identification of erk2-binding domain of ebitein1, a novel erk2-binding protein
TRANSCRIPT
Biochimica et Biophysica Acta 1784 (2008) 1319–1325
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Biochimica et Biophysica Acta
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Identification of ERK2-binding domain of EBITEIN1, a novel ERK2-binding protein
Kenji Miura ⁎, Junko ImakiDepartment of Developmental Anatomy and Regenerative Biology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan
⁎ Corresponding author. Tel.: +81 4 2995 1475; fax: +E-mail address: [email protected] (K. Miura).
1 Ebitein1 indicates the gene, mRNA or cDNA and EBI
1570-9639/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.bbapap.2008.05.015
a b s t r a c t
a r t i c l e i n f oArticle history:
We recently cloned a cDNA Received 7 February 2008Received in revised form 24 April 2008Accepted 27 May 2008Available online 11 June 2008Keywords:EBITEIN1Protein–protein interactionBinding proteinTwo-hybrid assayBinding domainEB domainECT domainTestisBioinformatics
encoding a novel extracellular signal-regulated kinase 2 (ERK2) binding protein,EBITEIN1, by yeast two-hybrid screening. In this study, we further characterized EBITEIN1. Bindingexperiments using various deletion mutants identified a 40-amino acid minimal sequence for binding ERK2.Binding experiments using substitution mutants indicated the crucial role of arginine residues in thissequence. Based on empirical and bioinformatic analyses, we propose two domains in EBITEIN1. One is theminimal sequence for binding ERK2 (EB domain) and the other is the EBITEIN1 C-terminal domain (ECTdomain). These results might pave the way for further empirical and bioinformatic analyses of EBITEIN1- andERK2-mediated events.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
We recently cloned and identified EBITEIN11 as a novel extra-cellular signal-regulated kinase 2 (ERK2) binding protein (DDBJ/EMBL/GenBank database under accession number AB359922) [1]. Immuno-histochemical results show that EBITEIN1 localizes in round sperma-tids, indicating that EBITEIN1 is first translated after meiosis, reachinga maximum at the stage of round spermatids and decreases toundetectable levels when the flagellum of spermatozoon is generated.Based on these results, we propose that EBITEIN1 is an interactor ofERK2, which is involved in the morphogenesis or differentiation ofround spermatids to spermatozoa [1].
Specific protein–protein interactions is one of the key regulatorymechanisms for the specific and accurate responses of cells [2–7].Therefore, identification of protein–protein interactions might lead tothe exact and detailed understanding of the function of interactingproteins. ERK2 has been previously shown to bind several proteins,including RSK1 [8], RSK2 [9], RSK3 [9], MEK [10], MNK1 [11], MNK2[11], GAB1 [12], mitogen-activated protein kinase phosphatase 1 [13],mitogen-activated protein kinase phosphatase 3 [14], IEX-1 [15], Tob[16], Nef-associated factor 1α [17], vinexin [18], phosphoproteinenriched in astrocytes [19], death-associated protein kinase [20],glycogen synthase kinase 3B [21], dual specificity protein phospha-
81 4 2996 5185.
TEIN1 indicates the protein.
l rights reserved.
tase 5 [22], vimentin [23], microphthalmia-associated transcriptionfactor [24], and ArhGAP9 [25]. ERK2 is a serine/threonine proteinkinase that is activated by phosphorylation in response to receptortyrosine kinases, G-protein coupled receptors, and integrins-mediated stimuli. ERK2 reportedly regulates cell proliferation, cellcycle, differentiation, and survival in many cell types [26–29]. Thespatio-temporal distribution of ERK1/2 is reported to be regulated byprotein–protein interactions [5,30–32]. ERK2-binding proteins maybe important for regulating fidelity and preventing crosstalk inintracellular signaling pathways [33,34].
Amino acid sequences for molecular recognition might be one oftheminimal functional domains of proteins. In this study, we analyzedthe minimal binding sequence of EBITEIN1 with ERK2 and identifiedthis sequence. Further, we discussed the sequence of EBITEIN1,especially the ERK2-binding domain, through comparisons withother EBITEIN sequences from other species.
