FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2085095382> ?p ?o ?g. }

• W2085095382 endingPage "7203" @default.
• W2085095382 startingPage "7197" @default.
• W2085095382 abstract "The mitogen-activated protein kinase (MAPK) also known as extracellular signal-regulated kinase (ERK) plays a crucial role in various signal transduction pathways. ERK is activated by its upstream activator, MEK, via threonine and tyrosine phosphorylation. ERK activity in the cell is tightly regulated by phosphorylation and dephosphorylation. Here we report the cloning and characterization of a novel dual specific phosphatase, HVH2, which may function in vivo as a MAP kinase phosphatase. The deduced amino acid sequence of HVH2 shows significant identity to the VH1-related dual specific phosphatase family. In addition, the N-terminal region of HVH2 also displays sequence identity to the cell cycle regulator, Cdc25 phosphatase. Recombinant HVH2 phosphatase exhibited a high substrate specificity toward activated ERK and dephosphorylated both threonine and tyrosine residues of activated ERK1 and ERK2. Immunofluorescence studies with an epitope-tagged HVH2 showed that the enzyme was localized in cell nucleus. Transfection of HVH2 into NIH3T3 cells inhibited the v-src and MEK-induced transcriptional activation of serum-responsive element containing promoter, consistent with the notion that HVH2 promotes the inactivation of MAP kinase. HVH2 mRNA showed an expression pattern distinct from CL100 (human homologue of mouse MKP1) and PAC1, two previously identified MAP kinase phosphatases. Our data suggest a possible role of HVH2 in MAP kinase regulation. The mitogen-activated protein kinase (MAPK) also known as extracellular signal-regulated kinase (ERK) plays a crucial role in various signal transduction pathways. ERK is activated by its upstream activator, MEK, via threonine and tyrosine phosphorylation. ERK activity in the cell is tightly regulated by phosphorylation and dephosphorylation. Here we report the cloning and characterization of a novel dual specific phosphatase, HVH2, which may function in vivo as a MAP kinase phosphatase. The deduced amino acid sequence of HVH2 shows significant identity to the VH1-related dual specific phosphatase family. In addition, the N-terminal region of HVH2 also displays sequence identity to the cell cycle regulator, Cdc25 phosphatase. Recombinant HVH2 phosphatase exhibited a high substrate specificity toward activated ERK and dephosphorylated both threonine and tyrosine residues of activated ERK1 and ERK2. Immunofluorescence studies with an epitope-tagged HVH2 showed that the enzyme was localized in cell nucleus. Transfection of HVH2 into NIH3T3 cells inhibited the v-src and MEK-induced transcriptional activation of serum-responsive element containing promoter, consistent with the notion that HVH2 promotes the inactivation of MAP kinase. HVH2 mRNA showed an expression pattern distinct from CL100 (human homologue of mouse MKP1) and PAC1, two previously identified MAP kinase phosphatases. Our data suggest a possible role of HVH2 in MAP kinase regulation. INTRODUCTIONA group of protein serine/threonine kinases, known as mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseMEKMAPK or ERK kinaseHVH2human VH1 homologous phosphatase 2MKP1MAPK phosphatase 1GSTglutathione S-transferaseSREserum-response elementMBPmyelin basic proteinEGFepidermal growth factorkbkilobase(s)PAGEpolyacrylamide gel electrophoresisCMVcytomegalovirusIBMX3-isobutyl-1-methylxanthine. or extracellular signal-regulated kinase (ERK), is acutely stimulated by various extracellular signals, including mitogenic growth factors such as insulin, epidermal growth factor (EGF), and phorbol esters (for review, see (1Cobb M.H. Boulton T.G. Robbins D.J. Cell Regul. 1991; 2: 965-978Crossref PubMed Scopus (426) Google Scholar, 2Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 3Pelech S.L. Sanghera J.S. Science. 