Molecular Cloning of a Human Transmembrane-type Protein

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1994 by The American Society for Biochemistry and Mol& Biology, Inc.

Vol. 269, No. 3, Islue of January 21, pp. 2076-2081.1994 Printed in U.S.A.

Molecular Cloning of a Human Transmembrane-type Protein Tyrosine Phosphatase and Its Expression in Gastrointestinal Cancers*

(Received for publication, May 20, 1993, and in revised form, August 11, 1993)

Takashi MatozakiS, Toshiya Suzuki, Tohru Uchida, Johji InazawaQ, Takeshi AriyamaQ, Kohei Matsuda, Kazutsugu Horita, Hitoshi Noguchi, Hisashi Mizuno, Choitsu Sakamoto, and Masato Kasuga From the Second Department of Internal Medicine, Kobe University School of Medicine, Kusurwki-cho, Chuo-ku, Kobe 650 and the $Department of Hygiene, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, Japan

To determine the expression of various protein-tyrosine phosphatases (PTPs) in human gastric cancers, cDNAs encoding conserved PTP domains were amplified by reverse transcriptase polymerase chain reaction from KATO-I11 cell mRNA and sequenced. Among 72 polymerase chain reaction clones, one of the cDNA sequences encoded a novel potential PTP (stomach cancer-associated PTP, SAP-1). The full length (3.9 kilo- bases) of the SAP-1 cDNA was further isolated from the KATO-I11 cell cDNA library and the WiDr cell cDNA library. The predicted amino acid sequence of the SAP- 1 cDNA showed that mature SAP- 1 consisted of 1093 amino acids and a transmembrane-type PTP, which possessed a single PTP-conserved domain in the cytoplasmic region. The extracellular region of SAP- 1 consisted of eight fibronectin type 111-like structure repeats and contained multiple N-glycosylation sites. These data suggest that SAP-1 is structurally similar to HPTPB and that SAP-1 and HPTPB represent a subfamily of transmembrane-type PTPs. SAP-1 was mainly expressed in brain and liver and at a lower level in heart and stomach as a 4.2-kilobase mRNA, but it was not detected in pancreas or colon. In contrast, among cancer cell lines tested, SAP-1 was highly expressed in pancreatic and colorectal cancer cells. The bacterially expressed SAP-1 fusion protein had tyrosine-specific phosphatase activity. Immunoblot- ting with anti-SAP-1 antibody showed that SAP-1 is a 200-kDa protein. In addition, transient transfection of SAP-1 cDNA to COS cells resulted in the predominant expression of a 200-kDa protein recognized by anti- SAP-1 antibody. SAP-1 is mapped to chromosome 19 region q13.4 and might be related to carcinoembryonic antigen mapped to 19q13.2.

The phosphorylation of tyrosine residues of protein is a crucial event in the regulation of normal cellular processes such as proliferation and differentiation and is also involved in the malignant transformation of cells (1, 2). In gastric

* This work was supported by a grant-in-aid for cancer research from the Ministry of Education, Science, and Culture of Japan, and a grant from the Osaka Cancer Research Foundation. The costa of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adver- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper hos been submitted to the GenBankTM/EMBL Data Bank with accession nurnber(s) 015049.

$ To whom correspondence should be addressed. Tel.: 011-81-78- 341-7451 (ext. 5526); Fax: 011-81-78-382-2080.

