Title: Molecular Cloning of Mouse ERK5/BMK1 Splice Variants and Characterization of ERK5 Functional Domains
Abstract: The mitogen-activated protein kinases (MAPKs) play important roles in regulation of cell growth and survival. Human MAPK 5 (ERK5) or Big MAP kinase 1 (BMK1) is a recently cloned member of the MAPK family. To identify ERK5-related kinases, we searched the GenBank™ expressed sequence tag (EST) data base for mouse cDNAs with homology to human ERK5. A full-length mouse cDNA that was highly homologous to the human ERK5 was identified. Further analysis of ERK5 polymerase chain reaction products generated from mouse embryo cDNA yielded three mouse ERK5 cDNAs (mERK5a, mERK5b, and mERK5c). Sequence analysis showed that these cDNAs are alternative splice products of the mouse ERK5 gene. Interestingly, expressed mERK5b and mERK5c act as dominant negative inhibitors based on inhibition of mERK5a kinase activity and mERK5a-mediated MEF2C transactivation. However, the physiological significance of mERK5b and mERK5c is not fully understood. Further investigation using these mouse ERK5 splice variants and other constructed mutants identified functional roles of several regions of mERK5, which appear to be important for protein-protein interaction and intracellular localization. Specifically, we found that the long C-terminal tail, which contains a putative nuclear localization signal, is not required for activation and kinase activity but is responsible for the activation of nuclear transcription factor MEF2C due to nuclear targeting. In addition, the N-terminal domain spanning amino acids (aa) 1–77 is important for cytoplasmic targeting; the domain from aa 78 to 139 is required for association with the upstream kinase MEK5; and the domain from aa 140–406 is necessary for oligomerization. Taken together, these observations indicate that ERK5 is regulated by distinct mechanisms determined by its unique structure and presumably the presence of multiple splice variants. The mitogen-activated protein kinases (MAPKs) play important roles in regulation of cell growth and survival. Human MAPK 5 (ERK5) or Big MAP kinase 1 (BMK1) is a recently cloned member of the MAPK family. To identify ERK5-related kinases, we searched the GenBank™ expressed sequence tag (EST) data base for mouse cDNAs with homology to human ERK5. A full-length mouse cDNA that was highly homologous to the human ERK5 was identified. Further analysis of ERK5 polymerase chain reaction products generated from mouse embryo cDNA yielded three mouse ERK5 cDNAs (mERK5a, mERK5b, and mERK5c). Sequence analysis showed that these cDNAs are alternative splice products of the mouse ERK5 gene. Interestingly, expressed mERK5b and mERK5c act as dominant negative inhibitors based on inhibition of mERK5a kinase activity and mERK5a-mediated MEF2C transactivation. However, the physiological significance of mERK5b and mERK5c is not fully understood. Further investigation using these mouse ERK5 splice variants and other constructed mutants identified functional roles of several regions of mERK5, which appear to be important for protein-protein interaction and intracellular localization. Specifically, we found that the long C-terminal tail, which contains a putative nuclear localization signal, is not required for activation and kinase activity but is responsible for the activation of nuclear transcription factor MEF2C due to nuclear targeting. In addition, the N-terminal domain spanning amino acids (aa) 1–77 is important for cytoplasmic targeting; the domain from aa 78 to 139 is required for association with the upstream kinase MEK5; and the domain from aa 140–406 is necessary for oligomerization. Taken together, these observations indicate that ERK5 is regulated by distinct mechanisms determined by its unique structure and presumably the presence of multiple splice variants. mitogen-activated protein kinase MAPK kinase MAPK kinase kinase extracellular signal-regulated kinase 1 and 2 MAPK/ERK kinase nuclear localization signal amino acid(s) c-Jun N-terminal kinase stress-activated protein kinase