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• W2047994580 abstract "Mitogen-activated protein (MAP) kinase lies at the convergence of various extracellular ligand-mediated signaling pathways. It is activated by the dual-specificity kinase, MAP kinase kinase or MEK. MAP kinase inactivation is mediated by dephosphorylation via specific MAP kinase phosphatases (MKPs). One MKP (MKP-1 (also known as 3CH134, Erp, or CL100)) has been reported to be expressed in a wide range of tissues and cells. We report the identification of a second widely expressed MKP, termed MKP-2, isolated from PC12 cells. MKP-2 showed significant homology with MKP-1 (58.8% at the amino acid level) and, like MKP-1, displayed vanadate-sensitive phosphatase activity against MAP kinase in vitro. Overexpression of MKP-2 in vivo inhibited MAP kinase-dependent gene transcription in PC12 cells. MKP-2 differed from MKP-1 in its tissue distribution and in its extent of induction by growth factors and agents that induce cellular stress, suggesting that these MKPs may have distinct physiological functions. Mitogen-activated protein (MAP) kinase lies at the convergence of various extracellular ligand-mediated signaling pathways. It is activated by the dual-specificity kinase, MAP kinase kinase or MEK. MAP kinase inactivation is mediated by dephosphorylation via specific MAP kinase phosphatases (MKPs). One MKP (MKP-1 (also known as 3CH134, Erp, or CL100)) has been reported to be expressed in a wide range of tissues and cells. We report the identification of a second widely expressed MKP, termed MKP-2, isolated from PC12 cells. MKP-2 showed significant homology with MKP-1 (58.8% at the amino acid level) and, like MKP-1, displayed vanadate-sensitive phosphatase activity against MAP kinase in vitro. Overexpression of MKP-2 in vivo inhibited MAP kinase-dependent gene transcription in PC12 cells. MKP-2 differed from MKP-1 in its tissue distribution and in its extent of induction by growth factors and agents that induce cellular stress, suggesting that these MKPs may have distinct physiological functions. INTRODUCTIONMitogen-activated protein kinases (MAP1 1The abbreviations used are: MAPmitogen-activated proteinEGFepidermal growth factorNGFnerve growth factorERKextracellular signal-regulated kinaseMKPMAP kinase phosphatasePCRpolymerase chain reactionbpbase pair(s)kbkilobase pairsCMVcytomegalovirus. kinases) mediate multiple cellular pathways regulating growth (1Pagés G. Lenormand P. L'Allenmain G. Chambard J.-C. Meloche S. Pouysségur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (923) Google Scholar) and differentiation(2Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1152) Google Scholar, 3Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar). In neuronal cells, MAP kinase activity mediates the actions of growth factors like EGF that stimulate cellular proliferation as well as factors like NGF that maintain neuronal survival and differentiation(4Chao M.V. Cell. 1992; 68: 995-997Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 5Nguyen T.T. Scimeca J.-C. Filloux C. Peraldi P. Carpentier J.-L. VanObberghen E. J. Biol. Chem. 1993; 268: 9803-9810Abstract Full Text PDF PubMed Google Scholar, 6Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (657) Google Scholar). Such ligand-activated signal transduction pathways involve activation of receptor-tyrosine kinases, which initiates a series of phosphorylation events that activate a cascade of serine/threonine kinases converging on the MAP kinase (also called extracellular signal-regulated kinase (ERK)) isoforms, ERK1 and ERK2 (7Alema S. Casalbore P. Agostini E. Tato F. Nature. 1985; 316: 557-559Crossref PubMed Scopus (197) Google Scholar, 8Thomas S.M. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (502) Google Scholar, 9Thomas S.M. Hayes M. D'Arcangelo G. Armstrong R.C. Meyer B.D. Zilberstein A. Brugge J.S. Halegoua S. Mol. Cell. Biol. 1991; 11: 4739-4750Crossref PubMed Scopus (49) Google Scholar).Activation of MAP kinase involves specific phosphorylations on threonine and tyrosine residues within the Thr-Glu-Tyr motif (10Lloyd E.D. Wooten M.W. J. Nerochem. 