We have identified seven ERK-related proteins (“ERPs”), including ERK2, that are stably associated in vivo with AP-1 dimers composed of diverse Jun and Fos family proteins. These complexes have kinase activity. We designate them as “class I ERPs.” We originally hypothesized that these ERPs associate with DNA along with AP-1 proteins. We devised a DNA affinity chromatography-based analytical assay for DNA binding, the “nucleotide affinitypreincubation specificity testrecognition” (NAPSTER) assay. In this assay, class I ERPs do not associate with AP-1 DNA. However, several new “class II” ERPs do associate with DNA. p41 and p44 are ERK1/2-related ERPs that lack kinase activity and associate along with AP-1 proteins with AP-1 DNA. Class I ERPs and their associated kinase activity thus appear to bind AP-1 dimers when they are not bound to DNA and then disengage and are replaced by class II ERPs to form higher order complexes when AP-1 dimers bind DNA. p97 is a class III ERP, related to ERK3, that associates with AP-1 DNA without AP-1 proteins. With the exception of ERK2, none of the 10 ERPs appear to be known mitogen-activated protein kinase superfamily members. We have identified seven ERK-related proteins (“ERPs”), including ERK2, that are stably associated in vivo with AP-1 dimers composed of diverse Jun and Fos family proteins. These complexes have kinase activity. We designate them as “class I ERPs.” We originally hypothesized that these ERPs associate with DNA along with AP-1 proteins. We devised a DNA affinity chromatography-based analytical assay for DNA binding, the “nucleotide affinitypreincubation specificity testrecognition” (NAPSTER) assay. In this assay, class I ERPs do not associate with AP-1 DNA. However, several new “class II” ERPs do associate with DNA. p41 and p44 are ERK1/2-related ERPs that lack kinase activity and associate along with AP-1 proteins with AP-1 DNA. Class I ERPs and their associated kinase activity thus appear to bind AP-1 dimers when they are not bound to DNA and then disengage and are replaced by class II ERPs to form higher order complexes when AP-1 dimers bind DNA. p97 is a class III ERP, related to ERK3, that associates with AP-1 DNA without AP-1 proteins. With the exception of ERK2, none of the 10 ERPs appear to be known mitogen-activated protein kinase superfamily members. activator protein-1 mitogen-activated protein mitogen-activated protein kinase extracellular signal-regulated kinase ERK-related protein nucleotide affinity preincubation specificity test recognition assay nuclear extract oligonucleotide wild type mutant Fos-related antigen polyacrylamide gel electrophoresis antibody 12-O-tetradecanoylphorbol-13-acetate fetal bovine serum gibbon ape leukemia virus glutathioneS-transferase Jun N-terminal kinase stress-activated protein kinase long terminal repeat myelin basic protein 4-morpholinepropanesulfonic acid C-terminal domain Regulatory transcription factors are of central importance in mediating cellular responses to environmental stimuli by coordinately regulating genes encoding proteins and enzymes that implement the responses. Activator protein-1 (AP-1)1 is a transcription factor that binds to and regulates genes containing TGAg/cTCA consensus cis-regulatory elements (referred to here as “AP-1 DNA-binding sites” or “AP-1 DNA”) (1Lee W. Mitchell P. Tjian R. Cell. 1987; 49: 741-752Abstract Full Text PDF PubMed Scopus (1359) Google Scholar, 2Angel P. Imagawa M. Chiu R. Stein B. Imbra R.J. Rahmsdorf H.J. Jonat C. Herrlich P. Karin M. Cell. 1987; 49: 729-739Abstract Full Text PDF PubMed Scopus (2146) Google Scholar) that generally lie within gene promoter regions. Regulation by AP-1 has been demonstrated in diverse cellular processes, including growth, differentiation, tissue remodeling, and apoptosis (Ref. 3Wisdom R. Exp. Cell Res. 1999; 253: 180-185Crossref PubMed Scopus (337) Google Scholar and references therein). Altered expression and DNA binding of AP-1 subunits, and inappropriate transactivation of AP-1-dependent effector genes, are events implicated in the pathogenesis of numerous cancers and in cardiovascular, neurological, and other disease states (4Denhardt D.T. Crit. Rev. Oncog. 