Big MAP kinase 1 (also known as ERK5) epidermal growth factor expressed sequence tag polymerase chain reaction reverse transcription green fluorescence protein Chinese hamster ovary Dulbecco's modified Eagles' medium phosphate-buffered saline polyacrylamide gel electrophoresis base pair(s) myelin basic protein dominant negative focal adhesion-associated protein tyrosine kinase Mitogen-activated protein kinases (MAPKs)1 are serine/threonine protein kinases that play important roles in signal transduction pathways activated by extracellular stimuli. MAPKs regulate many cellular processes, including cell proliferation, cell differentiation, cell death, and stress responses (1Kozak M. Cell. 1986; 44: 283-292Abstract Full Text PDF PubMed Scopus (3598) Google Scholar, 2Plath K. Engel K. Schwedersky G. Gaestel M. Biochem. Biophys. Res. Commun. 1994; 203: 1188-1194Crossref PubMed Scopus (18) Google Scholar). MAPKs constitute a superfamily of highly related serine/threonine kinases. At least seven family members of the MAPK family have been identified in mammals: ERK1/2 (extracellular signal-regulated kinase 1 and 2) (3Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4245) Google Scholar, 4Cobb M.H. Hepler J.E. Cheng M. Robbins D. Semin. Cancer Biol. 1994; 5: 261-268PubMed Google Scholar), JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) (5Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar, 6Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar, 7Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (506) Google Scholar), p38 (a mammalian equivalent of the yeast high-osmolarity glycerol kinase) (6Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar), Big MAP kinase 1 (BMK1, also known as ERK5) (8Lee J.D. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1995; 213: 715-724Crossref PubMed Scopus (289) Google Scholar, 9Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar), ERK6 (mitogen-activated protein kinase 6) (10Lechner C. Zahalka M.A. Giot J.F. Moller N.P. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (278) Google Scholar), and ERK7 (extracellular signal-regulated kinase 7) (11Abe M.K. Kuo W.L. Hershenson M.B. Rosner M.R. Mol. Cell. Biol. 1999; 19: 1301-1312Crossref PubMed Scopus (115) Google Scholar). MAPKs are activated by phosphorylation on threonine and tyrosine residues in a Thr-X-Tyr (TXY) motif involving upstream dual-specificity protein kinases (MAPK kinases) and phosphatases. The TXY activation motif is used to classify the MAPK superfamily into three main groups. The TEY (Thr-Glu-Tyr) group consists of extracellular signal-regulated kinases ERK1/2, ERK5, and ERK7. The TPY (Thr-Pro-Tyr) family consists of JNK/SAPK (5Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar). The TGY (Thr-Gly-Tyr) family includes p38 and ERK6. Each MAPK pathway generally consists of three kinase modules composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK) (12Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (867) Google Scholar). These kinase modules are differentially activated by a variety of cellular stimuli and contribute to distinct cellular function (13Takahashi M. Berk B.C. J. Clin. Invest. 1996; 98: 2623-2631Crossref PubMed Scopus (189) Google Scholar). The ERK1/2 module includes Raf isoforms, MEK1/2 and ERK1/2, which are highly responsive to mitogenic signals such as growth factors and cytokines. In contrast, JNK/SAPK and p38 are stress-sensitive pathways activated by MEK 4/7 and MEK3/6, respectively. ERK5 is specifically activated by MEK5 (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). Human ERK5 was recently cloned by two groups (8Lee J.D. