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• W2157776145 abstract "Induction of neuronal differentiation of the rat pheochromocytoma cell line, PC12 cells, by nerve growth factor (NGF) requires activation of the mitogen-activatedprotein (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-activatedprotein (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) 1The abbreviations used are: NGF, nerve growth factor; MAP, mitogen-activated protein; MKP-2, MAP kinase phosphatase 2; ERK, extracellular-regulated kinase; EGF, epidermal growth factor; PKI, protein kinase inhibitor; cPKI, cDNA-encoded PKI; cPKImut, inactive mutant of cPKI; sPKI, stearyl-modified PKI; PKA, protein kinase A; 8-CPT-cAMP, 8-(4-chlorophenylthio)-cyclic AMP; PBS, phosphate-buffered saline; RSV-β-gal; Rous sarcoma virus β-galactosidase; X-gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; H89,N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesylfonamide hydrochloride; MBP, myelin basic protein. 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 (1Shackleford G.M. Willert K. Wang J. Varmus H.E. Neuron. 1993; 11: 865-875Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 2Levi-Montalcini R. Angeletti P.W. Physiol. Rev. 1968; 48: 534-569Crossref PubMed Scopus (1465) Google Scholar, 3Levi-Montalcini R. Science. 1987; 237: 1156-1162Crossref Scopus (2701) Google Scholar). This differentiation has been examined extensively in the rat pheochromocytoma cell line, PC12 cells, a well studied model of growth factor actions (4Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4873) Google Scholar, 5Greene L.A. Tischler A.S. Adv. Cell. Neurobiol. 1982; 3: 373-414Crossref Google Scholar). In PC12 cells, neuronal differentiation by NGF requires activation of themitogen-activated protein (MAP) kinases (also called extracellular signal-regulated protein kinases, or ERKs) (6Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar). 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 (7Qiu M.-S. Green S.H. Neuron. 1991; 7: 937-946Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Ras activation triggers a cascade of phosphorylations on protein kinases that lie upstream of the ERKs (8Ohmichi M. Pang L. Decker S.J. Saltiel A.R. J. Biol. Chem. 1992; 267: 14604-14610Abstract Full Text PDF PubMed Google Scholar). 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 (9Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4245) Google Scholar). 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 (10Buday L. Warne P.H. Downward J. Oncogene. 1995; 11: 1327-1331PubMed Google Scholar). This loop uncouples Ras-dependent activation of ERKs from upstream activators (11Porfiri E. McCormick F. J. Biol. Chem. 1996; 271: 5871-5877Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). 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 (7Qiu M.-S. Green S.H. Neuron. 1991; 7: 937-946Abstract Full Text PDF PubMed Scopus (122) Google Scholar). 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 (12Vossler M. Yao H. York R. Rim C. Pan M.-G. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). 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. 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, cPKImut, were gifts of Richard 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. 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% CO2 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% CO2 before treatment with various reagents. 10 μm H89 was added to plates 15 min before treatment with NGF (50 ng/ml) or forskolin (10 μm). Lipid-modified PKI peptide (sPKI) was added at 5 μm, 10 min before treatment with NGF. 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 glycerol-shocked 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 μg/ml insulin, 100 μg/ml apotransferrin, 30 μm sodium selenite, 100 μm putrescine, and 20 nm progesterone (13Yao H. Labudda K. Rim C. Capodieci P. Loda M. Stork P.J.S. J. Biol. Chem. 1995; 270: 20748-20753Crossref PubMed Scopus (55) Google Scholar). After serum deprivation, cells were treated with the indicated reagents for 6 h before harvesting. Luciferase assay was performed as described previously (12Vossler M. Yao H. York R. Rim C. Pan M.