Vol. 189, No. 2, 1992 December 15, 1992

Cloning

BIOCHEMICAL

and characterization putative cytoplasmic

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1223-1230

of a human cDNA encoding a novel protein-tyrosine-phosphatasel

Mutsuhiro Takekawa, Fumio Itoh, Yuji Hinoda*, Yoshiaki Arimura, Minoru Toyota, Masuo Sekiya, Masaaki Adachi, Kohzoh Imai and Akira Yachi Department of internal medicine (Section I), Sapporo Medical College, Sl, W16, Chuo-ku, sapporo 060, Japan

Received

November

5,

1992

SUMMARY: We have cloned and characterized a human cDNA encoding a new member of the family of cytosolic type protein-tyrosine-phosphatase (PTP), designated as PTPGl, from an adult colon tissue cDNA library by using the PCR product as probe. We obtained 5 cDNA clones, which cover the predicted open reading frame encoding a 88-kDa protein composed of 780 amino acids, and it had no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein. The N-terminal region had a PTP catalytic domain that is 30-40% identical to previously reported human PTPs. This revealed that the enzyme composes an additional family of human PTPs. PTPGl was characterized by a long non-enzymatic domain located at the C-terminus, including PEST sequences which are characteristic for short half-life proteins in eukaryotes. Northern blot analysis of PTPGl mRNA showed a 4.6-kb transcript that was detected in a wide variety of cell lines to suggest its extensive expression. a 1992Academic mess, 1°C. Protein tyrosine phosphorylation proliferation,

differentiation,

plays a crucial role in signal transduction

of cell

and neoplastic transformation (1,2), and is dependent upon a

balance between protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activities. Previous studies have demonstrated that a number of oncogenes as well as growth factor receptors possess PTK activity. On the other hand, studies on PTPs are now rapidly growing and have revealed that PTPs play a central role in the regulation of cell function similar to PTKs (3). Furthermore, it has been suggested that they could be involved in neoplastic processes and act as tumor supressors (4,5). Their structural diversities and differences in catalytic activity suggest that these enzymes could form a large size of the gene family to participate in the control of a variety of biochemical pathways (6-8). To fully understand the physiological role of the PTPs, further analysis of new family members will obviously be required. 'Sequence GenBank

data from this article have been deposited Data Libraries under Accession No. D13380.

with

the

DDBJ/EMBL/

* To whom correspondence should be addressed. ABBREVIATIONS: PTK: protein tyrosine kinase; PTP: protein tyrosine phosphatase; LAR: leukocyte common antigen related molecule; PCR: polymerase chain reaction; cDNA: complementary DNA; PEP: PEST-domain phosphatase.

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0006-29 1X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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We have therefore isolated cDNAs encoding new members of the PTP family using PCR techniques (9) and report here three novel putative PTPs from human stomach tissues and the molecular cloning of one of them, designated as PTPGl. This gene encodes a cytosolic protein of 780 amino acids, which is characterized by a large carboxy-terminal domain with proline, serine/threonine and glutamate/aspartate-rich sequences (PEST sequences), which are indicative of the protein with rapid turnover within eukaryotic cells (10).