2. Materials and methods
2.1. Yeast two-hybrid assay for binding between two proteins
Yeast two-hybrid assays for binding between EBITEIN1 and ERK2were carried out using aMATCHMAKER Two-Hybrid System (ClontechLaboratories, Inc., Mountain View, CA) as described previously [1].Yeast strain AH109 (Clontech) was transformed with the pGBT9-ERK2or pGBKT7-ERK2 plasmid in which the entire coding region (codons 1to 359) of mouse ERK2 was fused to the C-terminus of the GAL4-DNA-
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binding domain. The yeasts were grown in synthetic dropout selectionmedium lacking tryptophan. Plasmid constructs of pACT2 fused totruncated-Ebitein1 were produced either by (i) in-frame insertion ofeach coding region of Ebitein1 into the pACT2 plasmid to fuse the C-terminus of the GAL4-DNA-activation domain sequence or (ii) cuttingthe pACT2-Ebitein1 constructs with appropriate restriction enzyme(s), treatment with T4 DNA polymerase (New England Biolabs,Ipswich, MA) or Klenow DNA polymerase (New England Biolabs)when necessary, and self-ligation. The pACT2-Ebitein1 constructswere transformed into yeast strain AH109 containing pGBT9-ERK2(codons 1–359) plasmid or pGBKT7-ERK2 (codons 1–359). Transfor-mants were plated on synthetic dropout selection medium lackingleucine, tryptophan, histidine, and adenine.
2.2. Antibodies
Anti-phospho-ERK1/2 antibody was purchased from InvitrogenCorporation (Carlsbad, CA). Anti-ERK1/2 antibody was prepared asdescribed previously [35]. This antibody recognizes both the nonpho-sphorylated andphosphorylated formsof ERK1/2. Anti-FLAGM2affinitygel was purchased from Sigma-Aldrich Corporation (St. Louis, MO).
2.3. Cell culture and preparation of cell extracts
COS-7 and TIG-1 cells were cultured in Dulbecco's modified Eagle'smedium (Invitrogen) supplemented with 25 mM HEPES (pH 7.2) and10% fetal bovine serum (Asahi Technoglass Corporation, Funabashi,Japan) at 37 °C in a humidified atmosphere containing 5% CO2. COS-7cells are fibroblast cells of African green monkey kidney transformedwith SV40 large T antigen [36] and TIG-1 cells are fibroblast cells fromnormal human fetal lung [37]. In the stimulation experiments,semiconfluent cells were cultured for 36 h in Dulbecco's modifiedEagle's medium supplemented with 25 mM HEPES (pH 7.4), 1 mg/mlbovine serum albumin, and 30 nM sodium selenite and thenstimulated with 20% fetal bovine serum. The cells were washed withice-cold phosphate-buffered saline and solubilized with lysis buffer(20 mM Tris–HCI [pH 7.4], 250 mM NaCl, 0.1 mM phenylmethylsulfo-nyl fluoride, 0.1 mM diisopropylfluorophosphate, 1 μg/ml leupeptin,10 μg/ml pepstatin A, 5 mM Na4P2O7, 10 mM NaF, 25 mM 2-glycerophosphate, 2 mM NaVO4, 1% Triton X-100, and 0.25% deoxy-cholic acid sodium salt). The extracts were sonicated briefly on ice andcentrifuged at 15,000 ×g for 30 min at 4 °C. The resulting supernatantswere used as cell lysates.
2.4. Binding assay using FLAG-tagged proteins
FLAG-Ebitein1 constructs were produced by in-frame insertion ofeach encoding region of Ebitein1 into pFLAG-CMV-2 plasmid vector(Sigma-Aldrich) to fuse it to the C-terminus of the FLAG. The FLAG-Ebitein1 constructs were transformed into Escherichia coli strainDH5α. Purified recombinant plasmids were transfected into COS-7cells using Fugene 6 (Roche Diagnostics, Basel, Switzerland) accordingto the manufacturer's instructions. COS-7 cell lysates were centri-fuged, and the supernatants were mixed with anti-FLAG M2-agarosefor 2 h at 4 °C with rotation. The immunoprecipitates were washedwith lysis buffer, eluted with FLAG peptide (Sigma-Aldrich), andanalyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis (PAGE), followed by immunoblotting.
2.5. Binding assay using GST fusion proteins
Glutathione S-transferase (GST)-Ebitein1 constructs were pro-duced by in-frame insertion of Ebitein1 (codons 59–111) intopGEX-2T plasmid vector (GE Healthcare, Little Chalfont, UK) togenerate N-terminal GST fusion proteins. GST-Ebitein1 (codons 59–111) constructs were transformed into E. coli strain BL21 Codon
plus (Stratagene, La Jolla, CA) and cultured in LB mediumsupplemented with ampicillin with shaking at 200 rpm at 18 °C,followed by 2 h in the presence of 0.25 mM isopropyl-β-D-thiogalactopyranoside to induce the expression of the fusionproteins. The bacteria were collected by centrifugation andresuspended in E. coli lysis buffer (20 mM Tris–HCl [pH 7.4],0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM diisopropylfluor-ophosphate, and 1% Triton X-100). Lysates were sonicated vigor-ously on ice and centrifuged for 1 h at 100,000 ×g for 1 h at 4 °C.The resulting supernatants were purified using glutathione-Sepharose 4B (GE Healthcare).