1996; 7: 261-291Crossref PubMed Scopus (50) Google Scholar, 5Chakraborti S. Chakraborti T. Cell. Signal. 1998; 10: 675-683Crossref PubMed Scopus (104) Google Scholar, 6Pennypacker K.R. J. Fla. Med. Assoc. 1995; 82: 551-554PubMed Google Scholar). AP-1 activation is a pivotal event in mediating cancer susceptibility and neoplastic transformation in response to endogenous and extracellular stimuli, including hormones, growth factors, tumor promoters, and many other carcinogenic agents (Refs. 7Bernstein L.R. Colburn N.H. Science. 1989; 244: 566-569Crossref PubMed Scopus (205) Google Scholar, 8Dong Z. Birrer M.J. Watts R.G. Matrisian L.M. Colburn N.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 609-613Crossref PubMed Scopus (367) Google Scholar, 9Young M.R. Li J.J. Rincon M. Flavell R.A. Sathyanarayana B.K. Hunziker R. Colburn N.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9827-9832Crossref PubMed Scopus (367) Google Scholar and see Ref. 3Wisdom R. Exp. Cell Res. 1999; 253: 180-185Crossref PubMed Scopus (337) Google Scholar for review). Many genes associated with neoplastic transformation harbor AP-1 sites, including genes encoding matrix-degrading enzymes, differentiation factors, and mitogenic agents. AP-1 is a dimer composed of proto-oncogene products encoded by thejun and fos families. The jun family consists of c-jun, junB, and junD, and the fos family consists of c-fos,fra-1, fra-2, and fosB. Dimers can be Jun family-Jun family homodimers or Jun family-Fos family heterodimers, generating 18 possible complexes via leucine zipper dimerization motifs within the Jun and Fos subunits. AP-1 dimers bind DNA via a basic region immediately adjoining the leucine zipper, forming a composite region in the proteins referred to as the “bZIP” motif (see Ref. 10Hurst H.C. Protein Profile, Transcription Factors 1: bZIP Proteins. 2. Academic Press, San Diego, CA1995: 105-176Google Scholarfor review). Gene expression is regulated by transactivation domains within the AP-1 subunits, which modulate the efficiency of RNA polymerase II transcriptional initiation. The transactivation and DNA binding activities of AP-1 are modulated by protein kinase cascades that terminate in phosphorylation of Jun and Fos family proteins. Members of the mitogen-activated protein kinase superfamily (“MAP kinases”) are thought to be responsible for phosphorylation of AP-1 proteins in vivo (see Ref. 11Karin M. J. Biol. Chem. 1996; 270: 16483-16486Abstract Full Text Full Text PDF Scopus (2239) Google Scholar for review). Ligand-activated receptors indirectly stimulate kinase activity of MAP kinase kinase kinases (MAPKKKs) that activate MAP kinase kinases (MAPKKs), which in turn activate MAPKs to phosphorylate their targets, often transcription factors such as Jun and Fos (see Refs. 12L'Allemain G. Prog. Growth Factor Res. 1994; 5: 291-334Abstract Full Text PDF PubMed Scopus (70) Google Scholar and 13English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar for review). The MAP kinase superfamily is composed of several subfamilies. Each subfamily consists of a discrete signaling module with distinct MAPKKK and MAPKK components. The most well characterized members of the ERK subfamily are ERK-1 and ERK-2 and, to a lesser degree, p63/ERK-3 and human p97/ERK3 (14Boulton T.G. Yancopoulos G.D. Gregory J.S. Slaughter C. Moomaw C. Hsu J. Cobb M.H. 