Proc. Natl. Acad. Sci. USAVol. 89, pp. 499-503, January 1992Biochemistry

Cloning and characterization of a mouse cDNA encoding acytoplasmic protein-tyrosine-phosphatase

(tyrosine phosphorylation/mouse embryo/human T-cell protein-tyrosine-phosphatase)

BEDRICH MOSINGER, JR.*, ULRICH TILLMANN, HEINER WESTPHAL, AND MICHEL L. TREMBLAYLaboratory 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-typephosphotyrosine phosphatase (PTP; EC 3.1.3.48) has beenisolated. The 1570-base-pair cDNA contains a single openreading frame that predicts a 382-amino acid protein with Mr44,640. The nucleic acid and amino acid sequences are homol-ogous to those of a previously described human T-cell PTP[Cool, D. E., Tonks, N. K., Charbonneau, H., Walsh, K. A.,Fischer, E. H. & Krebs, E. G. (1989) Proc. Nati. Acad. Sci.USA 86, 5257-5261]; however, the mouse and human 3'sequences diverge and predict markedly different proteincarboxyl termini. The mouse PTP gene is expressed as a1.9-kilobase message in several stages of murine embryonicdevelopment and in a variety of adult tissues. An additional1.3-kilobase message was found to be expressed specifically intestes. Finally, we report the isolation of a human T-cell PTPcDNA containing a 3' end sequence homologous to the mousePIP.

Protein phosphorylation has been shown to play a crucial rolein regulating fundamental cellular processes (1). Phosphory-lation of tyrosine residues in proteins in multicellular orga-nisms is associated mainly with the response of cells tostimuli mediated by hormone and growth factor receptors(2-5). Tyrosine phosphorylation is transient and is thought todepend upon the activity of both protein-tyrosine kinases(PTFKs) and protein-tyrosine-phosphatases (PTPs) (6). Alter-ation of tyrosine kinase activity can lead to cell transforma-tion as seen, for example, in infection of cells by certaintransforming retroviruses (3, 6-8). Similarly, when the de-phosphorylation of phosphotyrosine residues is prevented bythe use of PTP inhibitors, the effect mimics the overexpres-sion of tyrosine kinases and resembles an activated ortransformed cell state (9).

Like PTKs, the PTPs are thought to be involved in cellsignaling, cell growth and proliferation, and oncogenesis.Moreover, recent findings have shown that some receptor-type PTPs may be involved in cell regulatory processes thatare quite independent of those mediated by receptor PTKs(10). The identification of tyrosine kinases that play a role indevelopment (e.g., torso or flb gene product of Drosophilaand the murine c-kit oncogene product; refs. 11-13) suggeststhat PTPs could also act in concert to regulate complexmechanisms during embryogenesis.Members of the PTP gene family share a high degree of

homology in one particular domain (the PIP domain, about260 amino acids; refs. 14 and 15). The family can be dividedinto at least two subgroups: the first includes intracellularenzymes, whereas the second consists of membrane recep-tor-like molecules with large extracellular domains linked tointracellular PTP domains (16, 17).

We attempted to identify novel members of the PTP familythat could participate in mouse development and/or onco-genesis. In this paper we present the identification ofa widelydistributed intracellular mouse PTP (MPTP) that is expressedboth in the mouse embryo and in adult tissues. Our dataidentify this MPTP as a homologue of a previously describedhuman T-cell PTP (TC-PTP) (18); however, the mouse andhuman sequences differ completely in their 3' portions,predicting different protein carboxyl termini. A human TC-PTP cDNA was isolated that contained a 3' end sequencehomologous to MPTP.t

MATERIALS AND METHODSChemicals and Reagents. All restriction and modification

enzymes were obtained from Bethesda Research Laborato-ries or Boehringer Mannheim. The 11.5-day mouse embryocDNA library and the total human testis and human placentaRNAs were purchased from Clontech. A cDNA library of11.5-day embryonic mouse kidney was provided by G. R.Dressler (National Institutes of Health). A cDNA library wasprepared from mouse testis RNA in AZAPII by using thecDNA cloning kit from Stratagene. Oligonucleotides weresynthesized on a Milligen/Biosearch 8700 DNA synthesizer.Radionucleotides were purchased from Amersham. Thewheat germ in vitro translation system was purchased fromBoehringer Mannheim and used according to the manufac-turer's protocol. DNA sequences were determined by thedideoxy method (19) using 'the Sequenase kit from UnitedStates Biochemical.