1992; 257: 1355-1356Crossref PubMed Scopus (306) Google Scholar, 4Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (328) Google Scholar)). ERK activation is believed to play an essential role in mitogenic growth factor signal transduction. Evidence indicates that ERK can phosphorylate nuclear transcription factors (5Gille H. Sharrocks A.D. Shaw P.E. Nature. 1992; 358: 414-417Crossref PubMed Scopus (812) Google Scholar, 6Pulverer B.J. Kyriakis J.M. Avruch J. Nikolakaki E. Woodgett J.R. Nature. 1991; 353: 670-674Crossref PubMed Scopus (1187) Google Scholar, 7Seth A. Alvarez E. Gupta S. Davis R.J. J. Biol. Chem. 1991; 266: 23521-23524Abstract Full Text PDF PubMed Google Scholar), protein kinases(8Sturgill T.W. Ray L.B. Erikson E. Maller J.L. Nature. 1988; 334: 715-718Crossref PubMed Scopus (752) Google Scholar), cytoskeletal proteins(9Gotoh Y. Nishida E. Matsuda S. Shiina N. Kosako H. Shiokawa K. Akiyama T. Ohta K. Sakai H. Nature. 1991; 349: 251-254Crossref PubMed Scopus (315) Google Scholar), and proteins involved in regulation of cell growth(10Haycock J.W. Ahn N.G. Cobb M.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2365-2369Crossref PubMed Scopus (235) Google Scholar, 11Lin L-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar), suggesting an essential role in cellular signal transduction. ERK must be phosphorylated on both threonine and tyrosine residues to exert its full enzymatic activity(12Anderson N.G. Maller J.L. Tonks N.K. Sturgill T.W. Nature. 1990; 343: 651-653Crossref PubMed Scopus (793) Google Scholar, 13Ray L.B. Sturgill T.W. Proc. Natl. Acad. Sci U. S. A. 1988; 85: 3753-3757Crossref PubMed Scopus (260) Google Scholar). A single protein kinase, MEK, activates ERK2 by phosphorylating threonine 183 and tyrosine 185(14Boulton 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 (1477) Google Scholar, 15Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (836) Google Scholar, 16Rossomando A.J. Wu J. Michel H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5779-5783Crossref PubMed Scopus (54) Google Scholar). Constitutive activation of MEK can cause transformation in NIH3T3 cells and differentiation in PC12 cells(17Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 18Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1254) Google Scholar), demonstrating the importance of the MAP kinase pathway in signal transduction. In Swiss3T3 cells, MAP kinase reaches maximum activity approximately 5-10 min after EGF stimulation followed by a rapid inactivation(19Zheng C-F. Ohmichi M. Saltiel A.R. Guan K-L. Biochemistry. 1994; 33: 5595-5599Crossref PubMed Scopus (41) Google Scholar). Western blotting demonstrated that the amount of ERK protein did not change after mitogen stimulation, suggesting that ERK is inactivated by post-translational modifications(20Boulton T.G. Cobb M.H. Cell Regul. 1991; 2: 357-371Crossref PubMed Scopus (280) Google Scholar). Two protein phosphatases, MKP1/CL100 and PAC1, have been implicated in dephosphorylation of ERK (21Alessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020PubMed Google Scholar, 22Charles C.H. Sun H. Lau L.F. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5292-5296Crossref PubMed Scopus (183) Google Scholar, 23Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1022) Google Scholar, 24Ward Y. Gupta S. Jensen P. Wartmann M. Davis R.J. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar, 25Zheng C-F. Guan K-L. J. Biol. Chem. 1993; 268: 16116-16119Abstract Full Text PDF PubMed Google Scholar).Protein phosphatases are generally divided into Ser/Thr and Tyr phosphatases, based on the phosphoamino acid specificity. Unlike the protein kinases, protein Ser/Thr phosphatases share no sequence identity to the tyrosine-specific phosphatases. However, a growing number of phosphatases have recently been identified to dephosphorylate both Ser/Thr and Tyr residues (for review, see (26Guan K-L. Dixon J.E. Semin. Cell Biol. 1993; 4: 389-396Crossref PubMed Scopus (23) Google Scholar)). The prototype of this dual specific phosphatase is the VH1 phosphatase encoded by the vaccinia virus(27Guan K-L. Broyles S.S. Dixon J.E. Nature. 