TIG-1 cell lysates were mixed with glutathione-Sepharose 4B for2 h at 4 °C with rotation to remove endogenous GST and thencentrifuged at 15,000 ×g for 10 min at 4 °C. Next, GST-EBITEIN1(residues 59–111) fusion proteins and glutathione-Sepharose 4Bwere added to the supernatants and incubated for 2 h at 4 °C withrotary mixing. The glutathione-Sepharose 4B was collected bycentrifugation and washed with lysis buffer, and proteins wereeluted with SDS sample buffer, and analyzed by SDS-PAGE andimmunoblotting.
2.6. Site-specific mutagenesis and preparation of ERK2 construct withouta tag
Constructs containing site-directed mutations were prepared asfollows. Primers for site-directed mutation were phosphorylatedat their 5′-end by T4 polynucleotide kinase (Toyobo, Osaka,Japan). Polymerase chain reactions (PCRs) were performed usingthese 5′-phosphorylated primers, PfuTurbo DNA polymerase (Stra-tagene), and plasmid including the wild type sequence as atemplate. PCR products were purified, self-ligated, and transformedinto E. coli strain DH5α. Tag-free ERK2 constructs were prepared inthe same way to eliminate the GST sequence from pGEX-2T-ERK2. Aset of oligonucleotide primers corresponding to the region justoutside the GST sequence in pGEX-2T-ERK2 (codons 1 to 359) wassynthesized and phosphorylated on their 5′-ends by T4 polynucleo-tide kinase (Toyobo). PCR was performed using these primers, Pfu-Turbo DNA polymerase (Stratagene), and pGEX-2T-ERK2 plasmid asa template. The PCR products were purified, self-ligated, andtransformed into E. coli strain DH5α.
2.7. DNA sequence determination
The sequences of each expression construct used in this studywereconfirmed using an ABI PRISM® 310 Genetic Analyzer (AppliedBiosystems, Foster City, CA).
2.8. Western blotting
Proteins were separated by SDS-PAGE and electrophoreticallytransferred to polyvinylidene fluoride (PVDF) membranes. Themembranes were incubated with blocking solution containing skimmilk and bovine serum albumin for 1 h. The membranes were thenimmunoblotted with each antibody followed by a secondary antibodylabeled with alkaline phosphatase. Visualization was performed bychemiluminescence using CDP-Star (Tropix, Bedford, MA) or bycolorimetric detection using Western Blue Stabilized Substrate forAlkaline Phosphatase (Promega, Madison, WI).
2.9. Data Bank accession number
The accession numbers of the sequences used in this study are asfollows:mouseEBITEIN1 (testicular), AB359922 (DDBJ/EMBL/GenBank);rat EBITEIN1, BC098647 (DDBJ/EMBL/GenBank); rhesus monkey EBI-TEIN1, mcc:701303 (Kyoto Encyclopedia of Genes and Genomes(http://www.genome.ad.jp/)); chimpanzee EBITEIN1, ptr:451032
Fig. 1. Identification of the minimal ERK2-binding sequence of EBITEIN1. (A) Various regions of EBITEIN1 were tested for the ability to bind ERK2. pACT2 constructs including thesequences encoding various regions of EBITEIN1 were transformed into the yeast containing pGBT9-ERK2 (codons 1–359) plasmids. The yeasts were plated on synthetic dropoutselection media lacking leucine, tryptophan, histidine, and adenine. Interaction between the proteins was indicated by colony formation. (B) The ERK2-binding region of EBITEIN1was narrowed down by consecutive two-hybrid assays. Yeast strain AH109 containing the pGBKT7-ERK2 (codons 1–359) plasmid was transformed with truncated pACT2-Ebitein1plasmids and plated on synthetic dropout selectionmedia lacking leucine, tryptophan, histidine, and adenine. Interaction between the proteins was indicated by colony formation. Asthe first assay, residues 59–111, 71–111 and 83–111 were tested. As the second assay, residues 63–111 and 67–111 were tested. As the third assay, residues 64–111, 65–111 and 66–111were tested. As fourth assay, residues 64–76, 64–85 and 64–94 were tested. As fifth assay, residues 64–98, 64–102 and 64–106 were tested. As sixth assay, residues 64–103, 64–104and 64–105 were tested. The minimum binding sequence of EBITEIN1 with ERK2 was determined with repetition of these procedures. The determined minimal ERK2-bindingsequence (codons 64–103) is displayed in the black box. At least three repetitions were completed using each construct.