Amplification of a Conserved PETP Region of Human TC-PEP by Polymerase Chain Reaction (PCR). Human genomicDNA (2 ,ug) was used in the PCR with primers H1 and H2 in35 temperature cycles consisting of 80 sec at 940C, 1 min at550C, and 2 min at 720C in a Perkin-Elmer-Cetus PCR cycler.The PCR buffer was 50 mM KCI/1.5 mM MgCl2/0.1 mMdNTPs/15 mM Tris1HCl, pH 8.4. The nucleotide sequencesof primers H1 and H2 (generated from conserved regions ofthe PTP domain) were 5'-GCC-GAA-l'TC-GAA-GAG-GCA-CAA-AGG-AGT-TAC-ATC-3' and 5'-CCG-GGA-TCC-TTC-AGG-GAC-TCC-AAA-ATC-TOG-CC-3', respec-tively. The PCR-amplified fragment was isolated and clonedinto plasmid pGEM-4Z (Promega).

Isolation of the MPTP cDNA Clone from a Mouse EmbryoLibrary. The 350-base-pair (bp) PCR fragment generatedfrom human DNA was radiolabeled by random priming (20)and used to screen -500,Q00 plaques from an 11.5-day mouseembryo cDNA library in AgtlO. Filters were hybridized at60'C in 0.5 M NaH2PO4, pH 7.0/7% SDS/1 mM EDTA/0.5%

Abbreviations: PTK, protein-tyrosine kinase; PTP, protein-tyrosine-phosphatase; TC-PTP, T-cell PTP; MPTP, mouse PIP.*To whom reprint requests should be addressed.tThe sequences reported in this paper have been deposited in theGenBank data base (accession nos. M81477 and M81478).

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bovine serum albumin (21) and the final wash was performedwith 2x standard saline citrate (SSC)/0.1% SDS at roomtemperature.

Isolation of Additional MPTP cDNAs. Approximately500,000 independent clones of several cDNA libraries de-rived from mouse testes and mouse embryos were screenedwith the radiolabeled insert of the AgtlO phage. The hybrid-ization temperature was 65°C and the final stringent wash wasat 650C in 0.2x SSC/0.1% SDS.

Isolation of the 5' End of the MPTP cDNA. Total phageDNA of a random-primed 11.5-day embryonic mouse kidneycDNA library (2 ,g) was used in the PCR. Primers (25nucleotides long) corresponding to the T3 and T7 promotersequence in the pBluescript SK plasmid (Stratagene) wereused in combination with oligonucleotides derived from themost 5' sequences of the cDNA clones. The PCR conditionswere as described above. The products of six PCRs werepooled and subcloned and the DNAs of eight colonies weresequenced. All sequences were identical and overlapped the5' end of our longest previously identified mouse cDNAclone. The sequence contained a putative start codon andwas homologous to the 5' end of the human TC-PTP cDNA.The full-length cDNA was then assembled using the longestcDNA clone and the 5'-end PCR fragment (Fig. 1).

In Vitro Transcription and Translation of MPTP. MPTPmRNA was synthesized by T3 RNA polymerase from puri-fied plasmid DNA containing the entire MPTP coding region(22). mRNA was then translated in a wheat germ translationsystem with [35S]methionine according to the manufacturer's

protocol. One-fifth of each reaction mixture was electropho-resed in an SDS/12.5% polyacrylamide gel and the radioac-tive polypeptides were detected by fluorography.Northern Blot Analysis. Total RNA was extracted from

mouse embryos, adult mouse tissues, and cultured cell linesby using an RNA isolation kit (Stratagene) according to themanufacturer's protocol. Equal amounts (15 or 20 ,ug) of totalRNA were electrophoresed in a formaldehyde/form-amide/1% agarose gel, blotted, and hybridized to 32P-labeledprobes. Hybridization conditions were the same as thosedescribed for the screening of libraries. The final wash was in0.1x SSC/0.1% SDS at 650C.