1991; 350: 359-362Crossref PubMed Scopus (323) Google Scholar). Cellular proteins homologous to VH1 phosphatase have been identified. These include the cell cycle regulator Cdc25 (for review, see (28Millar J.B.A. Russell P. Cell. 1992; 68: 407-410Abstract Full Text PDF PubMed Scopus (193) Google Scholar)), the nitrogen-induced yeast YVH1(29Guan K-L Hakes D.J. Wang Y. Park H-D. Cooper T.G. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12175-12179Crossref PubMed Scopus (75) Google Scholar), and the human VHR(30Ishibashi T. Bottaro D.P. Chan A. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12170-12174Crossref PubMed Scopus (180) Google Scholar). KAP and Cdi1 were two dual specific phosphatases isolated by virtue of their interaction with the cyclin-dependent kinases(31Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1319) Google Scholar, 32Hannon G.J. Casso D. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1731-1735Crossref PubMed Scopus (110) Google Scholar). These enzymes have been implicated to play a role in cell cycle control.An immediate-early gene, 3CH134, induced by serum and growth factors in mouse fibroblasts, was isolated and shown to have significant amino acid sequence identity to VH1(33Charles C.H. Abler A.S. Lau L.F. Oncogene. 1992; 7: 187-190PubMed Google Scholar). The human homolog of 3CH134, CL100, was cloned by Keyse and Emslie (34Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (568) Google Scholar) as an immediate-early gene in response to oxidative stress and heat shock. Another VH1-related immediate-early gene, PAC1, was isolated from mitogen activated T-cells (35Rohan P.J. Davis P. Moskaluk C.A. Kearns M. Krutzsch H. Siebenlist U. Kelly K. Science. 1993; 259: 1763-1766Crossref PubMed Scopus (263) Google Scholar). CL100, 3CH134, and PAC1 have been demonstrated to specifically dephosphorylate threonine and tyrosine residues of ERKs(21Alessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020PubMed Google Scholar, 22Charles C.H. Sun H. Lau L.F. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5292-5296Crossref PubMed Scopus (183) Google Scholar, 23Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1022) Google Scholar, 24Ward Y. Gupta S. Jensen P. Wartmann M. Davis R.J. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar, 25Zheng C-F. Guan K-L. J. Biol. Chem. 1993; 268: 16116-16119Abstract Full Text PDF PubMed Google Scholar). A possible function of these mitogen-induced phosphatases may be to down-regulate ERK. Therefore, Sun et al.(23Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1022) Google Scholar) have suggested the name of MKP1 (map kinase phosphatase) for 3CH134 as an indication of its biological function. Genetic studies in yeast Saccharomyces cerevisiae identified a dual specific phosphatase, MSG5, which inactivated the FUS3 and KSS1 kinases, two MAP kinase homologs in the yeast mating pathway(36Doi K. Gartner A. Ammerer G. Errede B. Shinkawa H. Sugimoto K. Matsumoto K. EMBO J. 1994; 13: 61-70Crossref PubMed Scopus (204) Google Scholar).In this report, a novel dual specific phosphatase, HVH2 (for human VH1 homologous phosphatase 2), was isolated and characterized. The deduced HVH2 protein shares 62 and 55% sequence identity to CL100 and PAC1, respectively. Purified recombinant HVH2 specifically hydrolyzed the phosphothreonine and phosphotyrosine residues of the activated ERK1 and ERK2. HVH2 was found to be a nuclear protein and capable of blocking activation of a MAP kinase-regulated reporter gene expression.EXPERIMENTAL PROCEDURESCell CultureNIH3T3 and Swiss3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Hela cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfection was performed using Lipofectin (Life Technologies, Inc.) as described previously (37Zheng C-F. Guan K-L. J. Biol. Chem. 1994; 269: 19947-19952Abstract Full Text PDF PubMed Google Scholar). Hep G2 cells were cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum. For RNA induction, Hep G2 cells were starved in serum free medium for 24 h and then stimulated with one of the following reagents: insulin-like growth factor 1 (160 ng/ml), EGF (160 ng/ml), phorbol 12-myristate 13-acetate (500 nM), H2O2 (100 μM) or 3-isobutyl-1-methylxanthine (IBMX, 500 μM)/forskolin (25 μM). Cells were stimulated for 30 min, 1 and 3 h. RNA isolation and Northern hybridization were performed following standard procedures(38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).Cloning and Sequence AnalysisHVH2 cDNA was isolated by screening a human placenta cDNA library (Stratagene) using CL100 cDNA (39Kwak S.P. Hakes D.J. Martell K.J. Dixon J.E. J. Biol. Chem. 1994; 269: 3596-3604Abstract Full Text PDF PubMed Google Scholar) as a probe at moderate stringency. Hybridization was performed under conditions with 40% formamide, 5 × SSPE, 5 × Denharnt's solution, 0.1% SDS at 42°C(38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The filters were washed in 1 × SSC at 60°C. Plaques showing weak hybridization signals were isolated and purified by secondary and tertiary screening. Purified λ clones were converted into phagemid following manufacturer's instructions (Stratagene). Synthetic oligonucleotides were used to obtain the complete nucleotide sequence. Sequence alignment was performed using the Wisconsin Genetic Computation Group software.Expression and PurificationThe full-length HVH2 clone contained an insert of 2.3 kb. The cDNA was digested with NcoI, located at the initiation ATG, and SacI, located within the 3′-noncoding region. The 1.3-kb NcoI-SacI fragment containing the entire coding sequence was subcloned into pGEX-KG (40Guan K-L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1637) Google Scholar) digested with NcoI and SacI to produce pGEX-HVH2. This plasmid was then introduced into Escherichia coli strain TG-1 to express GST-HVH2 fusion protein. The GST-HVH2 was induced with 10 μM isopropylthio-β-galactoside at room temperature for 8-12 h. GST-HVH2 was purified as described (40Guan K-L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1637) Google Scholar) and stored at −80°C in 10% glycerol. Rat ERK2 was expressed with a histidine tag and purified by NAT-Ni affinity chromatography (Qiagen)(41Janknecht R. De Martynoff G. Lou J. Kipskind R.A. Nordheim A. Stunnenberg H.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8972-8976Crossref PubMed Scopus (414) Google Scholar). ERK1, GST-MEK2, and GST-CL100 were expressed and purified as described(25Zheng C-F. Guan K-L. J. Biol. Chem. 1993; 268: 16116-16119Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were determined by densitometric scanning of SDS-PAGE using bovine serum albumin as a standard.Phosphatase AssayThe p-nitrophenyl phosphate (pNPP) hydrolysis activity of GST-HVH2 was assayed in 200 μl of buffer A (50 mM HEPES, pH 7.5, 0.1% 2-mercaptoethanol) containing 20 mM pNPP at 37°C for 30 min. One unit of phosphatase activity was defined as the amount of enzyme required to hydrolyze 1 μmol of pNPP at 37°C in 1 min. Purified GST-HVH2 had a specific activity of 0.25 units/mg compared to 0.102 units/mg of GST-CL100(25Zheng C-F. Guan K-L. J. Biol. Chem. 1993; 268: 16116-16119Abstract Full Text PDF PubMed Google Scholar).Activated ERK1 and ERK2 were used as substrates for GST-HVH2. ERK (21.6 μg) was activated by GST-MEK2 (2.75 μg) in buffer B (18 mM HEPES, pH 7.5, 50 μM ATP, 10 mM magnesium acetate) at 30°C for 20 min. The GST-MEK2 was depleted by absorption to glutathione-agarose (Sigma). Under these conditions, ERK was usually activated by more than 100-fold. The activated ERK was then used for HVH2 inactivation assay in 10 μl of buffer A at 30°C for 10 min. Activity of HVH2-treated ERK was directly determined by the MBP kinase assay.32P-Labeled ERK1, ERK2, and GST-MEK2 were prepared by autophosphorylation in the presence of [γ-32P]ATP in buffer B. Casein was phosphorylated by either the catalytic subunit of protein kinase A or P43v-abl kinase as described(27Guan K-L. Broyles S.S. Dixon J.E. Nature. 1991; 350: 359-362Crossref PubMed Scopus (323) Google Scholar). ERK1 and ERK2 were also phosphorylated by GST-MEK2 in the presence of [γ-32P]ATP. Dephosphorylation of 32P-labeled proteins was performed in 20 μl of buffer A at 30°C for 10 min using 3.8 microunits of GST-HVH2 or 1,000 microunits of PTP1. Samples were analyzed by SDS-PAGE and visualized by autoradiography. Phosphoamino acid analysis was performed as described (42Kamps M.P. Sefton B.M. Anal. Biochem. 