MAP kinase activation was observed at a comparable dose of rottlerin (Fig. 2B). When cells were analyzed for phosphatidylinositol-3-kinase (PI-3-kinase) involvement, the opposite pattern was obtained. Pretreatment of cells with 200 nM wortmannin, an inhibitor of PI-3-kinase, resulted in complete inhibition of EGF-stimulated MAP kinase activity but had no effect on FGF-stimulated MAP kinase activity (Fig. 2C and D). These results indicate that FGF and EGF activate MAP kinase by distinct signaling pathways and are consistent with a role for PKC in the activation of ERKs by FGF.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Effects of rottlerin and wortmannin on FGF- and EGF-induced ERK activation in H19-7 cells. (A) H19-7 cells in N2 medium at 39°C were pretreated with the indicated dose of rottlerin and then stimulated with 10 ng of FGF per ml for 10 min. After lysis, equal protein aliquots were resolved by SDS-PAGE (10% polyacrylamide) and then immunoblotted with anti-phospho-ERK antibody. (B) Cells were treated as for panel A except that 10 ng of EGF per ml rather than FGF was used for stimulation. (C) H19-7 cells in N2 medium at 39°C were pretreated with the indicated dose of wortmannin for 15 min and then stimulated with 10 ng of FGF per ml for 10 min. After lysis, equal protein aliquots were resolved by SDS-PAGE (10% polyacrylamide) and then immunoblotted with anti-phospho-ERK antibody. (D) Cells were treated for panel C, except that 10 ng of EGF per ml rather than FGF was used for stimulation.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Effects of various inhibitors on FGF- and EGF-induced ERK activation in H19-7 cells. (A) H19-7 cells in N2 medium at 39°C were either untreated (CTRL) or were pretreated with 400 nM PDBu for 24 h, 1 µM Gö 6976 (Go) for 2 h, 5 µM rottlerin (Rott) for 6 h, 200 nM wortmannin (Wort) for 15 min, 1 µM chelerythrine chloride (CC) for 2 h, or 30 µM MEK inhibitor PD98059 (MI) for 15 min. Cells were then stimulated with 10 ng of FGF per ml for 10 min. After lysis, equal protein aliquots were resolved by SDS-PAGE (10% polyacrylamide) and then immunoblotted with anti-phospho-ERK antibody. (B) Cells were treated as in for panel A, except that they were stimulated with 10 ng of EGF per ml rather than FGF.

View larger version (33K): [in this window] [in a new window]   FIG. 4.   PKC is expressed in H19-7 cells and is constitutively associated with the membrane fraction. (A) Expression of PKC isozymes in H19-7 cells. H19-7 cells were lysed, and equal protein aliquots were resolved by SDS-PAGE (10% polyacrylamide). Samples were immunoblotted with antibodies to PKC, PKC, PKC, PKC, PKC, PKC, PKC, PKC, PKC, and PKC. (B) H19-7 cells were either untreated (CTRL) or were stimulated with 10 ng of FGF per ml or pretreated with 5 µg of rottlerin for 6 h prior to stimulation with 10 ng of FGF per ml for 10 min (Rott + FGF). The cells were lysed and fractionated into cytosolic (C) and membrane (M) fractions and immunoblotted with anti-PKC antibody as described in Materials and Methods.

View larger version (29K): [in this window] [in a new window]   FIG. 5.   Effects of various inhibitors on ERK activation in Raf-1:ER and PC12 cells. (A) Raf-1:ER cells were either untreated (CTRL) or were pretreated with 400 nM PDBu for 24 h, 1 µM Gö 6976 (Go) for 2 h, 5 µM rottlerin (Rott) for 6 h, or 1 µM chelerythrine chloride (CC) for 2 h and then stimulated with (+) or without () 1 µM estradiol for 30 min. After cell lysis, equal protein aliquots were resolved by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-phospho-ERK antibody. (B) PC12 cells were either untreated (CTRL) or were pretreated with 400 nM PDBu (PDBu) for 24 h or 5 µM rottlerin (Rott) for 6 h and then stimulated with 10 ng of EGF, FGF, or NGF per ml for 10 min. Samples were then processed as for panel A.

