Expression cloning of a human dual-specificity phosphatase.
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Journal: 1993/January - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 1281549
Abstract:
Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase. Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti-phosphotyrosine antibody. Among several clones showing decreased anti-phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro. The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1. The purified recombinant protein dephosphorylated several activated growth factor receptors, as well as serine-phosphorylated casein, in vitro. Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related (VHR) human protein. These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.
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Proc Natl Acad Sci U S A 89(24): 12170-12174
PMID: 1281549

Expression cloning of a human dual-specificity phosphatase.

Abstract

Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase. Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti-phosphotyrosine antibody. Among several clones showing decreased anti-phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro. The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1. The purified recombinant protein dephosphorylated several activated growth factor receptors, as well as serine-phosphorylated casein, in vitro. Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related (VHR) human protein. These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.

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Selected References

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  • Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. [PubMed] [Google Scholar]
  • Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. [PubMed] [Google Scholar]
  • Aaronson SA. Growth factors and cancer. Science. 1991 Nov 22;254(5035):1146–1153. [PubMed] [Google Scholar]
  • Fischer EH, Charbonneau H, Tonks NK. Protein tyrosine phosphatases: a diverse family of intracellular and transmembrane enzymes. Science. 1991 Jul 26;253(5018):401–406. [PubMed] [Google Scholar]
  • Hunter T. Protein-tyrosine phosphatases: the other side of the coin. Cell. 1989 Sep 22;58(6):1013–1016. [PubMed] [Google Scholar]
  • Kumagai A, Dunphy WG. The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system. Cell. 1991 Mar 8;64(5):903–914. [PubMed] [Google Scholar]
  • Gould KL, Nurse P. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature. 1989 Nov 2;342(6245):39–45. [PubMed] [Google Scholar]
  • Dunphy WG, Newport JW. Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase. Cell. 1989 Jul 14;58(1):181–191. [PubMed] [Google Scholar]
  • Yang XH, Seow KT, Bahri SM, Oon SH, Chia W. Two Drosophila receptor-like tyrosine phosphatase genes are expressed in a subset of developing axons and pioneer neurons in the embryonic CNS. Cell. 1991 Nov 15;67(4):661–673. [PubMed] [Google Scholar]
  • Tian SS, Tsoulfas P, Zinn K. Three receptor-linked protein-tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Cell. 1991 Nov 15;67(4):675–685. [PubMed] [Google Scholar]
  • Charbonneau H, Tonks NK, Kumar S, Diltz CD, Harrylock M, Cool DE, Krebs EG, Fischer EH, Walsh KA. Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5252–5256.[PMC free article] [PubMed] [Google Scholar]
  • Krueger NX, Streuli M, Saito H. Structural diversity and evolution of human receptor-like protein tyrosine phosphatases. EMBO J. 1990 Oct;9(10):3241–3252.[PMC free article] [PubMed] [Google Scholar]
  • Kornbluth S, Paulson KE, Hanafusa H. Novel tyrosine kinase identified by phosphotyrosine antibody screening of cDNA libraries. Mol Cell Biol. 1988 Dec;8(12):5541–5544.[PMC free article] [PubMed] [Google Scholar]
  • Guan KL, Broyles SS, Dixon JE. A Tyr/Ser protein phosphatase encoded by vaccinia virus. Nature. 1991 Mar 28;350(6316):359–362. [PubMed] [Google Scholar]
  • Miki T, Fleming TP, Bottaro DP, Rubin JS, Ron D, Aaronson SA. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science. 1991 Jan 4;251(4989):72–75. [PubMed] [Google Scholar]
  • Miki T, Matsui T, Heidaran MA, Aaronson SA. An efficient directional cloning system to construct cDNA libraries containing full-length inserts at high frequency. Gene. 1989 Nov 15;83(1):137–146. [PubMed] [Google Scholar]