1989; 176: 22-27Crossref PubMed Scopus (322) Google Scholar).The coding sequence of human ERK1 was subcloned into plasmid pALTER-1 (Promega) for site-directed mutagenesis. The catalytic essential lysine residue 71 of human ERK1 was mutated to arginine by oligonucleotide-directed mutagenesis (Promega) to produce a kinase-deficient ERK1∗. The threonine 202 and tyrosine 204 were independently mutated to alanine and phenylalanine, respectively, in the kinase-deficient ERK1∗. Mutations were confirmed by DNA sequencing and subcloned into pGEX-2T (43Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5035) Google Scholar) for expression and purification. ERK1∗, ERK1∗T202A, and ERK1∗Y204F were phosphorylated by GST-MEK2 as described above. Dephosphorylation of these mutant ERKs was performed as described for wild type ERK1. Dephosphorylation reactions were terminated by addition of SDS sample treatment buffer and resolved by SDS-PAGE. The samples were then transferred to nitrocellulose and quantitated by phosphoimaging or scintillation counting.Activated ERK1 or ERK2 (0.4 μg) was inactivated by 5.9 microunits of GST-HVH2 in 30 μl of buffer A at 30°C for 10 min. Sodium vanadate was added to inhibit HVH2. Half of the sample was directly used for MBP kinase assays. The other half was subjected to reactivation by 0.32 μg of GST-MEK2 in 20 μl of buffer B.Kinase AssayERK activity was determined as described(44Sturgill T.W. Ray L.B. Anderson N.G. Erikson A.K. Methods Enzymol. 1991; 200: 342-351Crossref PubMed Scopus (18) Google Scholar). In order to visualize the phosphorylated MBP, reactions were also directly analyzed by 15% SDS-PAGE and followed by autoradiography.ImmunofluorescenceThe myc epitope (45Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2157) Google Scholar) was incorporated into the C terminus of HVH2 by polymerase chain reaction. The epitope-tagged HVH2 cDNA was subcloned into pCMV4 vector (46Andersson S. Davis D.L. Dahlback H. Jornvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar) to produce pCMV-HVH2 myc. This plasmid was transfected into NIH3T3 or Hela cells by the Lipofectin method. Immunofluorescence of transfected cells with anti-myc antibody was performed following published methods(37Zheng C-F. Guan K-L. J. Biol. Chem. 1994; 269: 19947-19952Abstract Full Text PDF PubMed Google Scholar). Synthetic peptide, EQKLISEEDL, corresponding to the myc epitope was used for competition.Luciferase AssayThe HVH2 cDNA was subcloned into pCMV4 (46Andersson S. Davis D.L. Dahlback H. Jornvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar) to construct pCMV-HVH2. Human MEK1 cDNA (47Zheng C-F. Guan K-L. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar) was subcloned into the BamHI site of pCMV4 to produce pCMV-MEK. pJH2 (containing v-src) was a generous gift of Dr. Taparowsky (Purdue University). Plasmid SRE-luc containing the luciferase under the control of minimum promoter of thymidine kinase and serum-responsive element of c-fos was a generous gift of Dr. Pessin (University of Iowa)(48Yamauchi K. Holt K. Pessin J.E. J. Biol. Chem. 1993; 268: 14597-14600Abstract Full Text PDF PubMed Google Scholar). pCMV-luc containing the luciferase under the control of the CMV promoter was a gift of Dr. Cui (University of Michigan). Plasmid SRE-luc (0.2 μg) was transfected into NIH3T3 cells (60-mm plates) together with a different combination of plasmid pJH2 (1.0 μg) or pCMV-MEK (1.0 μg) and varying amounts of pCMV-HVH2 or pCMV-HVH2 myc in the presence of pCMV-SEAP (0.4 μg, for expressing alkaline phosphatase as an internal control)(49Cullen B.R. Malim M.H. Methods Enzymol. 1992; 216: 362-368Crossref PubMed Scopus (164) Google Scholar). Two days after transfection, cells were washed twice with ice-cold phosphate-buffered saline and harvested in 350 μl of cell lysis buffer(50Brasier A.R. Ausubel F.M. