Molecular and Cellular Biochemistry 118: 91-98, 1992. O 1992 KluwerAcademic Publishers. Printed in the Netherlands.

Molecular cloning, nucleotide sequence and expression of a cDNA encoding an intracellular protein tyrosine phosphatase, PTPase-2, from mouse testis and T-cells Hitoshi Miyasaka and Steven S.-L. Li Laboratory of Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA Received 25 May 1992; accepted 13 August 1992

Abstract The PTP-2 cDNA encoding an intracellular protein tyrosine phosphatase (PTPase-2) was isolated and sequenced from mouse testis and T-cell cDNA libraries. This PTP-2 cDNA was found to be homologous to human PTP-TC and rat PTP-S, and contained 1,551 nucleotides, including 1,146 nucleotides encoding 382 amino acids as well as 5' (61 nucleotides) and 3' (344 nucleotides) non-coding regions. Northern blot analysis indicated that PTP-2 mRNA of 1.9 Kb was most abundant in testis and kidney, although it was also present in spleen, muscle, liver, heart and brain. (Mol Cell Biochem 118: 91-98, 1992)

Key words: mouse, protein tyrosine phosphatase, cDNA cloning, nucleotide sequence, gene expression A bbreviations: PTPase - Protein Tyrosine Phosphatase (EC3.1.3.48); PTKase - Protein Tyrosine Kinase (EC2.7.1.112)

Introduction Protein tyrosine phosphorylation has been shown to play significant roles in the control of cell proliferation, differentiation, and neoplastic transformation [1, 2]. The level of cellular phosphotyrosine is determined by the relative activities of protein tyrosine kinases (PTKases, EC2.7.1.112) and protein tyrosine phosphatases (PTPase; EC3.1.3.48). Recent evidence indicates that PTPases not only simply reverse the effects of PTKases by dephosphorylating the protein substrates, but also play

important roles in cellular regulation [3, 4]. On the basis of molecular sizes, PTPases have been classified into two types: (i) low Mr intracellular enzyme containing a conserved PTPase domain, and (ii) high Mr receptorlinked transmembrane protein, which contains two tandemly conserved PTPase domains [5]. A low Mr PTPase was purified to homogeneity from human placenta and its amino acid sequence determined [6]. With the use of oligonucleotide probes/primers derived from this protein sequence, cDNAs encoding low Mr PTPases have been cloned from human placenta (PTP-1B), human T-cells (TC-PTP), human HeLa cells (PTPH1),

Address for offprints: S.S.-L. Li, Laboratory of Genetics, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709, USA

92 human breast carcinoma (PTP-1C), rat brain (PTP-1), rat spleen (PTP-S), and rat striatum (STEP) [7-14]. The leukocyte common antigen CD45 has been shown to exhibit sequence homology with PTP-1B and to have PTPase activity [15, 16]. Several cDNAs encoding receptor-linked PTPases have been cloned [17-21] and the human genes coding for receptor-linked PTPasegamma and L A R were further reported to be putative tumor suppressor genes [22, 23]. We are studying the developmental regulation of gene expression during mammalian spermatogenesis [24, 25]. Several PTK genes such as c-abl, pim-1 and ferT have been reported to exhibit stage-specific expression during spermatogenesis [26-30]. To investigate expression and regulation of PTP genes during spermatogenesis, we reported the PTP-1 cDNA encoding a low Mr intracellular PTPase-1 from mouse testis [31]. This paper describes the cloning, nucleotide sequence and deduced amino acid sequence of PTP-2 cDNA from mouse testis and T-cells as well as the results of Northern blot analysis.

Materials and methods Materials The rat PTP-1 cDNA probe was kindly provided by Drs. K. Guan and J. Dixon [8]. The lambda gt]l cDNA library was prepared by oligo(dT) priming of mRNAs from a mixture of germ cells isolated from adult CD-1 male mice, and was generously given by Drs. J. Welch and E.M. Eddy [32]. The lambda ZAP II cDNA library of EL4 T-cells derived from C57BL/6N mouse was obtained from Stratagene.