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(Kyoto Encyclopedia of Genes and Genomes); human EBITEIN1,hsa:84953 (Kyoto Encyclopedia of Genes and Genomes); mouse renalEBITEIN, BC031386 (DDBJ/EMBL/GenBank).
3. Results and discussion
3.1. Identification of the minimal ERK2-binding sequence of EBITEIN1
The ERK2-binding domain of EBITEIN1 was determined using ayeast two-hybrid method. Various constructs of the truncated Ebitein1fused to GAL4-DNA-activation domain (Fig. 1A) were transformed into
yeasts containing the constructs of full-length ERK2 (codons 1–359)fused to GAL4-DNA-binding domain, and the yeasts were grown onsynthetic dropout selection medium lacking leucine, tryptophan,histidine, and adenine. Binding by the two proteins was indicated bycolony formation. In this way, we found that the ERK2-binding domainof EBITEIN1 is contained within a 62-amino acid stretch includingresidues 59 to 120 (Fig. 1A).
We synthesized oligonucleotides encoding various truncations ofresidues 59 to 120 of EBITEIN1 by PCR and inserted them in-frame intopACT2. The constructswere then transformed into yeasts containing theconstructs of full-length ERK2 (codons 1–359) fused to GAL4-DNA-
Fig. 2. Effect of site-directed mutations on coprecipitation of EBITEIN1 and ERK2. (A) Amino acid sequences of the putative ERK2-binding domain (EB domain) deduced from thenucleotide sequences of cloned cDNAs and/or genomic DNAs were aligned. The residues are numbered from the amino to the carboxyl terminus. Residues nonidentical to that ofmouse EBITEIN1 are shown as white letters on black backgrounds. (B) COS-7 cells were transfected with pFLAG-CMV-2-Ebitein1 (codons 1–680) plasmids. The cells were starvedby cultivation for 36 h in serum-free medium. The cells were stimulated by replacing the medium with one containing 20% fetal bovine serum. After the indicated times, the cellswere lysed. The lysates were then centrifuged, and the supernatants were immunoprecipitated with anti-FLAG antibody. The immunoprecipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting (IB) and visualization by chemiluminescence with CDP-Star. Molecular markers were stained with Coomassie Brilliant Blue R-250 (CBB-R250). (C) Full-length ERK2 (residues 1 to 359) protein was expressed without a tag in E. coli strain BL21. The lysates were then centrifuged, and the supernatants were precipitatedusing GST-EBITEIN1 (residues 59–111). The coprecipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting (IB) followed by colorimetric visualization usingWestern Blue Stabilized Substrate for Alkaline Phosphatase. Molecular markers were stained with CBB-R250. At least three repetitions were completed using each construct.
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binding domain, and binding between the two proteinswas assessed bycolony formation. Constructs resulting in colony formationwere furthertruncated and tested in the next round of two-hybrid assays. In thisway,we identified the minimal ERK2-binding sequence as a 40-amino acidstretch from residue 64 to 103 (Fig. 1B). Residues 65 to 103 of EBITEIN1do not bind with ERK2, although residues 64 to 103 of EBITEIN1 bindwith ERK2. Residue 64 of mouse EBITEIN1, a threonine residue, isconserved in rat, rhesus monkey, chimpanzee and human (Fig. 2A).Therefore, residue 64 might be very important for binding of EBITEIN1with ERK2.
3.2. Analysis of the ERK2-binding domain of EBITEIN1 by site-directedmutagenesis
Amino acid sequence alignment of the putative ERK2-bindingdomain of EBITEIN1 is shown in Fig. 2A. Consensus sequence derivedfrom this alignment is also shown. Because arginine residues are
Fig. 3. Alignment of deduced amino acid sequences of EBITEIN1. (A) Alignment of deducedhuman. EBITEIN1 sequences fromvarious species were aligned using the ClustalW algorithmand in red boxes. The amino acid residues are numbered from the amino to the carboxyl tdomain of EBITEIN1 (ECT domain) are displayed on pale yellow and green backgrounds, respeis shown in black boxes. (B) Comparison of the deduced amino acid sequences of testicular EEBITEIN1 are indicatedwith black letters. Amino acid residues not conserved are indicatedwiare numbered from the amino to the carboxyl terminus. The ERK2-binding domain (EB domgreen backgrounds, respectively. Putative leucine zipper (corresponding to residues 571–59
thought to play an important role in the binding with ERK2 [8,33], wecarried out site-directed mutagenesis experiments to examine theirrole in EBITEIN1. The FLAG-Ebitein1 (RRAR, residues 69–72) constructand the substituted construct (AAAA, residues 69–72) were trans-fected into COS-7 cells, and their interaction with ERK2 was analyzedby coimmunoprecipitation experiments. Replacement of the RRARsequence in the ERK2-binding domain of EBITEIN1 eliminatedcoprecipitation of ERK2 and FLAG- EBITEIN1 (Fig. 2B), indicating thatarginine residues play a crucial role in ERK2 binding by EBITEIN1.