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York1989: 9.6.1-9.6.14Google Scholar). Cell lysates (50-100 μl) were directly used for luciferase assays following standard protocol(50Brasier A.R. Ausubel F.M. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York1989: 9.6.1-9.6.14Google Scholar). Cultured media from transfected cells were heated at 65°C for 5 min and directly used for alkaline phosphatase assays(49Cullen B.R. Malim M.H. Methods Enzymol. 1992; 216: 362-368Crossref PubMed Scopus (164) Google Scholar).RESULTSCloning of HVH2 cDNATo test the possibility that new ERK-specific phosphatases may exist, low stringency hybridization was performed to isolate new members of the dual specific phosphatases. Using CL100 as a probe(39Kwak S.P. Hakes D.J. Martell K.J. Dixon J.E. J. Biol. Chem. 1994; 269: 3596-3604Abstract Full Text PDF PubMed Google Scholar), 10 moderate hybridizing clones were isolated from a human placenta cDNA library. Restriction digestion with EcoRI followed by Southern hybridization revealed that three of the 10 clones contained an internal EcoRI site which is absent in CL100 cDNA. DNA sequencing analysis demonstrated that all three cDNAs encoded the same protein. The longest clone of 2.3 kb, designated as HVH2, encoded an open reading frame of 394 amino acid residues (Fig. 1).The deduced amino acid sequence of HVH2 displays 62 and 55% overall sequence identity to the complete sequences of CL100 and PAC1, respectively (Fig. 1)(34Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (568) Google Scholar, 35Rohan P.J. Davis P. Moskaluk C.A. Kearns M. Krutzsch H. Siebenlist U. Kelly K. Science. 1993; 259: 1763-1766Crossref PubMed Scopus (263) Google Scholar). The highest sequence conservation occurs in the C-terminal half of the molecules, where the catalytically essential cysteine (Cys287 for HVH2) found in all protein tyrosine phosphatases is located. This C-terminal domain also shares significant sequence identity to the active site region of dual specific phosphatases such as VH1, YVH1, KAP, and Cdi1. In contrast, the N-terminal 181 residues of HVH2 share only 33 and 25% sequence identity to the corresponding regions of CL100 and PAC1, respectively (Fig. 1). Interestingly, the N-terminal region of HVH2 showed significant sequence identity with the cell cycle regulator Cdc25 phosphatases. This sequence similarity has been observed in CL100 and MKP1(39Kwak S.P. Hakes D.J. Martell K.J. Dixon J.E. J. Biol. Chem. 1994; 269: 3596-3604Abstract Full Text PDF PubMed Google Scholar, 51Keyse S.M. Ginsburg M. Trends Biochem. Sci. 1993; 18: 377-378Abstract Full Text PDF PubMed Scopus (89) Google Scholar). It is worth noting that the catalytically essential cysteine in Cdc25 is absent in the N-terminal regions of both CL100 and HVH2, suggesting that the catalytic domain of HVH2 phosphatase resides in the C-terminal and not the N-terminal region of the polypeptide.Dephosphorylation and Inactivation of ERKsTo determine the substrate specificity of HVH2, several phosphoproteins were tested, including protein kinase A-phosphorylated casein (on serine), v-abl-phosphorylated casein (on tyrosine), autophosphorylated ERK1 (on tyrosine and serine), autophosphorylated GST-MEK2 (on serine and threonine), MBP (on threonine), and activated ERK1 (on serine, threonine, and tyrosine). Under the same assay conditions, GST-HVH2 dephosphorylated the activated ERK1 while no significant dephosphorylation was observed with all the other phosphoproteins tested (data not shown). These results indicated a high substrate specificity of HVH2 toward ERK. The efficiency of GST-HVH2 to inactivate ERK1 and ERK2 were determined and compared with that of PTP1 (52Guan K-L. 