Amplification of the 3' End of the Human TC-PTP cDNA.The protocol for PCR amplification of the 3' end of cDNAs(rapid amplification of cDNA ends, RACE) has been de-scribed by Frohman et al. (23). Briefly, a cDNA was preparedfrom 5 ,ug of total human placental RNA by using a (dT)17primer and murine reverse transcriptase. The product wasthen amplified by PCR using an oligo(dT) primer-linker andthe specific primer H1 (described above). This PCR productwas reamplified further using a 3' linker sequence and asecond, internal-specific primer (derived from positions 422-442 in the TC-PTP sequence). A single prominent fragment of1.2-kilobases (kb) was isolated, cloned into pBluescript SK,and sequenced.

RESULTSIsolation of a PTP Gene from a Mouse Embryo cDNA

Library. Several different oligonucleotide primers derived

GlyGluAspValAsnValLysGlnLeuLeuLeuAsnMetArgLysTyrArgMetGlyLeu 25860 GGAGAGGArAA73AAACAATTATTAC3GAATA~uAGAAGTATCGAAAGMGACTr 840

GCCGCCAIGTCGGCAACCATCGAGCGGGAGC GAGGAACGGATGCTCAGTGTCGCTGG

CAGCCGTTATACGAAAATrCGAAATGAATCCCATGACTATCCTCATAGAGTGGCCAAG

18 IleGlnThrProAspGlnLeuArgPheSerTyrMetAlaIleIleGluGlyAlaLysTyr 278120 ATTCAGACACCGGACCAAC3CAGAT7tTCCTACA3CGCCATAATAGAAGGAGCAACTAC 900

38180

PheProGluAsnArgAsnArgAsnArgTyrArgAspValSerProTyrAspHisSerArg 58TTrCCAGAAAcAGAAAccGAACAGATACAGAGTAGCCCATATGA TCACAGTCGT 240

ThrLysGlyAspSerAsnIleGlnLysArgTrpLysGluLeuSerLysGluAspLeuSerACAAAAGGAGAIAAATATACAGAAACGG'flGAAAGAAC¶ITI'CTAAAGAAGTTATCT

CCTT~TA~AACGGAIGI3GATCA[3GAAAT

298960

3181020

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

AlaGlnArgserTyrIleLeuThrGlnGlyProLeuProAsnThrCysCysHisPheTrp 98 SerSerGluSerIleLeuArgLysArgIleArgGluAspArgLysAlaThrThrAlaGln 358AAACACAGGGCCACTTC CGAACACATCISCCATTTICTGG 360 AGCAGTGAGAGCATrCTACGGAAACGTA1rCGAGAGGATAGAAAGGCTACGACGGCAG 1140

LeuMetValTrpGlnGlnLysThrLysAlaValValMetLeuAsnArgThrValGluLys 118C TAAACCGAACTGTAGAAAAA 420

GluSerValLysCysAlaGlnTyrTrpProThrAspAspArgGluMetValPheLysGlu 138GAA7CGG3TAAATTCACAGTACTG TCAACGGCTACAGAGAAACMTG'ITAAGGAA 480

ThrGlyPheSerValLysLeuLeuSerGluAspValLysSerTyrTyrThrValHisLeu 158ACGGGATrCAG'GAGCATI:ATCTGAAGAGTAAAATCATATTATACAGTACATCTA 540

c_ TACCGATTCTrcAATACC178600

ThrTrpProAspPheGlyValProGluSerProAlaSerPheLeuAsnPheLeuPheLys 198ACCTG CCAGAQ5=GTTCC TCAGCTICATTTAAACrTICTT TAAA 660

ValArgGluSerGlyCysLeuThrProAspHisGlyProAlaValIleHisCysSerAlaGlrAGAsAAltIlCCCIIGACCCACCA CCAGTGA7CCATTGCAG=r.