cancers and colon cancers, it has been shown that the expression of genes encoding growth factor receptors such as erb- B2 (3) and K-Sam (4), a gene encoding a fibroblast growth factor receptor family, is elevated (5). In addition, the level of tyrosine phosphorylated proteins is increased in gastric cancer cells (6). Since the level of tyrosine phosphorylation is determined by the balance between the actions of both protein- tyrosine kinases and PTPs' (7, €9, not only the unregulated activation of protein-tyrosine kinases but also the inactivation of PTPs may be involved in the malignant transformation of gastrointestinal cells. In fact, it has been suggested that the inactivation of HPTPr may be involved in the pathogenesis of small cell lung cancer, since the deletion of chromosome 3p21, where HPTPy is mapped, is often associated with this type of cancer (9). However, a recent study (10) has indicated that the contribution of PTPs to tumor suppression may not be simple, since it has been shown that the overexpression of HPTPa, a transmembrane-type PTP, induces cell transformation through the dephosphorylation of pp60'"" kinase (lo), suggesting that malactivation of PTP may be one of the steps to oncogenic transformation. Thus, various types of PTPs may be involved in the oncogenesis of various cancers in different manners. Since little is known of the role of PTPs in the pathogenesis of gastric cancers, we have studied the PTPs expressed in a gastric cancer cell line, KATO-111, by PCR amplification of cDNAs with oligonucleotide primers to the conserved regions of the known PTPs. Among PCR clones amplified from KATO-I11 cDNA, we have identified a gene encoding a novel transmembrane-type PTP, SAP-1 (gtomach cancer-associated PTE). This PTP is a 200-kDa glycosylated protein with a single PTP catalytic domain in the cytoplasmic region and FN type 111-like structures in the extracellular region. It is of interest that this PTP is highly expressed in colon cancers and pancreatic cancers but not in the corre- sponding normal tissues. Further, we have shown that human SAP-1 is located on the long arm of chromosome 19 at 13.4.

MATERIALS AND METHODS

Cloning of cDNAs for Humnn SAP-1-PCR amplification of cDNA was used to obtain portions of potential PTPs with PCR primers to conserved sequences of known PTPs, as described previously (11,12). Briefly, total RNA was extracted from KATO-I11 by the standard guanidium thiocyanate/cesium chloride method (13). Complementary DNA (cDNA) was then synthesized by incubating -2 pg of total RNA of KATO-I11 with 100 pmol of an antisense PCR primer and 60 units of avian myeloblastosis virus reverse transcriptase (Boehringer

The abbreviations used are: PTP, protein-tyrosine phosphatase; Ab, antibody; PCR, polymerase chain reaction; GST, glutathione S- transferase; pNPP, p-nitrophenyl phosphate, FN, fibronectin; MES, 4-morpholineethanesulfonic acid; SAP, stomach cancer-associated PTP CEA, carcinoembryonic antigen; kb, kilobase(s).

2075

2076 Cloning of a PTP Expressed in Gastrointestinal Cancers

Mannheim) for 60 min at 42 "C (13). PCR amplification was performed in a 100-pl reaction volume using one-tenth of the cDNA and 100 pmol of each oligonucleotide primer for 30 cycles, as described (13, 14). Each cycle included denaturation at 94 "C for 1.5 min, annealing at 37 "C for 3 min, and primer extension at 72 "C for 3

CAAGTGTGACCAGTACTGGCC and 5'-CCAACTCCCGCACTGC- min. Sense and antisense PCR primers used were 5"CGAATT-

AGTG, respectively. The primers containing flanking sequences for EcoRI or PstI sites to facilitate subsequent cloning. The PCR products were then digested with EcoRI and PstI, ligated to pUC 19 (Takara), and sequenced by dideoxy termination methods (15) using the Se- quenase kit (U. S. Biochemical Corp.). Among 72 independent PCR clones sequenced, one novel cDNA encoding a PTP domain was isolated (SAP-1). To isolate full-length cDNA of each PTP, a 260- base pair cDNA fragment of SAP-1 was excised, labeled with [a-"P] dCTP (3000 Ci/mmol) (Amersham Corp.) by random primer methods (15), and used to screen a cDNA library of KATO-111 cells (generously provided by Dr. Terada, National Cancer Research Institute). Three independent cDNA clones were subcloned to a pBluescript and the nucleotide sequence was determined in both directions as described above. Because the cDNA clones initially isolated did not contain the complete coding sequence, further cDNA clones were isolated from the cDNA library of WiDr cells, a colon cancer cell line (Japanese Cancer Research Resources Bank) in which SAP-1 mRNA was abun- dantly expressed.

