Activation of the Protein Kinase ERK5/BMK1 by Receptor Tyrosine Kinases
1999; Elsevier BV; Volume: 274; Issue: 37 Linguagem: Inglês
10.1074/jbc.274.37.26563
ISSN1083-351X
AutoresSachiko Kamakura, Tetsuo Moriguchi, Eisuke Nishida,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoERK5 (also known as BMK1), a member of the mitogen-activated protein kinase (MAPK) superfamily, was known to be activated strongly by oxidant and osmotic stresses. Here we have found that ERK5 is strongly activated by epidermal growth factor and nerve growth factor, whose receptors are tyrosine kinases. The activation of ERK5 was inhibited by expression of dominant-negative Ras and induced by expression of active Ras in PC12 cells, indicating a requirement for Ras in ERK5 activation. The epidermal growth factor-induced activation of ERK5 was found to be inhibited by PD98059 and U0126 inhibitors, which were previously thought to act specifically on classical MAPK kinase (also known as MEK1) and readily reversed by CL100 and MKP-3 dual-specificity phosphatases for which classical MAPKs were previously shown to serve as preferred substrates. The reporter assays demonstrated that the serum-induced enhancement of transcription from serum response element was significantly inhibited by expression of a dominant-negative form of MEK5, which was a direct and specific activator for ERK5 and that transcription from serum response element mediated by the Ets-domain transcription factor Sap1a, but not by Elk1, was stimulated by coexpression of ERK5 and active MEK5. In addition, Sap1a was shown to be phosphorylated by ERK5 in vitro and by the activation of the ERK5 pathway in cells. Moreover, the serum-induced c-Fos expression was markedly inhibited by expression of dominant-negative MEK5. These results reveal a novel signaling pathway to the nucleus mediated by ERK5 that functions downstream of receptor tyrosine kinases to induce immediate early genes, in parallel with the classical MAPK cascade. ERK5 (also known as BMK1), a member of the mitogen-activated protein kinase (MAPK) superfamily, was known to be activated strongly by oxidant and osmotic stresses. Here we have found that ERK5 is strongly activated by epidermal growth factor and nerve growth factor, whose receptors are tyrosine kinases. The activation of ERK5 was inhibited by expression of dominant-negative Ras and induced by expression of active Ras in PC12 cells, indicating a requirement for Ras in ERK5 activation. The epidermal growth factor-induced activation of ERK5 was found to be inhibited by PD98059 and U0126 inhibitors, which were previously thought to act specifically on classical MAPK kinase (also known as MEK1) and readily reversed by CL100 and MKP-3 dual-specificity phosphatases for which classical MAPKs were previously shown to serve as preferred substrates. The reporter assays demonstrated that the serum-induced enhancement of transcription from serum response element was significantly inhibited by expression of a dominant-negative form of MEK5, which was a direct and specific activator for ERK5 and that transcription from serum response element mediated by the Ets-domain transcription factor Sap1a, but not by Elk1, was stimulated by coexpression of ERK5 and active MEK5. In addition, Sap1a was shown to be phosphorylated by ERK5 in vitro and by the activation of the ERK5 pathway in cells. Moreover, the serum-induced c-Fos expression was markedly inhibited by expression of dominant-negative MEK5. These results reveal a novel signaling pathway to the nucleus mediated by ERK5 that functions downstream of receptor tyrosine kinases to induce immediate early genes, in parallel with the classical MAPK cascade. mitogen-activated protein kinase extracellular signal-regulated kinase MAPK kinase MAPK/ERK kinase epidermal growth factor nerve growth factor serum response element serum response factor ternary complex factor hemagglutinin myelin basic protein 12-O-tetradecanoylphorbol-13-acetate polymerase chain reaction Dulbecco's modified Eagle's medium dominant-negative Ras c-Jun N-terminal kinase stress-activated protein kinase The classical mitogen-activated protein kinase (MAPK,1 also known as ERK) cascade is an evolutionarily conserved module that mediates the signaling from various extracellular stimuli to the nucleus (1Ahn N.G. Seger R. Krebs E.G. Curr. Opin. Cell Biol. 1992; 4: 992-999Crossref PubMed Scopus (228) Google Scholar, 2Cobb M.H. Boulton T.G. Robbins D.J. Cell Regul. 