The cyclic adenosine monophosphate-dependent protein kinase (PKA) is required for the sustained activation of mitogen-activated kinases and gene expression by nerve growth factor.

Induction of neuronal differentiation of the rat pheochromocytoma cell line, PC12 cells, by nerve growth factor (NGF) requires activation of the mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK). cAMP-dependent protein kinase (protein kinase A (PKA)) also can induce differentiation of these cells. Like NGF, the ability of PKA to differentiate PC12 cells is associated with a sustained activation of ERKs. Here we show that maximal sustained activation of ERK1 by NGF requires PKA. Inhibitors of PKA partially blocked activation of ERK1 by NGF but had no effect on activation of ERK1 by EGF. Inhibition of PKA also reduced the ability of NGF and cAMP, but not EGF, to activate the transcription factor Elk-1, reduced the induction of both immediate early and late genes after NGF treatment, and blocked the nuclear translocation of ERK1 induced by NGF. We propose that PKA is an important contributor to the activation of ERK1 by NGF and is required for maximal induction of gene expression by NGF.

Induction of neuronal differentiation of the rat pheochromocytoma cell line, PC12 cells, by nerve growth factor (NGF) requires activation of the mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK). cAMP-dependent protein kinase (protein kinase A (PKA)) also can induce differentiation of these cells. Like NGF, the ability of PKA to differentiate PC12 cells is associated with a sustained activation of ERKs. Here we show that maximal sustained activation of ERK1 by NGF requires PKA. Inhibitors of PKA partially blocked activation of ERK1 by NGF but had no effect on activation of ERK1 by EGF. Inhibition of PKA also reduced the ability of NGF and cAMP, but not EGF, to activate the transcription factor Elk-1, reduced the induction of both immediate early and late genes after NGF treatment, and blocked the nuclear translocation of ERK1 induced by NGF. We propose that PKA is an important contributor to the activation of ERK1 by NGF and is required for maximal induction of gene expression by NGF.
Nerve growth factor (NGF) 1 promotes the differentiation of sympathetic and sensory neurons that is characterized by morphological features of neuronal differentiation (neurite formation) and changes in gene expression including the late gene transin (1)(2)(3). This differentiation has been examined extensively in the rat pheochromocytoma cell line, PC12 cells, a well studied model of growth factor actions (4,5). In PC12 cells, neuronal differentiation by NGF requires activation of the mitogen-activated protein (MAP) kinases (also called extracellular signal-regulated protein kinases, or ERKs) (6). The mechanisms by which NGF activates ERKs have been the subject of many studies. Upon NGF binding, activation of the NGF receptor, TrkA, triggers the assembly of a multimeric protein complex that includes the small monomeric G protein Ras (7). Ras activation triggers a cascade of phosphorylations on protein kinases that lie upstream of the ERKs (8). Epidermal growth factor (EGF) also induces ERK activation via Ras. Unlike that of NGF, the EGF activation of Ras and ERK triggers a mitogenic program within PC12 cells. The ability of NGF to trigger neuronal differentiation instead of proliferation is thought to depend, in part, on its ability to activate ERKs for long, sustained periods. Sustained activation of ERKs may be required for the translocation of ERKs into the nucleus where they induce a distinct set of gene expression (9). In contrast, ERK activation after EGF stimulation is transient. This is a consequence of the rapid termination of signals to ERK via a short feedback loop involving an ERK-dependent phosphorylation of the Ras activator SOS (10). This loop uncouples Ras-dependent activation of ERKs from upstream activators (11). The activity of this feedback loop is reflected in the transient activation of Ras by EGF. Interestingly, although NGF induces a sustained activation of ERKs, Ras activation after NGF treatment of PC12 cells is terminated rapidly (7). Since NGF activation of ERK is sustained despite the rapid inactivation of Ras, NGF may utilize Ras-independent pathways that are not inactivated rapidly to allow the sustained activation of ERKs.
Here we test this hypothesis by examining the requirement of PKA, cAMP-dependent protein kinase, for NGF activation of ERK1.