1992; 59: 1099-1109Crossref PubMed Scopus (22) Google Scholar) by MAP kinase kinase (MAP kinase and ERK kinase or MEK)(2Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1152) Google Scholar, 11Crews C.M. Erikson R.L. Cell. 1993; 74: 215-217Abstract Full Text PDF PubMed Scopus (296) Google Scholar). Phosphorylation of both these residues is required for MAP kinase activation(11Crews C.M. Erikson R.L. Cell. 1993; 74: 215-217Abstract Full Text PDF PubMed Scopus (296) Google Scholar, 12Zheng C.-F. Guan K.-L. J. Biol. Chem. 1993; 268: 23933-23939Abstract Full Text PDF PubMed Google Scholar). It has been suggested that the inactivation of MAP kinase is a critical event that regulates the physiological response to MAP kinase activation(13Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1022) Google Scholar). This inactivation is mediated, in part, by dephosphorylation of MAP kinases by dual specificity phosphatases called MAP kinase phosphatases (MKPs) that dephosphorylate both the threonine and tyrosine residues phosphorylated by MEK (13Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1022) Google Scholar, 14Allessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020PubMed Google Scholar, 15Ward Y. Gupta S. Jensen P. Wartmann M. Davis R.J. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar, 16Zheng C.-F. Guan K.-L. J. Biol. Chem. 1993; 268: 16116-16119Abstract Full Text PDF PubMed Google Scholar). The activation of MAP kinase appears to be tightly regulated through the coordinate action of MEK and MKPs. By regulating the extent of MAP kinase activation, these MKPs may dictate the choice of differentiation or proliferation within a developing cell(17Traverse S. Gomez N. Paterson H. Marshall C. Cohen P. Biochem. J. 1992; 288: 351-355Crossref PubMed Scopus (804) Google Scholar).The prototype dual-specificity phosphatase, VH1, was identified in vaccinia and showed similarity to cdc25, a protein that controls cell entry into mitosis(18Kumagai A. Dunphy W.G. Cell. 1992; 70: 139-151Abstract Full Text PDF PubMed Scopus (335) Google Scholar). VH1 homologues from human (PAC-1, CL100, and most recently B23), mouse (MKP-1 (3CH134 or Erp)), and yeast (Yop51, MSG5) have also been isolated(19Charles C.H. Abler A.S. Lau L.C. Oncogene. 1992; 7: 187-190PubMed Google Scholar, 20Doi K. Garner A. Ammerer G. Errede B. Shinkawa H. Sugimoto K. Matsumoto K. EMBO J. 1994; 13: 61-70Crossref PubMed Scopus (204) Google Scholar, 21Guan K. Haun R.S. Watson S.J. Geahlen R.L. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1501-1505Crossref PubMed Scopus (153) Google Scholar, 22Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (568) Google Scholar, 23Rohan P.J. Davis P. Moskaluk C.A. Kearns M. Krutzch H. Siebenlist U. Kelly K. Science. 1993; 259: 1763-1766Crossref PubMed Scopus (263) Google Scholar, 24Ishibashi T. Bottaro D.P. Michieli P. Kelley C.A. Aaronson S.A. J. Biol. Chem. 1994; 269: 29897-29902Abstract Full Text PDF PubMed Google Scholar). All are dual-specificity phosphatases that specifically dephosphorylate MAP kinase in vitro(25Sun H. Tonks N.K. Bar-Sagi D. Science. 1994; 266: 285-288Crossref PubMed Scopus (206) Google Scholar) and in vivo(13Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1022) Google Scholar, 15Ward Y. Gupta S. Jensen P. Wartmann M. Davis R.J. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar, 26Noguchi T. Metz R. Chen L. Mattéi M.-G. Carrasco D. Bravo R. Mol. Cell. Biol. 1993; 13: 5195-5205Crossref PubMed Scopus (168) Google Scholar). MKP-1 (also called 3CH134 or Erp) was discovered as an immediate early gene whose rapid transcription and subsequent translation are suggested to provide a feedback loop to terminate growth factor signals(13Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1022) Google Scholar, 19Charles C.H. Abler A.S. Lau L.C. Oncogene. 1992; 7: 187-190PubMed Google Scholar, 26Noguchi T. Metz R. Chen L. Mattéi M.-G. Carrasco D. Bravo R. Mol. Cell. Biol. 1993; 13: 5195-5205Crossref PubMed Scopus (168) Google Scholar). Overexpression of mouse MKP-1 was shown to dramatically inhibit fibroblast proliferation, suggesting that the inactivation of MAP kinase in vivo by MKP-1 has a profound negative effect on cellular proliferation(25Sun H. Tonks N.K. Bar-Sagi D. Science. 