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Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar). A system of several parallel MAPK signal transduction pathways has thus emerged. MAPKs regulate a number of transcription factors, including Elk/TCF, NFAT, ATF, and AP-1. ERKs and JNK/SAPKs mediate activity of AP-1 in response to mitogenic and stress factors, respectively. ERKs phosphorylate c-Jun, JunD, Fra-1, Fra-2, and FosB, and JNKs phosphorylate c-Jun and JunD (22Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Dent T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2941) Google Scholar, 23Kyriakis 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 (2403) Google Scholar, 24Gruda M.C. Kovary K. Metz R. Bravo R. Oncogene. 1994; 9: 2537-2547PubMed Google Scholar, 25Kallunki T. Deng T. Hibi M. Karin M. 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The diverse array of regulatory molecules that interact with Jun-Fos complexes underscores the exquisite circuitry dedicated to controlling AP-1 in its pivotal position as a regulator of essential cellular functions. Previously we identified several ERK-related proteins (ERPs), including the ERK2 MAP kinase, that bind in vivo to AP-1 complexes containing c-Jun and c-Fos (39Bernstein L.R. Ferris D.K. Colburn N.H. Sobel M.E. J. Biol. Chem. 1994; 269: 9401-9404Abstract Full Text PDF PubMed Google Scholar). In the present study we have found that five Jun and Fos family members form complexes with seven distinct ERK2-related ERPs, and these ERP·AP-1 complexes have associated kinase activity. To account for the multiplicity and stability of these complexes, we hypothesized that in addition to a simple enzyme-substrate relation, ERP·AP-1 complexes could interact with genes containing transcription regulatory AP-1 DNA recognition sequences. If this is the case, we should be able detect multicomponent ERP·AP-1·DNA complexes. To test this hypothesis, we devised a simple DNA affinity chromatography-based analytical assay, the “NAPSTER” assay, for specific association with DNA sequences. By these means we identified p41 and p44, two additional ERPs related to ERK2 that associate with the AP-1 DNA along with AP-1 proteins. In contrast, the first seven ERPs bind AP-1 proteins only when they are not bound to the DNA. We also identified p97, a 10th ERP related to ERK3, that associates with AP-1 DNA without AP-1 proteins. We therefore describe three distinct classes of ERPs as follows: seven class I ERPs that bind AP-1 dimers without DNA, two class II ERPs that associate along with AP-1 with DNA, and one class III ERP that associates with AP-1 DNA without AP-1 dimers. Antibodies and corresponding peptide antigen competitor peptides used for immunoblotting and immunodepletions included αc-Jun, αJunB, αJunD, αc-Fos, αFra-1, αFra-2, αFosB, αp38α, αJNK, αERK1, αERK5/BMK1, αERK6, and αERK3 D23 (which also recognizes human p97/ERK3) against amino acids 303–325 in rat ERK3, all of which were from Santa Cruz Biotechnology. Unless otherwise noted, antibodies used in experiments were from Santa Cruz Biotechnology. Other antibodies included anti-rat ERK3 “C” against amino acids 427–442 of rat ERK3, anti-human ERK3 “F” against amino acids 543–721 of human ERK3, and anti-rat ERK3 “I15” against amino acids 353–367 of rat ERK3. Antibodies from other sources include αJunD (“KG”) against full-length mouse JunD (40Gardner K. Moore T.C. Davis-Smyth T. Krutzsch H. Levens D. J. Biol. Chem. 1994; 269: 32963-32971Abstract Full Text PDF PubMed Google Scholar), αc-Jun-(948-4) against the C-terminal 82 amino acids of avian c-Jun (41Frame M.C. Wilkie N.M. Darling A.J. Chudleigh A. Pintzas A. Lang J.C. Gillespie D.A.F. Oncogene. 