ERK5 Activity Is Required for Nerve Growth Factor-induced Neurite Outgrowth and Stabilization of Tyrosine Hydroxylase in PC12 Cells
2009; Elsevier BV; Volume: 284; Issue: 35 Linguagem: Inglês
10.1074/jbc.m109.027821
ISSN1083-351X
AutoresYutaro Obara, Arata Yamauchi, Shin Takehara, Wataru Nemoto, Maho Takahashi, Philip A. Stork, Norimichi Nakahata,
Tópico(s)Melanoma and MAPK Pathways
ResumoExtracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt2cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 μm, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt2cAMP, but BIX02188 and BIX02189, at 30 μm, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt2cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt2cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process. Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt2cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 μm, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt2cAMP, but BIX02188 and BIX02189, at 30 μm, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt2cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt2cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process. ERKs 2The abbreviations used are: ERKextracellular signal-regulated kinasePC12pheochromocytoma cellsNGFnerve growth factorBt2cAMPdibutyryl cAMPMAPKmitogen-activated protein kinaseEGFepidermal growth factorBDNFbrain-derived neurotrophic factorGPCRG-protein-coupled receptorHRPhorseradish peroxidaseDMEMDulbecco's modified Eagle's mediumCREBcAMP-response element-binding proteinGSK3βglycogen synthase kinase-3βm.o.i.multiplicity of infectionsiRNAsmall interfering RNAGFPgreen fluorescent proteinEGFPenhanced GFP. or MAPKs are involved in proliferation, differentiation, migration, and gene expression. ERK1/2 is activated by variety of stimuli, and the signaling pathway leading to ERK1/2 activation has been well characterized (1.Goldsmith Z.G. Dhanasekaran D.N. Oncogene. 2007; 26: 3122-3142Crossref PubMed Scopus (324) Google Scholar, 2.Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (964) Google Scholar, 3.Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar). ERK5 is approximately double the molecular size of ERK1/2. The kinase domain is encoded by its amino-terminal half and shares ∼50% homology with ERK1/2, whereas its unique carboxyl terminus encodes two proline-rich regions, a nuclear export domain and a nuclear localization domain (4.Wang X. Tournier C. Cell. Signal. 2006; 18: 753-760Crossref PubMed Scopus (222) Google Scholar, 5.Nishimoto S. Nishida E. EMBO Rep. 2006; 7: 782-786Crossref PubMed Scopus (347) Google Scholar). Recently, it was reported that the autophosphorylated carboxyl terminus of ERK5 plays a critical role in activating transcription (6.Morimoto H. Kondoh K. Nishimoto S. Terasawa K. Nishida E. J. Biol. Chem. 2007; 282: 35449-35456Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The threonine and tyrosine residues on ERK5 are phosphorylated by MEK5 but not MEK1/2. In contrast, ERK1/2 is not phosphorylated by MEK5 but is phosphorylated by MEK1/2 (7.English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 8.Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). extracellular signal-regulated kinase pheochromocytoma cells nerve growth factor dibutyryl cAMP mitogen-activated protein kinase epidermal growth factor brain-derived neurotrophic factor G-protein-coupled receptor horseradish peroxidase Dulbecco's modified Eagle's medium cAMP-response element-binding protein glycogen synthase kinase-3β multiplicity of infection small interfering RNA green fluorescent protein enhanced GFP. Several physiological roles of ERK5 have been reported (4.Wang X. Tournier C. Cell. Signal. 2006; 18: 753-760Crossref PubMed Scopus (222) Google Scholar, 5.Nishimoto S. Nishida E. EMBO Rep. 2006; 7: 782-786Crossref PubMed Scopus (347) Google Scholar, 9.Cavanaugh J.E. Eur. J. Biochem. 