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 /mouse embryo/ 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--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- 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 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). is transient and is thought to synthesized on a Milligen/Biosearch 8700 DNA synthesizer. depend upon the activity of both protein-tyrosine Radionucleotides were purchased from Amersham. The (PTFKs) and protein-tyrosine- (PTPs) (6). Alter- wheat germ in vitro translation system was purchased from ation of tyrosine 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, 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 278 GCCGCCAIGTCGGCAACCATCGAGCGGGAGC GAGGAACGGATGCTCAGTGTCGCTGG 120 ATTCAGACACCGGACCAAC3CAGAT7tTCCTACA3CGCCATAATAGAAGGAGCAACTAC 900

38 ThrLysGlyAspSerAsnIleGlnLysArgTrpLysGluLeuSerLysGluAspLeuSer 298 CAGCCGTTATACGAAAATrCGAAATGAATCCCATGACTATCCTCATAGAGTGGCCAAG 180 ACAAAAGGAGAIAAATATACAGAAACGG'flGAAAGAAC¶ITI'CTAAAGAAGTTATCT 960

PheProGluAsnArgAsnArgAsnArgTyrArgAspValSerProTyrAspHisSerArg 58 318 TTrCCAGAAAcAGAAAccGAACAGATACAGAGTAGCCCATATGA TCACAGTCGT 240 CCTT~TA~AACGGAIGI3GATCA[3GAAAT 1020

ValLysLeuGlnSerThrGluAsnAspTyrIleAsnAlaSerLeuValAspIleGluGlu 78 GlySerGluAspGluLysLeuThrGlyLeuProSerLysValGlnAspThrValGluGlu 338 GTTAAAC A TAAAA TA CAG TA C 300 G A= A ICAA_ 1080

AlaGlnArgserTyrIleLeuThrGlnGlyProLeuProAsnThrCysCysHisPheTrp 98 SerSerGluSerIleLeuArgLysArgIleArgGluAspArgLysAlaThrThrAlaGln 358 AAACACAGGGCCACTTC CGAACACATCISCCATTTICTGG 360 AGCAGTGAGAGCATrCTACGGAAACGTA1rCGAGAGGATAGAAAGGCTACGACGGCAG 1140

LeuMetValTrpGlnGlnLysThrLysAlaValValMetLeuAsnArgThrValGluLys 118 LysValGlnGlnMetLysGlnArgLeuAsnGluThrGluArgLysArgLysArgProArg 378 C TAAACCGAACTGTAGAAAAA 420 AGGTGCAGCAGA3UAAACAGAGGCTAAATGAAACTGAACGAAAAAGAAAAAGGCCAAGA 1200

GluSerValLysCysAlaGlnTyrTrpProThrAspAspArgGluMetValPheLysGlu 138 LeuThrAspThrEnd 382 GAA7CGG3TAAATTCACAGTACTG TCAACGGCTACAGAGAAACMTG'ITAAGGAA 480 TTGACAGACACCTAAATGTTCATIGACTIG-AGACTA=TIGCAGCTATAAT . CT 1260

ThrGlyPheSerValLysLeuLeuSerGluAspValLysSerTyrTyrThrValHisLeu 158 TUGAIGTGCAAAGCAAGACCTGAAGCCCACTCCGGAAACTAAAGTGAGGCTCI;CTAACCC 1320 ACGGGATrCAG'GAGCATI:ATCTGAAGAGTAAAATCATATTATACAGTACATCTA 540 TGTAGATGCCTCACAAGTTCTGT1'rACAAAGTAGCTTCCATCCAGGGGATGAAGA 1380 178 c_ TACCGATTCTrcAATACC 600 ACGCCACCAGCAGAAGACT'GCAAACCCTITAATTTGATGTATii3TITrrAACA3GT6 1440

ThrTrpProAspPheGlyValProGluSerProAlaSerPheLeuAsnPheLeuPheLys 198 ACCTG CCAGAQ5=GTTCC TCAGCTICATTTAAACrTICTT TAAA 660 A~AAA~TAGAAGA3~3TAAAGGAAT&AAA _ GGAGCGACTACrTiGTAIT7GTACTGC 1500