We have recently identified a novel Ras-independent pathway by which cAMP induces sustained activation of ERKs in PC12 cells (12). This pathway involves the Ras-related small G protein Rap1 as well as the cAMP-dependent protein kinase, PKA. In this study, we examine the possibility that PKA also participates in NGF signaling to the MAP kinase cascade by providing NGF a Ras-independent pathway to ERKs. We show that inhibition of PKA can inhibit signaling of NGF to ERK, to the transcription factor Elk-1, and to a specific marker gene of differentiation and can block the nuclear translocation of ERK1 induced by NGF. In addition, we demonstrate that NGF as well as PKA can activate the small G protein Rap1 and that this activation is blocked by the PKA inhibitor PKI. Therefore, we propose that PKA participates in NGF signaling to ERKs, in part via the activation of Rap1, and that this pathway contributes to the sustained activation of ERKs that characterizes NGF signaling.

EXPERIMENTAL PROCEDURES
Materials-PC12-GR5 cells were kindly provided by Rae Nishi (Oregon Health Sciences University, Portland, Oregon). A126-1B2 cells, PKA-deficient PC12 cells, and stromelysin-1 (transin) cDNA were provided by Gary Ciment (Oregon Health Sciences University). Plasmids encoding Elk-1/Gal-4, 5xGal4-E1b/luciferase, protein kinase inhibitor (cPKI), and loss-of-function mutant of PKI, cPKI mut , were gifts of Rich-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ard Maurer (Oregon Health Sciences University). Agarose-conjugated ERK1 (c-16) used in immunoprecipitations was purchased from Santa Cruz Biotechnology Inc. NGF was from Boehringer Mannheim. EGF was from Sigma. Forskolin, H89, and 8-CPT-cAMP were purchased from CalBiochem.
Cell Culture-PC12 cells and A126-1B2 cells were maintained in Dulbecco's modified Eagle's medium plus 10% horse serum and 5% fetal calf serum on 100-mm plates to 50 -60% confluence at 37°C in 5% CO 2 before harvesting. For immune complex assays and Northern blotting, cells were deprived of serum and maintained in Dulbecco's modified Eagle's medium for 16 h at 37°C in 5% CO 2 before treatment with various reagents. 10 M H89 was added to plates 15 min before treatment with NGF (50 ng/ml) or forskolin (10 M). Lipid-modified PKI peptide (sPKI) was added at 5 M, 10 min before treatment with NGF.
Transient Transfections and Luciferase Assay-60 -80% confluent PC12 cells were co-transfected with the indicated cDNAs using a calcium phosphate transfection kit (Life Technologies, Inc.) according to the manufacturer's instructions. The vector pcDNA3 (Invitrogen Corp.) was added to each set of transfections to ensure that each plate received the same amount of DNA. Four h after transfection, cells were glycerolshocked and allowed to recover in serum-containing media overnight. Cells were then starved overnight in supplemented serum-free media (N2) that contained Dulbecco's modified Eagle's medium with 5 g/ml insulin, 100 g/ml apotransferrin, 30 M sodium selenite, 100 M putrescine, and 20 nM progesterone (13). After serum deprivation, cells were treated with the indicated reagents for 6 h before harvesting. Luciferase assay was performed as described previously (12). Briefly, cells were washed twice in phosphate-buffered saline (PBS), scraped in PBS, spun at low speed to collect cells, and lysed by freeze-thawing three times in 100 M K 2 PO 4 (pH 7.8). For determination of Elk-1 activity, cells were transfected with 5 g of Elk-1/Gal4 and 5 g of 5XGal4-E1B-luciferase and other plasmids as indicated. Luciferase activity was assayed using a luminometer (AutoLumat LB953), as described previously (12). Cells transfected with pcDNA3 in the absence of reporter plasmids provided a base-line value that was subtracted from all subsequent measurements. Luciferase activity was reported as the -fold increase above the basal levels reached in untreated cells that were transfected with the reporter plasmids alone.
Histological Detection of ␤-Galactosidase-The expression of ␤-galactosidase was used to identify transfectants. PC12 cells were maintained in Dulbecco's modified Eagle's medium with 10% horse serum and 5% fetal calf serum on collagen-coated plates. Plasmids were transfected along with RSV-␤-gal (3 g/plate) using LipofectAMINE (Life Technologies, Inc.) in serum-free media. After 4 h, 10% serum was reintroduced. Sixteen h later the cells were washed and placed in serum-free N2 media. The transfected cells were exposed to NGF (50 ng/ml) or 8-CPT-cAMP (175 M) for 2 days before fixation. PC12 cells were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde for 5 min, after which cells were washed in PBS and subjected to a ␤-galactosidase assay. Cells were incubated in PBS containing 2 M MgCl 2 , 5 M ferric cyanide, 5 M ferrous cyanide, and 0.1% X-gal in overnight at 37°C. Transfected cells, identified as those staining blue, were then counted to determine the percent of blue cells with neurites in each set of transfections. Each set of transfections was done in duplicate, and at least 200 cells were counted for each experimental condition.