1994; 266: 285-288Crossref PubMed Scopus (206) Google Scholar, 26Noguchi T. Metz R. Chen L. Mattéi M.-G. Carrasco D. Bravo R. Mol. Cell. Biol. 1993; 13: 5195-5205Crossref PubMed Scopus (168) Google Scholar).MAP kinase activation by growth factors has been extensively studied in PC12 cells(27Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1433) Google Scholar). PC12 cells originate from a rat pheochromocytoma and retain many features of neural crest-derived cells, most notably the ability to undergo neuronal differentiation upon stimulation by NGF (28Greene L.A. Tischler A.S. Adv. Cell. Neurobiol. 1982; 3: 373-414Crossref Google Scholar). Transfection with activated forms of the oncogenes ras, raf-1, and src into PC12 cells is sufficient for differentiation in the absence of NGF stimulation(6Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (657) Google Scholar, 8Thomas S.M. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (502) Google Scholar, 29Thomas K.R. Musci T.S. Neumann P.E. Capecchi M.R. Cell. 1991; 67: 969-976Abstract Full Text PDF PubMed Scopus (205) Google Scholar). As each of these genes has been shown to converge on MAP kinase activation, this implies that components of the MAP kinase cascade are required for neuronal differentiation. More recently it has been shown that the activation of MAP kinase kinase is required and sufficient for PC12 cell differentiation(3Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar). Despite our understanding of MAP kinase activation in neuronal differentiation, we know relatively little about MAP kinase inactivation. Initial studies presented here characterize a family of MKPs expressed in PC12 cells.We report the cloning of a novel MKP, MKP-2, that is highly expressed in PC12 cells. MKP-2 mRNA is expressed at moderate levels in nearly all tissues and cells and encodes a phosphatase that inactivates MAP kinase in vitro and MAP kinase-dependent gene transcription in vivo. Its distribution in the central nervous system and regulation in the hippocampus suggest a potential role for this phosphatase in neuronal signaling pathways.EXPERIMENTAL PROCEDURESMaterialsRestriction and modification enzymes were purchased from New England Biolabs (Beverly, MA), Boehringer Mannheim, and Promega. Superscript reverse transcriptase was from Life Technologies, Inc.; Taq DNA polymerase from Perkin-Elmer and Sequenase from U. S. Biochemical Corp. All enzymes were used according to the instructions from the manufacturer. [α-32P]dATP (3000 Ci/mmol), 35S-dATP, 35S-UTP (1500 Ci/mmol), [α-32P]ATP (800 Ci/mmol), [35S]cysteine (1075 Ci/mmol), and [α-32P]UTP (800 Ci/mmol at 40 mCi/ml) were purchased from Dupont NEN. Oligonucleotides were synthesized by a core facility at Oregon Health Sciences University. Antisera to MKP-1 was purchased from Santa Cruz Biotechnology Inc. An anitibody to phosphotyrosine (clone 4G10) was kindly provided by Brian Druker (OHSU, Portland, OR)(30Druker B. Okuda K. Matulonis U. Salgia R. Roberts T. Griffin J.D. Blood. 1992; 79: 2215-2220Crossref PubMed Google Scholar).Reverse Transcriptase PCR AmplificationOne μg of total RNA from PC12 cells was used to generate first strand cDNA after an initial annealing reaction to 0.1 μg of random hexamers at 70°C for 10 min. Following equilibration to ambient temperatures, a buffer containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 500 μM of each of four dNTPs, and 200 units/μg of Superscript reverse transcriptase was added, and the mix was incubated at 37°C for 1 h. The reaction was terminated by placing the tubes on ice, and the cDNA was recovered by ethanol precipitation. The pellet was washed with 70% ethanol and resuspended in 100 μl of 5 mM Tris, 0.5 mM EDTA mix. Five μl of this cDNA was used as a template for PCR amplification. Initially, two degenerate oligonucleotides were synthesized that generated a 204-bp cDNA fragment. The 5′ primer corresponded to the conserved WFNEAI sequence present in MKP-1 (26Noguchi T. Metz R. Chen L. Mattéi M.