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). GST-HVH2 effectively inactivated both ERK1 and ERK2 while PTP1 did not (Fig. 2A). GST-HVH2 also displayed a specific activity three times higher than GST-CL100 in an ERK1 inactivation assay. As expected, vanadate (2 mM) completely inhibited HVH2 activity while okadaic acid (5 μm) had no effect (not shown).Figure 2A, inactivation of ERK1 and ERK2 by GST-HVH2. The activated ERK1 was inactivated by GST-HVH2 (closed circles) or PTP1 (open circles). The activated ERK2 was inactivated by GST-HVH2 (open triangles). ERK activity was determined by the MBP kinase assay. B, tyrosine and threonine phosphatase activity of HVH2. ERK1 was phosphorylated by GST-MEK2 before dephosphorylation (lane 1), or dephosphorylated by GST-HVH2 (3.8 μU, lane 2) or PTP1 (1 milliunit, lane 3). Positions of free phosphate, phosphoserine, phosphothreonine, phosphotyrosine, and origin are denoted by P, pS, pT, pY, and O, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)If HVH2 dephosphorylated ERK on the same threonine and tyrosine residues which were recognized by MEK, the HVH2-dependent ERK inactivation should be reversible. To demonstrate this point, the HVH2-inactivated ERK2 was subjected to reactivation by MEK2 in the presence of 2 mM sodium vanadate, which inhibited the HVH2 activity. We observed that the HVH2-inactivated ERK2 could be quantitatively reactivated by MEK (not shown), indicating that HVH2 and MEK recognized the same residues on ERK.Dual Specific Phosphatase ActivityThe MEK2-activated ERKs contained phosphoserine, threonine, and tyrosine (Fig. 2B). Phosphoamino acid analysis revealed that GST-HVH2 dephosphorylated the threonine and tyrosine residues, which were phosphorylated by MEK, but not the autophosphorylated serine residue of the activated ERK1, while PTP1 dephosphorylated tyrosine only (Fig. 2B). GST-HVH2 could not completely dephosphorylate ERK1 because the autophosphorylated serine in ERK1 was resistant to the HVH2. Similar observations were obtained with the activated ERK2 (not shown).An intriguing difference between the activated ERK and other phosphoproteins was that the activated ERK contained adjacent phosphothreonine and phosphotyrosine. It is possible that the two neighboring phosphorylated residues serve as a recognition determinant for HVH2. To test this hypothesis, ERK1 phosphorylated on either threonine (ERK1∗Y204F) or tyrosine alone (ERK1∗T202A) was utilized as a substrate for HVH2. Threonine 202 and tyrosine 204 in ERK1 (53Charest D.L. Mordret G. Harder K.W. Jirik F. Pelech S.L. Mol. Cell. Biol. 1993; 13: 4679-4690Crossref PubMed Scopus (73) Google Scholar) correspond to threonine 183 and tyrosine 185 in ERK2 which are the activation-phosphorylation sites by MEK(14Boulton 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 (1477) Google Scholar, 15Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (836) Google Scholar, 16Rossomando A.J. Wu J. Michel H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5779-5783Crossref PubMed Scopus (54) Google Scholar). ERK1∗, a kinase-deficient mutant, was phosphorylated on both threonine and tyrosine by MEK2 (Fig. 3B). ERK1∗T202A, having threonine 202 substituted by an alanine, was phosphorylated only on tyrosine while ERK1∗Y204F, having tyrosine 204 substituted by a phenylalanine, was phosphorylated only on threonine (Fig. 3B). GST-HVH2 dephosphorylated all three ERK1∗ mutants (Fig. 3A), suggesting that double phosphorylations of adjacent threonine and tyrosine were not a prerequisite for HVH2 recognition. However, HVH2 dephosphorylated ERK1∗ and ERK1∗T202A more efficiently than ERK1∗Y204F (Fig. 3A), indicating that HVH2 preferred phosphotyrosine over phosphothreonine. Interestingly, MEK also phospho" @default.
• W2085095382 created "2016-06-24" @default.
• W2085095382 creator A5016834516 @default.
• W2085095382 creator A5068847647 @default.
• W2085095382 date "1995-03-01" @default.
• W2085095382 modified "2023-09-27" @default.
• W2085095382 title "Isolation and Characterization of a Novel Dual Specific Phosphatase, HVH2, Which Selectively Dephosphorylates the Mitogen-activated Protein Kinase" @default.
• W2085095382 cites W1479869774 @default.
• W2085095382 cites W1482432683 @default.
• W2085095382 cites W1509609109 @default.
• W2085095382 cites W1516078935 @default.
• W2085095382 cites W1572416479 @default.
• W2085095382 cites W1573821584 @default.
• W2085095382 cites W1613040252 @default.
• W2085095382 cites W1640618846 @default.
• W2085095382 cites W1782499902 @default.