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1995; 213: 715-724Crossref PubMed Scopus (289) Google Scholar, 9Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). Human ERK5 contains 816 amino acid residues with a primary structure distinct from other MAPK members. ERK5 has a unique long C-tail and a distinct loop-12 domain. ERK5 is activated by reactive oxygen species (15Abe 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), hyperosmolarity (16Yan C. Takahashi M. Okuda M. Lee J.D. Berk B.C. J. Biol. Chem. 1999; 274: 143-150Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), and fluid shear stress (16Yan C. Takahashi M. Okuda M. Lee J.D. Berk B.C. J. Biol. Chem. 1999; 274: 143-150Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Most recently, it has been shown that ERK5 is required for EGF-induced cell proliferation and progression through the cell cycle (17Kato 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). Although ERK5 has a TEY motif in its dual phosphorylation site similar to ERK1/2 and ERK7, several studies have shown that ERK5 has different upstream activators and downstream substrates compared with other MAPKs. In the present study, we report the identification of three alternatively spliced mouse ERK5 cDNAs, termed mERK5a, mERK5b, and mERK5c. The putative protein sequences deduced from these three cDNAs are identical except in their N-terminal regions. mERK5b and mERK5c lack N-terminal 69 amino acids and 139 amino acids, respectively, compared with mERK5a. It is likely that the variations in N-terminal sequences are produced by alternatively choosing different splicing donors and acceptors within a single intron. More interestingly, mERK5b and mERK5c, which are catalytically inactive, act as dominant negative kinases. Finally, by using N- or C-terminal truncated mERK5, we found that three N-terminal domains spanning aa 1–77, aa 78–139, and aa 140–406 are important for cytoplasmic targeting, association with the upstream kinase MEK5, and oligomerization, respectively. The C-terminal tail is essential for the biological activity of ERK5 in vivo by mediating nucleus translocation, which is dependent upon the NLS in the C-terminal region. These observations should help us understand the biological reasons for this diversity in ERK5 and may provide new approaches to modify ERK5 signaling. The GenBank™ expressed sequence tag (EST) data base was searched using the program BLAST with amino acid sequences corresponding to the human ERK5. Several EST fragments displayed high degrees of amino acid homology. Among them, a full-length mouse clone was obtained from Genome Systems Inc (GenBank™ accession number AA288345). A manual sequencing method with the Sequetherm Cycle sequencing kit (Epicentre Technologies Corp.) was used to sequence this clone according to the manufacturer's protocols. Several PCR fragments were found in the first-strand cDNAs of a mouse embryo (purchased from CLONTECH) and were subcloned into the TA vector (Invitrogen) for sequencing. Combining analysis of mouse ERK5 RT-PCR clones with mouse EST clones, three mouse ERK5 cDNAs were identified. To determine the mechanism for generating three mouse ERK5 isoforms, a mouse genomic DNA fragment encompassing the mouse ERK5 isoforms splicing junction was isolated by PCR amplification using oligonucleotides 5′-ACGAGTACGAGATCATCGAGACC-3′ and 5′-GGTCACCACATCAAAAGCATTAGG-3′. Clones for mouse ERK5a (nucleotides 10–2776), ERK5b (nucleotides 280–2826), and ERK5c (nucleotides 837–3196) were subcloned into the multiple cloning regions of the pcDNA3.1/His vectors (Invitrogen). All isoforms were fused in-frame with an N-terminal Xpress tag. mERK5a(-tail) (aa 1–406) was constructed by removing aa 407–806 with PstI digestion. GFP-tail and GFP-NLS were generated by ligation of the PCR-amplified C-tail (aa 407–806) and NLS fragment (aa 505–539), respectively, containing the artificial restriction sites BamHI andEcoRI to the pEGFP-C3 (CLONTECH) cut with BamHI and EcoRI. mERK5a(-NLS), lacking aa 505–539, was generated by PCR with the Expand High-Fidelity PCR system (Roche Molecular Biochemicals). Briefly, an antisense primer matching the DNA sequence upstream of the codon aa 505 and a sense primer downstream of the codon aa 539 were generated. PCR reaction was performed using the entire mERK5a in the PcDNA3.1/His vector as the template, and the PCR product was treated with T4 DNA polymerase to create a blunt end. Purified PCR product was further treated with polynucleotide kinase and T4 ligase and