Northern Blotting-For Northern blotting, poly(A)+ RNA (2-5 pg) extracted from human organs or cultured cells was electrophoresed on 1.2% agarose/formaldehyde gel and transferred to a nylon filter as described previously (14). The blot was hybridized with a 32P- labeled 3.0-kb fragment of SAP-1 cDNA in 50% formamide, 5 X SSC, 5 X Denhardt's solution, 50 mM NaPO, (pH 7.0), 0.1% SDS, and 100 pg/ml salmon sperm DNA overnight a t 42 "C. The blots were then washed as described previously (14). The filter was then rehybridized with a '*P-labeled mouse ,%actin probe.

Expression and Purification of SAP-1 and PTP Assays-For amplification of cDNA encoding the PTP domain of SAP-1, PCR amplification was performed essentially as described previously (14) by using a sense primer (5'-ATTGGATCCCAGGGGACATCCCAG- CTGAAG) (nucleotides 2435-2457), an antisense primer (5"CTAG- AATTCCTCATACGGGACTTCCTTCTCG) (nucleotides 3310- 3333), and a 3-kb SAP-1 cDNA as a template. The amplified PCR fragment was digested with BamHI and EcoRI and inserted into in- frame BamHI and EcoRI sites of pGEX-ST (Pharmacia LKB Bio- technology Inc.). Glutathione S-transferase (GST) fusion protein expression and purification were performed as described previously (16, 17). Fresh overnight cultured Escherichia coli (JM 109) trans- formed with pGEX-2T or pGEX-2T-SAP1 recombinant was diluted 1:20 in LB medium containing ampicillin (100 pg/ml) and incubated at 37 "C. After a 1.5-h incubation, isopropyl-l-thio-/%D-galactopyran- oside was added to a final concentration of 0.1 mM, and this was followed by a 5-h incubation. For fusion protein recovery on glutathione-Sepharose beads (Pharmacia), 10 ml of bacterial culture was centrifuged at 3000 rpm for 15 min at 4 "C and resuspended in 3 ml of NETN (20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing 1 mM phenylmethylsulfonyl fluoride and 10 pg/ml aprotinin. The bacteria was then lysed on ice by sonication for 30 s and centrifuged at 10,000 X g for 15 min at 4 "C. Aliquots of bacterial supernatant were then rocked for 30 min at 4 "C with 50 pl of glutathione-Sepharose (Pharmacia) suspended in phosphate-buff- ered saline (l:l, v/v). After centrifugation, beads were washed twice with 1 ml of WG buffer (50 mM Hepes pH 7.6, 150 mM NaCl, 0.1% Triton X-100) and twice with PTP assay buffer containing 40 mM MES (pH 5.0) and 1.6 mM dithiothreitol. The assay mixture (200 pl) containing assay buffer with 25 mM pNPP and GST fusion proteins bound to glutathione-Sepharose beads was incubated at 30 "C for 30 min. The reaction was terminated by the addition of 200 p1 of 1 N NaOH, and the absorbance at 410 nm was determined (18). The assay of the PTP activity of GST-SAP-1 fusion protein was also performed using the 32P-labeled synthetic peptide Raytide (18, 19). In the PTP assay, the assay mixtures (100 pl) containing the fusion protein, 25 mM imidazole (pH 7.2), 0.1 mg/ml bovine serum albumin, 10 mM dithiothreitol, and 1 pl of 32P-labeled Raytide (approximately 10,000 cpm/pl) were incubated at 30 "C for 30 min. The labeling of Raytide with [y3'P]ATP and ~60""" tyrosine kinase was carried out as described previously (18, 20). The reaction was terminated by the addition of 0.75 ml of an acidic charcoal mixture containing 0.9 M HCl, 90 mM sodium pyrophosphate, 2 mM NaH2P0,, and 4% Norit A. After centrifugation in a microcentrifuge, the amount of radioac-

tivity in 0.5 ml of supernatant was measured by a liquid scintillation counter.