RNA Isolation, Riboprobe Synthesis, and Northern Blot Analysis-RNA was isolated using RNAzol B (TEL-TEST, Inc. Friendswood, Texas) per the instructions of the manufacturer. Stromelysin (transin) riboprobes used to detect transin mRNA were synthesized after linearizing pGEM-TR1 with HindIII by using T7 RNA polymerase to make antisense RNA transcripts. Northern blotting using transin riboprobe has been previously described (13). MKP-2 riboprobe synthesis and Northern blotting using this cRNA probe were done as described previously (14). All filters were scanned and quantitated using a Molecular Dynamics PhosphorImager 445SI.
ERK Immune Complex Assay-Treated or untreated cells were lysed in a lysis buffer containing 10% sucrose, 1% Nonidet P-40, 20 M Tris-HCl (pH 8.0), 137 M NaCl, 10% glycerol, 2 M EDTA, 1 M phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 M sodium vanadate, and 10 M sodium fluoride. The lysates were spun at low speed to remove debris, and the supernatant was assayed for ERK1 activity. One hundred g of protein (as determined by Bradford Assay) from the supernatant was immunoprecipitated with an agarose-coupled antibody to ERK-1(C-16) (Santa Cruz Biotechnology Inc.) overnight at 4°C. The immunoprecipitates were washed three times in lysis buffer and assayed for kinase activity by incubating with 25 g of myelin basic protein (MBP) and 10 Ci of [␥- 32  PKA Assay-After treatment with the indicated reagents, PC12 cells were harvested, and soluble PKA was measured as described previously (15). Kinase reactions were incubated for 2 min at 30°C in the presence or absence of 10 M cAMP.
GTP Loading-For GTP loading studies, PC12 cells were transfected with 15 g of poly-histidine-tagged Rap1b together with or without cPKI using the calcium phosphate transfection kit (Life Technologies, Inc.). After transfection, cells were labeled with [ 32 P]orthophosphate as described (12). Rap1 was precipitated with nickel nitrilotriacetic acidagarose, and GTP loading was assayed as described (16) with the addition of a preclearing step using activated charcoal. Nucleotide samples were spotted on a polyethyleneimine-cellulose chromatography plate along with GTP and GDP standards (Sigma), resolved in 1 M KH 2 PO 4 (pH 3.4) at room temperature, and analyzed using a Molecular Dynamics PhosphorImager 445SI. The GTP fraction was calculated as follows: Immunofluorescence-PC12 cells were seeded onto Permanox chamber slides (Nunc, Inc.) at 30 -40% confluency, serum-starved for 16 h, and treated with or without NGF (50 ng/ml) for 90 min. H89 (10 M) was added 15 min before NGF, as indicated. Cells were fixed in 70% ethanol, 50 M glycine (pH 2.0) at Ϫ20°C for 20 min. The cells were incubated with ERK1 antisera (1:400 dilution) or normal rabbit sera (1:400 dilution) in PBS containing 0.5% goat serum for 1 h at room temperature. After five washes with PBS, cells were incubated with rhodamine-conjugated anti-rabbit IgG (1:800 dilution) for 1 h at room temperature and visualized using a Leitz DMRB microscope (Leica, Inc.).

Activation of ERK1 by cAMP and NGF Is Reduced by the
Inhibitor of PKA, H89 -Both NGF and agents that activate PKA, including forskolin and the cAMP analog 8-CPT-cAMP, induce sustained activation of ERK1 in PC12 cells (Fig. 1). ERK1 activation by forskolin peaked by 20 min and remained elevated for at least one h. 8-CPT-cAMP induced a similar induction of ERK1 (Fig. 1A). The activation of ERK1 by forskolin was completely blocked by an inhibitor of PKA, H89 (17) (Fig. 1A), suggesting that the actions of cAMP on ERK1 require PKA. NGF activation of ERK1 was inhibited at multiple time points (20,40, and 60 min of stimulation) in the presence of the PKA inhibitor H89 (Fig. 1, B and C). In contrast, EGF activation of ERKs was not blocked by H89 at 5 or 20 min (and only minimally blocked at 10 min) in the data presented in Fig. 1D, suggesting that H89 was preferentially acting on kinases downstream of NGF at this concentration.