subsequently transformed into the competent bacterial DH5α. CHO-K1 were maintained in DMEM/F-12 medium (Life Technologies, Inc) supplemented with 10% calf serum, 50 units/ml penicillin and 50 μg/ml streptomycin. Cells were transiently transfected at 50–80% confluence using LipofectAMINE reagent (Life Technologies, Inc.) and harvested 48 h after transfection. Cells were washed with phosphate-buffered saline (PBS) and 0.2 ml of TME lysis buffer (10 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm EDTA, 25 mm NaF) containing fresh 100 μm Na3VO4, 20 μg/ml leupeptin, 1 μg/ml pepstatin A, 4 μg/ml aprotinin, and 1 mmdithiothreitol (18Liao D.F. Monia B. Dean N. Berk B.C. J. Biol. Chem. 1997; 272: 6146-6150Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Cell lysates were prepared by scraping, sonication, and centrifugation. Mouse embryos (15 day) were homogenized in 2.0 ml of lysis buffer (50 mm sodium pyrophosphate, 50 mm NaF, 50 mm NaCl, 5 mm EDTA, 5 mm EGTA, 100 μm Na3VO4, 10 mmHEPES, pH 7.4, 0.1% Triton X-100, 500 μmphenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin). Cell fractionation was performed by extraction with different buffer and sequential centrifugation. First, cells were lysed in the TME lysis buffer contain 0.1% Triton X-100, and the cell lysate was centrifuged at 10,000 × g for 1 h. The supernatant was collected as the cytoplasmic fraction. The pellet was then resuspended in the lysis buffer containing 1% Triton X-100 and centrifuged at 10,000 × g for 1 h. The supernatant was collected as the nuclear fraction. The pellet was further resuspended in the RIPA buffer (20 mm Tris-HCl, pH 7.5, 2.5 mm EDTA, 1% Triton X-100, 10% glycerol, 1% deoxycholic acid, 0.1% SDS, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and centrifuged at 10,000 × g for 1 h. The supernatant was collected as the cytoskeleton fraction, and the pellet was resuspended in sample buffer as the nuclear fraction. Cell lysates, cellular fractionates, or tissue extracts were boiled in the presence of 1× sample buffer (50 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 4% β-mercaptoethanol, 0.02% bromphenol blue) and subjected to SDS-PAGE, and proteins were then transferred to nitrocellulose. The membrane was blocked for 1–2 h at room temperature with a commercial blocking buffer (Life Technologies, Inc.). The blot was incubated for 1 h at room temperature with the primary antibody (anti-Xpress antibody from Invitrogen), followed by incubation for 1 h with secondary antibody (horseradish peroxidase-conjugated). Immunoreactive bands were visualized by chemiluminescence (ECL, Amersham Pharmacia Biotech). Immune complex kinase assays were performed with ectopically expressed tagged ERK5 proteins from CHO-K1 cells as previously described (19Levesque M.J. Nerem R.M. J. Biomech. Eng. 1985; 107: 341-347Crossref PubMed Scopus (611) Google Scholar), except 2 μg of MBP was used per reaction in kinase buffer. Proteins were separated by 15% SDS-PAGE, transferred to a nitrocellulose, and subjected to autoradiography. The presence of epitope-tagged proteins in immunoprecipitates was verified by Western analysis with antibody against the tag. Total RNA was prepared from multiple adult mouse tissues and mouse embryos (15 days) using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Two degenerate oligonucleotide primers were designed corresponding to splicing junction sequences of three cDNAs: the sense primer 5′-ACGAGTACGAGATCATCGAGACC-3′ and antisense primer 5′-GGTCACCACATCAAAAGCATTAGG-3′. The first-strand cDNA was synthesized by Superscript II reverse-transcriptase (Life Technologies, Inc.) with antisense primer. The amplification was carried out in a 100-μl mixture containing 2 μl of the first-strand cDNA product, 10 μm each of the sense and antisense primer, and 5 units of Taq DNA polymerase (Life Technologies, Inc.). The PCR reaction was performed as follows: initial denaturation at 94 °C for 3 min and 30 cycles of amplification (denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min, extension at 72 °C for 2 min), followed by a final