-G. Carrasco D. Bravo R. Mol. Cell. Biol. 1993; 13: 5195-5205Crossref PubMed Scopus (168) Google Scholar) and PAC-1 (23Rohan P.J. Davis P. Moskaluk C.A. Kearns M. Krutzch H. Siebenlist U. Kelly K. Science. 1993; 259: 1763-1766Crossref PubMed Scopus (263) Google Scholar) (5′-TGG-TT(T/C)-AA(T/C)-GA(G/A)-GC(G/A/T/C)-AT-3′), while the 3′ primer corresponded to the conserved NFSFMG sequence present in MKP-1 (26Noguchi T. Metz R. Chen L. Mattéi M.-G. Carrasco D. Bravo R. Mol. Cell. Biol. 1993; 13: 5195-5205Crossref PubMed Scopus (168) Google Scholar) and PAC-1 (23Rohan P.J. Davis P. Moskaluk C.A. Kearns M. Krutzch H. Siebenlist U. Kelly K. Science. 1993; 259: 1763-1766Crossref PubMed Scopus (263) Google Scholar) (5′-C-CAT-(A/G)AA-(G/A/T/C)(G/C)(A/T)-(A/G)AA-(A/G)TT-3′) (see Fig. 2). Two additional oligonucleotides were made to confirm the novelty of MKP-2. The 5′ primer was a degenerate primer corresponding to the conserved YDQGGP sequence (5′-TA(T/C)-GA(T/C)-CA(A/G)-GG(G/A/T/C)-GG(G/A/T/C)-CC-3′), while the 3′ primer was specific for MKP-2 (5′-ATGAAGAAACGGGTGCGG-3′) corresponding to MKKRVR sequence (Fig. 1B and 2). This set of primers generated a 336-bp cDNA fragment corresponding to nucleotides 628-963 (Fig. 1B). The PCR reaction consisted of 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 15 mM Tris-HCl, pH 8.4, and 0.5 μg of each primer pair. PCR was allowed to proceed for 30 cycles. Each cycle consisted of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C in a thermocycler (Perkin-Elmer Cetus). The PCR products were purified and subcloned into pBluescript (SK-) (Stratagene) using restriction enzymes engineered at the ends of each of the primers.Figure 1:Restriction map and sequence of MKP-2 cDNA. A, the 4.8-kb MKP-2 cDNA was digested with various restriction enzymes, and a schematic representation of some of these sites is shown. The following abbreviations are used: RI, EcoRI; Pst, PstI; A, ApaI; H, HindIII; RV, EcoRV; Sma, SmaI; Bam, BamHI; 5′ UT, 5′-untranslated region; 3′ UT, 3′-untranslated region; ATG, translation start site; TAG, translation stop site. The coding region of MKP-2 is shown as a rectangularbox with two different domains highlighted. The stippled box represents CH2 domains (42Kwak S.P. Hakes D.M. Martell K.J. Dixon J.E. J. Biol. Chem. 1994; 269: 3596-3604Abstract Full Text PDF PubMed Google Scholar) while the hatched box represents the catalytic domain. B, nucleotide sequence and the encoded amino acid sequence of rat MKP-2 cDNA is shown. The translation start site is denoted as +1. The consensus catalytic site, the AU-sequence motifs in the 3′ untranslated region, and the putative polyadenylation signal are underlined. The 5′- and 3′-untranslated regions are depicted in lower case letters.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Screening of the PC12 cDNA Library and Isolation of the Full-length CloneThe 336-bp PCR fragment generated by using the specific MKP-2 primer (described above) was labeled by random primed synthesis and used to screen a PC12 oligo(dT)-primed cDNA library in λgt10 with 5 × 106 individual recombinants that had been size-selected prior to ligation for clones greater than 2 kb. The library was plated onto 20 LB plates and allowed to grow at 42°C to a concentration of 50,000 recombinants/plate. The plaques were then transferred onto nitrocellulose filters in duplicate. The filters were soaked in prehybridization/hybridization buffer (6 × SSC, 5 × Denhardt's solution, 1% SDS, 0.01 M EDTA, 50% formamide, 100 μg/ml denatured salmon sperm DNA) at 42°C for 1-2 h with gentle agitation. Random primed probe was made as described in the Boehringer Mannheim kit. One to two million cpm/ml of boiled MKP-2 specific probe was added directly to the prehybridization/hybridization mix, and hybridization was allowed to proceed at 42°C for 24 h. The filters were washed in 2 × SSC and 1% SDS for 2 h at 65°C with frequent changes in the wash solution. The final wash was in 1 × SSC and 1% SDS after which the filters were air dried and put on film. After the tertiary screen, three positive plaques were obtained. Phage DNA was isolated