NGF Activation of ERK1 Is Reduced by the Protein Kinase Inhibitor PKI-PKA can be inhibited by the protein kinase inhibitor (PKI), a physiological inhibitor of PKA (18,19). To test whether this specific inhibitor of PKA could alter NGF activation of ERK1, we treated wild type PC12 cells with a peptide corresponding to PKI sequences from amino acids 5 to 22 that had been shown to be a specific inhibitor of PKA (18,19). This peptide was modified by the addition of a stearyl group at the amino terminus (sPKI) to allow penetration into the cell (20). In vivo, sPKI inhibited NGF stimulation of ERK1 minimally at early time points but showed significant inhibitory effects at later time points (20 and 40 min) compared with NGF-treated cells not receiving peptide ( Fig. 2A). The addition of sPKI in vitro completely inhibited 8-CPT-cAMP-stimulated PKA activity ( Fig. 2A, right panel), demonstrating that the addition of the stearyl group did not interfere with the ability of PKI to inhibit PKA catalytic activity. Unrelated peptides that contained the stearyl modification did not alter the ability of NGF to activate ERK1 at the time points examined, 2 suggest-ing that the inhibition by sPKI was not due to a toxic effect of peptide. sPKI completely inhibited the activation of ERK1 by forskolin at 20 min ( Fig. 2A).
NGF Induction of Sustained Activation of ERKs Is Reduced in PKA-deficient PC12 Cells-The requirement of PKA in NGF activation of ERKs was also examined in a PC12 line that is deficient in cAMP responses, A126-1B2 cells (21). A126-1B2 cells contain near wild type levels of type I PKA but have greatly reduced levels of type II PKA. Furthermore, these mutant cells display altered PKA type I and type II regulatory subunits, as judged by ion-exchange chromatography. These alterations, as well as alterations in specific PKA-anchoring proteins (22), greatly reduce the response of the cell to cAMP (21). In these cells, NGF activation of ERK1 was reduced at 20, 40, and 80 min compared with wild type cells but appeared unchanged at early time points (2, 5, and 10 min) (Fig. 2B, upper and middle panels). The activation of ERK1 by 8-CPT-cAMP in these cells was blunted (Fig. 2B, lower panel). Western blotting demonstrated that the decrease in ERK1 activation after NGF stimulation in A126-1B2 cells was not due to the altered expression of ERK1. 2 These results, together with the previous data, demonstrate that PKA is required for maximal activation of ERK1 by NGF, particularly at later time points.
Maximal Activation of Elk-1 by NGF Requires PKA-The transcriptional effects of ERKs are mediated, in part, by its activation of Elk-1, a member of the Ets family of transcription factors and a component of the serum response factor (23)(24)(25).
To determine whether PKA was required for activation of Elk-1 by NGF, we examined the activation of Elk-1 directly in a mammalian two-hybrid system that measured the transactivation of a 5xGal4-E1b/luciferase gene by an Elk-1/Gal4 fusion protein (12,26). These plasmids were co-transfected with or without cDNA encoding the PKA inhibitor (cPKI) or an inactive mutant of PKI (cPKI mut ) (18). The maximal activation of Elk-1 by both NGF and 8-CPT-cAMP was blocked by PKI but not by the expression of cPKI mut (Fig. 3A). In contrast, the activation of Elk-1 by EGF was not blocked by either wild type or mutant PKI. Therefore, PKI specifically interferes with the activation of Elk-1 by NGF and 8-CPT-cAMP, but not EGF, demonstrating that NGF activation of Elk-1 is mediated in part by PKA.