LysValGlnGlnMetLysGlnArgLeuAsnGluThrGluArgLysArgLysArgProArgAGGTGCAGCAGA3UAAACAGAGGCTAAATGAAACTGAACGAAAAAGAAAAAGGCCAAGA

LeuThrAspThrEndTTGACAGACACCTAAATGTTCATIGACTIG-AGACTA=TIGCAGCTATAAT . CT

TUGAIGTGCAAAGCAAGACCTGAAGCCCACTCCGGAAACTAAAGTGAGGCTCI;CTAACCC

TGTAGATGCCTCACAAGTTCTGT1'rACAAAGTAGCTTCCATCCAGGGGATGAAGA

ACGCCACCAGCAGAAGACT'GCAAACCCTITAATTTGATGTATii3TITrrAACA3GT6

A~AAA~TAGAAGA3~3TAAAGGAAT&AAA _ GGAGCGACTACrTiGTAIT7GTACTGC

218 CATICCTAATGTACTATACTI'rrGCAGCATAAAT ATTAAATAGAAAAA720

AAAAAAAAAA

FIG. 1. Nucleic acid and deduced amino acid sequence of the MPTP cDNA. Asterisks indicate the 3' end sequence that differs between theMPTP and the published human TC-PTP. Underlined are areas of the sequence used to derive the oligonucleotides H1 and H2.

3781200

3821260

1320

1380

1440

1500

1560

1570GlyIleGlyArgSerGlyThrPheSerLeuValAspThrCysLeuValLeuMetGluLys 238GSCA CGG3GCGCT3GCACCT7CTC7CTITlAGATACC IGAMGAAMA 780

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from the conserved sequences of published PTP genes wereused in an attempt to identify MPTP genes by PCR. Thetemplates were mouse cDNA libraries as well as mouse andhuman genomic DNA. With primers H1 and H2 (see Mate-rials and Methods), a PCR fragment of 350 bp was amplifiedfrom human genomic DNA. The nucleotide sequence re-vealed that the fragment was composed of two conservedsequences both of which 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-stringencyconditions. From this library two identical phage clones wereisolated that contained a 1.2-kb insert complementary to the3' end ofa transcript homologous to the human TC-PTP gene.Additional cDNA clones isolated by hybridization to theL-2-kb cDNA from different cDNA libraries were also trun-cated at the 5' end. We used the PCR to amplify the missingregion from cDNA libraries.The full-length MPTP cDNA (1570 bp) is 88.8% identical

with the human TC-PTP cDNA in the conserved PTP do-main. The single open reading frame in the MPTP sequencepredicts a protein of 382 amino acid residues (Fig. 1). Theputative start codon is preceded by the consensus sequencefor translation initiation (CCCGCCGCC; ref. 24). A polya-denylylation signal was found 90 bp upstream of the polya-denylylation site. Downstream of position 1196 in the mousecDNA sequence the homology to the human TC-PTP cDNAends abruptly, so that the predicted carboxyl terminus of themouse protein is shorter and differs significantly from that ofthe predicted carboxyl terminus of the TC-PTP (18) (Fig. 2;see also Fig. 6). In addition, the noncoding 3' end of theMPTP cDNA was found to be considerably shorter than thehuman counterpart. In comparison to the TC-PTP, the MPTPhas a 15-bp deletion (after bp 1041) near the 3' end of theregion of homology that in the TC-PTP encodes the penta-peptide DRCTG (see Fig. 2). The PTP domain (96.5% iden-tical to the TC-PTP) contains an invariant cluster of aminoacids, HCSAGXGRXG, that is known to be essential forenzymatic activity and possibly constitutes a part of the PTPcatalytic site (14, 25).The full-length cDNA was transcribed in vitro from the T3

promoter. The mRNA was then translated using a wheatgerm translation system. The translation product closelyapproximates the predicted size of the full-length MPTP(44,640 Da) (Fig. 3). The MPTP cDNA was also expressed inEscherichia coli. The product, of Mr 45,000, has PTPaseactivity that is inhibited by sodium vanadate (U.T., unpub-lished data).

Expression of MPTP mRNA in the Mouse Embryo and inAdult Tissues. Northern blot analysis of total RNA isolatedfrom different tissues revealed that the MPTP gene encodes

Proc. Natl. Acad. Sci. USA 89 (1992) 501

M 1 2 3 4

974-5 '' ' .': ,

39- _

46- i

FIG. 3. In vitro translation of MPTP mRNA. The full-lengthMPTP cDNA was transcribed from the T3 promoter. Increasing

amounts of MPTP mRNA were translated with wheat germ in vitro

translation kit (Boehringer Mannheim) and the product was electro-

phoresed in an SDS/12.5% polyacrylamide gel. Lanes: M, markers

(Mr X 10-3); 1, no RNA; 2, 0.1 g.tg; 3, 0.2p:g; 4, 0.3 jltg.