by standard methods and digested with EcoRI to release the insert. The inserts obtained were cloned into pBluescript (SK-) and subjected to restriction enzyme mapping and sequencing.SequencingMKP-2 cDNA inserts obtained were sequenced on both strands by the method of Sanger (31Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52363) Google Scholar) using Sequenase reagents (U.S. Biochemical Corp.) according to the protocol suggested by the manufacturer. Multiple internal primers were made to allow sequencing of the complete 800-bp insert. The 4-kb insert was also sequenced using multiple internal primers on both strands through the termination codon and partially into the 3′-untranslated region. Sequences proximal to the polyadenylation signal were also obtained as shown (Fig. 1B).Cell Culture and Drug TreatmentsPC12-GR5 cells (courtesy of Rae Nishi, OHSU, Portland, OR) were grown at 5% CO2 in Dulbecco's modified Eagle's medium containing 5% fetal calf serum, 10% horse serum, and L-glutamine. Prior to drug treatments, the cells were serum-starved for 24 h with Dulbecco's modified Eagle's medium containing no serum and treated with either 100 ng/ml NGF or 20 ng/ml EGF for the indicated times.RNA IsolationTotal cellular RNA was isolated using RNAzol™ B (Biotecx Lab, Inc.) according to the manufacturer's protocol. Briefly, cells were grown to 30-50% confluency in a 100-mm plate, rinsed with cold phosphate-buffered saline, and scraped into 1 ml of RNAzol B. After vortexing, 0.1 ml of chloroform was added and incubated on ice for 15 min. The suspension was centrifuged, and the RNA was precipitated from the upper aqueous layer with an equal volume of isopropyl alcohol. After pelleting, the RNA was resuspended in water, quantitated at A260, and used directly for reverse transcriptase PCR or Northern analysis.Northern Blot AnalysisTen μg of total RNA was electrophoresed through a 1.2% agarose formaldehyde gel and transferred onto Magna NT filter (MSI, Westboro, MA) using standard methodology in 6 × SSC. Filters were prehybridized in 3 ml of hybridization buffer (5% SDS, 400 mM NaPO4 pH 7.2, 1 mM EDTA, 1 mg/ml bovine serum albumin, 50% formamide) at 65°C for 4 h in a rotating hybridization oven. 2-5 × 107 cpm/ml of the antisense riboprobe was then directly added to the hybridization buffer, and hybridization was allowed to proceed for 24 h. The next day, filters were initially washed in 1 × SSC at room temperature for 15 min and then washed in (0.05 × SSC, 0.1% SDS, 5 mM EDTA, pH 8) at 70°C for 3-4 h. Filters were autoradiographed at −70°C on Kodac XAR-5 film using Dupont intensifying screens. Quantitations were performed using a Molecular Dynamics PhosphorImager 445 SF, and all signals were normalized to the 18 S and cyclophilin signals respectively.Riboprobe SynthesisThe 336-bp cDNA fragment generated by using the specific MKP-2 primer (described under “Reverse Transcriptase PCR Amplification”) was subcloned in pBluescript (SK-) and was used to synthesize antisense riboprobes by linearizing the plasmid with SalI that was engineered into the 5′ primer. Full-length MKP-1 cDNA (kindly provided by Nicholas Tonks, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) was subcloned into pBluescript (SK-) and then linearized with BamHI to generate a 1.9-kb MKP-1 riboprobe. Rat cyclophilin (pSP65 1B15) and 18 S ribosomal RNA (18SpSP65) plasmids (kindly provided by James Douglass, Vollum Institute, Portland, OR) were linearized using PstI and HindIII respectively to generate linear antisense riboprobes. Antisense riboprobes were synthesized as described previously(32Misra-Press A. Cooke N.E. Liebhaber S.A. J. Biol. Chem. 1994; 269: 23220-23229Abstract Full Text PDF PubMed Google Scholar). Briefly, 1 μg of template DNA was incubated in transcription reaction mix (40 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, 20 units of RNasin, 0.5 mM each of rATP, GTP, and CTP, 12 μM rUTP, 50 μM [α-32P]UTP (800 Ci/mmol), and 15 units of the appropriate RNA polymerase) at 37°C for 60 min. The reaction was stopped by the addition of 2 units of RNase-free DNase and incubated at 37°C for 15 min. 