extension step of 10 min at 72 °C. Reactions were electrophoresed on a 1.5% agarose gel. The MEF2C fusion activator vector, encoding the GAL4-binding domain fused to MEF2C activation domain (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), was cotransfected into CHO-K1 cells along with the GAL4-responsive reporter plasmid pG5E1bLUC, which contains five GAL4 sites cloned upstream of a minimal promoter driving a luciferase gene (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). For transfection, CHO-K1 cells (0.2 × 106 cells per well) were seeded into 24-well plates the day before transfection. Cells were transfected with 0.5 μg of DNA in total per well using LipofectAMINE Plus (Life Technologies). After 5 h, the transfection was stopped by adding equal volume of DMEM/F-12 (10% fetal bovine serum). After 24 h, the medium was changed to a serum-free DMEM/F-12 for an additional 24 h. Then cells were collected for luciferase assay. In the cases of testing the endogenous ERK5, 10% serum was added and the cells were incubated for an additional 4 h before harvesting. A green fluorescence protein (GFP) expression vector (pEGFP-N1, from CLONTECH) was used to control for transfection efficiency. The total amount of DNA for each well was kept constant using the empty vector pcDNA3.1/His (Invitrogen). Luciferase assays were performed with a Luciferase Reporter Gene Assay kit (Roche Molecular Biochemicals) as instructed. Briefly, cells were washed twice with PBS and lysed in 200 μl of lysis buffer at room temperature for 15 min with shaking. 50 μl of cell extracts was transferred into a 96-well microtiter plate. The fluorescence intensity of GFP was measured using a Wallace multicounter (Wallace). 50 μl of luciferase substrate were then added to the cell lysates, and the luciferase activities were determined by measuring luminescence intensity using the same Wallace multicounter. To correct for transfection efficiency, the luciferase activity was divided by the green fluorescence intensity. CHO-K1 cells, grown on LabTek II chamber slides, were cotransfected with Xpress-tagged ERK constructs in the presence of either pCDNA3 vector or MEK5(D). Following the transfection for 48 h, the cells were then fixed in 10% formalin in PBS for 15 min, washed, blocked, and incubated with anti-Xpress antibody at a 1:2000 dilution. After a 1-h incubation at room temperature, the cells were washed and then incubated with fluorescein-conjugated anti-mouse antibody (Vector) at a 1:200 dilution. The stained cells were analyzed under an Olympus Fluoview confocal microscope. To identify mouse ERK5 cDNAs, the EST data base was searched, and several EST clones with high homology to human ERK5 were found. Among these, a full-length EST cDNA clone (AA288345) was purchased and sequenced. Comparing this sequence with the published human ERK5 sequence (8Lee J.D. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1995; 213: 715-724Crossref PubMed Scopus (289) Google Scholar, 9Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar), we found that this cDNA clone contained an insert as well as an in-frame stop codon within the insert. This suggested that multiple ERK5 cDNAs might exist. To confirm the presence of diverse species of ERK5 cDNAs in the mouse, mouse embryo first-strand cDNA (fromCLONTECH) was used for PCR with primers from conserved regions of human and mouse ERK5. Sequence analysis showed that at least three different ERK5 cDNAs were present, which we will designate as mERK5a, mERK5b, and mERK5c (Fig.1 A). mERK5a is the mouse cDNA most homologous to human ERK5. mERK5b and mERK5c cDNAs contain one or two inserts compared with mERK5a, respectively. Although mERK5b has a 50-bp insert (I-1) (Fig. 1 A), mERK5c, which corresponds to the mouse EST clone mentioned above, contains both the 50-bp (I-1) insert found in mERK5b and a 91-bp (I-2) insert (Fig. 1 A). Both inserts start with consensus splice donor "gt," and the second insert (I-2) ends with an acceptor sequence "ag," suggesting that they are likely to be intron sequences that were not spliced out. To