FI.9-kb mRNA. Since the cloned cDNA is 1.57 kb long, thefull-length transcript probably contains a considerably longer

5' untranslated sequence and/or a longer poly(A) tail. The

single 1.9-kb transcript was present in each of the adult

mouse tissues examined. The highest expression was found

in the ovaries, testes, thymus, and kidneys (Figs. 4 B and 5).

The expression ofMPTP was low in embryonic stem cells and

increased during later stages of development (Fig. 4A). A

second RNA transcript, of c1.3 kb, was detected exclusivelyin the testes (Fig. 4B). The smaller transcript did not hybrid-ize to a probe corresponding to bp 1195-1431 of the 3' MPTP

cDNA sequence, suggesting that this transcript was trun-cated at the 3' end (Fig. 5).

Identification of Two Human TC-PTP Transcripts. We

attempted to elucidate the observed sequence divergencebetween mouse and human cDNA termini. Total RNA iso-

lated from human placenta, human testes, and the humanendometrial carcinoma cell line RL95-2 (27) was hybridizedto the unique 3' sequence of the MPTP cDNA and to a probe

derived from the conserved PTP domain. The unique 3'

sequence detected an RNA of the same size and abundance

as that recognized by the PTP-specific probe (Fig. 5), sug-

gesting that the major human TC-PTP transcript contained

sequences homologous to the 3' terminus of the mouse

MPTP. We then used the RACE protocol (23) to isolate the

3' end sequence of the human TC-PTP cDNA. Analysis of

eight clones revealed that four contained a sequence highly

homologous (89.1% identical) to the MPTP cDNA, while the

other four contained a sequence identical to the published

TC-PTP but terminating at position 1535 of the TC-PTPcDNA (Fig. 6). Both sequences had a putative polyadenyly-

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

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

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

MPTP TAQKVQQMKQRLNETERKRKR PRLTDT* 382TC-PTPa --------------N------ ------* 387TC-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.

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A

28S-a

18S-a-

ES 8.5 10 I1.5

"lwi:q s? _

BTH TE KI LLJ MU SP

4.40 -

BR HR LI

Fi-

2 37 - -- a,

41.35-

0.24 -

FIG. 4. Expression of MPTP mRNA shown by Northern blotanalysis of total RNA (20 ikg per lane) extracted from mouse tissuesand embryonic stem cells. Autoradiograms were exposed for 4 dayswith intensifying screens. (A) RNA from undifferentiated mouseembryonic stem (ES) cell line D3 (26) and from 8.5-, 10.6-, and11.5-day mouse embryo. Positions of 28S (4.7 kb) and 18S (1.9 kb)ribosomal RNAs are indicated. (B) RNA from adult mouse thymus(TH), testis (TE), kidney (KI), lung (LU), skeletal muscle (MU),spleen (SP), brain (BR), heart (HR), and liver (LI). Positions ofRNAsize markers (kb) from Bethesda Research Laboratories are at left.

lation signal, located 92 and 21 bp, respectively, before theend of the transcripts.

DISCUSSIONA cDNA encoding a mouse non-receptor-type PTP (MPTP)is described. The cDNA sequence predicts a protein con-

taining 382 amino acid residues with Mr 44,640. A protein ofsimilar size was also obtained by in vitro translation. TheMPTP gene is transcribed throughout development in avariety of adult tissues, with highest expression in the ova-

ries, testes, thymus, and kidneys.In addition to the major MPTP transcript (1.9 kb), a

second, testes-specific message (1.3 kb) was detected. Theshorter transcript does not hybridize to a probe generatedfrom the 3' end of the MPTP cDNA and is therefore likely tobe truncated at the 3' end, although it could conceivably beencoded by a different gene. No second message was de-tected in RNA of human testes.The similarity between MPTP and human TC-PTP at both

the nucleic acid (88.8%) and the amino acid (93.2%) levelindicates that they are indeed homologs. However, the twocDNAs differ markedly at their 3' termini. There is a putativesplice donor site (AGGT) present in the published TC-PTPcDNA at the position where the sequence diverges from theMPTP, suggesting the possibility of an alternative splicingevent.