We further examined the interaction of these fusion proteins incells stimulated with fetal bovine serum. The position of the ERK2bands coprecipitating with EBITEIN1 was shifted upward by serumstimulation (Fig. 2B), indicating that both nonphosphorylated andphosphorylated forms of ERK2 bind with EBITEIN1.
In addition, we performed coprecipitation experiments using tag-free ERK2 expressed in E. coli and GST-EBITEIN1. Changing the RRARsequence (residues 69–72) to AAAR, ARAR, or RAAR eliminated the
amino acid sequences of EBITEIN1 from mouse, rat, rhesus monkey, chimpanzee, and[39]. Amino acid residues identical to that of mouse EBITEIN1 are shownwith black dotserminus. The ERK2-binding domain (EB domain) and the highly conserved C-terminalctively. Putative leucine zipper (corresponding to residues 571–599 of mouse EBITEIN1)BITEIN (i.e., EBITEIN1) and renal EBITEIN. Amino acid residues identical to that of mouseth red letters (testicular EBITEIN) or blue letters (renal EBITEIN). The amino acid residuesain) and EBITEIN1 C-terminal domain (ECT domain) are displayed on pale yellow and9 of mouse EBITEIN1) is shown in black boxes.
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Table 1Identity of EBITEIN1 from mouse and other species
Species Query sequence (mouse EBITEIN1)
Full lengtha EB domainb ECT domainc
Rat 86.8% 87.5% 96.9%Rhesus monkey 58.7% 55.0% 87.0%Chimpanzee 59.1% 60.0% 86.4%Human 58.4% 60.0% 86.4%
Percent identities were determined using the algorithm by Lipman and Pearson [40].a Residues 1–680 of mouse EBITEIN1.b ERK2-binding domain, residues 64–103 of mouse EBITEIN1.c EBITEIN1 C-terminal domain, residues 519–680 of mouse EBITEIN1.
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coprecipitation of ERK2 with EBITEIN1 (Fig. 2C). These results furthersupport the idea that arginine residues play a critical role in ERK2binding by EBITEIN1.
3.3. Bioinformatic examination of amino acid sequence of EBITEIN1
A cDNA from rat that may encode a protein homologous to mouseEBITEIN1 was cloned from testis and sequenced by the NIH MGCProject Team [38], but analysis has not yet been completed. Althoughthe cloning and sequencing of homologues of Ebitein1 has not beenreported in the DDBJ/EMBL/GenBank databases, genome sequenceand other data for possible homologues have been reported. EBITEIN1sequences from various species were aligned using the ClustalWalgorithm [39] (Fig. 3A).
On the basis of our observations and comparisons of proteinshomologous to mouse EBITEIN1, we suspect that EBITEIN1 iscomposed of at least two domains (Fig. 3A). The first is 40 aminoacid long, consists of residues corresponding to 64 to 103 of mouseEBITEIN1, and mediates ERK2 binding. We therefore named this theERK2-binding domain (EB domain). When compared with mouseEBITEIN1 using the algorithm by Lipman and Pearson [40], the aminoacid sequences of the putative EB domains of rhesus monkey,chimpanzee, and human forms are 55.0%, 60.0%, and 60.0% identical,respectively (Table 1). These values are almost the same as those forthe full-length proteins (Table 1). The EB domain of rat EBITEIN1, incontrast, has a higher identity (87.5%). On the basis of thesecomparisons, the consensus sequence of the putative EB domainwas determined (Fig. 2A). The sequence included 21 identical aminoacids, which corresponds to 52.5% identity with mouse EBITEIN1. Thisconsensus sequence has a conserved tandem pair of arginine residues(RR motif), which is likely essential for binding ERK2 (Fig. 2B and C).
The second domain of EBITEIN1 is located at its carboxyl terminusand consists of a highly conserved amino acid sequence (Fig. 3A andTable 1). This domain, which we named the EBITEIN1 carboxyl-terminus domain (ECT domain), is composed of 162 amino acids(corresponding to residues 519–680 of mouse EBITEIN1). The identityof this domain between mouse EBITEIN1 and EBITEIN1 from the otherspecies listed in Fig. 3A and Table 1 was over 86.4%, whereas theidentity for the full-length protein was only 58.7% for rhesus monkey,59.1% for chimpanzee, and 58.4% for human. The overall identity fromrat EBITEIN1, in contrast, was 86.8%.