2004; 271: 2056-2059Crossref PubMed Scopus (72) Google Scholar). For example, ERK5 regulates S-phase entry by epidermal growth factor (EGF) in HeLa cells (10.Kato Y. Tapping R.I. Huang S. Watson M.H. Ulevitch R.J. Lee J.D. Nature. 1998; 395: 713-716Crossref PubMed Scopus (360) Google Scholar). In dorsal root ganglia, ERK5 is activated by nerve growth factor (NGF) during retrograde transport of TrkA, and this action prevents apoptosis (11.Watson F.L. Heerssen H.M. Bhattacharyya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (384) Google Scholar). In developing cortical neurons, ERK5 plays a critical role in survival promoted by brain-derived neurotrophic factor (BDNF) (12.Liu L. Cavanaugh J.E. Wang Y. Sakagami H. Mao Z. Xia Z. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8532-8537Crossref PubMed Scopus (144) Google Scholar). Neuronal differentiation is reduced by ERK5 knockdown using antisense morpholino oligonucleotides in Xenopus laevis (13.Nishimoto S. Kusakabe M. Nishida E. EMBO Rep. 2005; 6: 1064-1069Crossref PubMed Scopus (35) Google Scholar). Also, ERK5 is required for generation of neurons from cortical progenitors (14.Liu L. Cundiff P. Abel G. Wang Y. Faigle R. Sakagami H. Xu M. Xia Z. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9697-9702Crossref PubMed Scopus (51) Google Scholar). In animal models, ERK5 gene knockout is lethal at E9.5–10.5 because of cardiovascular defects, indicating the involvement of ERK5 in heart development (15.Hayashi M. Lee J.D. J. Mol. Med. 2004; 82: 800-808Crossref PubMed Scopus (142) Google Scholar). Pathophysiological roles for ERK5 have been proposed for tumor development and cardiac hypertrophy (4.Wang X. Tournier C. Cell. Signal. 2006; 18: 753-760Crossref PubMed Scopus (222) Google Scholar). Thus, ERK5 plays essential physiological roles in addition to ERK1/2. In rat pheochromocytoma cells (PC12), EGF and NGF activate ERK5 via the small G-protein Ras (16.Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). But it has been shown that another neurotrophin, BDNF, activates ERK5 through Rap1 in cortical neurons (17.Wang Y. Su B. Xia Z. J. Biol. Chem. 2006; 281: 35965-35974Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In the mink lung epithelial cell line, interaction of Lck-associated adapter with MEKK2 is involved in activation of ERK5 (18.Sun W. Wei X. Kesavan K. Garrington T.P. Fan R. Mei J. Anderson S.M. Gelfand E.W. Johnson G.L. Mol. Cell. Biol. 2003; 23: 2298-2308Crossref PubMed Scopus (78) Google Scholar), whereas in bone marrow-derived mast cells, protein kinase C mediates FcϵRI-induced ERK5 activation and cytokine production (19.Li G. Lucas J.J. Gelfand E.W. Cell. Immunol. 2005; 238: 10-18Crossref PubMed Scopus (22) Google Scholar). Heterotrimeric G-protein-coupled receptor (GPCR) signals also activate ERK5. For example, GPCR agonists such as carbachol and thrombin activate ERK5 via Gαq/11 and Gα12/13 families of heterotrimeric G-proteins (1.Goldsmith Z.G. Dhanasekaran D.N. Oncogene. 2007; 26: 3122-3142Crossref PubMed Scopus (324) Google Scholar, 20.Fukuhara S. Marinissen M.J. Chiariello M. Gutkind J.S. J. Biol. Chem. 2000; 275: 21730-21736Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The ERK5 activation by carbachol and thrombin is not blocked by dominant-negative mutants of Ras and Rho nor the C3 toxin that inactivates Rho-mediated functions (20.Fukuhara S. Marinissen M.J. Chiariello M. Gutkind J.S. J. Biol. Chem. 2000; 275: 21730-21736Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In contrast, prostaglandin E2 and forskolin, both of which promote cAMP production, attenuate ERK5 phosphorylation induced by EGF via protein kinase A (21.Pearson G.W. Earnest S. Cobb M.H. Mol. Cell. Biol. 2006; 26: 3039-3047Crossref PubMed Scopus (32) Google Scholar). In addition, we recently reported that βγ subunits of Gi/o activated by lysophosphatidic acid blocked ERK5 activation by EGF and NGF in PC12 cells (22.Obara Y. Okano Y. Ono S. Yamauchi A. Hoshino T. Kurose H. Nakahata N. Cell. Signal. 