ValArgGluSerGlyCysLeuThrProAspHisGlyProAlaValIleHisCysSerAla 218 CATICCTAATGTACTATACTI'rrGCAGCATAAAT ATTAAATAGAAAAA 1560 GlrAGAsAAltIlCCCIIGACCCACCA CCAGTGA7CCATTGCAG=r. 720 AAAAAAAAAA 1570 GlyIleGlyArgSerGlyThrPheSerLeuValAspThrCysLeuValLeuMetGluLys 238 GSCA CGG3GCGCT3GCACCT7CTC7CTITlAGATACC IGAMGAAMA 780

FIG. 1. Nucleic acid and deduced amino acid sequence of the MPTP cDNA. Asterisks indicate the 3' end sequence that differs between the MPTP and the published human TC-PTP. Underlined are areas of the sequence used to derive the oligonucleotides H1 and H2. Downloaded by guest on September 26, 2021 Biochemistry: Mosinger et al. Proc. Natl. Acad. Sci. USA 89 (1992) 501

from the conserved sequences of published PTP genes were M 1 2 3 4 used in an attempt to identify MPTP genes by PCR. The

templates were mouse cDNA libraries as well as mouse and 97 4-5 '' ' .': , human genomic DNA. With primers H1 and H2 (see Mate- 39- _ rials and Methods), a PCR fragment of 350 bp was amplified from human genomic DNA. The nucleotide sequence re- vealed that the fragment was composed of two conserved 46- i sequences both ofwhich very similar (95% and 98% identity) to that of the human TC-PTP cDNA (data not shown). We used the 350-bp PCR fragments as probes to screen an 11.5-day mouse embryo cDNA library under low-stringency conditions. From this library two identical phage clones were isolated that contained a 1.2-kb insert complementary to the 3' end ofa transcript homologous to the human TC-PTP gene. FIG. 3. In vitro translation of MPTP mRNA. The full-length Additional cDNA clones isolated by hybridization to the MPTP cDNA was transcribed from the T3 promoter. Increasing amounts of MPTP mRNA were translated with wheat germ in vitro L-2-kb cDNA from different cDNA libraries were also trun- translation kit (Boehringer Mannheim) and the product was electro- cated at the 5' end. We used the PCR to amplify the missing phoresed in an SDS/12.5% polyacrylamide gel. Lanes: M, markers region from cDNA libraries. (Mr X 10-3); 1, no RNA; 2, 0.1 g.tg;p:g;3, 0.2 4, 0.3 jltg. The full-length MPTP cDNA (1570 bp) is 88.8% identical with the human TC-PTP cDNA in the conserved PTP do- FI.9-kb mRNA. Since the cloned cDNA is 1.57 kb long, the main. The single open reading frame in the MPTP sequence full-length transcript probably contains a considerably longer predicts a protein of 382 amino acid residues (Fig. 1). The 5' untranslated sequence and/or a longer poly(A) tail. The putative start codon is preceded by the consensus sequence single 1.9-kb transcript was present in each of the adult for translation initiation (CCCGCCGCC; ref. 24). A polya- mouse tissues examined. The highest expression was found denylylation signal was found 90 bp upstream of the polya- in the ovaries, testes, thymus, and kidneys (Figs. 4 B and 5). denylylation site. Downstream of position 1196 in the mouse The expression of MPTP was low in embryonic stem cells and cDNA sequence the homology to the human TC-PTP cDNA increased during later stages of development (Fig. 4A). A ends abruptly, so that the predicted carboxyl terminus of the second RNA transcript, of c1.3 kb, was detected exclusively mouse protein is shorter and differs significantly from that of in the testes (Fig. 4B). The smaller transcript did not hybrid- the predicted carboxyl terminus of the TC-PTP (18) (Fig. 2; ize to a probe corresponding to bp 1195-1431 of the 3' MPTP see also Fig. 6). In addition, the noncoding 3' end of the cDNA sequence, suggesting that this transcript was trun- MPTP cDNA was found to be considerably shorter than the cated at the 3' end (Fig. 5). human counterpart. In comparison to the TC-PTP, the MPTP Identification of Two Human TC-PTP Transcripts. We has a 15-bp deletion (after bp 1041) near the 3' end of the attempted to elucidate the observed sequence divergence