  • Miki T, Fleming TP, Crescenzi M, Molloy CJ, Blam SB, Reynolds SH, Aaronson SA. Development of a highly efficient expression cDNA cloning system: application to oncogene isolation. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5167–5171.[PMC free article] [PubMed] [Google Scholar]
  • Macgregor PF, Abate C, Curran T. Direct cloning of leucine zipper proteins: Jun binds cooperatively to the CRE with CRE-BP1. Oncogene. 1990 Apr;5(4):451–458. [PubMed] [Google Scholar]
  • Guan KL, Dixon JE. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. [PubMed] [Google Scholar]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed] [Google Scholar]
  • Bottaro DP, Rubin JS, Ron D, Finch PW, Florio C, Aaronson SA. Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors. J Biol Chem. 1990 Aug 5;265(22):12767–12770. [PubMed] [Google Scholar]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467.[PMC free article] [PubMed] [Google Scholar]
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed] [Google Scholar]
  • Hakes DJ, Dixon JE. New vectors for high level expression of recombinant proteins in bacteria. Anal Biochem. 1992 May 1;202(2):293–298. [PubMed] [Google Scholar]
  • Tonks NK, Diltz CD, Fischer EH. Characterization of the major protein-tyrosine-phosphatases of human placenta. J Biol Chem. 1988 May 15;263(14):6731–6737. [PubMed] [Google Scholar]
  • Boyle WJ, van der Geer P, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. [PubMed] [Google Scholar]
  • Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148.[PMC free article] [PubMed] [Google Scholar]
  • Charles CH, Abler AS, Lau LF. cDNA sequence of a growth factor-inducible immediate early gene and characterization of its encoded protein. Oncogene. 1992 Jan;7(1):187–190. [PubMed] [Google Scholar]
  • Streuli M, Krueger NX, Thai T, Tang M, Saito H. Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR. EMBO J. 1990 Aug;9(8):2399–2407.[PMC free article] [PubMed] [Google Scholar]
  • Strausfeld U, Labbé JC, Fesquet D, Cavadore JC, Picard A, Sadhu K, Russell P, Dorée M. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature. 1991 May 16;351(6323):242–245. [PubMed] [Google Scholar]
  • Lau LF, Nathans D. Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells. EMBO J. 1985 Dec 1;4(12):3145–3151.[PMC free article] [PubMed] [Google Scholar]
  • Lau LF, Nathans D. Expression of a set of growth-related immediate early genes in BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1182–1186.[PMC free article] [PubMed] [Google Scholar]
  • Millar JB, Russell P. The cdc25 M-phase inducer: an unconventional protein phosphatase. Cell. 1992 Feb 7;68(3):407–410. [PubMed] [Google Scholar]
  • Dunphy WG, Kumagai A. The cdc25 protein contains an intrinsic phosphatase activity. Cell. 1991 Oct 4;67(1):189–196. [PubMed] [Google Scholar]
  • Galaktionov K, Beach D. Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins. Cell. 1991 Dec 20;67(6):1181–1194. [PubMed] [Google Scholar]
  • Lindberg RA, Quinn AM, Hunter T. Dual-specificity protein kinases: will any hydroxyl do? Trends Biochem Sci. 1992 Mar;17(3):114–119. [PubMed] [Google Scholar]
  • Nakielny S, Cohen P, Wu J, Sturgill T. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase. EMBO J. 1992 Jun;11(6):2123–2129.[PMC free article] [PubMed] [Google Scholar]
  • Seger R, Ahn NG, Posada J, Munar ES, Jensen AM, Cooper JA, Cobb MH, Krebs EG. Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. J Biol Chem. 1992 Jul 15;267(20):14373–14381. [PubMed] [Google Scholar]
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD 20892.
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD 20892.
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Abstract
Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase. Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti-phosphotyrosine antibody. Among several clones showing decreased anti-phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro. The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1. The purified recombinant protein dephosphorylated several activated growth factor receptors, as well as serine-phosphorylated casein, in vitro. Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related (VHR) human protein. These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.
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