-G. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). 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 μmK2PO4 (pH 7.8). For determination of Elk-1 activity, cells were transfected with 5 μg of Elk-1/Gal4 and 5 μg of 5XGal4-E1B-luciferase and other plasmids as indicated. Luciferase activity was assayed using a luminometer (AutoLumat LB953), as described previously (12Vossler M. Yao H. York R. Rim C. Pan M.-G. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). 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. The expression of β-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-β-gal (3 μg/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 μm) 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 β-galactosidase assay. Cells were incubated in PBS containing 2 μm MgCl2, 5 μmferric cyanide, 5 μm 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 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 (13Yao H. Labudda K. Rim C. Capodieci P. Loda M. Stork P.J.S. J. Biol. Chem. 1995; 270: 20748-20753Crossref PubMed Scopus (55) Google Scholar). MKP-2 riboprobe synthesis and Northern blotting using this cRNA probe were done as described previously (14Misra-Press A. Rim C.S. Yao H. Roberson M.S. Stork P.J.S. J. Biol. Chem. 1995; 270: 14587-14596Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). All filters were scanned and quantitated using a Molecular Dynamics PhosphorImager 445SI. Treated or untreated cells were lysed in a lysis buffer containing 10% sucrose, 1% Nonidet P-40, 20 μm Tris-HCl (pH 8.0), 137 μm NaCl, 10% glycerol, 2 μm EDTA, 1 μmphenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μmsodium vanadate, and 10 μm sodium fluoride. The lysates were spun at low speed to remove debris, and the supernatant was assayed for ERK1 activity. One hundred μg 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 μg of myelin basic protein (MBP) and 10 μCi of [γ-32P]ATP in 60 μl of buffer containing 40 μm Hepes (pH 7.4), 40 μm MgCl2, 0.1 μm ATP, 4 μm sodium vanadate, and 10 μm sodium fluoride for 30 min at 30 °C. Reactions were terminated by the addition of 60 μl of 2× Laemmli sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis. Quantitations were performed by scanning the gel using a Molecular Dynamics PhosphorImager 445SI. After treatment with the indicated reagents, PC12 cells were harvested, and soluble PKA was measured as described previously (15Carr D.W. DeManno D.A. Atwood A. Hunzicker-Dunn M. Scott J.D. J. Biol. Chem. 1993; 268: 20729-20732Abstract Full Text PDF PubMed Google Scholar). Kinase reactions were incubated for 2 min at 30 °C in the presence or absence of 10 μm cAMP. For GTP loading studies, PC12 cells were transfected with 15 μg 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 [32P]orthophosphate as described (12Vossler M. Yao H. York R. Rim C. Pan M.-G. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Rap1 was precipitated with nickel nitrilotriacetic acid-agarose, and GTP loading was assayed as described (16Urano T. Emkey R. Feig L.A. EMBO J. 1996; 15: 810-816Crossref PubMed Scopus (300) Google Scholar) 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 mKH2PO4 (pH 3.4) at room temperature, and analyzed using a Molecular Dynamics PhosphorImager 445SI. The GTP fraction was calculated as follows: (GTP counts/3)/[(GTP counts/3) + (GDP counts/2)]. 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 μm) was added 15 min before NGF, as indicated. Cells were fixed in 70% ethanol, 50 μm glycine (pH 2.0) at −20 °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.). 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.1 A). The activation of ERK1 by forskolin was completely blocked by an inhibitor of PKA, H89 (17Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar) (Fig. 1 A), 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. 1 D, suggesting that H89 was preferentially acting on kinases downstream of NGF at this concentration. PKA can be inhibited by the proteinkinase inhibitor (PKI), a physiological inhibitor of PKA (18Day R.N. Walder J.A. Maurer R.A. J. Biol. Chem. 1989; 264: 431-436Abstract Full Text PDF PubMed Google Scholar, 19Patten S.M.V. Howard P. Walsh D.A. Maurer R. Mol. Endocrinol. 