A search of the DDBJ/EMBL/GenBank databases indicates that theremay be splice variant(s) of EBITEIN1. The full-length protein, which iscomposed of 612 amino acids (annotated based on the nucleotidesequence of DDBJ/EMBL/GenBank accession number BC031386 thatwas cloned from kidney), shares 530 consecutive identical amino acidresidues corresponding to residues 1 to 530 of mouse EBITEIN1 (Fig.3B). This renal form of EBITEIN lacks most of the ECT domain (residues519–680 of mouse EBITEIN1). Consequently, the renal form of EBITEINlacks the function mediated by the ECT domain. Although the ECTdomain contains putative leucine zipper (corresponding to residues571–599 ofmouse EBITEIN1), the function of this domain remains to be
determined. Further empirical and in silico structural and functionalstudies are necessary to clarify the functions of EBITEINs in detail.
In conclusion, we identified the minimal sequence of EBITEIN1 forbinding ERK2 and the crucial role for arginine residues in thissequence. These results might pave the way for further empirical andbioinformatic analyses of EBITEIN1-mediated events.
Acknowledgment
We thank Mrs. Yuko Nakamura for her technical support.
References
[1] K. Miura, J. Imaki, Molecular cloning of Ebitein1: A novel extracellular signal-regulated kinase 2-binding protein in testis, Biochem. Biophys. Res. Commun. 368(2008) 336–342.
[2] J. Vazquez-Prado, P. Casas-Gonzalez, J.A. Garcia-Sainz, G protein-coupled receptorcross-talk: pivotal roles of protein phosphorylation and protein–protein interac-tions, Cell. Signal. 15 (2003) 549–557.
[3] T. Tanoue, E. Nishida, Molecular recognitions in the MAP kinase cascades, Cell.Signal. 15 (2003) 455–462.
[4] T. Pawson, P. Nash, Assembly of cell regulatory systems through proteininteraction domains, Science 300 (2003) 445–452.
[5] R.M. Biondi, A.R. Nebreda, Signalling specificity of Ser/Thr protein kinases throughdocking-site-mediated interactions, Biochem. J. 372 (2003) 1–13.
[6] D. Barsyte-Lovejoy, A. Galanis, A.D. Sharrocks, Specificity determinants in MAPKsignaling to transcription factors, J. Biol. Chem. 277 (2002) 9896–9903.
[7] D. Auerbach, S. Thaminy, M.O. Hottiger, I. Stagljar, The post-genomic era ofinteractive proteomics: facts and perspectives, Proteomics 2 (2002) 611–623.
[8] J.A. Smith, C.E. Poteet-Smith, K. Malarkey, T.W. Sturgill, Identification of anextracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, asequence critical for activation by ERK in vivo, J. Biol. Chem. 274 (1999)2893–2898.
[9] Y. Zhao, C. Bjorbaek, D.E. Moller, Regulation and interaction of pp90(rsk) isoformswith mitogen-activated protein kinases, J. Biol. Chem. 271 (1996) 29773–29779.
[10] M. Fukuda, Y. Gotoh, E. Nishida, Interaction of MAP kinase with MAP kinasekinase: its possible role in the control of nucleocytoplasmic transport of MAPkinase, EMBO J. 16 (1997) 1901–1908.
[11] A.J. Waskiewicz, A. Flynn, C.G. Proud, J.A. Cooper, Mitogen-activated proteinkinases activate the serine/threonine kinases Mnk1 and Mnk2, EMBO J. 16 (1997)1909–1920.
[12] B. Roshan, C. Kjelsberg, K. Spokes, A. Eldred, C.S. Crovello, L.G. Cantley, ActivatedERK2 interacts with and phosphorylates the docking protein GAB1, J. Biol. Chem.274 (1999) 36362–36368.
[13] D.N. Slack, O.M. Seternes, M. Gabrielsen, S.M. Keyse, Distinct binding determinantsfor ERK2/p38alpha and JNK map kinases mediate catalytic activation andsubstrate selectivity of map kinase phosphatase-1, J. Biol. Chem. 276 (2001)16491–16500.
[14] M. Muda, A. Theodosiou, C. Gillieron, A. Smith, C. Chabert, M. Camps, U. Boschert, N.Rodrigues, K. Davies, A. Ashworth, S. Arkinstall, The mitogen-activated proteinkinase phosphatase-3 N-terminal noncatalytic region is responsible for tightsubstrate binding and enzymatic specificity, J. Biol. Chem. 273 (1998) 9323–9329.