Maximal Activation of the Immediate Early Gene MKP-2 by NGF Requires PKA-To examine further the requirement of PKA for NGF-regulated gene expression, we chose to study the immediate early gene MKP-2. It has been previously shown that MKP-2 expression is rapidly increased in response to NGF in PC12 cells (14). Its gene product, MAP kinase phosphatase 2, dephosphorylates and inactivates multiple members of the MAP kinase family including JNKs (c-Jun N-terminus Kinase) and p38 in vitro and in vivo (14,27,28). As JNKs and p38 trigger cell death in PC12 cells (29), these actions of MKP-2 have been proposed to mediate the neurotrophic action of these agents (27). MKP-2 expression in PC12 cells is also induced by a variety of other neurotrophic agents that activate ERKs, including fibroblast growth factor, insulin, and cAMP. 3 (n ϭ 4). B, time course of NGF activation of ERK1, and blockade by the PKA inhibitor H89. PC12 cells were treated with NGF (50 ng/ml) (white squares, n ϭ 6) or NGF and H89 (10 M) (black diamonds, n ϭ 4) for the time indicated. ERK1 kinase assay and quantitation were performed as described in A. The results are reported as -fold increase over basal. S.E. is indicated. C, an example of a single experiment shows the phosphorylation of activation, we examined the role of PKA in this process. The level of MKP-2 mRNA after NGF stimulation of wild type PC12 cells was reduced significantly by H89 (Fig. 3B, left panel). In addition, in the PKA-deficient cell line A126-1B2, MKP-2 induction by NGF was also reduced compared with wild type PC12 cells (Fig. 3B, right panel).
Maximal Induction of the Late Gene Transin by NGF Requires PKA-The expression of the metalloprotease transin (stromelysin) is stimulated by NGF but not EGF and has been used as a marker for neuronal differentiation of PC12 cells (1,30,31). Its induction by NGF is dependent on Ras, the MAP kinase kinase kinase Raf, and ERKs (13,32). It has been shown that this induction requires multiple transcription factors, including those of the Ets family (33,34). NGF induces low levels of transin mRNA in PC12 cells within 4 h (30), and by 24 h of stimulation, expression levels could be easily detected by Northern blot (Fig. 3C). 8-CPT-cAMP could not stimulate the expression of transin in the absence of additional agents (30) (Fig. 3C). However, 8-CPT-cAMP dramatically enhanced the ability of NGF to stimulate transin (data not shown), suggesting that NGF and PKA are synergistic in their induction of transin gene expression. The synergistic action of NGF and 8-CPT-cAMP on transin expression reflects the action of these agents on neurite outgrowth as well (35)(36)(37). The participation of PKA signaling in NGF induction of transin mRNA was examined using H89. Preincubation with H89 blocked the induction of transin mRNA by NGF, suggesting that PKA activity was required for NGF induction of this gene (Fig. 3C). Neither treatment with 8-CPT-cAMP or H89 alone stimulated transin mRNA to detectable levels (Fig. 3C). These data demonstrate that maximal induction of specific immediate early and late genes by NGF may require PKA.
NGF Does Not Stimulate Detectable PKA Kinase Activity in PC12 Cells-Since we observed the involvement of PKA in NGF signaling, we determined whether NGF could stimulate total cellular PKA activity directly. Based on the kinetics studies shown in Fig. 2, we treated cells with NGF over a time course extending from 5 to 40 min and examined total PKA activity retained within lysates prepared from those cells. We could not detect increases in PKA activity after NGF treatment at any of the time points examined (Fig. 4A). Similar results were seen after EGF treatment (Fig. 4B). In contrast, 8-CPT-cAMP induced a 5-fold increase in PKA activity at the time points examined (Fig. 4B). The activity within all lysates could be stimulated in vitro by cAMP except cells pretreated with H89, demonstrating the presence of PKA and the action of H89 on PKA in this experiment (Fig. 4B). These results are consistent with previous reports showing that adenylyl cyclase and PKA activities are not significantly increased after NGF stimulation of these cells (38,39). Taken together, these data raise the possibility that a fraction of the total cellular pool of PKA is activated by NGF. Alternatively, basal activity of PKA may play a permissive role in NGF regulation of ERKs.
NGF Activation of the B-Raf Activator Rap1 Requires PKA-The PKA activation of ERK in PC12 cells requires Rap1, a small GTP-binding protein in the Ras superfamily. Rap1 is a selective activator of one of the isoforms of MAP kinase kinase kinase, B-Raf, and the expression of B-Raf is required for Rap1 to activate ERKs (12). We show data that suggest that NGF utilizes PKA to activate ERK. It is possible that PKA participates in the NGF stimulation of ERK via its actions on Rap1. To test this hypothesis, we examined the GTP loading of transfected histidine-tagged Rap1 after transfection in PC12 cells. Both 8-CPT-cAMP and NGF increased GTP loading of Rap1 in this assay (Fig. 5), raising the possibility that Rap1 participates in NGF signaling. Activation of Rap1 by both NGF and 8-CPT-cAMP was reduced to basal levels by the co-transfection of cPKI, suggesting that PKA contributes to the activation of Rap1 by both NGF and 8-CPT-cAMP. Since Rap1 mediates the ability of PKA to activate ERKs in PC12 cells (12), the activation of Rap1 by PKA may contribute to NGF activation of ERKs in these cells. 4 PKI Can Block the Induction of Neurites Induced by 8-CPT-cAMP but Not NGF-We have previously shown that Rap1 activation is not required for the elaboration of neurites seen after NGF treatment of PC12 cells (12). However, it is possible that PKA may have other actions in NGF-treated cells that are required for the differentiation phenotype. To determine whether neuronal differentiation by NGF was dependent on 4 R. H. York, H. Yao, and P. J. S. Stork, manuscript in preparation.