MATERIALS

AND METHODS

Polymerase

chain reaction and subcloning of PTP domains. Degenerate mixed oligonucleotides for PCR were derived from two highly conserved regions within the catalytic domain of the PTP family. The sense and antisense primers, corresponding to the amino acid sequences GSDYINA (S-GGITCIGAT/cTAT/cATIAAT/&C-3’, primer 1) and KCDQYWP (S-GGCCAA/~TAT/cTGIG/TCA/&AT/cTT-3’, primer 2), respectively, were used (11). Total RNA was prepared from a surgically resected gastric cancer tissue and its adjacent normal mucosa by the previously reported procedure (12). The RNA was converted to single strand cDNA with random hexamers and Moloney murine leukemia virus reverse transcriptase. This cDNA was used as a template. The PCR was carried out through 30 cycles. The early 7 cycles were for lmin and 20s at 94’C (denaturation), 2min at 48’C (annealing), and 2min at 72“C (extension). The following cycles were carried out under the same conditions except for the annealing temperature at 52°C. The PCR products were subcloned into pBlueScript SK(-) plasmid (Stratagene) and sequenced with a Sequenase kit using 7-deaza-2’deoxy-CTP (USB). Isolation of cDNA clones. A cDNA library from adult human colon tissue (Clontech) was then screened with a [32P]labeled 0.2kb PTPGl PCR product. Plaques were transferred to nitrocellulose filters (Schleicher & Schuell) and screened by hybridization at 42’C in a solution containing 50% formamide/2xSSC (1xSSC is 0.15M NaCl/O.O015M trisodium citrate, pH 7.0), SxDenhardt’s solution, O.l%SDS, 10% dextran sulfate, and denatured salmon sperm DNA (lOOug/ml). Hybridizing phages were purified, and the phage DNAs were prepared and subcloned into pBlueScript SK(-) plasmid using standard techniques. DNA sequence analysis. Closed circular plasmid DNA was purified by alkaline extraction procedure. To obtain the complete sequence on both strands, overlapping deletion mutants were generated. The double strand DNA was denatured and the DNA sequence was determined by the dideoxynucleotide chain-termination method using manufacturer’s primers. The both GenBank and Swiss Prot data bases of sequence information was searched with FASTA program to identify proteins with amino acid sequence similar to PTPGl. Northern Blot Analysis. Total RNA from human various cell lines was prepared by the guanidium/CsCl procedure. Approximately 101.18of total RNAs were separated in 1.0% agarose formaldehyde gels by electrophoresis and were blotted to nitrocellulose filters. The filters were hybridized overnight with [32P]labeled random primed 1.2 kb cDNA fragment from AGl-Cl. Hybridization conditions were the same as those described for the screening of libraries. The final wash was in O.lxSSC/O. l%SDS at 55’C. The filters were exposed to film for 3 days with intensifier screens at -70°C. Southern Blot Analysis. Ten gg of human genomic DNA was digested with the restriction endonucleases Hi&III and BamHI. The blot was hybridized to the [32P]labeled 1.3kb cDNA fragment from 3’ side clone, hGl-C4, which does not contain any region of the conserved catalytic domain, and subjected to autoradiography for 3 days.

RESULTS

Isolation

of PTP related cDNA from

human gastric tissues. To identify the PTP

genes expressed in gastric mucosa, we utilized PCR amplification with degenerate primers derived from two regions highly conserved among known PTPs. Since these reactions resulted 1224

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in multiple bands, we isolated the major band of 191-bp corresponding to the expected intraprimer distance in known PTPs and some more longer bands (data not shown). Sequence analysis revealed that 46 out of 75 amplified cDNA clones encoded consensus amino acid sequences of PTPs, but the remaining 29 clones did not. These PTP-related products were classified into five different cDNA sequences. Two were identical to the known PTP genes, T cell PTP (36 clones were obtained) and PTPIB (1 clone), whereas the other three clones encoded novel putative protein tyrosine phosphatases, termed PTPGl (1 clone), PTPG2 (4 clones) and PTPG3 (4 clones), respectively (Fig. 1). To determine the primary structure of PTPGl, first of all, we screened an adult human colon cDNA library with the [s2P]labeled PCR product of PTPGl, since the transcript was detected in both gastric and colonic tissues, and the sequences of the cDNA clones were determined. Structure

of PTPGl.

The nucleotide and deduced amino acid sequences of PTPGl is

shown in Fig. 2. The overlapping 5 cDNA clones contained full length of a single long open reading frame of 2340 bp (Fig. 3). The initiation codon at the beginning of the open reading frame was flanked by a sequence that conforms to the strong consensus site of Kozak (13) for translation initiation (AGGATGG). The open reading frame terminated at position 2359 followed by a TGA termination codon. The sequence predicted a 780-amino acid polypeptide with a predicted molecular size of approximately 88 kDa. Hydropathy analysis indicates that PTPGl contains no hydrophobic segments appropriate for signal peptides or transmembrane domains and is thus also predicted to be an intracellular protein. The sequence coded for one phosphatase domain containing the conserved sequence motif, [HC231SAGCGRT],

found in

all known PTPs that is thought to be essential for phosphatase activity (6), located near the predicted amino terminus at residues 26 to 292. Therefore, there is a very large carboxyterminal extension of 488 amino acids. The carboxy-terminal domain contained approximately 470-amino-acids from positions 323 to 780 with enriched sequence for proline (11.4%), serine/ threonine (21.2%), glutamate/aspartate (15.5%). Within this area, furthermore, there were imperfect proline-rich repeats located at positions 331 to 351 and 672 to 690, and were four separate regions located at positions 361 to 385, 506 to 521, 565 to 583 and 666 to 679 in conformity to the criteria for PEST sequences which are characteristic for proteins with a rapid turnover rate (10). The PEST scores for these regions were significantly high (2.65, 14.03, 6.73 and 14.17, respectively), since PEST scores greater than 0 are indicative of potential rapid degradation (10). When comparing the amino acid sequence of PTPGl with known human PTPs on the catalytic domain, there was a significant sequence similarity in PTPP (37%), PTW domain 1 (370/o),