Isolation and characterization of cDNA clones The EcoRI-SacII D N A fragment containing the protein-coding sequence of rat PTP-1 cDNA was used as a probe to screen the mouse germ cell cDNA library using standard procedures [33]. The Genescreen membrane was hybridized at 60°C overnight in 1M NaC1/1% SDS/10% dextran/50 mM Tris-HCl, pH 7.5/200/~g/ml salmon sperm DNA, and washed twice with 2 × SSC (0.3 M sodium chloride/0.03 M sodium citrate, pH 7) at room temperature. The isolated PTP-2 cDNA of mouse germ cells was used as a template for PCR-amplification to obtain a cDNA probe of the catalytic domain,

and this probe was then used to screen the mouse T-cell cDNA library. The cDNA insert from the plaque-purified lambda gtll phages was isolated and subcloned in pBluescript phagemid. The cDNA insert from lambda ZAP II vector was excised to the Bluescript SK ( - ) phagemids. The nucleotide sequence of cDNA inserts was determined by the dideoxy chain-termination method using a T7 DNA polymerase [34]. Oligonucleotides synthesized according to the newly determined sequence were used as primers to obtain additional sequence. Both strands of the cDNA inserts were completely sequenced.

Northern blot analysis Tissues including testis, spleen, muscle, liver, kidney, heart and brain, of CD-1 mice (six weeks old) were quickly frozen in liquid nitrogen, and total RNAs were extracted by the guanidinium thiocyanate-phenol-chloroform method using the RNAzol B kit (Biotecx Laboratories, Inc.). Poly(A)+ RNAs were purified by oligo(dT) cellulose using the FastTrack kit (Invitrogen Corp.). Ten micrograms of poly(A)+ RNAs were electrophoresed on a 1.2% agarose/2.1% formaldehyde gel, transferred to a Nytran membrane (Schleicher & Schuell) by capillary blotting with 10 x SSC (1.5M sodium chloride/0.15 M sodium citrate, pH 7), and UVcross-linked as described [23]. The Northern blot was hybridized overnight at 42°C in 50% Formamide/5 × Denhardt's solution/5 x SSPE (0.75M sodium chloride, 50mM sodium phosphatate, 5mM EDTA, pH 7.4)/1% SDS/salmon sperm DNA (200/~g/ml) with randomly 32P-labeled cDNA probes of PTP-2. The filters were rinsed twice at room temperature for 15 min each and washed twice at 65° C for 15 rain each with 1 × SSPE/0.1% SDS. Autoradiograms were obtained by exposing the blot to Kodak XAR-5 film with intensifying screen at - 70 ° C for various lengths of times.

Computer analyses of amino acid sequences The deduced amino acid sequences of mouse PTP-1 [31] and PTP-2 (this report), human PTP-1B [6, 9, 10] and PTP-TC [7], and rat PTP-1 [8] and PTP-S [11] were compared and analyzed using GCG programs developed by The University of Wisconsin Genetics Computer Group [35].

93 PTP-2

Results

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Isolation and sequence analysis of PTP-2 cDNA Several strongly and two weakly positive cDNA clones were identified and plaque-purified from a library prepared from germ cells of male mice by screening with the rat PTP-1 probe [8]. The cDNA inserts from these strongly positive clones were reported to contain PTP-1 cDNA [31]. The cDNA inserts from the two weakly positive clones were isolated and partially characterized in this investigation. The complete nucleotide sequence of clone mY257 cDNA was determined (fig. 1) and found to contain a cDNA insert of 1,551 nucleotides, including a protein-coding sequence of 1,146 nucleotides and 5' (61 nucleotides) and 3' (344 nucleotides) non-coding regions (Fig. 2). The deduced sequence of 382 amino acids from clone mY257 exhibited only 51% identity to that of mouse PTPase-I reported previously [31]. However, this PTPase-2 had 91%, 90%, and 83% identity to the sequence of rat PTPase-S [11], human PTPase-TCa [36], and human PTPase-TCb [7], respectively. Thus, clone mY257 contained a cDNA encoding the mouse homologue (PTPase-2) of rat PTPase-S, human PTPase-TCa and human PTPase-TCb. Mouse PTPase-2 has a calculated Mr of 44,522 and its composition of 382 amino acids is: 14 Ala, 30 Arg, 17 Asn, 21 Asp, 8 Cys, 19 Gln, 33 Glu, 16 Gly, 10 His, 17 Ile, 32 Leu, 28 Lys, 10 Met, 12 Phe, 16 Pro, 28 Ser, 26 Thr, 6 Trp, 14 Tyr, and 25 Val. The PTPase-2 from mouse testis appeared to lack the COOH-terminus of 23 hydrophobic amino acids present in human PTPase-TCb from T-cells [7]. This difference might be due to alternative splicing of PTP-2 transcripts in mouse testis and T-cells. Two clones (mY502 and mY504) were then isolated from a library prepared from a T-cell line of the C57BL/6N mouse and the nucleotide sequences of their cDNA inserts determined. The cDNA insert of clone mY502 contained the entire protein-coding sequence of 1,146 nucleotides and 5' (3 nucleotides) and 3' (316 nucleotides) non-coding regions. The 5' sequence of the cDNA insert from clone mY504 was truncated at the 5th codon, while its 3' non-coding region had 319 nucleotides. The cDNA sequences of T-cell (C57B1/6) clones mY502 (1,465 nucleotides) and mY504 (1,451 nucleotides) were found to be identical to that of germ cell (CD-1) clone mY257, except for three polymorphic differences, that is, a silent change from a T to C at codon 12 (nucleotide no. 95