1991; 2: 965-978Crossref PubMed Scopus (429) Google Scholar, 3Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (964) Google Scholar, 4Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (329) Google Scholar). Several other members of the MAPK family including c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/MPK2 have been identified. Each member of MAPKs is activated by both tyrosine and threonine phosphorylation catalyzed by a distinct upstream kinase, a member of the MAPK kinase family (1Ahn N.G. Seger R. Krebs E.G. Curr. Opin. Cell Biol. 1992; 4: 992-999Crossref PubMed Scopus (228) Google Scholar, 3Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (964) Google Scholar, 5Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 6Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 7Kyriakis J.M. Avruch J. Bioessays. 1996; 18: 567-577Crossref PubMed Scopus (661) Google Scholar). Classical MAPK has a Thr-Glu-Tyr (TEY) sequence in the dual phosphorylation motif and is activated by mitogenic stimuli such as growth factors mainly through the receptor tyrosine kinase-Ras pathway. JNK and p38, which have Thr-Pro-Tyr (TPY) and Thr-Gly-Tyr (TGY) sequences, respectively, respond to a variety of cellular stresses and pro-inflammatory cytokines and may function in apoptosis, immune response, and differentiation. Most recently, ERK5/BMK1 was identified as a novel member of the MAPK family (8Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 9Lee J.D. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1995; 213: 715-724Crossref PubMed Scopus (289) Google Scholar). Although ERK5 has a TEY sequence in the activation phosphorylation site like classical MAPK, it is strongly activated by stresses such as oxidant and hyperosmolarity, like JNK and p38 (10Abe J. Kusuhara M. Ulevitch R.J. Berk B.C. Lee J.D. J. Biol. Chem. 1996; 271: 16586-16590Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Classical MAPK, JNK, and p38 are known to phosphorylate many of transcription factors to activate their transcriptional activity, which is required for the immediate early gene expression and various cellular responses (11Karin M. Hunter T. Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 12Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1165) Google Scholar). ERK5 has also been shown to phosphorylate transcription factors such as c-Myc and MEF2C, a member of the MADS box transcription factors (13English J.M. Pearson G. Baer R. Cobb M.H. J. Biol. Chem. 1998; 273: 3854-3860Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 14Kato Y. Kravchenko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (498) Google Scholar). The best characterized signaling target of MAPK, JNK, and p38 involves the serum response element (SRE) on the c-fos promoter. At the SRE, the dimer of serum response factor (SRF) forms a ternary complex with a ternary complex factor (TCF) to bring full SRE function. Two TCF proteins, Elk1 and Sap1a, which belong to the Ets transcription factor family, are phosphorylated and activated by classical MAPKs, JNK, and p38 (11Karin M. Hunter T. Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 15Treisman R. Curr. Opin. Genet. Dev. 1994; 4: 96-101Crossref PubMed Scopus (622) Google Scholar, 16Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar, 17Price M.A. Cruzalegui F.H. Treisman R. EMBO J. 1996; 15: 6552-6563Crossref PubMed Scopus (301) Google Scholar, 18Janknecht R. Hunter T. J. Biol. Chem. 1997; 272: 4219-4224Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). It has remained unclear, however, whether ERK5 functions at the element, although serum stimulation was shown to induce ERK5 activation (14Kato Y. Kravchenko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (498) Google Scholar). MEK5, a member of the MAPKK family, is a direct and specific activator of ERK5 (8Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 14Kato Y. Kravchenko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (498) Google Scholar, 19English 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). It has been reported that ERK5 somehow senses the signals from Src and Ras (13English J.M. Pearson G. Baer R. Cobb M.H. J. Biol. Chem. 1998; 273: 3854-3860Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 20Abe J. Takahashi M. Ishida M. Lee J.D. Berk B.C. J. Biol. Chem. 1997; 272: 20389-20394Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). However, activation of ERK5 by Ras or Raf-1, a downstream target of Ras, has not been demonstrated (13English