1991; 6: 205-209PubMed Google Scholar), mouse monoclonal anti-ERK1/2 antibody 1B3B9 (Upstate Biotechnologies, Inc., Lake Placid, NY), αFos-(75–155) against amino acid residues 75–155 of avian v-Fos (Upstate Biotechnologies, Inc.), and αFra-1-(1–276) against amino acids 1–276 of Fra-1 (42Kovary K. Bravo R. Mol. Cell. Biol. 1991; 11: 4466-44672Crossref PubMed Scopus (392) Google Scholar), all of which were used in immunoprecipitations and two-dimensional gels. Anti-ERK1-III is against amino acids 63–98 in subdomain III of rat ERK1 (Upstate Biotechnologies, Inc.). Reagents and supplies not described herein were purchased from vendors cited in Bernstein and Walker (43Bernstein L.R. Walker S.E. Biochim. Biophys. Acta. 1999; 1489: 263-280Crossref PubMed Scopus (21) Google Scholar). Human HT29 adenocarcinoma cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). For routine passage, HT29 cells were plated at 1:20 in T25 flasks in Dulbecco's modified Eagle's medium (Mediatech Cellgro, Herndon, VA) containing 10% heat-inactivated fetal bovine serum (FBS, Summit Biotechnology, Inc., Fort Collins, CO) and passaged every week with media changes after 4 days. JB6 Cl307b cells were grown and passaged as described (44Watts R.G. Ben-Ari E.T. Bernstein L.R. Birrer M.J. Winterstein D. Wendel E. Colburn N.H. Mol. Carcinog. 1995; 13: 27-36Crossref PubMed Scopus (33) Google Scholar). HT29 cells for nuclear extracts (“NE”) were plated in 150-mm tissue culture dishes (Nunc, Inc., Naperville, IL) at a density of 6 × 106 cells per dish in Dulbecco's modified Eagle's medium with 10% FBS and allowed to grow for 5 days with one media change 4 days after plating. Cells were routinely treated for 90 min with 10 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma) in fresh media containing 10% FBS, unless otherwise noted. Cells were then harvested, and NE was prepared as described (43Bernstein L.R. Walker S.E. Biochim. Biophys. Acta. 1999; 1489: 263-280Crossref PubMed Scopus (21) Google Scholar). Biotinylated AP-1 oligonucleotide (“oligo”) derived from nucleotides from the gibbon ape leukemia virus-long terminal repeat (GALV-LTR), contained a wild type AP-1 sequence (5′-agccagagaaatagatgagtcaacagc-3′). In this paper we refer to this sequence as the AP-1 DNA-binding site or AP-1 DNA. This sequence and the inverse complementary oligo were custom-synthesized by Macromolecular Resources (Fort Collins, CO). Binding of annealed, double-stranded biotinylated oligo to streptavidin beads (Roche Molecular Biochemicals, Indianapolis, IN) was performed according to the manufacturer's instructions. AP-1 DNA affinity chromatography was performed as described by Lee et al. (1Lee W. Mitchell P. Tjian R. Cell. 1987; 49: 741-752Abstract Full Text PDF PubMed Scopus (1359) Google Scholar) with major modifications designed to achieve rapid analytical scale isolation of labile and multicomponent protein-DNA complexes. For small scale experiments, as little as 30 µg of input NE protein and 6 µg of DNA on beads were used (1:5 ratio of DNA:input protein). For large scale experiments, 3–5 mg of NE protein and 150–250 µg of DNA on beads were used (1:20 ratio of DNA:input protein). Whole nuclear extracts (NE) were dialyzed into Buffer Z (0.1 m KCl, 25 mm HEPES, pH 7.8, 12.5 mm MgCl2, 1 mm dithiothreitol, 20% glycerol, v/v, 0.1% v/v Nonidet P-40, 0.1 µm ZnCl2, 5 mm NaF, 1 mm sodium orthovanadate, 0.1 mmphenylmethylsulfonyl fluoride). AP-1 and associated proteins were then isolated by single-step batchwise AP-1 DNA affinity chromatography to promote maximal stability and detection of associated proteins. Dialyzed NE was incubated for 75 min at 4 °C on a rotating clip wheel in the presence of 6 µg/ml poly(dI/dC) with AP-1 affinity beads. Beads were centrifuged for 1 min in an Eppendorf centrifuge at 4 °C, and the supernatant was removed. For small scale assays, beads were washed three times with Buffer Z; bound material was eluted by boiling the beads in SDS sample buffer, and the boiled material was directly loaded in SDS-PAGE. For larger scale assays, beads were washed five times in Buffer Z and then AP-1 and associated proteins eluted in Buffer Z containing 1 m KCl by twirling on a rotating clip wheel for 30 min at 4 °C. To assess specificity of binding to the AP-1 