1992; 6: 2114-2122PubMed Google Scholar). 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 (18Day R.N. Walder J.A. Maurer R.A. J. Biol. Chem. 1989; 264: 431-436Abstract Full Text PDF PubMed Google Scholar, 19Patten S.M.V. Howard P. Walsh D.A. Maurer R. Mol. Endocrinol. 1992; 6: 2114-2122PubMed Google Scholar). This peptide was modified by the addition of a stearyl group at the amino terminus (sPKI) to allow penetration into the cell (20Vijayaraghavan S. Goueli S.A. Davey M.P. Carr D.W. Biol. Reprod. 1997; 57: 1517-1523Crossref PubMed Scopus (36) Google Scholar). 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. 2 A). 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, 2H. Yao and P. J. S. Stork, unpublished observations. suggesting 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. 2 A). 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 (21VanBuskirk R. Corcoran T. Wagner J.A. Mol. Cell. Biol. 1985; 5: 1984-1992Crossref PubMed Scopus (77) Google Scholar). 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 (22Cassano S. Gallo A. Buccigrossi V. Porcellini A. Cerillo R. Gottesman M.E. Avvedimento E.V. J. Biol. Chem. 1996; 271: 29870-29875Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), greatly reduce the response of the cell to cAMP (21VanBuskirk R. Corcoran T. Wagner J.A. Mol. Cell. Biol. 1985; 5: 1984-1992Crossref PubMed Scopus (77) Google Scholar). 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. 2 B, upper and middle panels). The activation of ERK1 by 8-CPT-cAMP in these cells was blunted (Fig. 2 B, 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. 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 (23Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1108) Google Scholar, 24Janknecht R. Ernst W.H. Pingound V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (508) Google Scholar, 25Gille H. Strahl T. Shaw P.E. Curr. Biol. 1995; 5: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). 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 (12Vossler M. Yao H. York R. Rim C. Pan M.-G. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 26Roberson M.S. Misra-Press A. Laurence M.E. Stork P.J.S. Maurer R.A. Mol. Cell. Biol. 1995; 17: 3531-3539Crossref Google Scholar). These plasmids were co-transfected with or without cDNA encoding the PKA inhibitor (cPKI) or an inactive mutant of PKI (cPKImut) (18Day R.N. Walder J.A. Maurer R.A. J. Biol. Chem. 1989; 264: 431-436Abstract Full Text PDF PubMed Google Scholar). The maximal activation of Elk-1 by both NGF and 8-CPT-cAMP was blocked by PKI but not by the expression of cPKImut (Fig.3 A). 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. 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 thatMKP-2 expression is rapidly increased in response to NGF in PC12 cells (14Misra-Press A. Rim C.S. Yao H. Roberson M.S. Stork P.J.S. J. Biol. Chem. 1995; 270: 14587-14596Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Its gene product, MAP kinase phosphatase 2, dephosphorylates and inactivates multiple members of the MAP kinase family including JNKs (c-Jun N-terminusKinase) and p38 in vitro and in vivo(14Misra-Press A. Rim C.S. Yao H. Roberson M.S. Stork P.J.S. J. Biol. Chem. 1995; 270: 14587-14596Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 27Hirsch D.D. Stork P.J.S. J. Biol. Chem. 1997; 272: 4568-4575Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 28Enslen H. Tokumitsu H. Stork P.J.S. Davis R.J. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10803-10808Crossref PubMed Scopus (261) Google Scholar). As JNKs and p38 trigger cell death in PC12 cells (29Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5045) Google Scholar), these actions of MKP-2 have been proposed to mediate the neurotrophic action of these agents (27Hirsch D.D. Stork P.J.S. J. Biol. Chem. 1997; 272: 4568-4575Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). 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. 3A. Misra-Press, H. Yao, and P. J. S. Stork, unpublished observations. Since the induction of MKP-2 mRNA by NGF may require ERK 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. 