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Cytoplasmic Protein-Tyrosine-Phosphatase

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Cytoplasmic Protein-Tyrosine-Phosphatase Proc. Natl. Acad. Sci. USA Vol. 89, pp. 499-503, January 1992 Biochemistry Cloning and characterization of a mouse cDNA encoding a cytoplasmic protein-tyrosine-phosphatase (tyrosine phosphorylation/mouse embryo/human T-cell protein-tyrosine-phosphatase) BEDRICH MOSINGER, JR.*, ULRICH TILLMANN, HEINER WESTPHAL, AND MICHEL L. TREMBLAY Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Building 6, Bethesda, MD 20892 Communicated by Igor B. Dawid, September 27, 1991 ABSTRACT A mouse cDNA encoding a non-receptor-type We attempted to identify novel members ofthe PTP family phosphotyrosine phosphatase (PTP; EC 3.1.3.48) has been that could participate in mouse development and/or onco- isolated. The 1570-base-pair cDNA contains a single open genesis. In this paper we present the identification ofa widely reading frame that predicts a 382-amino acid protein with Mr distributed intracellular mouse PTP (MPTP) that is expressed 44,640. The nucleic acid and amino acid sequences are homol- both in the mouse embryo and in adult tissues. Our data ogous to those of a previously described human T-cell PTP identify this MPTP as a homologue of a previously described [Cool, D. E., Tonks, N. K., Charbonneau, H., Walsh, K. A., human T-cell PTP (TC-PTP) (18); however, the mouse and Fischer, E. H. & Krebs, E. G. (1989) Proc. Nati. Acad. Sci. human sequences differ completely in their 3' portions, USA 86, 5257-5261]; however, the mouse and human 3' predicting different protein carboxyl termini. A human TC- sequences diverge and predict markedly different protein PTP cDNA was isolated that contained a 3' end sequence carboxyl termini. The mouse PTP gene is expressed as a homologous to MPTP.t 1.9-kilobase message in several stages of murine embryonic development and in a variety of adult tissues. An additional AND 1.3-kilobase message was found to be expressed specifically in MATERIALS METHODS testes. Finally, we report the isolation of a human T-cell PTP Chemicals and Reagents. All restriction and modification cDNA containing a 3' end sequence homologous to the mouse enzymes were obtained from Bethesda Research Laborato- PIP. ries or Boehringer Mannheim. The 11.5-day mouse embryo cDNA library and the total human testis and human placenta Protein phosphorylation has been shown to play a crucial role RNAs were purchased from Clontech. A cDNA library of in regulating fundamental cellular processes (1). Phosphory- 11.5-day embryonic mouse kidney was provided by G. R. lation of tyrosine residues in proteins in multicellular orga- Dressler (National Institutes ofHealth). A cDNA library was nisms is associated mainly with the response of cells to prepared from mouse testis RNA in AZAPII by using the stimuli mediated by hormone and growth factor receptors cDNA cloning kit from Stratagene. Oligonucleotides were (2-5). Tyrosine phosphorylation is transient and is thought to synthesized on a Milligen/Biosearch 8700 DNA synthesizer. depend upon the activity of both protein-tyrosine kinases Radionucleotides were purchased from Amersham. The (PTFKs) and protein-tyrosine-phosphatases (PTPs) (6). Alter- wheat germ in vitro translation system was purchased from ation of tyrosine kinase activity can lead to cell transforma- Boehringer Mannheim and used according to the manufac- tion as seen, for example, in infection of cells by certain turer's protocol. DNA sequences were determined by the transforming retroviruses (3, 6-8). Similarly, when the de- dideoxy method (19) using'the Sequenase kit from United phosphorylation ofphosphotyrosine residues is prevented by States Biochemical. the use of PTP inhibitors, the effect mimics the overexpres- Amplification of a Conserved PETP Region of Human TC- sion of tyrosine kinases and resembles an activated or PEP by Polymerase Chain Reaction (PCR). Human genomic transformed cell state (9). DNA (2 ,ug) was used in the PCR with primers H1 and H2 in Like PTKs, the PTPs are thought to be involved in cell 35 temperature cycles consisting of 80 sec at 940C, 1 min at signaling, cell growth and proliferation, and oncogenesis. 