region of homology that in the TC-PTP encodes the penta- between mouse and human cDNA termini. Total RNA iso- peptide DRCTG (see Fig. 2). The PTP domain (96.5% iden- lated from human placenta, human testes, and the human tical to the TC-PTP) contains an invariant cluster of amino endometrial carcinoma cell line RL95-2 (27) was hybridized acids, HCSAGXGRXG, that is known to be essential for to the 3' of the MPTP cDNA and to a enzymatic activity and possibly constitutes a part of the PTP unique sequence probe catalytic site (14, 25). derived from the conserved PTP domain. The unique 3' sequence detected an RNA of the same size and abundance The full-length cDNA was transcribed in vitro from the T3 as the (Fig. promoter. The mRNA was then translated using a wheat that recognized by PTP-specific probe 5), sug- germ translation system. The translation product closely gesting that the major human TC-PTP transcript contained approximates the predicted size of the full-length MPTP sequences homologous to the 3' terminus of the mouse (44,640 Da) (Fig. 3). The MPTP cDNA was also expressed in MPTP. We then used the RACE protocol (23) to isolate the . The product, of Mr 45,000, has PTPase 3' end sequence of the human TC-PTP cDNA. Analysis of activity that is inhibited by sodium vanadate (U.T., unpub- eight clones revealed that four contained a sequence highly lished data). homologous (89.1% identical) to the MPTP cDNA, while the Expression of MPTP mRNA in the Mouse Embryo and in other four contained a sequence identical to the published Adult Tissues. Northern blot analysis of total RNA isolated TC-PTP but terminating at position 1535 of the TC-PTP from different tissues revealed that the MPTP gene encodes cDNA (Fig. 6). Both sequences had a putative polyadenyly-

MPTP MSATIEREFEELDAQCRWQPLYLEIRNESHDYPHRVAKFPENRNRNRYRDVSPYDHSRVKLQSTENDYINASLVDIEEAQRSYILTQGPLPNTCCHFWLMVWQQKTKAVVMLNRTVEKES 12 0 TC-PTPa -PT ------T-R ------NA------I ----- 120 TC-PTPb -PT------T-R------NA ------I ----- 120

MPTP VKCAQYWPTDDREMVFKETGFSVKLLSEDVKSYYTVHLLQLENINTGETRTISHFHYTTWPDFGVPESPASFLNFLFKVRESGCLTPDHGPAVIHCSAGIGRSGTFSLVDTCLVLMEKGE 240 TC-PTPa ------Q--L------S------S-N------D 240 TC-PTPb ------Q--L------S------S-N------D 240

MPTP DVNVKQLLLNMRKYRMGLIQTPDQLRFSYMAIIEGAKYTKGDSNIQKRWKELSKEDLSPICDHSQNRVMVEKYNGKRIGSEDEKLTG LPSKVQDTVEESSESILRKRIREDRKAT 360 TC-PTPa ---I--V------CI ----S------AF --- P-KI-T ----N---L-E ---- DRCTG-S--M---M--N---A------360 TC-PTPb ---I--V------C CI-----S------A-----AF---P-KI-T------L-E- DRCTG-S--M---M--N---A------360

MPTP TAQKVQQMKQRLNETERKRKR PRLTDT* 382 TC-PTPa ------N------* 387 TC-PTPb ------N------WLYWQ- I- -KMGFMSVILVGAFVGWRLFFQQNAL* 415 FIG. 2. Amino acid comparison of the MPTP and the two TC-PTP variants (TC-PTPb is identical to the published TC-PTP; ref. 18). Identical residues are indicated by dashes. Alignments were created by the VAX version 7 computer program PILEUP. Downloaded by guest on September 26, 2021 502 Biochemistry: Mosinger et al. Proc. Natl. Acad. Sci. USA 89 (1992) A ES 8.5 10 I1.5

28S-a 18S-a- "lwi

:q? _ s B TH TE KI LLJ MU SP BR HR LI

4.40 - F

i- 2 37 - -- a,

41.35-

- 0.24 a.....