View larger version (26K): [in this window] [in a new window]   FIG. 6.   Rottlerin does not block FGF- or EGF-induced ERK activation in NIH 3T3 cells. (A) Effect of FGF or EGF on ERK phosphorylation. NIH 3T3 cells were either untreated (CTRL) or were stimulated with 10 ng of EGF or FGF per ml for 10 min in the presence or absence of 5 µM rottlerin (Rott). Cells were lysed, resolved by SDS-PAGE (10% polyacrylamide), and then immunoblotted with anti-phospho-ERK. (B) Expression of PKC in NIH 3T3 cells. Cells were treated as for panel A, lysed, resolved by SDS-PAGE (10% polyacrylamide), and then immunoblotted with anti-PKC antibody.

View larger version (32K): [in this window] [in a new window]   FIG. 7.   PKC but not PKC is required for FGF-induced ERK activation. (A) Antisense (AS) PKC phosphorothioate ODNs block PKC expression and ERK activation in H19-7 cells. Cells were pretreated with either sense or antisense PKC ODNs as described in Materials and Methods and then either left untreated (CTRL) or were stimulated with 10 ng of FGF per ml for 10 min. Cells were then lysed, and the lysates were resolved by SDS-PAGE (10% polyacrylamide) and assayed for PKC expression by immunoblotting with anti-PKC antibody. MAP kinase activation was assayed by immunoblotting with anti-phospho-ERK antibody. (B) Antisense PKC phosphorothioate oligonucleotides block PKC expression, but do not inhibit ERK activation in H19-7 cells. Cells were pretreated with either sense or antisense PKC oligonucleotides as described in Materials and Methods and then either were left untreated or were stimulated with 10 ng of FGF per ml for 10 min. Cells were then lysed, and the lysates were resolved by SDS-PAGE (10% polyacrylamide) and assayed for PKC expression by immunoblotting with anti-PKC antibody. MAP kinase activation was assayed by immunoblotting with anti-phospho-ERK antibody. (C) Antisense PKC phosphorothioate oligonucleotides block PKC expression and ERK activation by NGF in PC12 cells. PC12 cells were treated and analyzed as for panel A, except that 50 ng of NGF per ml was used instead of FGF. (D) Antisense PKC phosphorothioate oligonucleotides block PKC expression but do not inhibit ERK activation by NGF in PC12 cells. PC12 cells were treated and analyzed as for panel B, except that 50 ng of NGF per ml was used instead of FGF.

Rottlerin does not inhibit c-Raf activation or Raf kinase activity. At what point in the pathway is PKC acting? The observation that rottlerin suppresses ERK stimulation by constitutively activated Raf suggests that the inhibitory step is downstream of Raf. To investigate this possibility further, we focused on H19-7 cells and determined whether Raf kinase activity is suppressed by rottlerin by using three different approaches. First, H19-7 cells were transfected with FLAG-tagged c-Raf. After exposure of cells to the PKC inhibitor prior to stimulation, Raf was immunoprecipitated with anti-FLAG antibody and then assayed for kinase activity with kinase-dead MEK as a substrate. As shown in Fig. 8A, pretreatment with rottlerin actually enhanced c-Raf activity in response to FGF. Second, we directly assayed endogenous c-Raf that was immunoprecipitated from H19-7 cells treated with FGF in the presence or absence of rottlerin (Fig. 8B). The results clearly indicate that c-Raf kinase activity is not suppressed by rottlerin and in fact seems to be further stimulated, probably due to suppression of feedback inhibition by downstream effectors (49). To ensure that the suppression of ERK activity by rottlerin is not due to modulation of Raf kinase activity, we determined the effect of rottlerin on Raf-1:ER by immunoprecipitating the Raf fusion protein with anti-ER antibody from estradiol-stimulated cells. As shown in Fig. 8C, rottlerin had no significant effect on Raf-1:ER activity. These results indicate that PKC is required for activation of the signaling pathway downstream of Raf.