mY502

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mY504

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Fig. 1. Sequencing strategy of mouse PTP-2 cDNA clones. The protein-coding sequence is shown by open box. The 5' and 3' non-coding regions are indicated by solid lines. Clone mY257 was isolated from a lambda gtll library prepared with mixed germ cells from male mice [32], while clones mY502 and mY504 were identified from lambda ZAPII library prepared from a mouse T-cell line. The direction and length of each sequencing run are indicated by horizontal arrows. The sequence of the complementary strands of the cDNA inserts from clone mY257 was determined completely.

of mY257 cDNA), a replacement of G by A and a deletion of C at nucleotide no. 1477-8 in the 3' noncoding region of mY502 and mY504. Therefore, mouse PTP-2 mRNAs in testis and T-cells are transcribed and processed similarly from the same PTP-2 gene.

Expression of PTP-2 poly(A) + RNA The results of Northern blot analysis of poly(A)+ RNAs isolated from mouse testis, spleen, muscle, liver, kidney, heart and brain are given in Fig. 3. The single PTP-2 mRNA was detected to be approximately 1.9 Kb. Clones mY257, mY502 and mY504 contained PTP-2 cDNA inserts of 1,551, 1,465 and 1,451 nucleotides, respectively, and all of them lacked polyadenylation signal and poly(A)-tail. Therefore, their 3' noncoding regions were truncated. The mRNA of PTP-2 appeared to be abundant in testis and kidney, and was also present in other tissues investigated. Rat PTP-S mRNAs of 1.7 Kb were found in spleen, thymus, macrophage and brain [11]. Human PTP-TCb mRNAs of 2.3 Kb were present in T-cells and placenta [7].

Discussion Mammalian PTPases have been classified into two families: a low Mr intracellular PTPase and a receptorlinked transmembrane PTPase [5]. Human PTPase-IB [6, 9, 10], PTPase-Tca [36] and PTPase-TCb [7], mouse PTPase-1 [31] and PTPase-2 (this report), and rat