• W2085095382 cites W1821277660 @default.
• W2085095382 cites W1826524642 @default.
• W2085095382 cites W1968474526 @default.
• W2085095382 cites W1971334343 @default.
• W2085095382 cites W1977302628 @default.
• W2085095382 cites W1977472414 @default.
• W2085095382 cites W1979502921 @default.
• W2085095382 cites W1979881407 @default.
• W2085095382 cites W1984903682 @default.
• W2085095382 cites W1987428363 @default.
• W2085095382 cites W1989480480 @default.
• W2085095382 cites W1995858338 @default.
• W2085095382 cites W1996223704 @default.
• W2085095382 cites W1999157780 @default.
• W2085095382 cites W2001521752 @default.
• W2085095382 cites W2006650852 @default.
• W2085095382 cites W2006868309 @default.
• W2085095382 cites W2011245371 @default.
• W2085095382 cites W2011590971 @default.
• W2085095382 cites W2022506262 @default.
• W2085095382 cites W2022658303 @default.
• W2085095382 cites W2024847198 @default.
• W2085095382 cites W2033303857 @default.
• W2085095382 cites W2034419242 @default.
• W2085095382 cites W2037426608 @default.
• W2085095382 cites W2039202467 @default.
• W2085095382 cites W2040611554 @default.
• W2085095382 cites W2043573783 @default.
• W2085095382 cites W2053286013 @default.
• W2085095382 cites W2053849194 @default.
• W2085095382 cites W2063213781 @default.
• W2085095382 cites W2068446924 @default.
• W2085095382 cites W2071993841 @default.
• W2085095382 cites W2072404407 @default.
• W2085095382 cites W2084376502 @default.
• W2085095382 cites W2085495372 @default.
• W2085095382 cites W2086725260 @default.
• W2085095382 cites W2087965346 @default.
• W2085095382 cites W2093621392 @default.
• W2085095382 cites W2115701574 @default.
• W2085095382 cites W2140566131 @default.
• W2085095382 cites W2146388445 @default.
• W2085095382 cites W2312445582 @default.
• W2085095382 cites W894556329 @default.
• W2085095382 cites W934662886 @default.
• W2085095382 cites W9946280 @default.
• W2085095382 doi "https://doi.org/10.1074/jbc.270.13.7197" @default.
• W2085095382 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7535768" @default.
• W2085095382 hasPublicationYear "1995" @default.
• W2085095382 type Work @default.
• W2085095382 sameAs 2085095382 @default.
• W2085095382 citedByCount "174" @default.
• W2085095382 countsByYear W20850953822012 @default.
• W2085095382 countsByYear W20850953822013 @default.
• W2085095382 countsByYear W20850953822014 @default.
• W2085095382 countsByYear W20850953822015 @default.
• W2085095382 countsByYear W20850953822016 @default.
• W2085095382 countsByYear W20850953822017 @default.
• W2085095382 countsByYear W20850953822018 @default.
• W2085095382 countsByYear W20850953822019 @default.
• W2085095382 countsByYear W20850953822020 @default.
• W2085095382 countsByYear W20850953822021 @default.
• W2085095382 countsByYear W20850953822022 @default.
• W2085095382 countsByYear W20850953822023 @default.
• W2085095382 crossrefType "journal-article" @default.
• W2085095382 hasAuthorship W2085095382A5016834516 @default.
• W2085095382 hasAuthorship W2085095382A5068847647 @default.
• W2085095382 hasBestOaLocation W20850953821 @default.
• W2085095382 hasConcept C11960822 @default.
• W2085095382 hasConcept C124952713 @default.
• W2085095382 hasConcept C132149769 @default.
• W2085095382 hasConcept C142362112 @default.
• W2085095382 hasConcept C178666793 @default.
• W2085095382 hasConcept C184235292 @default.
• W2085095382 hasConcept C185592680 @default.
• W2085095382 hasConcept C2775941552 @default.
• W2085095382 hasConcept C2780429522 @default.
• W2085095382 hasConcept C2780980858 @default.
• W2085095382 hasConcept C55493867 @default.
• W2085095382 hasConcept C60644358 @default.
• W2085095382 hasConcept C86803240 @default.
• W2085095382 hasConcept C95444343 @default.

• next