View larger version (41K): [in this window] [in a new window]   FIG. 8.   Rottlerin (Rott) does not block FGF-induced c-Raf, FLAG-Raf, or estradiol-induced Raf-1:ER kinase activity. (A) H19-7 cells were transfected with either control vector or an expression vector for FLAG-tagged c-Raf. The cells were then left untreated (CTRL), pretreated with 5 µM rottlerin for 6 h, and/or stimulated with 10 ng of FGF per ml for 10 min. Following treatment, cells were lysed, and FLAG-Raf was immunoprecipitated with anti-FLAG antibody. The samples were resolved by SDS-PAGE (10% polyacrylamide) and assayed for Raf kinase activity by using inactive MEK as a substrate as described in Materials and Methods. The immunoprecipitated Raf was quantitated by immunoblotting with anti-FLAG antibody. DMSO, dimethyl sulfoxide. (B) H19-7 cells were left untreated, pretreated with 5 µM rottlerin for 6 h, and/or stimulated with 10 ng of FGF per ml for 10 min. Following treatment, cells were lysed, and endogenous c-Raf was immunoprecipitated with anti-c-Raf antibody. The samples were resolved by SDS-PAGE (10% polyacrylamide) and assayed for Raf kinase activity by using inactive MEK as a substrate as described in Materials and Methods. The immunoprecipitated Raf was quantitated by immunoblotting with anti-c-Raf antibody. (C) Raf-1:ER cells were either untreated or were stimulated with 1 µM estradiol for 30 min. The cells were then lysed, and the Raf-1:ER was immunoprecipitated with anti-ER antibodies and assayed for Raf kinase activity by using inactive MEK as a substrate as described in Materials and Methods. Immunoprecipitated Raf-1:ER was quantitated by immunoblotting with anti-ER antibody.

PKC is required for MEK activation. Since rottlerin inhibits ERK activity, it is likely that either activation of MEK or activation of ERK is being suppressed. To monitor the activation state of MEK, we analyzed MEK phosphorylation at the key serine residues in the activation loop by using an anti-phospho-MEK antibody. The results indicate that rottlerin and chelerythrine chloride block MEK activation by FGF but not EGF in both H19-7 and PC12 cells (Fig. 9). Consistent with our previous observations, the inhibitor of the cPKCs had no effect. Similar results were obtained when Raf-1:ER cells stimulated with estradiol or PC12 cells stimulated with FGF or EGF in the presence or absence of the PKC inhibitors were evaluated (Fig. 9B and C). To determine whether activation of ERK by MEK requires PKC, H19-7 cells were cotransfected with expression vectors for constitutively activated MEK (MEK-2E) and HA-ERK2. Cells were either untreated or exposed to rottlerin, and then the HA-ERK2 was immunoprecipitated and assayed for kinase activity with MBP as a substrate. As shown in Fig. 9D, rottlerin had no significant effect on the activation of ERK by MEK-2E. However, rottlerin was able to inhibit FGF-induced ERK activation in the same experiment (7). Taken together, these results indicate that PKC is required for the phosphorylation and activation of MEK.