CGGATAGAGCGGGGCCCGAGCCTGTCCGCTGTGGTAGTTCCGCTCGCGCTGCCCCGCCGCC

1381 1501 1551

CAGCAGAAGACTTGCAAACCCTTTAATTTGATGTATTGTTTTTTAACATGTGTATGAAATGTAGAAAGATGTAAAGGAAATAAATTAGGAGCGACTGCCTTTGTATTGTACTGCCATTCC

TAATGTATTTTTATACCTTTTTGGCAGCATTAAATATTTTTATTAAATAG

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AT G T C G G C A A C C A T C G A G C G G G A G T T C G A G G A A T T G G A T G C T C A G T G T C G C T G G C A G C C G T T A T A C T TGGAAATTCGAAATGAATCCCATGACTATCCTCATAGAGTGGCCAAGTTTCCA M S A T I E R E F E E L D A Q C R W Q P L Y L E I R N E S H D Y P H R V A K F P GAAAACAGAAACCGAAACAGATACAGAGATGTAAGCCCATATGATCACAGTCGTGTTAAACTGCAAAGTAC TGAAAATGATTATATTAATGCCAGCT TAGTTGACATAGAAGAGGCACAA E N R N R N R Y R D V S P Y D H S R V K L Q S T E N D Y I N A S L D V I E E A Q AGAAGTTACATCTTAACACAGGGCCCACTTCCGAACACATGCTGCCATTTCTGGCTCATGGTGTGGCAGCAAAAGACCAAAGCAGTTGTCATGCTAAACCGAACTGTAGAAAAAGAATCG R S Y I L T Q G P L P N 'T C C H F W L M V W Q Q K T K A V V M L N R T V E K E S G TTAAATGTGCACAGTACTGGCCAACGGATGACAGAGAAATGGTGTTTAAGGAAACGGGATTCAGTGTGAAGCTCTTATCTGAAGATGTAAAATCATATTATACAGTACATC TACTACAG V K C A Q Y W P T D D R E M V F K E T G F $ V K L L S E D V K S Y Y T V H L L Q TTAGAAAATATCAATACTGGTGAAACCAGAACCATATC TCAC T TCCATTATACCACCTGGCCAGATTTTGGGGTTCCAGAGTCACCAGCTTCATTTCTAAACTTCT TGTTTAAAGTTAGA L E N I N T G E T R T I S H F H Y T T W P D F G V P E S P A S F L N F L F K V R GAATCTGGTTGTTTGACCCCTGACCATGGACCTGCAGTGATCCATTGCAGTGCGGGCATCGGGCGCTCTGGCACCTTCTC TCTTGTAGATACCTGTC T TGTTCTGAT GGAAAAAGGAGAG E S G C L T P D H G P A V I H C S A G I G R S G T F S L V D T C L V L M E K G E GATGTTAATGTGAAACAATTATTACTGAATATGAGAAAGTATCGAATGGGACTTATTCAGACACCGGACCAACTCAGATTCTCCTACATGGCCATAATAGAAGGAGCAAAGTACACAAAA D V N V K Q L L L N M R K Y R M G L I Q T P D Q L R F S Y M A I I E G A K Y T K GGAGATTCAAATATACAGAAACGGTGGAAAGAACTTTCTAAAGAAGATTTATCTCCTATTTGTGATCATTCACAGAACAGAGTGATGGTTGAGAAGTACAATGGGAAGAGAATAGGTTCA G D S N I Q K R W K E L S K E D L S P I C D H S Q N R V M V E K Y N G K R I G S GAAGATGAAAAGTTAACAGGGCTTCCTTCTAAGGTGCAGGATACTGTGGAGGAGAGCAGTGAGAGCATTCTACGGAAACGTATTCGAGAGGATAGAAAGGCTACGACGGCTCAGAAGGTG E D E K L T G L P S K V Q D T V E E S S E S I L R K R I R E D R K A T T A Q K V CAGCAGATGAAACAGAGGC TAAATGAAACTGAACGAAAAAGAAAAAGGCCAAGATTGACAGACACCTAAATGTTCATGAC T TGAGAC TATTCTGCAGCTATAAAAT T TGAACC T TTGATG

PTP-2

deduced amino acid sequence is given below its nucleotide sequence.

Fig. 2. The nucleotide and deduced amino acid sequences of mouse PTP-2 cDNA. The complete nucleotide sequence of mouse PTP-2 (clone mY257) is presented, and the