Using the PCR technique, we have isolated two variants ofthe human TC-PTP cDNA sequence containing different 3'

|.1

FIG. 5. Expression of human TC-PTP and MPTP mRNA shownby Northern blot analysis of total RNA (20 ,ug per lane) from human(HU) endometrial carcinoma cell line RL95-2 (27) (RL), testis (TE),and placenta (PL) and from mouse (MO) kidney (KI), testis (TE), andovary (OV). (A) The filter was hybridized with a probe derived fromthe conserved domain of mouse MPTP cDNA (see text). (B) Thesame filter was stripped and rehybridized with the probe correspond-ing to the MPTP 3'-end sequence. The hybridization temperature was600C and the final wash was 30 min in 1 x SSC at 600C. Autoradio-grams were exposed for 2 days with intensifying screens. Positionsof 28S and 18S ribosomal RNAs are marked.

end sequences. One sequence (referred to as TC-PTPa) ishighly homologous (89.1%) to the mouse MPTP sequence andpredicts a protein with a carboxyl terminus identical to thatof the MPTP. The second sequence (TC-PTPb) is identical tothe published TC-PTP cDNA but terminates at position 1535.Both cDNA clones contain a consensus polyadenylylationsignal and correspond well to the size ofthe mouse cDNA andhuman mRNA (detected on Northern blots), respectively.Our data suggest that the TC-PTPa mRNA could in fact be

the major TC-PTP transcript in human tissues. Northernanalysis showed similar signal intensity in the major 1.9-kbhuman transcript when probes corresponding either to theconserved PTP domain or to the MPTP 3' end were used.Also, 16 independently isolated mouse MPTP clones con-tained the same 3' end sequence.

Despite recent advances in the identification ofPTPs, theirfunctions are largely unknown. The PTPs could target thesame substrates as PTKs and therefore act as negativeregulators. Alternatively, they could target and/or dephos-phorylate tyrosine residues within the PTKs themselves,which has been shown to increase the activity of some PTKs(7, 28-30). Involvement of PTPs in cell signaling could bemodulated at a more subtle level by proteins that containSH2/SH3 Src-homology domains. These proteins have re-cently been shown to bind phosphotyrosine residues and to

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Proc. Natl. Acad. Sci. USA 89 (1992) 503

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

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

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

MPTPAGATGTAAAGGA_1ATTAGGAGCGACTACTTTGTATTGTACTGCCATTCCTAATGTATTTTTATACTTTTTGGCAGCATTAAATATTTTTATTAAATAGA53AA 1573TC-PTPa -C-----C-A--------------- A--T--------------------------C-C-----------------------------------G-------- TCAAAAAAAAAAAAAAAA 1574TC- PTPb AGGACTCTTGACTGCAGGTTCCTCTGAACCCCAAACTGTAAATGGCTGTCTAAAT GACATTCATGTTTGT ._ 1573

FIG. 6. Comparison of the 3' end sequences ofMPTP cDNA and the two TC-PTP variants (TC-PTP sequence is identical to TC-PTPb). Theconsensus 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 tyrosinekinase receptors (31). This possibility has been stronglysupported by the recent isolation of a cytoplasmic PTPcontaining an SH2 domain (32).

Since upregulated PTKs are capable of promoting celltransformation, it is plausible that downregulation of PTPscould lead to similar effects. Interestingly, in some humanlung and renal cancers a region of chromosome 3 (3p21)harboring a receptor-type PTPy gene is frequently deleted(33).

Proteins homologous to the cytoplasmic PTPs were alsofound to be encoded by "YOP H" genes present on plasmidsof several bacterial strains of Yersinia and by the VH1 geneof vaccinia virus. When expressed in Escherichia coli, theseproteins were shown to have PTP enzymatic activity. More-over, in Yersinia strains, the presence of YOP H genes hasbeen shown to be obligatory for pathogenesis (34, 35).Although we cannot predict how many cytosolic PTPs

remain to be discovered, the low number of these enzymesdetected to date, together with their ubiquitous expression,suggests a general role in cellular regulation and perhapsinvolvement in the pathogenesis of certain diseases.

We are grateful to Drs. P. E. Love and Ch. Y. Huang for criticalreview of the manuscript and to Drs. G. R. Dressler and K. Mahonfor the mouse kidney cDNA library and technical advice. This workwas in part supported by the German Academic Exchange CouncilGene Technology Program (U.T.).

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