2008; 20: 1275-1283Crossref PubMed Scopus (17) Google Scholar). Thus, the mechanism of ERK5 regulation is complex, and the detailed mechanisms remain unclear. PC12 cells are a good in vitro model of immature neurons. In response to NGF, they differentiate toward sympathetic neurons (23.Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 2424-2428Crossref PubMed Scopus (4873) Google Scholar). We have shown that NGF and cAMP promote ERK1/2 phosphorylation via Ras or Rap1 in PC12 cells, and ERK1/2 activation is essential for neurite outgrowth by NGF or cAMP (24.Obara Y. Labudda K. Dillon T.J. Stork P.J. J. Cell Sci. 2004; 117: 6085-6094Crossref PubMed Scopus (115) Google Scholar). Although the role of ERK5 in neural differentiation is suggested (13.Nishimoto S. Kusakabe M. Nishida E. EMBO Rep. 2005; 6: 1064-1069Crossref PubMed Scopus (35) Google Scholar, 14.Liu L. Cundiff P. Abel G. Wang Y. Faigle R. Sakagami H. Xu M. Xia Z. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9697-9702Crossref PubMed Scopus (51) Google Scholar), the detailed molecular mechanism in neuronal differentiation remains unclear. Therefore, in this study, we attempted to clarify the physiological role of ERK5 in the neural differentiation process, especially focusing on neurite outgrowth and neuron-specific gene expression such as tyrosine hydroxylase. NGF, Bt2cAMP, D-luciferin, Hoechst-33258, and antibodies against FLAG M2 and β-actin were purchased from Sigma. EGF was purchased from PeproTech EC. (London, UK). Cycloheximide and SB203580 were purchased from Wako Pure Chemicals (Tokyo, Japan). U0126, PD98059, antibodies against phospho-ERK1/2, ERK1/2, phospho-ERK5, ERK5, and tyrosine hydroxylase and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody were purchased from Cell Signaling (Beverly, MA). This phospho-specific ERK5 antibody also recognizes phospho-ERK1/2. Antibodies against ERK2, Rap1 and C3G, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence assay kit and HRP-conjugated anti-mouse IgG were purchased from GE Healthcare. Lipofectamine 2000 was purchased from Invitrogen. Phosphotyrosine hydroxylase (Ser-31) antibody was from Chemicon (Temecula, CA). BIX02188 and BIX02189 were kindly provided from Boehringer Ingelheim (Ridgefield, CT). DNA plasmid encoding tyrosine hydroxylase promoter-driven luciferase was kindly provided by Dr. Bistra Nankova (New York Medical College). DNA plasmids for measuring transcriptional activity of myocyte-enhancer factor (MEF) 2 (MEF2C/Gal4 and Gal4/Luciferase) were kindly given by Dr. Zhengui Xia (University of Washington) (25.Cavanaugh J.E. Ham J. Hetman M. Poser S. Yan C. Xia Z. J. Neurosci. 2001; 21: 434-443Crossref PubMed Google Scholar). DNA plasmid encoding mycERK5, MEK5A (S311A/T315V), and MEK5D (S311D/T315D) were from Dr. Eisuke Nishida (Kyoto University). mycERK5 kinase-dead mutant (K83M) was created from mycERK5 in this laboratory by using the QuikChange site-directed mutagenesis kit (Stratagene, Cedar Creek, TX). Adenovirus encoding RasS17N (RasN17) was provided by Dr. Dennis Stacey (Cleveland Clinic Institute). Adenovirus encoding FLAG-Rap1GAP1 was constructed using FLAG-Rap1GAP1 (26.Carey K.D. Dillon T.J. Schmitt J.M. Baird A.M. Holdorf A.D. Straus D.B. Shaw A.S. Stork P.J. Mol. Cell. Biol. 2000; 20: 8409-8419Crossref PubMed Scopus (73) Google Scholar). C3G siRNA (sense, GGACUUUGAUGUUGAAUGUtt; antisense, ACAUUCAACAUCAAAGUCCtg) was purchased from Ambion (Austin, TX) (27.Wang Z. Dillon T.J. Pokala V. Mishra S. Labudda K. Hunter B. Stork P.J. Mol. Cell. Biol. 2006; 26: 2130-2145Crossref PubMed Scopus (145) Google Scholar). PC12 cells were obtained from the Japanese Cancer Research Bank (Tokyo, Japan). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (Cell Culture Laboratory, Cleveland, OH), 5% horse serum (Invitrogen), penicillin (50 units/ml), and streptomycin (50 μg/ml) in an incubator containing 5% CO2 at 37 °C. PC12 cells that stably overexpress dominant-negative MEK5 were cultured in the presence of G418 (Invitrogen). To obtain PC12 cells that stably overexpress EGFP-tagged Rap1GAP (28.Meng J. Casey P.J. J. Biol. Chem. 2002; 277: 43417-43424Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the cells were transfected with EGFP-tagged Rap1GAP and cultured in the presence of G418. Then the EGFP-positive and -negative cells were selected by cell sorting using FACSAria (BD Biosciences). HEK293 cells were grown in DMEM supplemented with 10% fetal calf serum and the above antibiotics. Electrophoresis was performed on 8–11% acrylamide gels. Proteins were transferred electrically from the gel onto polyvinylidene difluoride membrane (Millipore, Bedford, MA) by the semi-dry blotting method. The blots were blocked for 1 h with 5% low fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature and incubated with primary antibodies overnight at 4 °C. The blots were washed several times and incubated with HRP-conjugated anti-rabbit or anti-mouse IgG antibody as a secondary antibody in TBST containing 5% low fat milk at room temperature for 2 h. After rinsing with TBST, blots were developed using a chemiluminescence assay kit and visualized by the exposing the chemiluminescence from the membrane to the Hyperfilm enhanced chemiluminescence. The densities of the bands corresponding to tyrosine hydroxylase were analyzed by densitometry (ImageJ 1.36b, National Institutes of Health). Activated Rap1 was isolated from cell lysates using a protocol adapted from that of Franke et al. (29.Franke B. Akkerman J.W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). Treated cells were lysed in 300 μl of ice-cold lysis buffer (10% glycerol, 1% Nonidet P-40, 50 mm Tris-HCl (pH 8.0), 200 mm NaCl, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 1 μm leupeptin, 10 μg/ml soybean trypsin inhibitor, 10 mm NaF, 0.5 mm aprotinin, 1 mm Na3VO4). Lysates were clarified by low speed centrifugation, and supernatants containing 500–1000 μg of total protein were incubated with 40 μg of GST-RalGDS fusion protein (gift of Dr. J. L. Bos, Utrecht University, Utrecht, The Netherlands) for 40 min, followed by incubation with glutathione-agarose beads for an additional 40 min at 4 °C. Beads were rinsed three times with lysis buffer, and protein was eluted from the beads with Laemmli sample loading buffer. The amount of Rap1 bound to the beads was detected by Western blotting using antibody to Rap1. The neurite extension from PC12 cells was utilized as an index of neuronal differentiation. The cells were fixed with 4% paraformaldehyde and then the nuclei were stained with Hoechst-33258. The photographs were taken with CELAVIEW-RS100 (Olympus, Tokyo, Japan). The number of nuclei and total length of neurites were calculated with the CELAVIEW software (Olympus, Tokyo, Japan), and then the value of total neurite length divided by nucleus number was expressed as neurite length per cell (μm/cell). For transfection experiments (FIGURE 6, FIGURE 7), the number of nuclei and total neurite length in GFP-positive cells were selectively counted by using the software. Data are expressed as means ± S.E. of the values of three wells (30.Obara Y. Aoki T. Kusano M. Ohizumi Y. J. Pharmacol. Exp. Ther. 2002; 301: 803-811Crossref PubMed Scopus (67) Google Scholar). The DNA plasmids were transfected into PC12 cells using the transfection reagent Lipofectamine 2000. Briefly, the cells were seeded onto 24-well plates at 1 × 105 (cells/well) and