3 B, 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. 3 B,right panel). 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 (1Shackleford G.M. Willert K. Wang J. Varmus H.E. Neuron. 1993; 11: 865-875Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 30Machida C.M. Scott J.D. Ciment G. J. Cell Biol. 1991; 114: 1037-1048Crossref PubMed Scopus (38) Google Scholar, 31Nordstrom L.A. Lochner J. Ciment G. Mol. Cell Neurosci. 1995; 6: 56-58Crossref PubMed Scopus (79) Google Scholar). Its induction by NGF is dependent on Ras, the MAP kinase kinase kinase Raf, and ERKs (13Yao H. Labudda K. Rim C. Capodieci P. Loda M. Stork P.J.S. J. Biol. Chem. 1995; 270: 20748-20753Crossref PubMed Scopus (55) Google Scholar, 32D'Arcangelo G. Halegoua S. Mol. Cell. Biol. 1993; 13: 3146-3155Crossref PubMed Scopus (136) Google Scholar). It has been shown that this induction requires multiple transcription factors, including those of the Ets family (33deSouza S. Lochner J. Machida C.M. Matrisian L.M. Ciment G. J. Biol. Chem. 1995; 270: 9106-9114Crossref PubMed Scopus (25) Google Scholar, 34Kirstein M. Sanz L. Quiñones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). NGF induces low levels of transin mRNA in PC12 cells within 4 h (30Machida C.M. Scott J.D. Ciment G. J. Cell Biol. 1991; 114: 1037-1048Crossref PubMed Scopus (38) Google Scholar), and by 24 h of stimulation, expression levels could be easily detected by Northern blot (Fig. 3 C). 8-CPT-cAMP could not stimulate the expression of transin in the absence of additional agents (30Machida C.M. Scott J.D. Ciment G. J. Cell Biol. 1991; 114: 1037-1048Crossref PubMed Scopus (38) Google Scholar) (Fig. 3 C). 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 (35Connolly J.L. Greene S.A. Greene L.A. J. Cell Biol. 1984; 98: 457-465Crossref PubMed Scopus (86) Google Scholar, 36Gunning P.W. Landreth G.E. Bothwell M.A. Shooter E.M. J. Cell Biol. 1981; 89: 240-245Crossref PubMed Scopus (188) Google Scholar, 37Heidemann S.R. Joshi H.C. Schechter A. Fletcher J.R. Bothwell M. J. Cell Biol. 1985; 100: 916-927Crossref PubMed Scopus (107) Google Scholar). 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.3 C). Neither treatment with 8-CPT-cAMP or H89 alone stimulated transin mRNA to detectable levels (Fig. 3 C). These data demonstrate that maximal induction of specific immediate early and late genes by NGF may require PKA. 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.4 A). Similar results were seen after EGF treatment (Fig. 4 B). In contrast, 8-CPT-cAMP induced a 5-fold increase in PKA activity at the time points examined (Fig. 4 B). 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. 4 B). These results are consistent with previous reports showing that adenylyl cyclase and PKA activities are not significantly increased after NGF stimulation of these cells (38Race H.M. Wagner J.A. J. Neurochem. 1985; 44: 1588-1592Crossref PubMed Scopus (27) Google Scholar,39Balbi D. Allen J.M. Mol. Brain Res. 1994; 23: 310-316Crossref PubMed Scopus (30) Google Scholar). 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. 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 (12Vossler M. Yao H. York R. Rim C. Pan M.-G. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). 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 (12Vossler M. Yao H. York R. Rim C. Pan M.-G. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar), the activation of Rap1 by PKA may contribute to NGF activation of ERKs in these cells. 4R. H. York, H. Yao, and P. J. S. Stork, manuscript in preparation. We have previously shown that Rap1 activation is not required for the elaboration of neurites seen after NGF treatment of PC12 cells" @default.
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• W2157776145 date "1998-04-01" @default.
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• W2157776145 title "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" @default.
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• W2157776145 doi "https://doi.org/10.1074/jbc.273.14.8240" @default.
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