DNA sequence, we developed an assay that we termed thenucleotide affinity preincubationspecificity test recognition assay (the NAPSTER assay). The NAPSTER assay consists of a matched set of three samples. For sample I, whole NE is directly chromatographed batchwise with AP-1 DNA beads. For sample II, NE is preincubated for 15 min at 4 °C with a 2.5-fold molar excess of wild type AP-1 DNA oligo (relative to moles of DNA on the beads) before batchwise DNA affinity chromatography. For sample III, NE is preincubated for 15 min at 4 °C with a 2.5-fold molar excess of mutant oligo (5′-agccagagaaatagaggagtctacagc-3′; mutant AP-1 core sequence GGAGTCT, mutations underlined), before chromatography. After chromatography, beads are washed and directly loaded in SDS-PAGE or subjected to elution procedures as described above. SDS-PAGE and immunoblotting were performed as described (39Bernstein L.R. Ferris D.K. Colburn N.H. Sobel M.E. J. Biol. Chem. 1994; 269: 9401-9404Abstract Full Text PDF PubMed Google Scholar, 43Bernstein L.R. Walker S.E. Biochim. Biophys. Acta. 1999; 1489: 263-280Crossref PubMed Scopus (21) Google Scholar). Peptide competition assays for immunoblotting and immunoprecipitations were performed using a 25-fold molar excess of peptide as described (39Bernstein L.R. Ferris D.K. Colburn N.H. Sobel M.E. J. Biol. Chem. 1994; 269: 9401-9404Abstract Full Text PDF PubMed Google Scholar, 43Bernstein L.R. Walker S.E. Biochim. Biophys. Acta. 1999; 1489: 263-280Crossref PubMed Scopus (21) Google Scholar). A full-length recombinant human p97/ERK3 cDNA sequence (“rhERK3”) was cloned into the pET3C vector (Stratagene, La Jolla, CA). Thirty cycles of polymerase chain reaction were performed to amplify a linearBamHI-EcoRI rhERK3 DNA fragment containing the full-length ERK3 gene from plasmid pGEX2T-ERK3, using forward primer 5′-agggtttccatatggcagagaaatttg-3′ and reverse primer 5′-tttgtgtgcatatgacatgccagttaa-3′. This method enabled in-frame insertion of the gene into the NdeI site of the vector. Recombinant hERK3 was expressed in BL21 Codon Plus bacteria (Stratagene, Inc., La Jolla, CA) according to the manufacturer's instructions. Glutathione S-transferase-JunD (GST-JunD; see Ref. 45Powers C. Krutzsch H. Gardner K. J. Biol. Chem. 1996; 271: 30089-30095Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), a mouse JunD fusion construct, was expressed in DH5α bacteria. Expressed JunD was purified from bacterial extracts by GST affinity chromatography with glutathione-agarose beads (Sigma) and eluted with 15 mm glutathione (Sigma). pGCTD, a GST fusion construct containing the mouse gene encoding the C-terminal domain of RNA polymerase II, (“CTD”; see Ref. 46Peterson S.R. Dvir A. Anderson C.W. Dynan W.S. Genes Dev. 1992; 6: 426-438Crossref PubMed Scopus (117) Google Scholar) was expressed, and GST-CTD fusion protein was purified on the GST affinity column. V8 protease digest analyses were performed on p97 protein isolated by batchwise DNA affinity chromatography from 20 mg of nuclear extract protein. Proteins isolated on affinity beads were eluted in 1m KCl and precipitated in 10% w/v trichloroacetic acid at 4 °C. Precipitated protein was run in denaturing and reducing SDS-PAGE, and the p97 band was excised from the gel. Recombinant human ERK3 was also run in SDS-PAGE and similarly excised for V8 analyses. Excised p97 and recombinant human ERK3 bands were subjected to V8 protease digestion with 0.04 units of endoproteinase Glu-C (V8 protease; Roche Molecular Biochemicals) per lane, according to procedures described previously (39Bernstein L.R. Ferris D.K. Colburn N.H. Sobel M.E. J. Biol. Chem. 1994; 269: 9401-9404Abstract Full Text PDF PubMed Google Scholar, 47Cleveland D.W. Fischer S.G. Kirschner M.W. Laemmli U.K. J. Biol. Chem. 1977; 252: 1102-1106Abstract Full Text PDF PubMed Google Scholar). Samples either underwent two rounds (“double