cultivated for a day. The DNA plasmids ((0.25 μg of Elk-1/Gal, 0.25 μg of 5× Gal4-E1B/Luciferase, 0.2 μg of β-galactosidase, and 0.3 μg of ERK5/MEK5 mutants or 0.1 μg of MEF2C/Gal, 0.1 μg of Gal-luciferase, 0.1 μg of β-galactosidase, and 1.2 μg of ERK5 mutants or 0.4 μg of tyrosine hydroxylase promoter/luciferase, 0.4 μg of ERK5 or MEK5 mutants, and 0.2 μg of β-galactosidase)) and transfection reagent (1 μl/tube) were mixed gently in DMEM (10 μl/tube) and incubated for 20 min at room temperature. After the addition of DMEM (40 μl/tube), this entire mixture was transferred to the cultured media (50 μl/well), which had been replaced with serum-free DMEM (200 μl). The cells were incubated for 4 h at 37 °C, and then the media were replaced with growth medium (500 μl) containing 10% fetal calf serum and 5% horse serum. For reporter gene assays, the cells were incubated with drugs at 37 °C for 6–8 h after serum starvation and subjected to luciferase assay. Cells were lysed in lysis buffer (1% Triton X-100, 110 mm K2HPO4, 15 mm KH2PO4 (pH 7.8)) (100 μl/well). Then after centrifuging the lysates to remove the cell debris, the supernatant (50 μl/tube) was mixed with 300 μl of assay buffer (25 mm Gly-Gly, 15 mm MgSO4, 5 mm ATP, 10 mm NaOH). The luciferase reaction was started by adding 100 μl of luciferin solution (150 μm), and luciferase activity was measured using a luminometer (GENE LIGHT 55, Microtech Nition, Funabashi, Japan). As an internal control, β-actin promoter-driven β-galactosidase activity was measured in the lysates to normalize for the transfection efficiency. PC12 cells were infected with adenoviruses encoding dominant-negative RasN17 and FLAG-Rap1GAP1 that block endogenous activities of Ras and Rap1, respectively (31.Obara Y. Horgan A.M. Stork P.J. J. Neurochem. 2007; 101: 470-482Crossref PubMed Scopus (49) Google Scholar). The infection was carried out for 3 days at 50 m.o.i. for RasN17 adenovirus. FLAG-Rap1GAP1 adenovirus at 50 m.o.i. was applied to cells together with transactivating virus at 250 m.o.i. Control cells were infected with transactivating virus alone. After infection for 3 days, the medium was replaced with serum-free medium, and the cells were stimulated with drugs. Data were expressed as the mean values ± S.E., and significant differences were analyzed using Tukey's method. To examine whether NGF or cAMP can activate ERK5 in PC12 cells, PC12 cells were stimulated with NGF (100 ng/ml) or Bt2cAMP (0.5 mm) for 3–360 min. Then phosphorylation of ERK5 and ERK1/2 was observed by Western blotting (Fig. 1). As shown previously, NGF induced the sustained phosphorylation of ERK5 and ERK1/2 (24.Obara Y. Labudda K. Dillon T.J. Stork P.J. J. Cell Sci. 2004; 117: 6085-6094Crossref PubMed Scopus (115) Google Scholar, 32.York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (761) Google Scholar). In contrast, although Bt2cAMP induced ERK1/2 phosphorylation, it did not induce ERK5 phosphorylation. Whereas it has been reported that MEK inhibitors, such as PD98059 and U0126, block MEK5 as well as MEK1/2 (16.Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar), it has been shown that these inhibitors do not block ERK5 signaling efficiently at low doses (33.Rovida E. Spinelli E. Sdelci S. Barbetti V. Morandi A. Giuntoli S. Dello Sbarba P. J. Immunol. 2008; 180: 4166-4172Crossref PubMed Scopus (51) Google Scholar, 34.Pollack V. Sarközi R. Banki Z. Feifel E. Wehn S. Gstraunthaler G. Stoiber H. Mayer G. Montesano R. Strutz F. Schramek H. Am. J. Physiol. Renal Physiol. 