J.M. Pearson G. Baer R. Cobb M.H. J. Biol. Chem. 1998; 273: 3854-3860Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Thus, the signaling pathways for the MEK5-ERK5 cascade are largely undefined. Here we have shown that ERK5 is strongly activated by epidermal growth factor (EGF) and nerve growth factor (NGF) through Ras and that the ERK5 pathway is blocked by several inhibitors known as the classical MAPK pathway inhibitors. The reporter gene assays have demonstrated that c-fos SRE is regulated by the MEK5-ERK5 cascade via Sap1a activation. In addition, Sap1a is shown to be phosphorylated by ERK5 in vitro and by the activation of the ERK5 pathway in cells. The serum-induced c-Fos expression is inhibited by expression of dominant-negative MEK5. These findings reveal a novel signaling pathway from the plasma membrane to the nucleus that is mediated by the MEK5-ERK5 pathway. A mouse brain cDNA library (Stratagene) was screened with a 1-kilobase pair-cDNA fragment encoding the kinase domain of the human ERK5/BMK1. Under high stringency washing conditions, a positive clone, mouse ERK5 cDNA, was isolated. The mouse MEK5 cDNA was obtained in a process of the MKK7 cDNA screening (21Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar). Each of the coding regions was subcloned into pcDL-SRα456 and pSRα-HA1 (22Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). The mutagenic primers (5′-CAGCTGGTGAATGCTATAGCCAAGGTGTATGTTGGAAC-3′ and 5′-GTTCCAACATACACCTTGGCTATAGCATTCACCAGCTG-3′) were used to obtain MEK5(A), in which Ser-311 and Thr-315 were replaced by Ala and Val, respectively. The primers (5′-CAGCTGGTGAATGATATAGCCAAGGACTATGTTGGAAC-3′ and 5′-GTTCCAACATAGTCCTTGGCTATATCATTCACCAGCTG-3′) were used to obtain MEK5(D), in which Ser-311 and Thr-315 were replaced by Asp as described (14Kato Y. Kravchenko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (498) Google Scholar). The mutagenic primers (5′-CAGCAGGTGGCCATCGATAAGATACCTAATGC-3′ and 5′-GCATTAGGTATCTTATCGATGGCCACCTGCTG-3′) were used to obtain a kinase-negative form of ERK5 (ERK5(KN)), in which Lys-84 was replaced by Asp, and the C-terminal half (458–806) of ERK5(KN) was deleted by digestion with Nae I to obtain ERK5(KNΔC). The ERK5(KNΔC) was subcloned into pGEX-6P (Amersham Pharmacia Biotech). RhoA, Rac1, H-Ras, and Cdc42 cDNAs were obtained by PCR from a HeLa cDNA library (Stratagene) and subcloned into pCR-Blunt (Invitrogen). Then PCR-based mutagenesis was performed to obtain the mutant series RhoV14, RhoN19, RacV12, dnRas(N17), and Cdc42V12, which were subcloned into pcDL-SRα456, except Cdc42V12, which was subcloned into pcDNA3 (Invitrogen). Elk1 and Sap1a cDNAs were amplified by PCR using the HeLa cDNA library (Stratagene) as a template and subcloned into pcDL-SRα456 and pGEX-6P. Ser-287 and Thr-291 in MKK7 (21Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar) were replaced by Asp and Glu, respectively, by PCR-based mutagenesis to obtain a constitutively active form of MKK7 (MKK7(DE)). The fragment encoding three copies of the Myc epitope with Bam HI and Bgl II sites was synthesized by PCR, digested with Bam HI and Bgl II, and inserted into the Bgl II cut pcDL-SRα456 to generate pSRα-Myc1. CL100 (23Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (571) Google Scholar) and MKP-3 (24Muda M. Boschert U. Dickinson R. Martinou J.C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar) cDNAs were subcloned into the pSRα-Myc1 vector. Other expression plasmids were as follows. pcDL-SRα456-RasV12 was kindly given by S. Hattori, pcDL-SRα456-MKK(A) was used for expression of a dominant-negative form of Xenopus MAPKK (MEK1) (25Gotoh I. Fukuda M. Adachi M. Nishida E. J. Biol. Chem. 1999; 274: 11874-11880Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), pSRαHA-MAPK was used for expression of HA-tagged Xenopus MAPK (ERK2) (21Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar), pSRαHA-JNK was used for expression of HA-tagged rat JNK2/SAPKα, pSRαHA-p38 was used for expression of HA-tagged human p38α (21Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar), pSRE-Luc and pSRF-Luc (Stratagene) were used for luciferase assays, and PEGFP-C1 (CLONTECH) was used for identification of transfected cells. COS7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. Rat PC12 cells were cultured in DMEM supplemented with 0.35% glucose, 5% heat-inactivated horse serum, and 10% fetal calf serum. HeLa and NIH3T3 cells were maintained in DMEM with 10% calf serum. All plasmids used in transfection were prepared with QIAGEN-tip 500. 