PTPGl PTPG2 PTPG3 Consensus

NFI KGVYGPKAYVATQGPLANTVIDFWRMVWEYNWIIVMACREFEMGRK SYI RIVNCGEEYFYIATQGPLLSTIDDFWQMVLENNSNVIAMITREIEGGII NIIMPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVFQ NSRLIVMTTKEVERGKS gsDYINA------------g------Yi--QGp--QGp---T--DFW~~~e------”M-~---E----KCdQyWp

Fig.. Sequence alignment of the PTPGl, G2, G3 PCR fragments. Deduced amino acid sequences of the PCR products as well as the consensus sequence are given in single-letter code. 122.5

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GCCGGGGGGACGCGGGAGGATGGAGCAAGTGGAGATCCTGAG~TT~TCCA~~GTCCAG~~TG~~GTCCTGAC MEQVEILRKFIQRVQAMKSPD CACAATGGGGAGGACAACTTCGCCCGGGACTTCATGCGGTT~G~~TTGTCTAC~TATAG~CAG~GATATATCCC ZZHNGEDrNFARDFMRLRRLSTKYRTEKIYP ACAGCCACTGGAGAAAAAGG~TGTTAAAAAGAACATG 50 TAT GE K E E N VK K N RY K D I L P F D H ACATTAAAGACTCCTTCACGATTCAGACTATATCAATG 76 T LK T P S Q D S D Y I NAN F I KGVY GP ACTCAAGGACCTTTAGCAAATACAGTAATAGATTTTTGGATTTTTGGA~ATGGTATGGGAGTAT~TGTTGTGATCATTGT~TGGCCT~ 106 T QGP LAN TV I D FWRMVWE Y NVV I CGAGAATTTGAGATGGGAAGGAAAAAA TGTGAGCGCTATTGGCCTTTGTATGGAGAAGACC~CATA 134 R E F EM GR K K C E RY WP LY GE D P I T ATTTCTTGTGAGGATGAACAAGCAAGAACAGACTACTTCATCAGGACACTCTTACTTG~TTTC~TG~TCTCGTAG~TG 162 I SC E D E Q AR T D YF I R T L LLEF QN TATCAGTTTCATTATGTGAACTGGCCAGACCAGACCATGATGTTCCTTCATCATTTGATTCTATTCTGGACATGAT~GCTT~TGA~ 190 Y QF HY VNWP DH DVP S S F D S I L D M AAATATCAAGAACATGAAGATGTTCCTATTTGTATTCATTGCAGTGCAGGCTGT~~G~CAGGTGCCATTTGTGCCATAGAT 218 KY Q E H E D VP I C I H C S AG C GRT GA TATACGTGGAATTTACTAGCTGGGAAAATACCAGAGG 246 Y TW N L LK AG K I P E EF NVFN L I QE CATTCTGCAGTACAAACAAAGGAGCAATATGAACTTGTTC 214HSAVQTKEQYELVHRAIA-dLFEKQLQLY GAAATTCATGGAGCTCAGAAAATTGCTGCTGATGGAGTG~TG~TT~CACTG~CATGGTCA~TCCATA~~CT~ 302 E I H GA QK I AD GVN E I NT E N MV S S CAAGATTCTCCTCCTCCAAAACCACCACCCGCAGTTGCCACCG