FIG. 2. ERK1 activation by NGF is blunted in A126-1B2 cells and inhibited by PKI peptide (sPKI) in PC12 cells.
A, inhibition of NGF-stimulated ERK activation by PKI peptide. PC12 cells were pretreated with or without sPKI peptide (20 M) for 20 min and then treated with NGF (50 ng/ml) or forskolin (Forsk., 10 M) for the time indicated. Cells were lysed at the indicated times, and ERK1 kinase assays were performed as described in Fig. 1A. Right panel, the inhibition of PKA in vitro by purified PKI protein and sPKI peptide is shown to control for the function of sPKI. H89 was utilized as a positive control for PKA inhibition. B, ERK1 activation in PKA-deficient cells. A126-1B2 cells were treated with NGF in the same way as with PC12 cells. Equal amounts of protein was assayed for ERK1 activity. The time course of ERK1 activation by NGF in these cells was compared with wild type (wt) PC12 cells. Note that NGF-induced ERK1 activity at later time points was reduced in A126-1B2 cells. The inability of 8-CPT-cAMP to activate ERKs in these cells is shown as a control.
PKA, we transfected cPKI into wild type PC12 cells along with the marker RSV-␤gal. The detection of ␤-galactosidase expression permitted the identification of transfected cells (stained blue) using histochemical methods. Cells were treated with 8-CPT-cAMP or NGF and assayed for neurite outgrowth and ␤-galactosidase expression as described (12,13). Both NGF and 8-CPT-cAMP induced neurites in cells that did not express ␤-galactosidase (unstained) (74 and 65%, respectively). The same percentages of neurite outgrowth were seen in NGF-or 8-CPT-cAMP-treated cells that had been transfected with RSV-␤gal in the absence of additional cDNAs 2 (12,13). The expression of PKI reduced the percentage of neurites after 8-CPT-cAMP treatment (65% to 10%), whereas the expression of PKI minimally reduced the percentage of neurite in NGF-treated cells (74 to 63%) (Fig. 6). This is consistent with previous reports demonstrating that NGF was able to differentiate the PKA-deficient A126-1B2 cells (40). Therefore, although PKA contributed to the sustained activation of ERK1 by NGF in PC12 cells, it was not required for neurite outgrowth induced by NGF in these cells.
H89 Blocks Nuclear Translocation of ERK1 by NGF-Nuclear translocation of ERKs in PC12 cells has also been associated with their sustained activation and can be detected by immunofluorescence after NGF but not EGF treatment of these cells (6). Nuclear localization of ERK1 was seen 90 min after NGF treatment (Fig. 7C). In untreated cells, ERK1 was mainly in cytoplasma (Fig. 7A). The nuclear staining seen after NGF treatment was blocked by pretreatment with H89 (Fig. 7E), suggesting that PKA was required for this action of NGF. Parallel samples prepared and incubated with normal rabbit serum showed no nonspecific staining (Fig. 7, B, D, and F). DISCUSSION The ability of NGF to stimulate differentiation of PC12 cells has been associated with a sustained activation of ERKs. In contrast, EGF activation of ERKs was rapidly terminated (9). The signaling pathways that are selectively activated by NGF to permit sustained activation of ERKs have not been fully elucidated. PKA also stimulates neuronal differentiation of PC12 cells and induces a sustained activation of ERKs (41). In this study we examined the possibility that PKA contributes to the sustained activation of ERKs seen after NGF treatment. We showed that maximal activation of ERKs by NGF was diminished in PC12 cells in three independent assays using two inhibitors of PKA, PKI and H89, and in a clonal isolate of PC12 cells that is deficient in cAMP signaling.