View larger version (26K): [in this window] [in a new window]   FIG. 9.   Activation of MEK but not ERK requires PKC. (A) PKC inhibitors block MEK activation by FGF but not EGF in H19-7 cells. Cells were either untreated (CTRL) or were pretreated with 1 µM Gö 6976 (Go) for 2 h, 5 µM rottlerin (Rott) for 6 h, 1 µM chelerythrine chloride (CC) for 2 h, or 30 µM MEK inhibitor PD98059 (MI) for 15 min. Cells were then stimulated with 10 ng of either FGF or EGF per ml for 10 min. After cell lysis, equal protein aliquots were resolved by SDS-PAGE (10% polyacrylamide). MEK activation was assayed by immunoblotting with anti-phospho-MEK (P-MEK) antibody. (B) PKC inhibitors block MEK activation by estradiol in Raf-1:ER cells. Cells were pretreated as for panel A and then exposed to 1 µM estradiol for 30 min. Samples were resolved by SDS and assayed for MEK activation as for panel A. (C) PKC inhibitors block MEK activation by FGF but not EGF in PC12 cells. PC12 cells were treated and processed as for panel A. (D) Rottlerin does not block activation of ERK by constitutively activated MEK. H19-7 cells were mock transfected or cotransfected with an expression vector for HA-ERK2 and MEK-2E, a constitutively activated MEK. Cells were then left untreated or were pretreated with 5 µM rottlerin for 6 h or stimulated with 10 ng of FGF per ml as indicated. Following treatment, cells were lysed, and HA-ERK was immunoprecipitated with anti-HA antibody. The immunoprecipitated HA-ERK was assayed for kinase activity by using MBP as a substrate as described in Materials and Methods, and the reaction products were resolved by SDS-PAGE (10% polyacrylamide). The immunoprecipitated ERK was quantitated by immunoblotting.

PKC mediates neurite outgrowth. The MAP kinase signaling pathway plays a critical role in neuronal differentiation by FGF and NGF in PC12 cells (8, 39) and by activated Raf in H19-7 cells (25, 26). To test whether PKC is also necessary for these processes, PC12 cells were stimulated with NGF in the presence or absence of rottlerin, and the cells were monitored for morphological differentiation. As shown in Fig. 10A, rottlerin inhibited neurite outgrowth in NGF-treated cells. Pretreatment with rottlerin also inhibited FGF-induced neurite outgrowth in PC12 cells (7). Similarly, rottlerin prevented estradiol-induced neurite outgrowth in Raf-1:ER cells (Fig. 10B), consistent with its ability to block ERK activation. In contrast, rottlerin did not block differentiation of H19-7 cells by FGF (7), consistent with our previous results demonstrating that FGF can promote neuronal differentiation via an MEK-independent pathway (26). The role of PKC in neurite outgrowth was confirmed by the use of antisense PKC oligonucleotides. As shown in Fig. 11A, pretreatment of PC12 cells with antisense but not sense PKC oligonucleotides suppressed neurite outgrowth in response to 5 days of NGF treatment. Under these conditions, the antisense oligonucleotides still blocked expression of PKC (Fig. 11B). These results indicate that PKC is an important physiological regulator of neurite outgrowth in at least two cell systems.

View larger version (92K): [in this window] [in a new window]   FIG. 10.   Rottlerin blocks neurite outgrowth in NGF-induced PC12 and estradiol-activated Raf-1:ER cells. (A) PC12 cells untreated or pretreated with 5 µM rottlerin (Rott) and then exposed to 50 ng of NGF per ml for 4 days. Original magnification, ×100. (B) Raf-1:ER cells untreated or pretreated with 5 µM rottlerin and then exposed to 1 µM estradiol for 1 day. Original magnification, ×40.

View larger version (101K): [in this window] [in a new window]   FIG. 11.   PKC antisense oligonucleotides block neurite outgrowth in NGF-induced PC12 cells. (A) PC12 cells either untreated (CTRL) or pretreated with sense (PKC-S) or antisense (PKC-AS) ODNs as described in Materials and Methods and then stimulated with 50 ng of NGF per ml (+ NGF) for 5 days as indicated. Original magnification, ×100. (B) Immunoblots of PKC from cells treated as described for panel A. Cell lysates were collected and resolved by SDS-PAGE (10% polyacrylamide) and assayed for PKC expression by immunoblotting with anti-PKC antibody.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

This paper describes the surprising finding that PKC is required for activation of the MAP kinase cascade by neurogenic factors FGF and NFG in two different neuronal cell systems: hippocampal H19-7 and pheochromocytoma PC12 cells. In contrast, EGF, which acts as a mitogen in these cells, activates MAP kinase by a different, wortmannin-sensitive pathway. This effect is cell type specific, since FGF does not require PKC for MAP kinase induction in NIH 3T3 fibroblasts. Finally, PKC is also required for neurite outgrowth in response to neurogenic agents that are dependent upon MEK for differentiation. Moreover, the mechanism by which PKC activates this pathway has not been previously described. Surprisingly, our data suggest that PKC acts downstream of or in parallel with Raf and upstream of MEK in the FGF signaling cascade.