depletion”) or three rounds (“triple depletion”) of immunodepletion. For double depletions, ∼3.2 mg of NE per sample was dialyzed against Buffer Z and incubated for 90 min with an antibody mix consisting of 20 µg of αc-Jun, 20 µg of αJunD (Santa Cruz Biotechnology), and 10 µl of αJunD (KG), all of which had been dialyzed against Buffer Z for 45 min at 4 °C. Samples were then incubated with 25 µl of protein A-Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C with constant mixing. The procedure was repeated a second time to obtain double depleted samples. For triple depletions, ∼5.1 mg of dialyzed NE was preincubated for 90 min at 4 °C with an antibody mix consisting of 20 µg of αc-Jun, 20 µg of αJunB, 20 µg of αJunD, 10 µl of αJunD (KG), 20 µg of αc-Fos, 20 µg of αFosB, 20 µg of αFra-1, and 20 µg of αFra-2, followed by incubation with protein A-Sepharose for 1 h at 4 °C. This procedure was repeated twice to obtain triple depleted samples. Control samples did not contain antibody mix but underwent depletion with protein A-Sepharose alone. Samples were then preadsorbed to reduce nonspecific binding by batchwise DNA affinity chromatography with beads containing streptavidin-linked mutant AP-1 oligo. Immunodepleted samples were subjected to the NAPSTER assay. For two-dimensional gels and immunodepletion assays, immunoprecipitations were performed as described (see above and Ref. 39Bernstein L.R. Ferris D.K. Colburn N.H. Sobel M.E. J. Biol. Chem. 1994; 269: 9401-9404Abstract Full Text PDF PubMed Google Scholar). For kinase assays and Western blots, Dynal protein A magnetic beads (Dynal, Oslo, Norway) were preadsorbed for 30 min at room temperature with 5 µg of antibody in 0.1m Phosphate Buffer, pH 8.2. Samples were incubated for 1 h with 400–500 µg of dialyzed NE at 4 °C with constant twirling and then washed 3 times with Dulbecco's phosphate-buffered saline (Life Technologies, Inc.), according to the manufacturer's instructions. Protein kinase assays were performed on immunoprecipitated and DNA affinity-purified proteins using a MAP kinase assay kit with the classic MAP kinase substrate myelin basic protein (MBP; Upstate Biotechnology, Inc.) with minor modifications of the manufacturer's protocol. Kinase assays of immunoprecipitated proteins bound to Dynal beads were performed directly after washing steps, without elution. For kinase assays of DNA-binding proteins, nuclear extracts were dialyzed, subjected to batchwise AP-1 DNA affinity chromatography with mutant AP-1 beads to eliminate nonspecifically associated material, and then subjected to batchwise DNA affinity chromatography with beads harboring a wild type AP-1 DNA sequence. Prior to assay, proteins bound to AP-1 DNA affinity beads were eluted with a 2.5-fold molar excess of wild type AP-1 oligo to maximize the specificity and the signal to noise ratio of the assay. Elution was performed in 30 µl of Kinase Assay Buffer (20 mm MOPS pH 7.2, 25 mm β-glycerophosphate (Sigma), 5 mm EGTA, 1 mm sodium orthovanadate, 1 mm dithiothreitol) containing 40 µg wt oligo at 4 °C for 30 min. Kinase assays were performed in a 50-µl reaction consisting of 10 µl of substrate (20 µg of MBP or 1 µg of recombinant c-Jun (Upstate Biotechnologies, Inc.), 1 µg of recombinant c-Fos, 10 µg of histone H1 (Roche Molecular Biochemicals), 10 µg of histone H3 (Roche Molecular Biochemicals), GST-CTD, or GST-JunD; 10 µl of [γ-32P]ATP mixture (1 µl of 10 mCi, 6000 Ci/mmol; Amersham Pharmacia Biotech) diluted with proteins eluted in 9 µl of 75 µm MgCl2 and 500 µm ATP in Assay Buffer), and 30 µl of Kinase Assay Buffer for 15 min at 30 °C. In vivo metabolic labeling of JB6 Cl30 7b cells with Translabel (ICN, Inc., Costa Mesa, CA) was performed as described (39Bernstein L.R. Ferris D.K. Colburn N.H. Sobel M.E. J. Biol. Chem. 1994; 269: 9401-9404Abstract Full Text PDF PubMed Google Scholar). In vivo metabolic labeling