24 h before transfection, 1.5 × 105 COS7 and PC12 cells were plated on a 60-mm dish, and 0.5 × 105 HeLa and NIH3T3 cells were plated on a 35-mm dish. For cell staining, cells were plated onto coverslips. Transfection for COS7, PC12, and NIH3T3 cells was carried out using a LipofectAMINE (Life Technologies, Inc.) with 4.5-μg total amounts of plasmids on 60-mm dish or 1.5 μg on a 35-mm dish. After 24 h, the complete medium was replaced by a low serum medium consisting of DMEM with 0.5% calf serum for COS7 and NIH3T3 cells or DMEM with 1% heat-inactivated horse serum for PC12 cells. Then the cells were incubated for 24 h further. HeLa cells were transfected using FuGENE6 (Roche Molecular Biochemicals) with 1.5-μg total amounts of plasmids on a 35-mm dish and incubated for 40 h in DMEM supplemented with 0.5% calf serum. The transfection efficiency was 8–12% in COS7 cells, 5–10% in PC12 and NIH3T3 cells, and 4–8% in HeLa cells. 48 h after transfection, COS7 or PC12 cells in the low serum medium were treated with or without each stimuli: 30 nm EGF, 10% fetal calf serum, and 100 ng/ml NGF for 5 min and 10 μm lysophosphatidic acid, 1 mmH2O2, 100 ng/ml TPA, 100 J/m2 UV, incubation at 45 °C (heat shock), 100 nm A23187, and 2 μg/ml anisomycin for 30 min. For kinase assays of endogenous ERK5, subconfluent cells on a 100-mm dish in the low serum medium were treated with or without the growth factors as described above. Then the cells were washed once in ice-cold Hepes-buffered saline, scraped in a lysis buffer consisting of 20 mm Tris-Cl (pH 7.5), 5 mm EGTA, 25 mm β-glycerophosphate, 1% Triton X-100, 2 mm dithiothreitol, 1 mm vanadate, 1 mm phenylmethylsulfonyl fluoride, and 1% aprotinin (200 μl of buffer/60-mm dish or 400 μl of buffer/100-mm dish) and extracted by pipetting 20 times, followed by centrifugation at 12,000 rpm for 20 min. The supernatant was incubated with 45 μl of a 1:1 slurry of protein A-Sepharose beads (Amersham Pharmacia Biotech) and antibody (10 μl (200 μg/ml) of rabbit anti-HA antibody Y-11 (Santa Cruz), 20 μl (100 μg/ml) of goat anti-ERK5 antibody C-20 (Santa Cruz), or 10 μl (200 μg/ml) of mouse anti-Myc antibody 9E10 (Santa Cruz)) for 2 h at 4 °C. The immune complex on beads was washed twice with a solution containing 20 mm Tris-Cl (pH 7.5), 500 mm NaCl, 2 mm dithiothreitol, and 0.05% Tween 20. Then, the immunoprecipitate was divided into two parts, and one part was used for the kinase assay, and the other was used for immunoblotting. To detect the kinase activity, the immune complex was washed once with a reaction buffer containing 20 mm Tris-Cl (pH 7.5), 2 mm EGTA, 2 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride and incubated for 30 min at 30 °C with the substrate in a buffer (final volume, 15 μl) containing 20 mm Tris-Cl (pH 7.5), 10 mmMgCl2 and 100 μm ATP (2 μCi of [γ-32P]ATP). 10 μg of myelin basic protein (MBP) and 10 μg of GST-ERK5(KNΔC) were used as substrate for ERK5 and MEK5, respectively. The immune complex kinase assays for classical MAPK, JNK, and p38 were carried out using 3 μg of MBP, GST-c-Jun (1–79), or GST-ATF-2 as described (21Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar). After SDS-polyacrylamide gel electrophoresis, the radioactivity was analyzed with an image analyzer (Bio-Rad). 48 h after transfection, NIH3T3 cells in DMEM with 0.5% calf serum were treated with or without 15% fetal calf serum for 2 h. Then the cells on the coverslip were fixed with 3.7% formaldehyde in phosphate-buffered saline for 10 min at 37 °C, permeabilized in 0.5% Triton X-100 in phosphate-buffered saline for 10 min, rinsed with phosphate-buffered saline containing 3% bovine serum albumin (Sigma) for 30 min, and then stained with rabbit anti-human c-Fos antibody (Upstate Biotechnology Inc.) for 16 h at 4 °C. After washing three times with phosphate-buffered saline, the cells were stained with 4,6-diamidino-2-phenylindole and tetramethyl rhodamine isothiocyanate-labeled anti-rabbit IgG antibody (Cappel) for 1 h at room temperature. 