2007; 293: F1714-F1726Crossref PubMed Scopus (52) Google Scholar, 35.Mody N. Leitch J. Armstrong C. Dixon J. Cohen P. FEBS Lett. 2001; 502: 21-24Crossref PubMed Scopus (226) Google Scholar). To examine the selectivity of these MEK inhibitors in our system, PC12 cells were preincubated with U0126 (10 or 30 μm) for 30 min and then further stimulated with NGF (100 ng/ml) or Bt2cAMP (0.5 mm) for 10 min. Although ERK1/2 phosphorylation by both reagents was abolished by pretreatment with U0126, ERK5 phosphorylation was not blocked at all (Fig. 2A). Another MEK inhibitor, PD98059, also suppressed ERK1/2 phosphorylation by NGF and Bt2cAMP but did not suppress ERK5 phosphorylation by NGF (Fig. 2B). Judging from these results, we used U0126 as a selective inhibitor for ERK1/2 signaling in this study. Recently, BIX02188 and BIX 02189 were developed as selective pharmacological inhibitors of the MEK5/ERK5 pathway (36.Tatake R.J. O'Neill M.M. Kennedy C.A. Wayne A.L. Jakes S. Wu D. Kugler Jr., S.Z. Kashem M.A. Kaplita P. Snow R.J. Biochem. Biophys. Res. Commun. 2008; 377: 120-125Crossref PubMed Scopus (121) Google Scholar). PC12 cells were pretreated with or without BIX02188 and BIX02189 (3–30 μm) for 30 min, and the cells were stimulated with NGF (100 ng/ml) for 5 min. Both inhibitors blocked ERK5 phosphorylation in a concentration-dependent manner, whereas phosphorylation levels of ERK1/2 were unaffected (Fig. 2C). Therefore, we used BIX02188 and BIX02189 as selective pharmacological inhibitors for ERK5 signaling in this study. Next, we attempted to confirm the selectivity of dominant-negative mutants of ERK5 or MEK5. HEK293 cells were co-transfected with the constitutively active MEK5 mutant (MEK5D) and empty vector, ERK5 wild-type (ERK5WT), or dominant-negative ERK5 mutant (ERK5KD). MEK5D caused phosphorylation of both ERK5WT and ERK5KD, but the band shift of ERK5KD was smaller because ERK5KD lacks its kinase activity and subsequent autophosphorylation (Fig. 3A). It has been established that ERK5 and p38 MAPK also phosphorylate MEF2C at Ser-387 that is critical for transcriptional activation. In addition, p90 ribosomal S6 kinase 2 activated by ERK1/2 phosphorylates MEF2C on Ser-192 that is also required for transcriptional activation (37.Wang Y. Liu L. Xia Z. J. Neurochem. 2007; 102: 957-966Crossref PubMed Scopus (18) Google Scholar). To confirm the inhibitory effect of ERK5KD on endogenous ERK5 activity, MEF2C-dependent transcription was examined. PC12 cells were stimulated by NGF (100 ng/ml) for 8 h in the presence of U0126 (10 μm) and a p38 MAPK inhibitor SB203580 (1 μm). NGF significantly promoted MEF2C-dependent transcription, and this effect was significantly blocked by ERK5KD (Fig. 3B). Because constitutively active Ras oncogenic mutant (RasV12) is a strong activator for ERK1/2 signaling (38.Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar), we examined whether ERK5KD or dominant-negative MEK5 mutant (MEK5A) interferes with ERK1/2 phosphorylation induced by RasV12 in PC12 cells. Although U0126 completely inhibited ERK1/2 phosphorylation, the phosphorylation was not affected by ERK5KD or MEK5A (Fig. 3C). Luciferase expression resulting from Elk1 phosphorylation by ERK1/2 was often regarded as an index of in vivo ERK1/2 kinase activity, and it has been shown that ERK5 does not phosphorylate Elk1 (16.Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 25.Cavanaugh J.E. Ham J. Hetman M. Poser S. Yan C. Xia Z. J. Neurosci. 2001; 21: 434-443Crossref PubMed Google Scholar). NGF (100 ng/ml, 6 h) robustly promoted ERK1/2 activity, and this effect was completely blocked by U0126 but not affected by ERK5KD or MEK5A (Fig. 3D). These results suggest that ERK5KD or MEK5A does not affect ERK1/2 signaling. Whereas it has been shown that Ras regulates ERK5 activity in PC12 cells and COS7 cells (16.Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar), there are reports demonstrating that Ras was not involved in ERK5 activation by growth factors or GPCR agonists such as carbachol and thrombin (10.Kato Y. Tapping R.I. Huang S. Watson M.H. Ulevitch R.J. Lee J.D. Nature. 