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95 PTPase-1 [8] and PTPase-S [11] belong to the low Mr intracellular PTPase family. The amino acid sequences of these PTPases are compared in Fig. 4. Mouse PTPase-1 exhibits 93% and 83% identity to rat PTPase-1 and human PTPase-IB, respectively, while mouse PTPase-2 have 91%, 90% and 83% identity to rat PTPase-S, human PTPase-TCa and human FFPase-TCb, respectively. These low Mr PTPase family can further be divided into two different subfamilies. There is only 48-52% identity between one subfamily containing mouse PTPase-1, rat PTPase-1 and human PTPase-IB and the other subfamily containing mouse PTPase-2, rat PTPase-S, human PTPase TCa and human PTPase-TCb. Therefore, mouse PTP-1, rat PTP-1 and human PTP-1B are closely homologous to each other, and they were evolved from the same ancestral PTP-1 gene. On the other hand, mouse PTP-2, rat PTP-S, human PTP-TCa and human PTP-TCb must have originated from another ancestral PTP-2 gene. These two subfamilies may simply be referred to as PTP-1 and PTP-2 gene subfamilies, and they probably duplicated and diverged before speciation of mouse, rat and human. As to human PTPase-TCa [36] and PTPase-TCb [7], both proteins contain the identical sequence of NH2-terminal 381 amino acids, and they differ at their COOH-termini of 6 or 34 residues. This difference at the COOH-terminus as well as 3' noncoding region might be due to alternative splicing at the presumably exon-intron junction between residues 381-382. On the basis of sequence variation, the protein structure of PTPase-1 and PTPase-2 may be subdivided into NH2-terminal, catalytic, and COOH-terminal domains (Fig. 4). The NH2-terminal domains of 39 or 41 amino acids exhibit 90-100% identities within the same subfamily and 39-41% of identities between different subfamilies. The catalytic domains of 249 or 245 amino acids exhibit the highest degree of identities: 94-97% within same subfamily and 71-73% between different subfamilies. It may be noted that the Cys at residue no. 215 or 216 was previously shown to be the active site and the basic sequence of NRNRYR at residues no. 42-47 or 44-49 was conserved in these PTPases. The COOHterminal domain is highly variable in sizes and sequences. PTPase-1 has 144 or 147 amino acids; PTPase-2 from rat PTP-S, mouse PTP-2, human PTP-TCa and human PTP-TCb has 77, 96,101 and 129 residues, respectively. There are 48-88% identities within the same subfamily, but only 9-21% identities are observed between subfamilies. It is also of interest to note that NH2-terminal, cata-

Fig. 3. Northern blot analysisof mouse PTP-2 poly(A)+ RNAs.

Lanes1, testis;2, spleen;3, muscle;4, liver(3/~g);5, kidney;6, heart; 7, brain; 8, testis (5/zg). Ten microgramsof poly(A)+ RNAs were loaded except where indicated. The blot was hybridizedwith the cDNAprobe containingCOOH-terminaland 3' non-codingregionof PTP-2. lytic and COOH-terminal domains of these PTPases have quite different properties. The NH2-terminal domain has net negative charge and acidic isoelectric point (pI of around 5), and the catalytic domain is nearly neutral (pI of about 7). The COOH-terminal domain of PTPase-1 has highly negative charge with acidic pI of approx. 5, while that of PTPase-2 has highly positive charge with a basic pI of around 11. As to the total protein, three PTPases-1 have net charge of - 9, - 11 or - 12, and acidic pI of about 6. On the contrary, all PTPase-2 have net charges of + 3 or + 4 and basic pI of approximately 8. It has been suggested that the COOH-terminal domain of the low Mr PTPases may have important regulatory roles such as providing signals for specific intracellular locations [5]. The COOH-terminal 114 amino acids of human PTPase-IB were truncated during the enzyme purification from placenta [6] and the COOH-terminus of 19 hydrophobic amino acids, including two conserved Cys residues, has been suggested to be responsible for localizing the full-length enzyme to the particulate membrane fraction through posttranslational modification of polyisoprenylation and/or palmi-

96 Human PTP-IB Rat PTP-I Mouse PTP-I Mouse PTP-2 Rat PTP-S Human PTP-TC

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Human Rat Mouse Mouse Rat Human

PTP-IB PTP-I PTP-I PTP-2 PTP-S PTP-TC

VSPFDHSRIKLHQEDNDYINASLIKMEEAQ RSYILTQGPL PNTCGHFWEM VSPFDHSRIKLHQEDNDYIN ASLIKMEEAQ RSYILTQGPL PNTCGHFWEM VSPFDHSRIKLHQEDNDYIN ASLIKMEEAQ RSYILTQGPL PNTCGHFWEM VSPYDHSRVKLQSTENDYIN ASLVDIEEAQ RSYILTQGPL PNTCCHFWLM VSPYDHSRVKLQSAENDYIN ASLVDIEEAQ RSYILTQGPL PNTCCHFWLM VSPYDHSRVKLQNAENDYIN ASLVDIEEAQ RSYILTQGPL PNTCCHFWLM

98 98 98 I00 i00 I00

Human PTP-IB Rat PTP-I Mouse PTP-I Mouse PTP-2 Rat PTP-S Human PTP-TC

VWEQKSRGVVMLNRVMEKGSLKCAQYWPQK EEKEMIFEDT NLKLTLISED VWEQKSRGVVMLNRIMEKGS LKCAQYWPQK EEKEMVFDDT NLKLTLISED VWEQKSRGVVMLNRIMEKGS LKCAQYWPQQ EEKEMVFDDT GLKLTLISED VWQQKTKAVVMLNRTVEKES VKCAQYWP.T DDREMVFKET GFSVKLLSED VWQQKTRAVVMLNRTVEKES VKCAQYWP.T DDREMVFKET GFSVKLLSED VWQQKTKAVVMLNRIVEKES VKCAQYWP.T DDQEMLFKET GFSVKLLSED