40 h after transfection, HeLa cells in DMEM with 0.5% calf serum were treated with or without 15% fetal calf serum and incubated for 8 h. Then the cells were washed once in ice-cold Hepes-buffered saline, scraped in 150 μl of reporter lysis buffer (Promega), and centrifuged at 12,000 rpm for 20 min after vortexing for 15 s. The supernatant was used as a cell extract to detect the luciferase activity. The luciferase assay was carried out using a luciferase assay system (Promega). In brief, 20 μl of the room-temperature cell extract was mixed with 100 μl of room temperature luciferase assay reagent containing the substrate. The reaction was performed and measured in a luminometer (LB9507, Berthold). The protein concentration was determined with a protein assay kit (Bio-Rad) and used for normalization of the luciferase assays. All the experiments were repeated at least three times. For in vitro analysis, GST-Sap1a and GST-Elk1 were prepared by using the expression vector pGEX-6P (Amersham Pharmacia Biotech), expressed in Escherichia coli, and purified by affinity chromatography on glutathione-Sepharose 4B (Amersham Pharmacia Biotech). For in vivo phosphorylation, 6 h after transfection of Myc-tagged Sap1a or Elk1, COS7 cells were labeled for 24 h with 0.5 mCi/ml [32P]orthophosphate in phosphate-free DMEM with 10% fetal calf serum. Then, cell lysates were prepared and subjected to immunoprecipitation with anti-myc antibody (Santa Cruz). We isolated the ERK5 and MEK5 cDNA clones from mouse brain cDNA library and determined the nucleotide sequences containing their open reading frames (Fig. 1). The mouse ERK5 is deduced to contain 806 amino acids and shows 92% identity with human ERK5 (8Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 9Lee J.D. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1995; 213: 715-724Crossref PubMed Scopus (289) Google Scholar). The kinase domain, which is located in the N-terminal half is highly conserved, including the dual phosphorylation site (Thr-219 and Tyr-221) in the TEY sequence (Fig.1 A). The mouse MEK5 is predicted to have 448 amino acids and displays 99 and 93% identity with the rat and human sequences, respectively (8Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 19English 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). The activating phosphorylation sites Ser-311 and Thr-315 are conserved in the species (Fig.1 B).Figure 1Primary structures of mouse ERK5 and mouse MEK5. A, sequence alignment of mouse ERK5 (mERK5) with human ERK5/BMK1 (hERK5) (8Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 9Lee J.D. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1995; 213: 715-724Crossref PubMed Scopus (289) Google Scholar). The GENETYX-MAC program was used to align the amino acid sequences.B, the predicted amino acid sequence of mouse MEK5 (mMEK5) was aligned with those of rat (rMEK5) and human MEK5 (hMEK5) (8Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 14Kato Y. Kravchenko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (498) Google Scholar, 19English 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). In both A and B, identical amino acid residues were linked with verticle lines. Activating phosphorylation sites and ATP-binding sites were denoted by asterisks and arrowheads, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the characteristics of ERK5 activation, we compared the kinase activity in response to several extracellular stimuli. The epithelial-like COS7 cells transfected with HA-tagged mouse ERK5 were exposed to various stimuli; EGF, lysophosphatidic acid, serum, phorbol ester TPA, UV, heat shock, A23187 Ca2+ ionophore, and anisomycin. As a control, the cells were stimulated by H2O2, which was previously reported to induce ERK5 activation (10Abe J. Kusuhara M. Ulevitch R.J. Berk B.C. Lee J.D. J. Biol. Chem. 1996; 271: 16586-16590Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). The immune complex kinase assays revealed that ERK5 was strongly activated by EGF, TPA, or lysophosphatidic acid as well as by H2O2 (Fig.2 A). Although fetal calf serum (10%) and heat shock at 45 °C activated ERK5 moderately, UV, A23187, or anisomycin did not. In the phaeochromocytoma PC12 cells, ERK5 activation by serum was very weak, whereas the activation by EGF or by NGF was strong (Fig. 2 B). The EGF-induced activation of HA-tagged ERK5 was seen in other cell lines, including C2C12 and NIH3T3 cells (data not shown). The EGF-induced activation of ERK5 in PC12 cells peaked at 5 min and then declined rather rapidly (Fig.2 B). The activation by NGF also peaked at about 5 min and persisted longer than that by EGF (Fig. 2 B). The activation of ERK5 by H2O2, TPA, or heat shock was slow and peaked at about 30 min or later (data not shown). We then measured the activity of endogenous ERK5 by an immune complex kinase assay with anti-ERK5 antibody C-20 (Santa Cruz) and found that endogenous ERK5 was activated by EGF in PC12 cells and COS7 cells and by NGF in PC12 cells (Fig. 2 C). The EGF-induced activation of endogenous ERK5 was also observed in NIH3T3 cells and HeLa cells (data not shown). Low molecular weight GTPases such as the Ras family and the Rho family proteins have been shown to function in the signaling pathways of growth factors or stress stimuli to activate the downstream kinase cascades (7Kyriakis J.M. Avruch J. Bioessays. 