Affinity Purification of a Polyclonal Ab to SAP-I-To generate a polyclonai Ab to SAP-1, GST protein or GST-SAP-1 fusion protein containing PTP domain was obtained from a 0.5-2-liter bacterial

washed twice with 10 ml of NETN and further incubated with 7 ml culture and bound to glutathione-Sepharose beads. Beads were then

of G buffer containing 50 mM Tris-HC1 (pH 9.6) and 10 mM glutathione (Sigma) for 30 min at 4 "C. The eluted proteins from the gels were dialyzed overnight against 1 liter of a solution containing 25 mM Tris-HC1 (pH 7.5), 1 mM EDTA, and 1 mM dithiothreitol and then concentrated by Cetriprep-10. For immunization, female rabbits were first injected with 0.4 mg of GST-SAP-1 fusion protein in Freund's complete adjuvant and boosted every 2 weeks with 0.2 mg of antigen in Freund's incomplete adjuvant. For affinity purification, 5 mg of either GST or GST-fusion protein was rocked with 400 mg of CNBr- activated Sepharose 4B (Pharmacia) at 4 "C overnight followed by blocking in 30 ml of 1 M Tris-HC1 (pH 8.0). Gels were then suspended in 1 ml of coupling buffer containing 0.2 M NaHC03 (pH 8.3) and 0.5 M NaCl and washed in 30 ml of elution buffer containing 100 mM glycine (pH 2.5). Rabbit serum was then subjected to a column containing gels prebound to GST. The pass was further subjected to a column containing gels prebound to GST-SAP-1 fusion protein, washed with 20 ml of 10 mM Hepes pH 7.5, and subsequently washed again with 20 ml of Tris-HC1 (pH 7.5) containing 0.5 M NaCl. The Ab bound to the gels was then eluted with 5 ml of elution buffer and the eluate was combined with 10 ml of 1 M Hepes pH 8.8.

Zmmunoblotting and Immunoprecipitation-For immunoblotting experiments, confluent 60-mm plates of either WiDr cells, HL-60 cells (the Japanese Cancer Research Resources Bank), or transfected COS-7 cells were lysed on ice in 0.5 ml (approximately 2 mg/ml protein) of ice-cold lysis buffer (50 mM Tris-HC1 (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100,0.5% deoxycholate, 0.1% SDS, 10% glycerol) containing 1 mM PMSF and 10 pg/ml aprotinin. The lysates were centrifuged at 10,000 X g for 15 min at 4 "C, and the resultant supernatants were used for immunoprecipitation and immunoblotting. Immunoprecipitation was performed by incubating 500 pl of cell lysate with 5 pg of either anti-SAP-1 Ab or preimmune serum prebound to Sepharose-protein A beads (Pharmacia) (20-4 beads) for 4 h at 4 "C. The beads with immunoprecipitated proteins were washed twice with 1 ml of WG buffer (50 mM Hepes pH 7.6, 150 mM NaC1, 0.1% Triton X-loo), and then solubilized in SDS- polyacrylamide gel electrophoresis sample buffer. Gel electrophoresis and immunoblotting were performed with a polyclonal anti-SAP-1 Ab as described previously (14, 17), except that visualization of immunoreactive protein bands was carried out by using ECL detec- tion kit (Amersham Corp.).

Transient Expression of SAP-1 in COS Cells-For transient expression of SAP-1, a full-length SAP-1 cDNA was inserted into the EcoRI site of pSRa (originally distributed by Dr. N. Arai). Semiconfluent COS-7 cells in 6-cm dishes were transfected with 3 pg of pSRa containing SAP-1 cDNA, or a vector alone as a control, by the calcium phosphate method (15). After transfection, the cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 3 days. Cells were then lysed as described above, and lysates were used for immunoprecipitation and immunoblotting.