FIG. 4. Expression of MPTP mRNA shown by Northern blot |.1 analysis of total RNA (20 ikg per lane) extracted from mouse tissues and embryonic stem cells. Autoradiograms were exposed for 4 days with intensifying screens. (A) RNA from undifferentiated mouse FIG. 5. Expression of human TC-PTP and MPTP mRNA shown embryonic stem (ES) cell line D3 (26) and from 8.5-, 10.6-, and by Northern blot analysis of total RNA (20 ,ug per lane) from human 11.5-day mouse embryo. Positions of 28S (4.7 kb) and 18S (1.9 kb) (HU) endometrial carcinoma cell line RL95-2 (27) (RL), testis (TE), ribosomal RNAs are indicated. (B) RNA from adult mouse thymus and placenta (PL) and from mouse (MO) kidney (KI), testis (TE), and (TH), testis (TE), kidney (KI), lung (LU), skeletal muscle (MU), ovary (OV). (A) The filter was hybridized with a probe derived from spleen (SP), brain (BR), heart (HR), and liver (LI). Positions ofRNA the conserved domain of mouse MPTP cDNA (see text). (B) The size markers (kb) from Bethesda Research Laboratories are at left. same filter was stripped and rehybridized with the probe correspond- ing to the MPTP 3'-end sequence. The hybridization temperature was lation signal, located 92 and 21 bp, respectively, before the 600C and the final wash was 30 min in 1 x SSC at 600C. Autoradio- end of the transcripts. grams were exposed for 2 days with intensifying screens. Positions of 28S and 18S ribosomal RNAs are marked. DISCUSSION end sequences. One sequence (referred to as TC-PTPa) is A cDNA encoding a mouse non-receptor-type PTP (MPTP) highly homologous (89.1%) to the mouse MPTP sequence and is described. The cDNA sequence predicts a protein con- predicts a protein with a carboxyl terminus identical to that taining 382 amino acid residues with Mr 44,640. A protein of of the MPTP. The second sequence (TC-PTPb) is identical to similar size was also obtained by in vitro translation. The the published TC-PTP cDNA but terminates at position 1535. MPTP gene is transcribed throughout development in a Both cDNA clones contain a consensus polyadenylylation variety of adult tissues, with highest expression in the ova- signal and correspond well to the size ofthe mouse cDNA and ries, testes, thymus, and kidneys. human mRNA (detected on Northern blots), respectively. In addition to the major MPTP transcript (1.9 kb), a Our data suggest that the TC-PTPa mRNA could in fact be second, testes-specific message (1.3 kb) was detected. The the major TC-PTP transcript in human tissues. Northern shorter transcript does not hybridize to a probe generated analysis showed similar signal intensity in the major 1.9-kb from the 3' end of the MPTP cDNA and is therefore likely to human transcript when probes corresponding either to the be truncated at the 3' end, although it could conceivably be conserved PTP domain or to the MPTP 3' end were used. encoded by a different gene. No second message was de- Also, 16 independently isolated mouse MPTP clones con- tected in RNA of human testes. tained the same 3' end sequence. The similarity between MPTP and human TC-PTP at both Despite recent advances in the identification ofPTPs, their the nucleic acid (88.8%) and the amino acid (93.2%) level functions are largely unknown. The PTPs could target the indicates that they are indeed homologs. However, the two same substrates as PTKs and therefore act as negative cDNAs differ markedly at their 3' termini. There is a putative regulators. Alternatively, they could target and/or dephos- splice donor site (AGGT) present in the published TC-PTP phorylate tyrosine residues within the PTKs themselves, cDNA at the position where the sequence diverges from the which has been shown to increase the activity of some PTKs MPTP, suggesting the possibility of an alternative splicing (7, 28-30). Involvement of PTPs in could be event. modulated at a more subtle level by proteins that contain Using the PCR technique, we have isolated two variants of SH2/SH3 Src-homology domains. These proteins have re- the human TC-PTP cDNA sequence containing different 3' cently been shown to bind phosphotyrosine residues and to Downloaded by guest on September 26, 2021 Biochemistry: Mosinger et al. Proc. Natl. Acad. Sci. USA 89 (1992) 503

MPTP CGGAAACGTATTCGAGAGGATAGAAAGGCTACGACGGCTCAGAAGGTGCAGCAGATGAAACAGAGGCTAAATGAAACTGAACGAAAAAGAAAAAGGCCAAGATTGACAGACACCTAAATG 1220 TC-PTPa ------C------C--C--A------G-A------A 1215 TC-PTPb ------C ------C--C--A------G-A ------.--GGTTATATTGGCAACCTATTCTC 1216

MPTP TTCATGACTTGAGACTATTCTGCAGCTATAAAATTTGAACCTTTGATGTGCAAAGCAAGACCTGAAGCCCACTCCGGAAACTAAAGTGAGGCTTGCTAACCCTGTAGATTGCCTCACAAG 1340 TC-PTPa ------A---A ------T ------A------C------C------1334 TC-PTPb ACTAAGATGGGGTTTATGTCAGTCATTTTGGTTGGCGCTTTTGTTGGCTGGAGACTGTTTTTTCAGCAAAATGCCCTATAAACAATTAATTTTGCCCAGCAAGCTTCTGCACTAGTAACT 1336