148 148 148 149 149 149

Human PTP-1B Rat PTP-I Mouse PTP-I Mouse PTP-2 Rat PTP-S Human PTP-TC

IKSYYTVRQLELENLTTQET REILHFHYTT WPDFGVPESP ASFLNFLFKV VKSYYTVRQLELENLATQEAREILHFHYTT WPDFGVPESP ASFLNFLFKV VKSYYTVRQLELENLTTKET REILHFHYTT WPDFGVPESP ASFLNFLFKV VKSYYTVHLLQLENINTGET RTISHFHYTT WPDFGVPESP ASFLNFLFKV VKSYYTVHLLQLENINSGET RTISHFHYTT WPDFGVPESP ASFLNFLFKV VKSYYTVHLLQLENINSGET RTISHFHYTT WPDFGVPESP ASFLNFLFKV

198 198 198 199 199 199

Human PTP-IB Rat PTP-I Mouse PTP-I Mouse PTP-2 Rat PTP-S Human PTP-TC

RESGSLSPEHGPVVVHCSAG IGRSGTFCLA DTCLLLMDKR KDPSSVDIKK RESGSLSPEHGPIVVHCSAG IGRSGTFCLA DTCLLLMDKR KDPSSVDIKK RESGSLSLEHGPIVVHCSAG IGRSGTFCLA DTCLLLMDKR KDPSSVDIKK RESGCLTPDHGPAVlHCSAG IGRSGTFSLV DTCLVLMEKG ED...VNVKQ RESGSLNPDHGPAVlHCSAG IGRSGTFSLV DTCLVLMEKG ED...VNVKQ RESGSLNPDHGPAVlHCSAG IGRSGTFSLV DTCLVLMEKG DD...INIKQ

248 248 248 246 246 246

Human PTP-1B Rat PTP-I Mouse PTP-I Mouse PTP-2 Rat PTP-S Human PTP-TC

VLLEMRKFRMGLIQTADQLR FSYLAVlEGA KFIMGDSSVQ DQWKELSHED VLLEMRRFRMGLIQTADQLR FSYLAVlEGA KFIMGDSSVQ DQWKELSHED VLLEMRRFRMGLIQTADQLR FSYLAVlEGA KFIMGDSSVQ DQWKELSRED LLLNMRKYRMGLIQTPDQLR FSYMAIIEGA KYTKGDSNIQ KRWKELSKED ILLSMRKYRMGLIQTPDQLR FSIMAIIEGA KYTKGDSNIQ . . . . . . . . . . VLLNMRKYRMGLIQTPDQLR FSYMAIIEGA KCIKGDSSIQ KRWKELSKED

298 298 298 296 286 296

Human Rat Mouse Mouse Rat Human

PTP-IB PTP-I PTP-I PTP-2 PTP-S PTP-TC

LEPPPEHIPPPPRPPKRILE PHNGKCREFFPNHQWVKEET QEDKDCPIKE LEPPPEHVPPPPRPPKRTLE PHNGKCKELFSNHQWVSEES CEDEDILARE LDLPPEHVPPPPRPPKRTLE PHNGKCKELFSSHQWVSEET CGDEDSLARE LSPICDH. . . . . SQNRVMVE KYNGK. . . . . . . . . . . . . . R IGSEDEKL.. .............. NRTMTE KYNGK. . . . . . . . . . . . . . R IGSEDEKL.. LSPAFDH. . . . . SPNKIMTE KYNGN. . . . . . . . . . . . . . R IGLEEEKLTG

348 348 348 325 306 327

Human PTP-IB Rat PTP-I Mouse PTP-I Mouse PTP-2 Rat PTP-S Human PTP-TC

EKGSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPA KGEPSLPEKD E.SRAPSlAV HSMSSMSQDTEVRKRMVGGGLQSAQASVPT EEELSPTEEE E.GRAQSSAMHSVSSMSPDTEVRRRMVGGGLQSAQASVPT EEELSSTEEE ...TGLPSKV QDTVEESSES ILRKRIREDR .KATTAQKVQ QMKQRLNETE ...TGLSSKV PDTVEESSES ILRKRIREDR .KATTAQKVQ QMRQRLNETE DRCTGLSSKMQDTMEENSESALRKRIREDR ,KATTAQKVQ QMKQRLNENE