PKA has been previously proposed to be required for a subset of NGF actions in PC12 cells (42). Inhibiting PKA blocks selected actions of NGF at transcriptional (43,44) as well as post-transcriptional levels (45,46). NGF can augment cAMP production in some neuronal cells (47) and has been reported to stimulate the production of cAMP in PC12 membranes (48,49). However, neither the direct demonstration of an increase in adenylyl cyclase nor in PKA activity by NGF in PC12 cells has been established (38,39). We were also not able to detect PKA activation by NGF in PC12 cells (at 5, 10, 15, 20, and 40 min), raising the possibility that only a fraction of the total cellular PKA might be activated by the NGF signaling complex. This may be achieved by the activation of a distinct subcellular pool of PKA, either directly or indirectly, through the regulation of specific adenylyl cyclases or phosphodiesterases (50). Alternatively, PKA might be recruited to specific subcellular compartments in response to NGF to form a signal complex with activators of downstream effectors of NGF, such as Rap1. Subcellular localization of PKA with specific signaling complexes may be achieved via specific targeting proteins. For example, PKA may be anchored to multienzyme complexes via a diverse family of PKA-anchoring proteins (20,51,52). In addition, the adapter molecule Grb2 has been implicated in targeting PKA to growth factor receptors (53). Other kinases are also targeted to components of the NGF receptor signaling complex (54,55). One of these kinases, protein kinase N, has been reported to activate PKA directly (56,57). Therefore, it is possible that PKA may participate in a signaling complex through a direct interaction with these proteins that associate with the NGF receptor/tyrosine kinase cascade. FIG. 3. Activation of transcription by NGF requires PKA. A, maximal activation of Elk-1 by NGF in PC12 cells requires PKA. NGF activation of Elk is blocked by transfection of a cPKI but not by cPKI mut . PC12 cells were transfected with plasmids encoding either cPKI (10 g), cPKI mut (10 g), or vector (10 g) and Elk-1/Gal-4 (3 g) and 5xGal4-E1b/luciferase (3 g) plasmids and subsequently treated with NGF (50 ng/ml), EGF (100 ng/ml), or 8-CPT-cAMP (175 M) as indicated. Elk-1 activation was monitored by luciferase activity. S.E. is shown (n ϭ 3). B, expression of MKP-2 mRNA by NGF is blocked by H89 and blunted in PKA-deficient cells. Both PC12 cells and A126-1B2 were starved overnight before NGF treatment. They were pretreated with or without 10 M H89 for 15 min before NGF treatment. Cells were then incubated with 50 ng/ml NGF for the indicated time period. RNA was prepared as described under "Experimental Procedures." 15 g of RNA was loaded on the gel and subsequently subjected to Northern blotting. Note that MKP-2 mRNA induced by NGF was blocked at 1.5 and 2 h by H89. MKP-2 expression after NGF treatment was also blunted in A126-1B2 cells at every time point examined. C, expression of transin mRNA by NGF is blocked by H89. PC12 cells were treated, and RNA was prepared as in B. RNA samples from each treatment were Northernblotted with transin probe. 10 M H89 was added onto plates 15 min before NGF or 8-CPT-cAMP treatment in samples indicated.
In PC12 cells, NGF induces a sustained activation of ERKs that is associated with neuronal differentiation (9). The inhibition of PKA by H89 or PKI in these cells or through the use of PC12-derived PKA-deficient cells blocked the sustained activation of ERKs by NGF significantly more than it blocked the rapid, initial portion of ERK activation. However, neither method of PKA inhibition interfered with the ability of NGF to induce differentiation (Fig. 6) (13, 43, 60, 61). Therefore, PKA does not appear to be required for NGF induction of neurites in PC12 cells. Furthermore, although sustained activation of ERK is sufficient for triggering differentiation, it is not necessary (58,59). Other signals than ERK activation might also be required for triggering this process, as suggested by others (62).