MPTP TTGTCTGTTTACAAAGTAAGCTTTCCATCCAGGGGATGAAGAACGCC ACCAGCAGAAGACTT GCAAACCCTTTAATTTGATGTATT GTTTTTTAACATGTGTATGAAATGTAGAA 1455 TC-PTPa ----T------A----A------G-A--C ------T---G-A ------G----- G--AAGTG------TGA------14 5 4 TC- PTPb GACAGTGCTACATTAATCATAGGGGTTTGTCTGCAGCAAACGCCTCATATCCCAAAAACGGTGCAGTAGAATAGACATCAACCAGATAAGTGATATTTACAGTCACAAGCCCAACATCTC 1456

MPTP AGATGTAAAGGA_1ATTAGGAGCGACTACTTTGTATTGTACTGCCATTCCTAATGTATTTTTATACTTTTTGGCAGCATTAAATATTTTTATTAAATAGA53AA 1573 TC-PTPa - C-----C-A------A--T------C-C------G------TCAAAAAAAAAAAAAAAA 1574 TC- PTPb AGGACTCTTGACTGCAGGTTCCTCTGAACCCCAAACTGTAAATGGCTGTCTAAAT GACATTCATGTTTGT ._ 1573 FIG. 6. Comparison of the 3' end sequences of MPTP cDNA and the two TC-PTP variants (TC-PTP sequence is identical to TC-PTPb). The consensus sequence for the splice donor site in the TC-PTP cDNA is underlined, as are the potential polyadenylylation signals. participate in the signaling pathways of several tyrosine Ricca, G., Jaye, M. & Schlesinger, J. (1990) Proc. Natl. Acad. kinase receptors (31). This possibility has been strongly Sci. USA 87, 7000-7004. supported by the recent isolation of a cytoplasmic PTP 11. Pawson, T. & Bernstein, A. (1990) Trends Genet. 6, 349-356. 12. Klein, R., Martin-Zanca, D., Barbacid, M. & Parada, L. F. containing an SH2 domain (32). (1990) Development 109, 845-850. Since upregulated PTKs are capable of promoting cell 13. Dubreuil, P., Rottapel, R., Reith, A. D., Forrester, L. & transformation, it is plausible that downregulation of PTPs Bernstein, A. (1990) Ann. N. Y. Acad. Sci. 599, 58-65. could lead to similar effects. Interestingly, in some human 14. Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C. D., lung and renal a region of chromosome 3 (3p21) Harrylock, M., Cool, D. E., Krebs, E. G., Fischer, E. H. & harboring a receptor-type PTPy gene is frequently deleted Walsh, K. A. (1989) Proc. Natl. Acad. Sci. USA 86,5252-5256. 15. Krueger, N. X., Streuli, M. & Saito, H. (1990) EMBO J. 9, (33). 3241-3252. Proteins homologous to the cytoplasmic PTPs were also 16. Tonks, N. K. & Charbonneau, H. (1989) Trends Biochem. Sci. found to be encoded by "YOP H" genes present on plasmids 14, 497-500. of several bacterial strains of Yersinia and by the VH1 gene 17. Alexander, D. R. (1990) New Biol. 2, 1049-1062. of vaccinia virus. When expressed in Escherichia coli, these 18. Cool, D. E., Tonks, N. K., Charbonneau, H., Walsh, K. A., proteins were shown to Fischer, E. H. & Krebs, E. G. (1989) Proc. Natl. Acad. Sci. have PTP enzymatic activity. More- USA 86, 5257-5261. over, in Yersinia strains, the presence of YOP H genes has 19. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. been shown to be obligatory for pathogenesis (34, 35). Acad. Sci. USA 74, 5463-5467. Although we cannot predict how many cytosolic PTPs 20. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, remain to be discovered, the low number of these enzymes 6-13. detected to date, together with their ubiquitous expression, 21. Church, G. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991-1995. suggests a general role in cellular regulation and perhaps 22. Tillmann, U., Gunther, R., Schweden, J. & Bause, E. (1987) involvement in the pathogenesis of certain diseases. Eur. J. Biochem. 162, 635-642. 23. Frohman, M. 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