1998; 395: 713-716Crossref PubMed Scopus (360) Google Scholar, 20.Fukuhara S. Marinissen M.J. Chiariello M. Gutkind J.S. J. Biol. Chem. 2000; 275: 21730-21736Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). On the other hand, Rap1, another small G-protein, is necessary for ERK5 activation by BDNF in cortical neurons (17.Wang Y. Su B. Xia Z. J. Biol. Chem. 2006; 281: 35965-35974Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Other studies examining Rap1 have shown contrasting results that Rap1 activated by exchange protein activated by cAMP inhibits ERK5 activity within protein complexes containing protein kinase A anchoring protein (39.Dodge-Kafka K.L. Soughayer J. Pare G.C. Carlisle Michel J.J. Langeberg L.K. Kapiloff M.S. Scott J.D. Nature. 2005; 437: 574-578Crossref PubMed Scopus (440) Google Scholar). Thus, the involvement of Ras and Rap1 in ERK5 activation is controversial. Therefore, we attempted to examine the involvement of these small G-proteins in ERK5 activation in PC12 cells. PC12 cells were infected with adenovirus encoding dominant-negative mutant of Ras (RasN17) for 3 days at 50 m.o.i. Then the cells were stimulated with NGF (100 ng/ml) or EGF (100 ng/ml) for 5 min. Although the Ras-dependent ERK1/2 phosphorylation by NGF and EGF was largely reduced by RasN17, ERK5 phosphorylation by these growth factors was resistant to RasN17, suggesting that Ras is not involved in ERK5 phosphorylation by NGF and EGF in PC12 cells (Fig. 4A). Similarly, FLAG-Rap1GAP1 that inactivates Rap1 activity was overexpressed by adenoviral infection at 50 m.o.i. for 3 days, and then PC12 cells were stimulated with NGF (100 ng/ml) for 5 min. ERK5 phosphorylation was resistant to Rap1GAP1 as shown in the case of RasN17 (Fig. 4B). In addition, Rap1 activity was completely blocked by infection of adenovirus encoding FLAG-Rap1GAP1 (data not shown). Also, a major Rap1 guanine nucleotide exchange factor, C3G, is known to mediate Rap1 activation by NGF (32.York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (761) Google Scholar, 40.Hisata S. Sakisaka T. Baba T. Yamada T. Aoki K. Matsuda M. Takai Y. J. Cell Biol. 2007; 178: 843-860Crossref PubMed Scopus (96) Google Scholar). Therefore, we attempted to block Rap1 activation by C3G knockdown. In the condition that C3G expression is diminished by siRNA, ERK5 phosphorylation was not largely reduced (Fig. 4C). Furthermore, ERK5 phosphorylation by NGF or EGF was examined in PC12 cells that stably overexpress EGFP-Rap1GAP. However, ERK5 phosphorylation was not blocked again by NGF (100 ng/ml) or EGF (100 ng/ml) (Fig. 4D). In EGFP-Rap1GAP cells, Rap1 activation by NGF was efficiently reduced, as determined by pulldown assay with GST-RalGDS (Fig. 4E). Taking these results together, ERK5 phosphorylation by NGF is Ras- and Rap1-independent. PC12 cells extend neurites in response to NGF or Bt2cAMP in an ERK1/2-dependent manner (24.Obara Y. Labudda K. Dillon T.J. Stork P.J. J. Cell Sci. 2004; 117: 6085-6094Crossref PubMed Scopus (115) Google Scholar). As shown previously, we confirmed that U0126 significantly attenuated the neurite outgrowth by both NGF and Bt2cAMP (Fig. 5A). Furthermore, both BIX02188 and BIX02189 (30 μm) significantly reduced neurite extension by NGF (100 ng/ml, 1 day) in PC12 cells (Fig. 5B), indicating ERK5 is required for the NGF-induced neurite outgrowth. Next, PC12 cells were co-transfected with GFP and empty vector or ERK5KD and then further stimulated with NGF (100 ng/ml, 1 day) or Bt2cAMP (0.5 mm, 1 day). When GFP-positive cells were examined, ERK5KD significantly reduced the NGF-induced neurite extension (Fig. 6A). However, the neurite outgrowth induced by Bt2cAMP was not attenuated by ERK5KD overexpression (Fig. 6A). Similarly, the neurite outgrowth induced by NGF (100 ng/ml, 1 day) but not
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