398 397 397 371 352 376

NKNRNRYRD NKNRNRYRD NKNRNRYRY NRNRNRYRD NRNRNRYRD NRNRNRYRD

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Human PTP-IB E.DHALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT. Rat PTP-I QKAHRPVHWKPFLVNVCMATALATGAYLCY RVCFH. . . . Mouse PTP-I HKAHWPSHWKPFLVNVCMATLLATGAYLCY RVCFH. . . . Mouse PTP-2 RKRKR. . . . . PRLTDT. . . . . . . . . . . . . . . . . . . . . . . Rat P T P - S RKRKR. . . . . PRLTDT. . . . . . . . . . . . . . . . . . . . . . . Human PTP-TCa RKRKR. . . . . PRLTDT. . . . . . . . . . . . . . . . . . . . . . . Human PTP-TCb RKRKRWLYWQ PILTKMGFMS VlLVGAFVGW TLFFQQNAL

435 432 432 382 363 387 415

Fig. 4. A m i n o acid sequence alignment of mouse PTPase-1 and PTPase-2, rat PTPase-1 and PTPase-S, and h u m a n PTPase-IB, PTPase-TCa and PTPase-TCb. A m i n o acid sequences of mouse PTPase-1 [31] and PTPase-2 (this report), h u m a n PTPase-IB [6, 9-10], PTPase-Tca [36] and PTPase-TCb [7], and rat PTPase-1 [8] and PTPase-S [11] were aligned by a G C G computer program [35]. Gaps are indicated by dots. These amino acid sequences were subdivided into NH2-terminal, catalytic and C O O H - t e r m i n a l domains at positions denoted by vertical arrows. The first boundary at residues no. 3 9 ~ 0 of h u m a n PTPase-IB was previously used to demarcate the conserved catalytic domain. The second boundary was defined at residues no. 286-7, because an intron in h u m a n PTP-1B gene as well as a deletion in rat PTP-S sequence is located.

97 toylation [8]. The COOH-terminal domain of PTPase-2 from mouse PTP-2, rat PTP-S and human PTP-TCa contains 96, 77 and 101 amino acids, respectively, and they possess the identical sequence of their last 12 amino acids. As stated above, the COOH-terminal sequence of 34 residues from human PTP-TCb [71 is totally different from those of human PTP-TCa, mouse PTP-2 rat PTP-S. The sequence of COOH-terminal 57 amino acids from rat PTPase-S was previously shown to exhibit relatively strong homology to the basic region of transcription factors Jun and Fos [11]. Further, the sequence of R K R K R at residues no. 377-381 of human PTPase-TCa and PTPase-TCb is conserved in mouse PTPase-2 and rat PTPase-S, but it is absent in three PTPase-l. This stretch of five basic amino acids had been previously identified as nuclear localization signals in certain protooncogenes [5]. Thus, it is tempting to speculate that PTPase-2 play an essential function in the nucleus, since the basic pI of 8 was found for all four PTPase-2, which suggests a general affinity for DNA. After the completion of this work, a mouse cDNA encoding the intracellular PTPase-TC was reported [36]. A comparison of the nucleotide sequences between the recently published PTP-TC and PTP-2 described in this manuscript reveals three differences, that is, single nucleotide deletions each at 5' and 3' noncoding regions in addition to a silent change at codon no. 169.

Acknowledgement We thank Drs. K. Guan and J. Dixon for providing rat PTP-1 cDNA probe, Drs. J. Welch and E.M. Eddy for giving us mouse germ cell cDNA library, and Drs. F. Johnson, E.M. Eddy, and J. Welch for reading the manuscript and making helpful suggestions, and Ms. N. Gore for preparing the manuscript.

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Molecular cloning, nucleotide sequence and expression of a cDNA encoding an intracellular protein tyrosine phosphatase, PTPase-2, from mouse testis and T-cells.

The PTP-2 cDNA encoding an intracellular protein tyrosine phosphatase (PTPase-2) was isolated and sequenced from mouse testis and T-cell cDNA librarie...
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