explore the mechanism for producing these three different mouse ERK5 cDNAs, PCR and sequencing of the mouse genomic DNA flanking the splicing region of the ERK5 gene was performed. The genomic structure of the splicing junction of mouse ERK5 gene is shown in Fig. 1 B. Sequence analysis of genomic DNA showed an additional 429-bp (I-3) insert with consensus "gt/ag" just after I-2. It is likely that the variations in N-terminal sequences are produced by alternatively choosing different splicing donors and acceptors within a single intron that is composed of I-1, I-2, and I-3. mERK5a is likely generated by use of the splicing donor D1 and the acceptor A2, whereas mERK5b is generated by use of D2 and A2 and mERK5c is generated by use of D3 and A2. The I-1 intron introduces a stop codon and causes mERK5b and mERK5c to have shorter N termini than mERK5a. mERK5a contains a putative open reading frame (ORF) from nucleotide 27 to nucleotide 2447 that encodes a protein of 806 amino acids with a predicted molecular mass of 88 kDa, whereas mERK5b (putative ORF: nucleotides 284–2497) contains 737 amino acid with a predicted molecular mass of 80 kDa and mERK5c (putative ORF: nucleotide 864–2867) consists of 667 amino acid residues with a predicted molecular mass of 73 kDa. The predicted N-terminal amino acid sequences for the three isoforms of ERK5 are shown in Fig.2 A. It is important to note that mERK5b and mERK5c lack the GXGXXG domain for ATP binding, which is present in mERK5a (underlined in Fig.2 A). Further characterization of the mouse ERK5 protein sequence with currently available profile data bases resulted in the identification of a proline-rich region and a bipartite nuclear localization signal (NLS) in the C-terminal domain (Fig. 2 B). The proline-rich region and the NLS are located at aa 578–690 and aa 505–539, respectively. Comparison of the deduced amino acid sequence of mouse ERK5a with human ERK5 showed 91% homology to human ERK5 (Fig.2 B). Many functional domains important for kinase activity, including the TEY phosphorylation site, are conserved between human and mouse ERK5. The major differences between human and mouse ERK5 occur in a small portion of the N terminus and the proline-rich region in the C terminus. To confirm the existence of the three forms of mouse ERK5 mRNAs, we performed RT-PCR with a different source of mouse embryo mRNA. Three bands corresponding to PCR products from the three recombinant cDNAs were detected (Fig.3 A). To determine the presence of three mouse ERK5 mRNAs in adult mouse tissues and examine tissue-specific expression, RT-PCR was performed for multiple adult mouse tissues. PCR products encoding the three mouse ERK5 mRNAs were detected in all mouse adult tissues examined (Fig.3 B). To confirm the presence of the endogenous protein products corresponding to the three splice variants, we used a polyclonal antibody against ERK5, which was made using amino acids EGHGMNPADIESLQREIQMDSPML of the human ERK5 as antigen. This human ERK5 peptide is 100% similar to the corresponding amino acids of the three mouse isoforms. Preliminary data showed that it recognized mouse and human ERK5 equally well. Immunoblotting of mouse embryonic proteins revealed three distinct bands (Fig. 3 C) with the molecular weights corresponding to mERK5a, mERK5b, and mERK5c, respectively. Relative protein levels of three splice variants are consistent with their relative mRNA levels, mERK5a > mERK5c > mERK5b. Because mERK5b and mERK5c lack the GXGXXG domain required for ATP binding, it is very likely that mERK5b and mERK5c have no kinase activity. To further explore the function of mERK5b and mERK5c, phosphorylation of myelin basic protein (MBP) was evaluated by anin vitro kinase assay. Immunoprecipitated mERK5a, isolated from the cells coexpressing constitutively active MEK5(D), rapidly phosphorylated MBP (Fig. 4). In contrast, mERK5b and mERK5c failed to phosphorylate MBP, which is similar to results using human dominant negative ERK5 (DN-hERK5) (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). To investigate the