Chromosomal Mapping of SAP-1 by Fluorescence in Situ Hybridi- zation-Determination of chromosomal location of SAP-1 was performed as described previously (21, 22). Briefly, normal male metaphase chromosomes were obtained by thymidine synchronizingfiro- modeoxyuridine release (23). The probe, pBluescript-SAP, containing a partial SAP-1 cDNA (3 kb), was labeled with biotin-16-dUTP (Boehringer Mannheim) by nick translation. Fluorescence in situ hybridization was performed as described previously (22). The fluorescence signals of hybridized probes were amplified with avidin- fluorescein isothiocyanate (Boehringer Mannheim) and biotinylated anti-avidin D (Vector) according to the protocols described by Pinkel et al. (24).

RESULTS

Molecular Cloning and Sequencing of SAP-1 cDNAs-To isolate PTPs, which are expressed in KATO-I11 cells, we used an approach based on sequence homology and PCR amplification. Sequencing of 72 amplified cDNA clones showed nine different cDNA sequences; these included P T P H l ( 5 0 clones) (25), PTPlB (4 clones) (26), TCPTP (1 clone) (271, H e P T P

Cloning of a PTP Expressed in Gastrointestinal Cancers 2077

(3 clones) (28), SAP-Z/SHPTPZ (3 clones) (29), HPTPa (2 clones) (30), HPTPy (4 clones) (30), LAR (3 clones) (31), and a novel putative PTP (SAP-1) (2 clones). The amplified cDNA for SAP-1 detected a 4.2-kb transcript in KATO-I11 cells and was used to isolate cDNA clones from the KATO- I11 cell cDNA library (provided by Dr. M. Terada, National Cancer Research Institute). Since cDNA clones initially isolated from the KATO-I11 cDNA library did not contain the complete coding sequence, further cDNA clones were isolated from the cDNA library of WiDr cells, a colon cancer cell line.

The complete nucleotide sequence of cloned cDNAs ex- tended to 3925 nucleotides and contained a single long open reading frame of 1118 amino acid residues (nucleotides 42- 3395) (Fig. 1). The first ATG codon, found in this open reading frame at position 42, matched the Kozak consensus sequence (32) for a translation initiation site. Therefore, the methionine at position 42-44 is presumed to be the translation initiation codon. The 3’-noncoding region contains a typical polyadenylation signal (ATTAAA) followed by a poly(A) tail. The amino-terminal 25 amino acids are highly hydrophobic and are a likely signal peptide (33). The predicted mature SAP-1 protein is composed of a 728-amino acid extracellular domain, a %-amino acid transmembrane region, and a 340- amino acid cytoplasmic region. The cytoplasmic region of the predicted protein sequence contains the conserved sequence motifs found in all PTPs so far (7,8) (Figs. 1 and 2 A ) . Thus, SAP-1 contains a single PTP domain in its cytoplasmic region as well as HPTPP (30) and DPTP 10 D (34,35), a drosophila homolog of HPTPB. The extracellular domain of SAP-1 contains 8 repeated amino acid sequences, each of which possesses a homologous structure to that found in the fibronectin type I11 repeats (Figs. 1 and 2B). In contrast with the cytoplasmic region, the extracellular region of SAP-1 contains 24 potential N-glycosylation sites (N-X-S or N-X-T), suggesting that SAP-1 might be a highly glycosylated transmembrane protein. Comparison of the amino acid sequence of the SAP-1 PTP domain with those of known PTPs shows that HPTPP appear to be most closely related to SAP-1, since their PTP domain is 49.6% identical. Furthermore, comparison of nucleotide sequences reveals that the SAP-1 PTP domain is similar to those of human LAR and HPTPj3, with highest degree of similarity 59.8 and 59.3%, respectively. In contrast to the PTP domain, comparison of each FN 111-like repeat of SAP- 1 reveals that 24% are similar to HPTPP, suggesting that the extracellular domains of SAP-1 and HPTPP may be function- ally different.