8

1

358 386 414 442 470 498 526 554 582 610 638 666 694 722 750 178

16 25 S

RVK

L 33

KAY

V

41 IV

MAC

FA

P

50 F

K

58 E

S

RRL

I

S

LMR

I

CA

67 75 I

D 83

MRT

QR 92 100

I

E

P

E P H P V P P I L T P S P P S AF P T V T T VW Q D AGATACCATCCAAAGCCAGTGTTGCATATGGTTTCATCAG~C~CATTCAGCAGACCTC~CAG~CTATAGT~TC~CA P K P V LH MV S S E QH S AD LN RN Y S K - R YH GAACTTCCAGGGAAAAATGAATCAACAATTGAACAGATAGATAAAAAA TTGGAACGAAATTTAAGTTTTGAGATTAAGAAGGTC ELPGKNESTIEQI DKKLERNLSFEIKKV CCTCTCCAAGAGGGACCAAAAAGTTTTTGATGGG~CACACTTTTG~TAGGGGACATGC~TT~TT~TCTGCTTCACCT PLQEGPK SFDGNTLLNRGHAIKIKSASP TGTATAGCTGATAAAATCTCTAAGCCACACAGG~TT~GTTCAGATCT~TGTCGGTGATACTTCCCAG~TTCTTGTGT~AC CIADKISKPQELSSDLNVGDTSQNSCVD TGCAGTGTAACACAATCAAACAAAGTTTCAGTTTCAGTTACTCCACCAG~G~TCCCAG~TTCAGACACACCTCC~G~CAGACC~ CSVTQSNKVSVTP PEESQNSDTPPRPDR TTGCCTCTTGATGAGAAAGGACATGTAACGTGGTCATTTCATGGACCTGAAAATGCCATACCATACCCATACCTGATTTATCTG~G~ LPLDEKGHVTWSFHGPENAIPIPDLSEG AATTCCTCAGATATCAACTATCAAACTAGGAAAACTGTGATCTT NSSDINYQTRKTVSLTPSPTTQVETPDL GTGGATCATGATAACACTTCACCACTCTTCAGAACACCCCCTCAGTTTTACT~TCCACTTCACTCTGATGACTCAGACTCAGAT VDHDNTSPLFRTPLSFTNPLHSDDSDSD GAAAGAAACTCTGATGGTGCTGTGACCCAGAATAAAACTATACT ERNSDGAVTQNKTNI STASATVSAATAT GAAAGCATTTCTACTAGGAGTATTGCCAATGTCCATTGTCCATTGCTAGACAT~TATAGCAGG~C~CACATTCA~TGCT~ E S I STRKVLPMSIARHN IAGTTHSGAEK GATGTTGATGTTAGTGAAGATTCACCTCCTCCCCTACCTG~G~CTCCTG~TCGTTTGTGTTAGC~GTG~CAT~TA~ D v D v s E ~~~~i:L~~~~~.~~~~:~~~~:~~~~~~~~~~~:~~~~~~~~~~~~~~~~:~~~~:~~~~,~~~~~~~~~~ H CCTGTAAGATCGGAATGGAGTGAACTTCAAAGTCAGGAACGATCTGAAC AAAAAAAGTCTGAAGGCTTGATAACCTCTGAAAAT PVRSEWSELQSQERSEQKKSEGLITSEN GAGAAATGTGATCATCCAGCGGGAGGTATTCACTATGAAA EKCDHPAGGIHYEMC IECPPTFSDKREQ ATATCAGAAAATCCAACAGAAGCCACAGATATTGGTTTTGGT~TCGATGTGG~CCC~GGACC~GAGATCCACCTT~ ISENPTEATDIGFGNRCGKPKGPRDPPS GAATGGACATGATTCAGGGAGCTAGAAGACACACTTT~GTTATACTGG~TTCAGGTGCCACTG~~CAGATTTATAGTAT E W T * TCCATCTTTAATATGTGGGACTAACAGCAGCAGTGTA~TTGTTACCTT~TATTTTTTGCTGGGACCATCTACCTGCCTTATACTA CACTTAGGAAAAAGTATTACATATGGTTTATTTTGAAACTTC~GTATTATTGCCTT~TGTCTCTT~CCCTGTTACAC~TG CTTGTAGACATGTTAATATAGTAATACCTTTACCTTTATGATATATTGAGTTT~GGACTACCCTTTTTCTGTTTTATCATGTATT~TT ATTTTGTATATGTACAGGGCAAGTAGGTAGGTATAT~TTTGAT~GTTGC~TTG~TATTATT~CAG~GATGT~G~TTT CTGCATGGTCTAAATCTTTGTGTACTTTATTTGTAAATTATTTGCCCT~AGTTTTAG~TAGTTTCTG~TTTT~CTTG CTGGATTCATGCAGCCAGCTTTGCAGGTTATCAGAGATCA

E

K 109

N

D

S

T

125 134 142 151 159 167 176 184 193 201 N

Fig. 2. Nucleotide and predicted amino acid sequences of PTPGl. The nucleotide sequence is numbered at right. The amino acid sequence is shown in single-letter code below the nucleotide sequence and is numbered at left. The PTPase domain is indicated by half-brackets. The four regions that match the consensus for a PEST sequence are underlined, and the imperfect proline-rich repeats are shaded.