1996; 18: 567-577Crossref PubMed Scopus (661) Google Scholar, 12Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1165) Google Scholar, 26Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (475) Google Scholar,27Hunter T. Cell. 1997; 88: 333-346Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). To test the effect of activation of these GTPases on the activation of ERK5, cells were cotransfected with plasmids expressing HA-ERK5 and each of dominant-active forms of GTPases: RhoV14, RacV12, Cdc42V12, or RasV12. The immune complex kinase assay revealed that ERK5 as well as classical MAPK (also known as ERK1 and ERK2, simply called MAPK hereafter) was strongly activated by RasV12 in PC12 cells (Fig.3 A) and in C2C12 cells (data not shown). In agreement with a recent report (13English J.M. Pearson G. Baer R. Cobb M.H. J. Biol. Chem. 1998; 273: 3854-3860Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), however, ERK5 was not markedly activated by RasV12 in COS7 cells, whereas MAPK was activated (Fig. 3 B). RacV12 and Cdc42V12, which were able to activate JNK in COS7 cells and PC12 cells (data not shown), were not potent activators of ERK5 (Fig. 3, A and B). RhoV14 also failed to activate ERK5. Because activation of Ras was found to be able to induce ERK5 activation, at least in several types of cells including PC12 cells, we next tested the requirement of Ras for ERK5 activation. PC12 cells were cotransfected with HA-ERK5 and dominant-negative Ras (dnRas). After NGF stimulation, the kinase activity of ERK5 was measured by immune complex kinase assay. The NGF-induced activation of ERK5 was inhibited by expression of dnRas (Fig. 3 C, left). The EGF-induced activation of ERK5 in PC12 cells (data not shown) and in COS7 cells (Fig. 3 C, right) was also inhibited by dnRas. Furthermore, to assess the requirement of Ras for endogenous ERK5 activation, we used a PC12 cell line in which dominant negative Ras (N17) was stably expressed (28Li H. Kawasaki H. Nishida E. Hattori S. Nakamura S. J. Neurochem. 1996; 66: 2287-2294Crossref PubMed Scopus (12) Google Scholar). In the N17Ras-expressing PC12 cell line, the EGF- or NGF-induced activation of endogenous ERK5 was markedly reduced, although the amount of endogenous ERK5 was almost equivalent to that in wild-type PC12 cells (Fig. 3 D). These results, therefore, indicate that Ras is important for the activation of ERK5 induced by NGF and EGF. The growth factor-induced Ras-dependent activation of ERK5 and its having the TEY sequence are the same as the characteristics of MAPK. We tested the effect of some well known inhibitors of the classical MAPK cascade on ERK5 activation. COS7 cells were pretreated with increasing concentrations of PD98059, a MAPKK/MEK1 inhibitor that does not inhibit the JNK or p38 pathway (29Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar, 30Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar), and stimulated by EGF. Another MAPKK inhibitor, U0126 (31Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2754) Google Scholar), was also tested. The kinase assay showed that PD98059 and U0126 inhibited the EGF-induced ERK5 activation in a concentration-dependent manner (Fig. 4 A). The inhibitory effect of these drugs on ERK5 activation was similar to, or slightly more potent than, that on MAPK activation (29Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar, 30Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar, 31Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2754) Google Scholar) (Fig.4 A). It is assumed that the target of PD98059 and U0126 may be MEK5, analogous to the action of the drugs on the MAPK cascade, because the MEK5-ERK5 cascade and the classical MAPKK-MAPK cascade are analogous but independent. In fact, expression of dominant-negative MAPKK, MKK(A), which inhibited MAPK activation, did not inhibit ERK5 activation. Similarly, the ERK5 activation, but not the activation of MAPK, JNK, or p38, was inhibited by expression of dominant-neg
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