of HT29 cells with Tran35S-label was performed as described (39Bernstein L.R. Ferris D.K. Colburn N.H. Sobel M.E. J. Biol. Chem. 1994; 269: 9401-9404Abstract Full Text PDF PubMed Google Scholar), except that labeling was done overnight in cells plated at 6 × 106 cells/150-mm tissue culture dish. Two-dimensional non-equilibrium pH gradient gel electrophoresis (NEPHGE two-dimensional gels) was performed as described (39Bernstein L.R. Ferris D.K. Colburn N.H. Sobel M.E. J. Biol. Chem. 1994; 269: 9401-9404Abstract Full Text PDF PubMed Google Scholar). pH 3.5 to 10 and pH 5.0 to 7.0 ampholines were from Pierce. To determine whether ERK-related proteins (ERPs) interact in vivo with AP-1 components, we performed immunoprecipitations of 35S-metabolically labeled mouse epidermal JB6 Cl30 7b and human HT29 colon adenocarcinoma whole cell extracts with MAP kinase and AP-1 antibodies, followed by comparative two-dimensional gel electrophoresis (two-dimensional gels). As shown in Fig. 1, the PAN-ERK antibody αERK1-III immunoprecipitates ERK2, since a major spot immunoprecipitated by αERK1-III has identical mobility to ERK2 immunoprecipitated by specific anti-ERK2 (compare protein spots in Fig.1, panels 1 and 2; see Ref. 39Bernstein L.R. Ferris D.K. Colburn N.H. Sobel M.E. J. Biol. Chem. 1994; 269: 9401-9404Abstract Full Text PDF PubMed Google Scholar). Immunoprecipitation of whole cell extracts with αERK1-III followed by Western blots with specific antibodies against
Regarding a recent article by Embree-Ku and Gruppuso, which appeared in the August 2005 issue of HEPATOLOGY,1 I am concerned the data presented in the paper do not justify the conclusion that the molecule nucleolin binds to the NFκB DNA binding motif. The authors determined in Fig. 2 that nucleolin binds to this motif. They did this by excising an NFκB oligo gel shift band from a native PAGE gel shift gel and identifying the protein by MALDI analyses. They sought to confirm their conclusion in Fig. 3 by showing that immunodepletion of nucleolin decreases the intensity of the gel shift signal. These data do not justify the conclusions because nuclear extract proteins often run in native PAGE gels with mobilities that are unaffected or minimally affected by the added DNA, even when bound to the DNA. Even if the authors were to have run their gel shift gel with nuclear extract and added NO DNA, nucleolin is likely to have had the same mobility. It is likely that the identification of nucleolin is a function of its mobility in the gel, not of its binding to DNA. The authors could have attempted a Western on the band they excised from the EMSA gel to confirm the identity of nucleolin, although this would not have proven that nucleolin binds to the DNA, for the reasons cited in the previous paragraph. But it would have directly shown that nucleolin was in the band analyzed by MALDI, a necessary exercise since MALDI is a matching method with many false positives, especially for very crude samples such as nuclear extracts. In Fig. 3, the authors did attempt to confirm the identity of nucleolin by comparing the intensities of the gel shift bands for anti nucleolin depleted samples to samples mock-depleted with non-immune IgG. However, nucleolin is such an abundant protein that it is hard to deplete; successful depletion should have been verified by immunoblotting of the supernatant with anti nucleolin to show that the amount of nucleolin was significantly reduced. Another reason this experiment is not conclusive is that the same result would have been obtained if nucleolin sequesters NFκB protein by complexing with it. In this scenario the reduced signal intensity in the gel shift would be attributable to reduced presence of NFκB, which of course binds to the NFκB motif. Nucleolin binds to a number of proteins including several other transcription factors and DNA binding proteins, so this is quite plausible.2-4 Taken together, the data presented do not justify the conclusion that nucleolin binds to the NFκB DNA motif. Nucleolin may bind to this motif, but additional data are needed before this conclusion is warranted. Lori R. Bernstein Ph.D.*, * Department of Pathology and Laboratory Medicine, Texas A & M University System Health Science Center, College Station, TX.