[15] J. Garcia, Y. Ye, V. Arranz, C. Letourneux, G. Pezeron, F. Porteu, IEX-1: a new ERKsubstrate involved in both ERK survival activity and ERK activation, EMBO J. 21(2002) 5151–5163.
[16] M. Maekawa, E. Nishida, T. Tanoue, Identification of the anti-proliferative proteinTob as a MAPK substrate, J. Biol. Chem. 277 (2002) 37783–37787.
[17] S. Zhang, M. Fukushi, S. Hashimoto, C. Gao, L. Huang, Y. Fukuyo, T. Nakajima,T. Amagasa, S. Enomoto, K. Koike, O.Miura, N. Yamamoto, N. Tsuchida, A newERK2binding protein, Naf1, attenuates the EGF/ERK2 nuclear signaling, Biochem.Biophys. Res. Commun. 297 (2002) 17–23.
[18] M. Mitsushima, A. Suwa, T. Amachi, K. Ueda, N. Kioka, Extracellular signal-regulated kinase activated by epidermal growth factor and cell adhesion interactswith and phosphorylates vinexin, J. Biol. Chem. 279 (2004) 34570–34577.
[19] E. Formstecher, J.W. Ramos, M. Fauquet, D.A. Calderwood, J.C. Hsieh, B. Canton, X.T.Nguyen, J.V. Barnier, J. Camonis, M.H. Ginsberg, H. Chneiweiss, PEA-15 mediatescytoplasmic sequestration of ERK MAP kinase, Dev. Cell. 1 (2001) 239–250.
[20] C.H. Chen, W.J. Wang, J.C. Kuo, H.C. Tsai, J.R. Lin, Z.F. Chang, R.H. Chen, Bidirectionalsignals transduced by DAPK-ERK interaction promote the apoptotic effect of DAPK,EMBO J. 24 (2005) 294–304.
[21] Q. Ding, W. Xia, J.C. Liu, J.Y. Yang, D.F. Lee, J. Xia, G. Bartholomeusz, Y. Li, Y. Pan, Z. Li,R.C. Bargou, J. Qin, C.C. Lai, F.J. Tsai, C.H. Tsai, M.C. Hung, Erk associates with andprimes GSK-3beta for its inactivation resulting in upregulation of beta-catenin,Mol. Cell 19 (2005) 159–170.
[22] M. Mandl, D.N. Slack, S.M. Keyse, Specific inactivation and nuclear anchoring ofextracellular signal-regulated kinase 2 by the inducible dual-specificity proteinphosphatase DUSP5, Mol. Cell. Biol. 25 (2005) 1830–1845.
[23] E. Perlson, S. Hanz, K. Ben-Yaakov, Y. Segal-Ruder, R. Seger, M. Fainzilber, Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve,Neuron 45 (2005) 715–726.
1325K. Miura, J. Imaki / Biochimica et Biophysica Acta 1784 (2008) 1319–1325
[24] D.M. Molina, S. Grewal, L. Bardwell, Characterization of an ERK-binding domaininmicrophthalmia-associated transcription factor anddifferential inhibition of ERK2-mediated substrate phosphorylation, J. Biol. Chem. 280 (2005) 42051–42060.
[25] B.K. Ang, C.Y. Lim, S.S. Koh, N. Sivakumar, S. Taib, K.B. Lim, S. Ahmed, G. Rajagopal,S.H. Ong, ArhGAP9, a novel MAP kinase docking protein, inhibits Erk and p38activation through WW domain binding, J. Mol. Signal. 2 (2007) 1–15.
[26] S.Meloche, J. Pouyssegur, The ERK1/2mitogen-activated protein kinase pathway as amaster regulator of the G1- to S-phase transition, Oncogene 26 (2007) 3227–3239.
[27] S. Torii, T. Yamamoto, Y. Tsuchiya, E. Nishida, ERK MAP kinase in G cell cycleprogression and cancer, Cancer Sci. 97 (2006) 697–702.
[28] G. Pearson, F. Robinson, T. Beers Gibson, B.E. Xu, M. Karandikar, K. Berman, M.H.Cobb, Mitogen-activated protein (MAP) kinase pathways: regulation andphysiological functions, Endocr. Rev. 22 (2001) 153–183.
[29] J.C. Chambard, R. Lefloch, J. Pouyssegur, P. Lenormand, ERK implication in cell cycleregulation, Biochim. Biophys. Acta 1773 (2007) 1299–1310.