Preferential Expression of SAP-1 Transcripts in Pancreatic and Colon Cancer Cells-To examine the pattern of expression of SAP-l,3.O-kb SAP-1 cDNA was used as a probe in North- ern blotting with various tissues and cultured cell lines. In tissues and cells tested so far, a major transcript of approximately 4.2 kb was observed. Among normal human tissues tested, SAP-1 was not ubiquitously expressed but was pri- marily present in brain and liver and at a low level in heart and stomach (Fig. 3). In lung, pancreas, colon, placenta (Fig. 3), and peripheral mononuclear cells (data not shown), SAP- 1 transcripts were not detected. Although SAP-1 transcripts were readily detected in KATO-I11 cells, a low level was observed in MKN45 and JR-1 cells, and no detectable level was observed in other gastric cancer cells. In contrast, SAP- 1 was highly expressed in two pancreatic cancer cell lines, Panc-1 and MIA-Paca 2, and in three colon cancer cell lines, WiDr, SW837, and COL0230 (Fig. 4). In addition, two other colon cancer cell lines, COL0320 and SW480, showed a detectable level of SAP-1 transcripts (Fig. 4). Among other cells tested, SAP-1 was expressed in the glioblastoma cell line,

A172, and at a low level in T97G, another glioblastoma cell line (Fig. 4). In both A431 cells, an epidermoid carcinoma cell line, and ZR-75-1 cells, a breast cancer cell line, a low level of transcripts was observed, while no SAP-1 transcripts were observed in Huh-7, a hepatoma cell line, or in Hela cells. In hematopoietic cells, such as HL-60, SKM, Raji, and SKNO- 1, SAP-1 transcripts were not detected (Fig. 4).

SAP-1 cDNA Encodes an Active PTP of 200 kDa-To determine the catalytic activity of SAP-1, the gene encoding a putative PTP domain of SAP-1 was amplified by PCR. The amplified DNA was inserted into the pGEX-2T vector, and then the GST-SAP-1 fusion protein was bacterially expressed; this was followed by purification. As shown in Fig. 5, GST- SAP-1 protein was expressed as a 65-kDa protein, with an apparent molecular mass consistent with the size of the PTP domain encoded by the cDNA insert of SAP-1 plus the 26 kDa contributed by the GST leader sequence. Although GST alone had no PTP activity, the GST-SAP-1 showed a significant PTP activity when either pNPP or 32P-labeled Raytide was used as a PTP substrate (Fig. 5).To further characterize SAP-1 protein, an affinity-purified polyclonal Ab against the GST-SAP-1 fusion protein was generated. Immunoblotting of WiDr cell lysate with anti-SAP-1 Ab recognized a single protein of 200 kDa (Fig. 6, lane I). In contrast, SAP-1 protein was not detected in HL-60 cell lysates (Fig. 6, lane 2). These data correspond well with results showing that SAP-1 mRNA was highly expressed in WiDr cells but not in HL-60 cells (see Fig. 4). Since the protein product predicted by SAP-1 cDNA would have an apparent M, of 120 after the removal of the signal peptide, the apparent size of SAP-1 seen in immunoblotting suggests a potential post-translational modifica- tion. In addition, significant PTP activity against either pNPP or labeled Raytide was detected in the immunoprecip- itates from WiDr cells (data not shown). To further confirm whether the isolated gene encoded SAP-1 protein, we tran- siently transfected COS-7 cells with a SAP-1 expression vector by the calcium phosphate method. The immunoblotting of transfected COS cell lysates with anti-SAP-1 Ab showed the expression of a 200-kDa protein, the size of which corre- sponded well to that of the SAP-1 protein detected in WiDr cells (Fig. 6, lanes 3 and 4 ) .