550C, and 2 min at 720C in a Perkin-Elmer-Cetus PCR cycler. Moreover, recent findings have shown that some receptor- The PCR buffer was 50 mM KCI/1.5 mM MgCl2/0.1 mM type PTPs may be involved in cell regulatory processes that dNTPs/15 mM Tris1HCl, pH 8.4. The nucleotide sequences are of those mediated by receptor PTKs of primers H1 and H2 (generated from conserved regions of quite independent the PTP domain) were 5'-GCC-GAA-l'TC-GAA-GAG-GCA- (10). The identification of tyrosine kinases that play a role in CAA-AGG-AGT-TAC-ATC-3' and 5'-CCG-GGA-TCC- development (e.g., torso or flb gene product of Drosophila TTC-AGG-GAC-TCC-AAA-ATC-TOG-CC-3', respec- and the murine c-kit oncogene product; refs. 11-13) suggests tively. The PCR-amplified fragment was isolated and cloned that PTPs could also act in concert to regulate complex into plasmid pGEM-4Z (Promega). mechanisms during embryogenesis. Isolation of the MPTP cDNA Clone from a Mouse Embryo Members of the PTP gene family share a high degree of Library. The 350-base-pair (bp) PCR fragment generated homology in one particular domain (the PIP domain, about from human DNA was radiolabeled by random priming (20) 260 amino acids; refs. 14 and 15). The family can be divided and used to screen -500,Q00 plaques from an 11.5-day mouse into at least two subgroups: the first includes intracellular embryo cDNA library in AgtlO. Filters were hybridized at enzymes, whereas the second consists of membrane recep- 60'C in 0.5 M NaH2PO4, pH 7.0/7% SDS/1 mM EDTA/0.5% tor-like molecules with large extracellular domains linked to intracellular PTP domains (16, 17). Abbreviations: PTK, protein-tyrosine kinase; PTP, protein-tyrosine- phosphatase; TC-PTP, T-cell PTP; MPTP, mouse PIP. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. M81477 and M81478). 499 Downloaded by guest on September 26, 2021 500 Biochemistry: Mosinger et al. Proc. Natl. Acad. Sci. USA 89 (1992) bovine serum albumin (21) and the final wash was performed protocol. One-fifth of each reaction mixture was electropho- with 2x standard saline citrate (SSC)/0.1% SDS at room resed in an SDS/12.5% polyacrylamide gel and the radioac- temperature. tive polypeptides were detected by fluorography. Isolation of Additional MPTP cDNAs. Approximately Northern Blot Analysis. Total RNA was extracted from 500,000 independent clones of several cDNA libraries de- mouse embryos, adult mouse tissues, and cultured cell lines rived from mouse testes and mouse embryos were screened by using an RNA isolation kit (Stratagene) according to the with the radiolabeled insert of the AgtlO phage. The hybrid- manufacturer's protocol. Equal amounts (15 or 20 ,ug) oftotal ization temperature was 65°C and the final stringent wash was RNA were electrophoresed in a formaldehyde/form- at 650C in 0.2x SSC/0.1% SDS. amide/1% agarose gel, blotted, and hybridized to 32P-labeled Isolation of the 5' End of the MPTP cDNA. Total phage probes. Hybridization conditions were the same as those DNA of a random-primed 11.5-day embryonic mouse kidney described for the screening oflibraries. The final wash was in cDNA library (2 ,g) was used in the PCR. Primers (25 0.1x SSC/0.1% SDS at 650C. nucleotides long) corresponding to the T3 and T7 promoter Amplification of the 3' End of the Human TC-PTP cDNA. sequence in the pBluescript SK plasmid (Stratagene) were The protocol for PCR amplification of the 3' end of cDNAs used in combination with oligonucleotides derived from the (rapid amplification of cDNA ends, RACE) has been de- most 5' sequences of the cDNA clones. The PCR conditions scribed by Frohman et al. (23). Briefly, a cDNA was prepared were as described above. The products of six PCRs were from 5 ,ug of total human placental RNA by using a (dT)17 pooled and subcloned and the DNAs of eight colonies were primer and murine reverse transcriptase. The product was sequenced. All sequences were identical and overlapped the then amplified by PCR using an oligo(dT) primer-linker and 5' end of our longest previously identified mouse cDNA the specific primer H1 (described above). This PCR product clone. The sequence contained a putative start codon and was reamplified further using a 3' linker sequence and a was homologous to the 5' end of the human TC-PTP cDNA. second, internal-specific primer (derived from positions 422- The full-length cDNA was then assembled using the longest 442 in the TC-PTP sequence). A single prominent fragment of cDNA clone and the 5'-end PCR fragment (Fig. 1). 1.2-kilobases (kb) was isolated, cloned into pBluescript SK, In Vitro Transcription and Translation of MPTP. MPTP and sequenced. mRNA was synthesized by T3 RNA polymerase from puri- fied plasmid DNA containing the entire MPTP coding region RESULTS (22). mRNA was then translated in a wheat germ translation Isolation of a PTP Gene from a Mouse Embryo cDNA system with [35S]methionine according to the manufacturer's Library. Several different oligonucleotide primers derived GlyGluAspValAsnValLysGlnLeuLeuLeuAsnMetArgLysTyrArgMetGlyLeu 258 60 GGAGAGGArAA73AAACAATTATTAC3GAATA~uAGAAGTATCGAAAGMGACTr 840 18 IleGlnThrProAspGlnLeuArgPheSerTyrMetAlaIleIleGluGlyAlaLysTyr
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