possibility that mERK5b and mERK5c act as dominant negative isoforms, mERK5b or mERK5c were cotransfected with mERK5a and kinase assays were performed. As shown in the last two lanesof Fig. 4, mERK5b inhibited mERK5a kinase activity. mERK5c behaved in a similar manner (data not shown). Because the transactivation of the transcription factor MEF2C is stimulated by human ERK5-induced phosphorylation (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), we measured MEF2C activity as a means to determine the kinase activities of different ERK5 isoforms. Utilizing fusion proteins containing the transactivation domain of MEF2C fused to the DNA binding domain of the yeast transcription factor GAL4, we were able to assess the effects of the mouse ERK5 isoforms on the activity of transcription factor MEF2C fusion protein. This was done by measuring the luciferase activity from CHO-K1 cells cotransfected with a construct containing five copies of the GAL4-binding site upstream of a luciferase reporter gene. As expected, MEF2C-dependent reporter gene expression was enhanced dramatically when mERK5a and MEK5(D) were cotransfected into CHO-K1 cells (Fig. 5 A). mERK5b and mERK5c, similar to DN-hERK5, did not stimulate MEF2C activity (Fig.5 A). The same results were observed after extracellular stimulation with 10% serum (Fig. 5 B). To further demonstrate that mERK5b and mERK5c behave as dominant negative forms, we coexpressed mERK5b, mERK5c, or DN-hERK5 with mERK5a. Introduction of mERK5b (Fig. 5 C) and mERK5c (Fig. 5 D), similar to DN-hERK5 (Fig. 5 E), inhibited mERK5a-induced MEF2C-dependent reporter gene expression in a dose-dependent manner. mERK5b and mERK5c also dose-dependently inhibited MEF2C activation by endogenous ERK5 after stimulation with 10% serum (Fig. 5 F). These results suggest that mERK5b and mERK5c may function as dominant negative inhibitors of the ERK5 signaling pathway. To determine the role of the N-terminal region, which is absent in mERK5b and mERK5c, we examined the subcellular distribution of different ERK5 isoforms by cell fractionation and Western blotting of lysates from cells expressing epitope-tagged mERK5s. In the unstimulated cells, we observed that mouse mERK5a and human hERK5 were present in both the cytoplasm and the nucleus with the majority in the cytoplasm (Fig.6). However, mERK5b and mERK5c were exclusively present in the nucleus. There was no detectable mERK5a, mERK5b, and mERK5c expression in the membrane and cytoskeleton fractions (data not shown). These results suggest that the N-terminal domain spanning aa 1–77 is important for cytoplasmic targeting of mERK5a. Because the protein sequence analysis of ERK5 uncovered several interesting domains, we further investigated the roles of these regions. ERK5 has a unique 400-amino acid long C-terminal tail whose function is not known. To determine whether the C-terminal domain of ERK5 plays a role in regulating ERK5 kinase activity, an Xpress-tagged ERK5 truncated at Gln-406, termed mERK5a(-tail), was generated. When mERK5a(-tail) and ERK2 amino acid sequences are aligned, the length of mERK5a(-tail) is comparable to that of ERK2. To test whether mERK5a(-tail) was catalytically active, an in vitro kinase assay using MBP as a substrate was performed. Using MEK5(D) to activate ERK5, mERK5a(-tail) was able to phosphorylate MBP in vitrosimilar to mERK5a (Fig. 7 A,upper panel). These results suggest that the kinase activity of mERK5a(-tail) is comparable to mERK5a. Another indication of kinase activity is autophosphorylation. The anti-Xpress antibody recognized both the full-length mERK5A and the truncated mERK5a(-tail) in cell lysates from transfected CHO-K1 cells, suggesting that mERK5a(-tail) protein was stably expressed (Fig.7 A, lower panel). Upon activation by coexpression of MEK5(D), mERK5a(-tail) exhibited an electrophoretically shifted band similar to the full-length mERK5a (Fig. 7 A, lower panel), suggesting that mERK5a(-tail) was phosphorylated by MEK5. The shifted band of the full-length ERK5 is consistent with the phosphorylation of ERK5 and is general