The ability of PKA to augment NGF signaling may be most important in the regulation of gene expression during differentiation. Previous reports have suggested that the induction of selected genes and proteins by NGF requires PKA (30, 39, 42, 44 -46). Much of the transcriptional actions of PKA in neuronal cells are mediated by specific sites present in the promoter of many cAMP-responsive genes, called cAMP-responsive elements (63). Transcriptional activation by cAMP can occur via phosphorylation of a PKA-responsive site within the transactivation domain of the cAMP-responsive element binding protein (64). Another transcription factor, Elk-1, can also be activated by PKA in neuronal cells via PKA activation of the MAP kinase cascade (12). Elk-1 is a member of the Ets family of transcription factors and is activated by ERKs by direct phosphorylation (23,24,65). Stimulation of ERK and Elk-1 is required for full activation of the serum response element within the c-fos promoter (66 -68) and other immediate early genes that are activated by NGF (69,70). We show here that maximal activation of Elk-1 by NGF but not EGF requires PKA. Therefore, PKA may contribute to the specificity of NGF transcriptional effects via its activation of Elk-1.
PKA modulates NGF induction of representative early and late genes as well. We show here that one immediate early gene, MKP-2, also requires PKA for its maximal induction by NGF. The induction of late genes by NGF may also be regulated by PKA. One example is transin, whose expression is a FIG. 5. GTP loading of Rap1 by cAMP and NGF is blocked by PKI. GTP loading was assayed in PC12 cells after transfection of 6ϫ His-Rap (15 g) in the presence or absence of cPKI (10 g). Cells were metabolically labeled with [ 32 P]orthophosphate and treated with NGF or 8-CPT-cAMP for 10 or 30 min as indicated. Lysates were prepared as described under "Experimental Procedures." His-Rap was precipitated with nickel nitrilotriacetic acid-agarose, and GTP and GDP content in the eluents were analyzed by thin layer chromatography. The GTP fraction of total guanine nucleotide is shown above each lane. marker for neuronal differentiation (1,30,71). This may be mediated in part by NGF activation of ERKs, since the activation of the ERK substrate, Ets, has been implicated in transin activation (13,34,72).
One mechanism by which PKA might influence NGF signals to the nucleus is via regulation of nuclear translocation of ERKs. We have previously demonstrated that forskolin, an activator of adenylyl cyclase, could promote the nuclear translocation of ERKs in EGF-treated PC12 cells (13). Here, we show data that suggest that PKA may be required for the translocation of ERKs by cells treated with NGF alone. Since nuclear translocation of ERKs correlates with their sustained activa-tion, the involvement of PKA in NGF signaling may be a consequence of its ability to augment the sustained activation of ERKs by NGF. It is also possible that PKA may have additional protein targets that help regulate the subcellular localization of ERKs in a manner that is independent of its action of ERK activity. In either case, the ability of PKA to increase the nuclear concentration of ERK proteins after NGF treatment may account, in part, for the PKA-dependent activation of Elk-1 by NGF as well as NGF induction of the genes encoding transin and MKP-2.
Although Ras was required for the actions of both NGF and EGF on ERKs in PC12 cells, the mechanisms that distinguish NGF and EGF signaling to ERKs are not known. In particular, it is not known how NGF can activate ERK for extended periods. The activation of Ras by both EGF and NGF is rapidly terminated, suggesting that sustained activation of ERKs by NGF involves pathways that are downstream or independent of Ras. We propose that the activation of PKA by NGF allows NGF to activate Ras-independent pathways that are not rapidly terminated. One potential Ras-independent pathway that is activated by PKA involves the small G protein Rap1. Rap1 has been recently shown to be required for the sustained activation of ERKs by PKA in PC12 cells (12). We show here that Rap1 is activated after NGF treatment and that this activation requires PKA. The ability of NGF to activate Rap1 distinguishes it from EGF (12). Rap1 activation stimulates ERKs in PC12 cells via its direct activation of the MAP kinase kinase kinase B-Raf, the only known effector of Rap1 (Fig. 6) (12). We suggest that the activation of Rap1 by NGF may account for the high level of B-Raf activity seen after NGF treatment of PC12 cells (73,74). We propose that NGF activation of ERK may be enhanced by its activation of Rap1 via the action of PKA.
FIG. 6. Inhibition of PKA does not interfere with neurite outgrowth. A, PC12 cells were transfected with RSV-␤gal and cPKI and treated with 8-CPT-cAMP or NGF for 2 days as indicated. Cells were then stained with X-gal as described under "Experimental Procedures." ␤-Galactosidase was used as a marker for transfected cells; therefore, blue cells represent transfected cells. Cells with neurites were counted from both blue and white cells. PKI and ␤-galactosidase-transfected cells were treated with 8-CPT-cAMP (left panel) or NGF (right panel). The percentage of neuritebearing cells is included in the text.