[30] W. Kolch, Coordinating ERK/MAPK signalling through scaffolds and inhibitors, Nat.Rev. Mol. Cell. Biol. 6 (2005) 827–837.
[31] A.J. Bardwell, M. Abdollahi, L. Bardwell, Docking sites on mitogen-activatedprotein kinase (MAPK) kinases, MAPK phosphatases and the Elk-1 transcriptionfactor compete for MAPK binding and are crucial for enzymic activity, Biochem. J.370 (2003) 1077–1085.
[32] J. Pouyssegur, V. Volmat, P. Lenormand, Fidelity and spatio-temporal control inMAP kinase (ERKs) signalling, Biochem. Pharmacol. 64 (2002) 755–763.
[33] T. Tanoue,M.Adachi, T.Moriguchi, E.Nishida,Aconserveddockingmotif inMAPkinasescommon to substrates, activators and regulators, Nat. Cell. Biol. 2 (2000) 110–116.
[34] C.J. Caunt, A.R. Finch, K.R. Sedgley, C.A. McArdle, Seven-transmembrane receptorsignalling and ERK compartmentalization, Trends Endocrinol. Metab. 17 (2006)276–283.
[35] K. Miura, J. Imaki, Phosphorylated extracellular signal-regulated kinase 1/2 islocalized to the XY body of meiotic prophase spermatocytes, Biochem. Biophys.Res. Commun. 346 (2006) 1261–1266.
[36] Y. Gluzman, SV40-transformed simian cells support the replication of early SV40mutants, Cell 23 (1981) 175–182.
[37] M. Ohashi, S. Aizawa, H. Ooka, T. Ohsawa, K. Kaji, H. Kondo, T. Kobayashi, T.Noumura, M. Matsuo, Y. Mitsui, S. Murota, K. Yamamoto, H. Ito, H. Shimada, T.Utakoji, A new human diploid cell strain, TIG-1, for the research on cellular aging,Exp. Gerontol. 15 (1980) 121–133.
[38] D.S. Gerhard, L. Wagner, E.A. Feingold, C.M. Shenmen, L.H. Grouse, G. Schuler, S.L.Klein, S. Old, R. Rasooly, P. Good, M. Guyer, A.M. Peck, J.G. Derge, D. Lipman, F.S.Collins, W. Jang, S. Sherry, M. Feolo, L. Misquitta, E. Lee, K. Rotmistrovsky, S.F.Greenhut, C.F. Schaefer, K. Buetow, T.I. Bonner, D. Haussler, J. Kent, M. Kiekhaus, T.Furey,M. Brent, C. Prange, K. Schreiber, N. Shapiro, N.K. Bhat, R.F. Hopkins, F. Hsie, T.Driscoll, M.B. Soares, T.L. Casavant, T.E. Scheetz, M.J. Brown-stein, T.B. Usdin, S.Toshiyuki, P. Carninci, Y. Piao, D.B. Dudekula, M.S. Ko, K. Kawakami, Y. Suzuki, S.Sugano, C.E. Gruber, M.R. Smith, B. Simmons, T. Moore, R. Waterman, S.L. Johnson,Y. Ruan, C.L.Wei, S.Mathavan, P.H. Gunaratne, J.Wu, A.M. Garcia, S.W.Hulyk, E. Fuh,Y. Yuan, A. Sneed, C. Kowis, A. Hodgson, D.M. Muzny, J. McPherson, R.A. Gibbs, J.Fahey, E. Helton, M. Ketteman, A. Madan, S. Rodrigues, A. Sanchez, M. Whiting, A.Madari, A.C. Young, K.D. Wetherby, S.J. Granite, P.N. Kwong, C.P. Brinkley, R.L.Pearson, G.G. Bouffard, R.W. Blakesly, E.D. Green, M.C. Dickson, A.C. Rodriguez, J.Grimwood, J. Schmutz, R.M. Myers, Y.S. Butterfield, M. Griffith, O.L. Griffith, M.I.Krzywinski, N. Liao, R. Morin, D. Palmquist, A.S. Petrescu, U. Skalska, D.E. Smailus, J.M. Stott, A. Schnerch, J.E. Schein, S.J. Jones, R.A. Holt, A. Baross, M.A. Marra, S.Clifton, K.A. Makowski, S. Bosak, J. Malek, The status, quality, and expansion of theNIH full-length cDNA project: the Mammalian Gene Collection (MGC), GenomeRes. 14 (2004) 2121–2127.
[39] J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22 (1994)4673–4680.
[40] D.J. Lipman, W.R. Pearson, Rapid and sensitive protein similarity searches, Science227 (1985) 1435–1441.