1226

A

T

209 218 226 235 243 251 26C 268 277 285 293

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domain

Regulatory

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PEST

3’

5’ -

PTPGl

PCR

’ hGl-Cl * XGl-C2 * hGl-C33

L

4 hGl-C36 1 )cGl-C4

I

I

0

1

I 2

I

3 (kb)

w Schematic diagram of the PTPGl mRNA and the relationship of six cDNA isolates. The open reading frame region is boxed, whereas thin lines at the both ends represent 5’ and 3’ untranslated regions. Relative positions of the PEST regions and the PTPase domain are indicated. The thin open bars show the portion of the sequence contained in each of the cDNA isolates.

LAR domain

1 (36%), PTPlB

(35%), T cell PTP (34%) and PTPHl

(32%). More

interestingly, the greatest similarity of the catalytic domain was revealed in murine PEP (PESTdomain phosphatase) (67%) which contains a large carboxy-terminal

extension with PEST

sequences and an imperfect proline-rich repeat as found in PTPGl (14). In addition, partial homology was found in both carboxy- and amino-terminus regions (located at positions 1 to 26 and 761 to 780 ) and in sequences of the proline-rich repeats (Fig. 4). However, the carboxyterminal domain and the extra-catalytic amineterminal region were unique for PTPGl.

A

B

C

PTPGl

(l-26)

PEP

(l-23)

PTPGl

(761-780)

PEP

(782-802)

PTPGl

(331-351)

;;;Gl

II'I;;;;;

MEQVEILRKFIQRVGAMKSPDHNGED :.: ::: . : : : .:. MDQREILQQLLKE AQKKKL NSEE GFGNRCGKPKGPRDPPSEWT : : ::: .:::::: ::: : GFGNRFSKPKGPRNPPSAWNM

[$j[~'~'

Fig. 4. Amino acid sequence alignment of homologous regions between PTPGI and murine PEP. (A) and (B) show comparison of N-terminal and C-terminal sequences, respectively. Double dots indicate identical residues; equivalent amino acids marked by single dots. (C) shows comparison of imperfect proline-rich repeat sequences within C-terminal domain of two PTPases. The conserved sequences are boxed. Numbers indicate positions of the first and last residues.

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12

A

0 1

2

3

4

3

4

5

6

7

8

c 5

6

7

a

9

-aBs

05

paccin

06

Hindlll digest

+

23.11

c c

0.41 6.61 4.41

c

2.01

BamHl digest

Fip. 5. Northern blot analysis of PTPGI mRNA. Total RNA from various cell lines were hybridized with a 1.2 kb fragment from Xl-Cl and P-actin. (A) Total RNA samples from hepatocellular carcinoma cell lines; PLC (Lane l), HLF (Lane 2), HLE (Lane 3), CHC-32 (Lane 4). (B) Those from a gastric cancer cell line; NUGC3 (Lane 5) and colorectal cancer cell lines; BM314 (Lane 6), COLO 201 (Lane 7). (C) Those from hematopoietic cell lines; PEER (Lane 8), KS62 (Lane 9). Positions of 28s and 18s rRNAs are indicated. Fig. 6. Southern blot analysis of PTPGl gene. Ten micrograms of human genomic DNA from a normal colonic mucous membrane (Lane 1 and 5), colonic cancer tissue (Lane 2 and 6) and colorectal cancer cell lines; DLDl (Lane 3 and 7), COLO 201 (Lane 4 and 8). DNA was digested with Hi&II (Lane l-4) and BarnHI (Lane 5-8). Numbers indicate the size of DNA markers in kb.