The mouse epidermal JB6 cell system consists of clonal genetic variants that are sensitive (P+) or resistant (P-) to the promotion of neoplastic transformation by phorbol esters and other tumor-promoting agents. P+ cells display AP-1-dependent phorbol-ester-inducible transactivation of gene expression, whereas P- cells have a defect in transactivation. Transfection of promotion sensitivity gene pro-1 into P- cells reconstituted both P+ phenotype and AP-1-dependent phorbol-ester-inducible transactivation. P- and P+ cells exhibited induction of c-jun and c-fos messenger RNA levels by phorbol ester, but P- cells had significantly lower basal and induced levels of jun mRNA than P+ cells. Basal and induced levels of c-jun protein were significantly lower in P- cells as well. Differences in levels the 80-kDa pI 4.5 protein p80 were also observed in JB6 cells as a function of preneoplastic progression; high levels of p80 protein and mRNA were observed in P- cells, intermediate levels in P+ cells, and negligible levels were observed in transformed derivatives of JB6 cells. Phorbol ester treatment induced phosphorylation but not synthesis of p80. These data are consistent with the hypotheses that AP-1 is required in the signal transduction pathway for promotion of neoplastic transformation by tumor promoter, that pro genes may control AP-1 activity, that threshold levels of Jun mRNA and protein may play a role in transactivation and promotion sensitivity, and that the p80 protein in JB6 cells may behave in vivo as a suppressor of cellular transformation.
We have long held this paper in high esteem for its breadth of arguments supporting the hypothesis that elevated FSH is a root cause of advanced maternal age oocyte aneuploidy (referred to here as the "Gonadotropin-Aneuploidy
The mouse epidermal JB6 cell system consists of clonal genetic variants that are sensitive (P+) or resistant (P-) to the promotion of neoplastic transformation by phorbol esters and other tumor-promoting agents. P+ cells display AP-1-dependent phorbol-ester-inducible transactivation of gene expression, whereas P- cells have a defect in transactivation. Transfection of promotion sensitivity gene pro-1 into P- cells reconstituted both P+ phenotype and AP-1-dependent phorbol-ester-inducible transactivation. P- and P+ cells exhibited induction of c-jun and c-fos messenger RNA levels by phorbol ester, but P- cells had significantly lower basal and induced levels of jun mRNA than P+ cells. Basal and induced levels of c-jun protein were significantly lower in P- cells as well. Differences in levels the 80-kDa pI 4.5 protein p80 were also observed in JB6 cells as a function of preneoplastic progression; high levels of p80 protein and mRNA were observed in P- cells, intermediate levels in P+ cells, and negligible levels were observed in transformed derivatives of JB6 cells. Phorbol ester treatment induced phosphorylation but not synthesis of p80. These data are consistent with the hypotheses that AP-1 is required in the signal transduction pathway for promotion of neoplastic transformation by tumor promoter, that pro genes may control AP-1 activity, that threshold levels of Jun mRNA and protein may play a role in transactivation and promotion sensitivity, and that the p80 protein in JB6 cells may behave in vivo as a suppressor of cellular transformation.
Abstract Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called “egg infertility.” A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women.