25 mM EDTA was then added, samples were phenol-chloroform extracted and ethanol-precipitated. Antisense riboprobes were resuspended in water at a concentration of 1-2 × 106 cpm/μl.In Situ HybridizationMale Sprague-Dawley rats (200-300 g) were anesthetized and perfused with 1000 ml of 4% paraformaldehyde in borate buffer, pH 9.5, at 4°C (fixation buffer). Brains were dissected and incubated in fixation buffer for 8 h and then incubated overnight in fixation buffer with 10% sucrose. Brains were sectioned serially into five series of 30-μm slices with a sliding microtome. Sections were prepared and hybridized as described previously(33Arriza J.L. Stoler M.H. Angerer R.C. Neuron. 1988; 1: 887-900Abstract Full Text PDF PubMed Scopus (510) Google Scholar). The 336-bp MKP-2 specific cDNA fragment was used to synthesize antisense and sense riboprobes. Sections were hybridized with 35S-labeled riboprobes (107 cpm/ml) in 66% formamide, 0.26 M NaCl, 1.32 × Denhardt's solution, 13.2 mM Tris, pH 8.0, 1.32 mM EDTA, 13.2% dextran sulfate, pH 8.0, at 65°C for 24 h. Slides were washed in 4 × SSC, digested with RNase A (20 μg/ml for 30 min at 37°C), and then rinsed in a stringent wash containing 0.1 × SSC at 65°C for 30 min. Sections were dehydrated, dipped in NTB-2 emulsion (Kodak), and developed after 21 days. Light- and darkfield photomicrographs were taken with a Dialux 22 EB at 32× magnification.In Vitro Transcription and Translation ReactionsFull-length MKP-1 and MKP-2 cDNAs were used in a coupled in vitro transcription and translation reaction using TNT™ coupled reticulocute lysate system (Promega) as per the manufacturer's instructions. Briefly, 1 μg of circular DNA was incubated with 27.5 μl of TNT rabbit reticulocyte lysate, 2 μl of TNT reaction buffer, 1 μl of T7 RNA polymerase, 1 μl of 1 mM amino acid mix minus cysteine, 2 μl of [35S]cysteine (1075 Ci/mmol at 11 mCi/ml), and 1 μl of RNAsin at 40 units/μl in a final volume of 50 μl. The reaction was incubated at 30°C for 1-2 h. The synthesized proteins were separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography.PC12 Transfection AssaysPC12 cells were grown to approximately 60% confluence prior to transfection. For transient transfection experiments, 3 μg of 5 × Gal4-E1B luciferase and 3 μg of cytomegalovirus (CMV) Gal4-Elk1 transactivation domain2 2Roberson, M. S., Misra-Press, A., Laurance, M. E., Stork, P. J. S., and Maurer, R. A. (1995) Mol. Cell. Biol., in press. were used in combination with either 6 μg of Rous sarcoma virus promoter coupled to globin (control) or constitutively active form of Raf kinase (Raf BxB) (34Bruder J.T. Heidecker G. Rapp U.R. Genes & Dev. 1992; 6: 545-556Crossref PubMed Scopus (396) Google Scholar) and 30 μg of either pCDNA3 (Invitrogen, Inc.) containing the CMV promoter, CMV MKP-1, or CMV MKP-2. Cells were transfected by calcium phosphate-mediated DNA transfer as described previously(35Howley P.M. Sarver N. Law M.F. Methods Enzymol. 1983; 101: 387-402Crossref PubMed Scopus (28) Google Scholar). Cell lysates were prepared 20-24 h following transfection, and luciferase activity was determined as described previously(36Luehrsen K.R. Wet J.R.d. Walbot V. Methods Enzymol. 1992; 216: 397-414Crossref PubMed Scopus (125) Google Scholar).Western Blotting of PC12 ProteinsPC12 cells were lysed in 200 μl of a 1% Nonidet P-40 lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 2 mM vanadate). Protein concentrations were determined by the method of Bradford(37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213510) Google Scholar). One hundred μg of total protein was resolved on a 12% SDS-polyacrylamide gel and transferred onto Immobilon P membrane. The membranes were probed with an MKP-1 antibody (diluted at 1:2000) (Santa Cruz Biotech., Inc.). A horseradish peroxidase-conjugated secondary antibody was used to allow detection of the appropriate bands using enhanced chemiluminescence (Amersham Corp.).Bacterial Expression of MKP-2The catalytic domain of MKP-2 encoded within a carboxyl-terminal 690-bp fragment (C-MKP-2), extending from amino acids 163-393, was subcloned into the PET 23b vector (Novagen) using