Many of the experiments presented here utilized the pharmacological agent rottlerin, a bisindolylmaleimide kinase inhibitor, as a potent inhibitor of PKC. Rottlerin also inhibits the activity of CaM kinase III (eEF2) at a similar concentration (IC₅₀, 5.3 µM). Since a 6-h pretreatment with rottlerin was necessary to fully block mitogen-induced ERK activation, we cannot rule out the possibility that rottlerin might also be acting indirectly through effects on protein translation. It should be noted, however, that similar pretreatment times have been utilized to suppress PKC activity in other cell systems (29). In addition, other lines of evidence confirm that PKC is required for MAP kinase activation. First, the nonselective PKC inhibitors PDBu and chelerythrine chloride produced the same results as those seen with rottlerin. In contrast, Gö 6976, a PKC inhibitor with specificity for the Ca²⁺-dependent  and I isoforms, did not mimic the effects of rottlerin. Second, we utilized an antisense strategy to demonstrate that blocking expression of the  but not the  isoform yielded the same effects as pretreatment with rottlerin. Taken together, these results indicate that PKC is necessary for ERK activation by FGF and NGF as well as neurite outgrowth induced by activated Raf or NGF in neuronal cells.

Some progress has been made recently in elucidating the signaling pathways mediated by FGF. Upon binding to the receptor, FGF interacts with heparin proteoglycan sulfates to promote dimerization and activation (40). Receptor tyrosine kinases undergo autophosphorylation upon stimulation; some of the phosphorylation sites act as anchors for other signaling molecules, and other sites within the catalytic domain are required for activity. There are at least seven autophosphorylation sites in FGFR1. A C-terminal site (Y766) recruits phospholipase C (PLC) and has been implicated in neurite outgrowth of cultured cerebellar neurons (19). However, a recent analysis of FGFR1 and FGFR3 chimeras demonstrated that the juxtamembrane region is primarily responsible for neurite outgrowth in PC12 cells; the PLC binding site induced only a very modest increase in neurite outgrowth (28). Elimination of all of these phosphorylation sites except the two within the catalytic domain does not prevent FGF from activating the MAP kinase cascade or inducing PC12 cell differentiation (35). Instead, FGFR interacts via the juxtamembrane domain with a lipid-linked docking protein, termed SNT/FRS2, which forms a complex with Grb2, Sos (the activator of Ras), and the tyrosine phosphatase Shp2 upon FGF stimulation (24). This complex appears to be the primary mechanism by which FGF stimulates MAP kinase and PC12 cell differentiation (18). Interestingly, SNT/FRS2 also interacts with the NGF receptor and mediates NGF-induced PC12 cell differentiation, consistent with a common signaling mechanism. The observation that EGF does not signal via the SNT/FRS2 adapter protein and activates ERK independently of PKC, whereas FGF and NGF both require PKC, suggests that PKC may also be a component of this common neuronal differentiation signaling pathway.

Several lines of evidence have supported a role for PKC in PC12 cell neurite outgrowth. PLC activation by FGFR1 has been reported to promote MAP kinase activation (21). Similarly, mutational analysis of the Trk receptor suggests that the PLC binding site is required for signaling (37, 43). Since PLC generates DAG, an activator of PKC, these studies are consistent with a role for PKC. More direct evidence comes from microinjection studies using a PKC-neutralizing antibody (1) and bryostatin-mediated down-regulation of PKC (42) to inhibit neurite outgrowth. Using the first variable domains of PKC and PKC as inhibitors, Messing and coworkers concluded that PKC, but not PKC, is required for enhancement of NGF responses by phorbol esters and ethanol in PC12 cells (23). Surprisingly, they did not observe any inhibition of NGF-induced MAP kinase activation or neurite outgrowth by the PKC fragment, presumably due to incomplete inhibition of PKC activity. Weinstein and coworkers have noted that PKC is selectively translocated in response to NGF but not EGF in PC12 cells, whereas PKC, -, and - are not changed (38), consistent with our observations with H19-7 cells. However, the studies reported herein provide the first direct evidence that PKC is the specific PKC isozyme responsible for neurite outgrowth in PC12 cells.