Distribution

of PTPGI mRNA. To evaluate

the tissue distribution

of PTPGI, Northern

blot analysis was performed. A -4.6 kb transcript was constantly detected in total RNAs from a variety of human cell lines (Fig. 5). Furthermore, PTPGl mRNA was also detected in MKN 45, KATO III (gastric cancer cell lines), SW1 116 (colon cancer cell lines), PK-1, Pant-I (pancreatic cancer cell lines), HuH7, cHc-4 (hepatocellular carcinoma cell lines), HO, TA (Bcell lines) and CEM (T-cell leukemia) (data not shown). These results suggested that PTPGl mRNA could be widely expressed in various tissues, although their levels of transcription showed variable. Southern Blot Analysis of the PTPGI Gene.

Southern blot analysis of PTPGl gene

was performed by using genomic DNAs from normal colon and colon cancer tissues with a [32P]labeled 1.3 kb fragment derived from hGl-C4 as probe (Fig. 6). A single band was observed for BarnHI, while several bands were detected with Hi&II. Since the cDNA probe contains no internal HindlII site, PTPGI may be a single-copy gene which consists of a several exons, although the genomic analysis of PTPG 1 will be required to confirm it.

DISCUSSION We have isolated a new putative cytoplasmic PTP cDNA whose sequences suggested it could be a protein with a rapid turnover. Such type of human cytoplasmic PTP has never been reported so far. Murine PEP (PEST-domain phosphatase) has very recently been obtained from a 7OZ/3 pre-B cell library by Matthews et al. (14). A high homology at the amino acid level (67%) was revealed only in the catalytic domain between PEP and PTPGl, indicating that 1228

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PTPGl is not a human homolog of PEP. Northern blot analysis demonstrated that PTPGl is widely expressed in various cell lines, whereas murine PEP is expressed primarily

in

hematopoietic tissues (14). These facts represent they make up of a new family of cytosolic type PTPs. Interestingly, imperfect proline-rich repeats were highly homologous between Cterminal domains of human PTPGl and murine PEP, suggesting that they play an important role in functional regulation of these PTPs. The most striking feature of the PTPGl protein is the presence of four PEST regions which are usually found in proteins with extremely short intracellular half-life. All the PEST proteins appear to be important regulatory molecules, and most of them, including ~53, c-Myc, c-Fos, v-Myb, ElA, Vpl and cyclins, are located in the nucleus (10,15,16). In this context, it should be noted that PTPGl contained a basic sequence (VKKNRYK)

at positions 59 to 65 that meets the qualifications

for one class of nuclear

localization signal (17,18). It is of interest that a number of putative phosphorylation sites were found in the C-terminal domain of PTPGl. There were two potential sites for phosphorylation by a cell cycle-regulated kinase, p34cdc2, located at positions 518 to 522 (DTPPR) and 692 to 696 (NTPVR). They conformed to the consensus motif for p34 cdc2 kinase, XS/TPXK/R (19,20), suggesting that PTPGl may be phosphorylated

in a cell-cycle dependent manner. In addition, PTPGl

contained one putative phosphorylation site (DDS- 606DSDE) for casein kinase II (21,22) and two sites for double-stranded DNA-dependent

kinase (ES-514Q, QS704Q) (23,24). Casein

kinase II, which phosphorylates several nuclear oncoproteins, is induced in response to mitogens. The concensus motifs for ds-DNA-dependent kinase, ET/SQ or QT/SQ, were also found in several nuclear phosphoproteins including the SV40 large T antigen and ~53. It has been reported that p34CdC2 kinase or its similar protein might regulate RB and ~53 in consequence of their phosphorylation (25,26). Particularly, ~53 is phosphrylated by abovementioned three kinases in vitro (24,26,27), raising the possibility that ~53 and PTPGl are regulated in a similar manner. Thus, PTPGl may play an important role in cell proliferation, differentiation and the cell-cycle as a key molecule. ACKNOWLEDGMENTS We thank Dr. K. Kimura in the Department of Microbiology, Sapporo Medical College, for protein homology search and Ms. Ohe for expert technical assistance. We are also grateful to the Japanese Cancer Research Bank for providing us with cell lines. This work was financed by grants for Cancer Research from the Ministry of Education, Science and Culture (A.Y, K.1, Y.H), and from the Ministry of Health and Welfare, Japan (A.Y, K.1). Note

added

in proof.