specific PCR oligonucleotides. This vector provides an amino-terminal epitope tag derived from the T7 capsid protein (T7 tag) that can be detected using specific antibodies (T7 antibody, Novagen). The frame of the resultant cDNA was confirmed by sequencing. This plasmid and the vector alone were used to transform BL21 bacteria (Novagen). A protein of the expected size (28 kDa) was induced upon incubation with 0.4 mM isopropyl-1-thio-β-D-galactopyranoside and was detected using anti-T7 capsid antibodies. Prior to phosphatase assays, bacterial extracts were prepared in lysis buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA) and sonicated. Insoluble debris was pelleted and the supernatant assayed directly.ERK2 Dephosphorylation AssayActivated ERK2 was prepared by incubating 10 ng of recombinant ERK2 (kindly provided by Dr. Edwin Krebs, University of Washington, Seattle, Washington) with 0.1 μg of active MAP kinase kinase (MEK) (Santa Cruz Biotechnology, Inc.) in 1 × MEK buffer (25 mM Hepes, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, and 50 μM [γ-32P]ATP) at 30°C for 30 min. The activation of ERK2 was confirmed by Western blot analysis using an antibody directed against phosphotyrosine(30Druker B. Okuda K. Matulonis U. Salgia R. Roberts T. Griffin J.D. Blood. 1992; 79: 2215-2220Crossref PubMed Google Scholar). Ten ng of activated ERK2 was incubated with 10 μg of bacterial lystates from MKP-2 expressing and nonexpressing cells in 1 × phosphatase buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10 mM dithiothreitol) for 15 min at 30°C. The reaction was stopped by the addition of an equal volume of 2 × Laemmli sample buffer, and the samples were separated by 13% SDS-polyacrylamide gel electrophoresis. The dephosphorylation of ERK2 was confirmed by Western blotting with the phosphotyrosine antibody using enhanced chemiluminescence for detection of the signal.Phosphotyrosine Phosphatase AssayThe synthetic peptide Raytide (Oncogene Sciences) was phosphorylated on a unique tyrosine using Src tyrosine kinase activity immunoprecipitated from C3H10T1/2 cells (kindly provided by Sally Parsons, University of Virginia) as described(38Stork P. Misra-Press A. Pan M.-G. Methods Neurosci. 1995; (in press)Google Scholar). Bacterial extracts containing 10-60 μg of bacterial proteins were incubated at 30°C for 30 min with labeled peptide (104 cpm) in 100 μl of 1 × phosphatase buffer (described above). Additional reagents (10 μM vanadate, 20 nM microcystin-leucine-arginine, and 1 μM okadaic acid) were added without prior incubation. The reaction was terminated by the addition of 0.75 ml of stop solution (2 mM NaHP04, 90 mM sodium pyrophosphate, 0.9 M HCl, 4% (v/v) Norit A). Following brief centrifugation, 400 μl of the supernatant was added to 2.5 ml of scintillant, and the released counts from phosphatase activity were measured on a scintillation counter. All phosphatase assays were performed in duplicate.RESULTSPC12 Cells Express Multiple MKPs: Identification and Cloning of a Novel MKPTo identify potential MKPs that are expressed in PC12 cells, a screening strategy involving reverse transcriptase PCR amplification was employed. Alignment of the sequences of the known members of the MKP family (human PAC-1, mouse PAC-1, mouse 3CH134, and VH1) showed areas of high sequence homology particularly surrounding the catalytic core consensus site (HCXAGXXR, where X is any amino acid) (Ref. 23Rohan P.J. Davis P. Moskaluk C.A. Kearns M. Krutzch H. Siebenlist U. Kelly K. Science. 1993; 259: 1763-1766Crossref PubMed Scopus (263) Google Scholar and Fig. 2). Degenerate primers were designed to the conserved amino acid sequence WFNEAI (5′ primer) and the conserved amino acid sequence NFSFMG (3′ primer) surrounding the catalytic core site (Fig. 2). Reverse transcriptase PCR on total RNA from PC12 cells with these two degenerate primers revealed the e" @default.
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• W2047994580 title "A Novel Mitogen-activated Protein Kinase Phosphatase. STRUCTURE, EXPRESSION, AND REGULATION" @default.
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