Chromosomal Localization of the Human SAP-1 Gene-To determine the chromosomal location of the SAP-1 gene, fluorescence in situ hybridization was performed using pBluescript bearing SAP-1 cDNA. A total of 100 metaphase cells were examined. Of them, 62% exhibited specific hybridization signals at 19q13.4 (Fig. 7). The distribution of the signals was as follows: double spots on both homologs of chromosome 19 (12.9%), double spots on one homolog and a single spot on the other (45.2%), and a single spot on both or one homolog (41.9%). Such double-spot signals were not detected on any other chromosome region. These results localized the human SAP-1 gene to 19q13.4.

DISCUSSION

In the present study, we have isolated a novel transmembrane-type PTP that contains a single PTP domain in the cytoplasmic region. Among transmembrane-type PTPs identified so far, only human HPTPp and DPTP 10 D of Dro- sophila contain a single PTP domain, whereas others have duplicated PTP domains. When the amino acid sequence of the PTP domain of SAP-1 was compared with those of known PTPs, HPTPP was found to be most closely related to SAP- 1. In addition, SAP-1 shows FN 111-like repeated structure but possesses no immunoglobulin-like domains in its extracellular region as do HPTPp and DPTP 10 D. Thus, SAP-1

2078 Cloning of a PTP Expressed in Gastrointestinal Cancers

119 1

239 41 359 81 419 12 1 599

719 161

201 839

appears to be structurally similar to HPTPp and DPTP 10 D and may belong to the Class I1 of PTP described by Krueger et al. (30). However, the amino acid sequence of each tandem repeat in the SAP-1 extracellular region is different from that seen in HPTPP, indicating that SAP-1 and HPTPp may have different functions and act at different sites. The size of SAP- 1 protein predicted by isolated cDNA is 120 kDa, whereas immunoblotting with anti-SAP-1 Ab showed that SAP-1 is a 200-kDa protein. Since there are multiple putative N-glycosylation sites in the extracellular region of SAP-1, it might be

post-translationally modified and highly glycosylated. Al- though the extracellular region of SAP-l might act as an adhesion molecule according to the predicted structure, the role of the intracellular PTP domain is largely unknown. The binding of the specific ligand to SAP-1 or cell-to-cell contact might regulate the PTP activity, thereby influencing the phosphorylation state of SAP-1 target protein.

It is interesting that SAP-1 is predominantly expressed in colon cancer cells and pancreatic cancer cells, whereas SAP- 1 transcripts are undetectable in normal colon and pancreas.


A SAP-1 ( 796) ASASENNAKNRYRNVLPYDWSRVPLKPIHEE PGSDY INASFME'GLWSPQEFIATQGPLPQTVGDFWRLVUEQQSHTLVMLTNCM HPTPP (1700) ALLPENRGKNRYNNILPYDATRVRLSNVDD DPCSDYINASYIPGNNFRREYIVTQGPLPGTKDDFWKMVWEQNVHNIVMVTQCV PTPlB (35) AKLPKNKNRNRYRDVSPFDHSRIIUHQ EDNDY INASLIKMEEAQRSYILTQGPIPNTCGHFWEMVWEQKSRGWMLNRVM LAR (1344) SNLEVNKPKNRYANVIAYDHSRVILTSIDG VPGSDY INANYIDGYMQNAYIATQGPLPETMGDFWRMVWEQRTATWMMTRME LCA DPTPlOD (1291) ADLPCNRPKNRFTNILPYDHSRFIUQPVDD DEGSDY INANYVPGHNSPREFIVTQGPLHSTRDDFWRMCWESNSRAIVHLTRCF