Morerecently, den Hertog et al. (28) has published areport describing the isolation of a,PTPase, P19-PTP, from a murine embryonal carcinoma cell line. Comparison of P19-PTP and PTPGl revealed high sequence similarity except for the sequence at positions 296 to 418. The nucleotide sequence of PTPGl corresponding to this region began with 9u2-GAAIAAAICAG and closed with 126*-GGG/AAA/AAT. A putative identical region in P19-PTP was from 9% GAA/AAC/AGC to 12g4-GCA/AAA/AGT. These strongly suggest that the frame shift occured in P19-PTP, giving rise to the totally different sequence. 1229

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REFERENCES Hunter, T. and Cooper, J. A. (1985) Annu. Rev. Biochem. 54, 897-930. Ulhich, A. and Schlessinger, J. (1990) Cell 61,203-212. Fischer, E. H., Charbonneau, H. & Tonks, N. K. (1991) Science 253,401X)6. LaForgia, S., Morse, B., Levy, J., Barnea, G., Cannizzaro, L. A., Li, F., Nowell, P. C., Boghosian-Sell, L., Glick, J., Weston, A., Harris, C. C., Drabkin, H., Patterson, D., Croce, C. M., Schlessinger, J. & Huebner, K. (1991) Proc. Nutl. Acud. Sci. USA 88, 5038-5040. 5. Brown-Shimer, S., Johnson, K. A., Hill, D. E. & Bruskin, A. M. (1992) Cancer Res. 52, 478-482. 6. Streuli, M., Krueger, N. X., Thai, T., Tang, M. & Saito, H. (1990) EMBO J. 9, 2399-2407. 7. Tonks, N. K., Diltz, C. D. & Fischer, E. H. (1988) J. Biol. Chem. 263, 6731-6737. Tonks, N. K., Diltz, C. D. & Fischer, E. H. (1990) J. Biol. Chem. 265, 10674-10680. t : Arimura, Y., Hinoda,Y., Itoh, F., Takekawa, M., Tsujisaki, M., Adachi, M., Imai, K. & Yachi, A. (1992) Tumor Biol. 13, 180-186. 10. Rogers, S., Wells, R. and Rechsteiner, M. (1986) Science 234, 364-368. 11. Nishi, M., Ohagi, S. & Steiner, D. F. (1990) FEBS LETT. 271, 178-180. 12. Chomczynsky, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. 13. Kozak, M. (1989) J. Cell Biol. 108,229-241. 14. Matthews, R. J., Bowne, D. B., Flares, E. and Thomas, M. L. (1992) Mol. Cell. Biol. 12,2396-2405. 15. McCarty, D. R., Hattori, T., Carson, C. B., Vashil, V., Laser, M. and Vasil, I. K. (1991) Cell 66, 895-905. 16. van Lohuizen, M., Verbeek, S., Scheijen, B., Wientjens, E., van der Gulden, H. and Bems, A. (1991) Cell 65,737-752. 17. Roberts, B. (1989) Biochem. Biophys. Actu 1008, 263-280. 18. Garcia-Bustos, J., Heitman, J. and Hall, M. N. (1991) Biochim. Biophys. Actu 1071, 83-101. 19. Peter, M., Nakagawa, J., Doree, M., Labbe, J. C. and Nigg, E. A. (1990) Cell 60, 791-801. 20. Shenoy, S., Choi, J. K., Bagrodia, S., Copeland, T. D., Maller, J. L. and Shalloway, D. (1989) Cell 57,763-774. 21. Pinna, L. A. (1990) Biochim. Biophys. Actu 1054, 267-284. 22. Kuenzel, E. A., Mulligan, J. A., Sommercorn, J. and Krebs, E. G. (1987) J. Biol. Chem. 262,9136-9140. 23. Lees-Miller, S. P., Chen, Y.-R. & Anderson, C. W. (1990) Mol. Cell. Biol. 10, 6472-648 1. 24. Wang, Y. and Eckhart, W. (1992) Proc. Nutl. Acud. Sci. USA 89, 4231-4235. 25. Lees, J. A., Buchkovich, K. J., Marshak, D. R., Anderson, C. W. & Harlow, E. (1991) EMBO J. 10,4279-4290. 26. Bischoff, J. R., Friedman, P. N., Marshak, D. R., Prives, C. & Beach, D. (1990) Proc. Nutl. Acud. Sci. USA 87,4766-4770. 27. Meek, D. W., Simon, S., Kikkawa, U. & Eckhart, W. (1990) EMBO J. 9,3253-3260. 28. den Hertog, J., Pals, C. E. G. M., Jonk, L. J.C. and Kruijer, W. (1992) Biochem. Biophys. Rese. Comun. 184, 1241-1249.

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