Very recently, PKC (27) and PKC (5, 27) have been shown to be activated by a wortmannin-sensitive, PI-3-kinase-dependent pathway via phosphorylation of PKC by PDK1. Surprisingly, although EGF stimulated ERK by a wortmannin-sensitive pathway, the activation was not suppressed by PKC inhibitors. However, we cannot rule out the possibility that the aPKC might play a role in the EGF signaling pathway. In contrast, the PKC-dependent ERK activation that we have observed in response to FGF or NGF is wortmannin insensitive. Thus, it appears that PDK1 is not a key regulator of PKC or ERK activation by FGF or NGF in the neuronal cells.

Previous studies of nonneuronal cells have shown that Raf can be activated by PKCs. Cooper and colleagues demonstrated that constitutively activated PKC and PKC can activate Raf, and the phorbol ester-sensitive PKCs are required for EGF stimulation of Raf in NIH 3T3 and COS cells (3). Interestingly, our results indicate that the phorbol ester-sensitive PKCs are not required for EGF stimulation of MAP kinase in either PC12 or H19-7 cells. Recently, Parker and coworkers (41) showed by transfection studies with constitutively active PKCs that PKC and PKC and presumably other classic and novel PKC isozymes can activate Raf in COS cells. In contrast to these studies, using a similar approach, Ueda et al. (47) concluded that PKC, but not PKC or PKC, could activate MEK in a Raf-dependent manner in COS cells. While the reason for this discrepancy is not clear, these studies demonstrate that PKC as well as other PKCs can activate Raf.

The mechanism by which PKC regulates MEK activation remains to be elucidated. The only PKC that has been shown to activate MEK directly is PKC (41), and this signaling pathway is Raf independent. The role of Raf in the action of PKC in H19-7 and PC12 cells is not completely clear. In H19-7 cells, FGF activates c-Raf, and dominant-negative c-Raf blocks activation of ERK (25, 26). The data presented here show that PKC is not required for c-Raf activation or Raf kinase activity. Although we cannot rule out a role for PKC in B-Raf activation, the data clearly demonstrate that activation of MEK by c-Raf or B-Raf requires PKC. It appears that ERK activation by MEK does not require PKC, since rottlerin treatment did not significantly alter ERK activation by constitutively activated MEK. One possible mechanism by which PKC might activate MEK is through direct phosphorylation of MEK or indirect phosphorylation via a mechanism such as inactivation of phosphatases. Alternatively, PKC may act as a scaffolding protein that brings MAP kinase and its activators together in the appropriate cellular location.

The difference between the EGF and FGF-NGF signaling pathways in the activation of MEK in PC12 and H19-7 cells may be a contributing factor in the different physiological responses to these stimuli. Several lines of evidence have shown that activation of MEK is both required and sufficient for neurite outgrowth in PC12 cells (8, 39). In both PC12 and H19-7 cells, EGF activates a transient MAP kinetics signal, whereas that from FGF is prolonged, and the difference between the actions of EGF and FGF has been ascribed to the difference in the kinetics (32). However, it has recently been shown that Rap1 is required for the prolonged ERK activation, but not for the initial MAP kinase signal or for the neurite outgrowth (50). Thus, it is likely that the mechanism responsible for the initial activation of MEK and ERKs by FGF relative to EGF, which we have now shown involves PKC, could regulate the signaling cascade leading to neuronal differentiation.