(647) ARKPFNQNKNRYVDILPYDYNRVELSEING DAGSNY INASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCE

SAP-1 (880) EAGRVKCEHYWP LDSQPCTHGH LRVTLVGEEVMENWTVRELLLLQVE EQKTLSVRQFHYQAWPDHGVPSSPDTLLAFWRMLR HPTPP (1784) EKGRVKCDHYWP ADQDSLYYGD LILQ~SESVLPEWTIREFKICGEEQLDAHRLIRHFHYTVWPDHGVPETTQSLIQFVRTVR PTPlB (115) EKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLT TQETREILHFHYTTWPDFGVPESPASFLNFLFKVR LAR (1428) EKSRVKCDQYWP ARGTETCGL IQVTLLDTVELATYTVRTFALHKSG SSEKRELRQFQFMAWPDHGVPEYPTPILAFLRRVK LCA (731) EGNRNKCAEYWPSMEEGTRAFGD VVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLFfRRVN DPTPlOD (1375) EKGREKCDQYWP NDTVPVFYGD IKVQILNDSHYADWVMTEFMLCR GSEQRILRHFHFTTWPDFGVPNPPQTLVRFVRAFR

SAP-1 (962 HPTPP (1867 PTPlB (200 LAR (1509 LCA (815 DPTPlOD (1455

QWLDQTMEGGPPIVHCSAGVGRTGTLIALDVLLRQLQSEGL LGPFSFVRKMRESRPLMVQTEAQYVFLHQCICGSSNSQPR DYINRSPGAGPTWHCSAGVGRTGTFIALDRILQQLDSKDS VDIYGAVHDLRLHRVHMVQTECQYVYLHQCVRDVLRARKL

ACNPL DAGPMVJHCSAGVGRTGCFIVIDAMLERMKHEKT VDIYGHVTCMRSQRNYMVQTETQYVFIHEALLEAATCGHT ESGSLSPEHGPVWHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKLE~KFRMGLIQTADQLRFSYLAVIEGAKFIMG

AFSNF FSGPIWHCSAGVGRTGTYIGIDAMLEGLEAENK VDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGET DRICA EQRPIWHCSAGVGRSGTFITLDRIIQQINTSDY VDIFGIWAMRKERVWMVQTEQQYIDIHQCLLAVLEGKEN

6 1. (3) 2. ( 9 2 ) 3. (181) 4. ( 2 5 2 ) 5 . (341) 6 . (430) 7. ( 5 2 0 ) 8 . ( 6 2 0 )

.... . . PAPN PGRN LTWTQTT SSISLS WEV PDGLDSQNSNYW VQCTGD GGTTETlWTTA TNvTvrG LGPGS LYTC SVWVEKD TAPN PVRN XRVEAW SSIALT WEV PLXPDPQNSTYG VEYTGD GGRAGTRSTAH TNI'IWG LEPGC LYAF mKN TAHN PVRW ESGGSDHQLHLPELGGPR WHR PTELDLZRTSAL EMVAEQ RLETQQ TPESPMG LGPGS LYE SWJVEKD TAPN PVRN LTvEAQ?N SSIALT WEV PLXPDPQNSTYG VEYED GGRAGTRSTAA TNITVDR LEPGC LYVF SVWVGM TAPN PVRN LAME- SSIACC WEV PDGPYPQDYTYW VGYTGD GGGTETRN'MN TSVTAER LEPGT LYTF SVWAEKN TVPN A W S LSXQDWIN STIALR WIA PQGPGQSSYSYW VSWVRE I3lTDPRTQSTSG TDITLKE LEAGS L Y E TVWAERN TAPN EVTD LQNE'IQTKN SVMLW WKA PGDPHSQLYVXW VQwnsK GHPRRGQDFQAWWQTSRT?E TwyRveA LEPGT LYNF TVWAERN TYPD TVTIT SCVSTSAGY GVNLI W S C PQGGYEAFELE VGGQR GSQDRSSCG EAVSVLG WAR SYPA TITPIWD

. . . . . . . . . GVNS s v m GINS SRETRNAT GVNS SWRLWST GINS SRETRNAT GARG SRQNVSIS EVRG YNSTLTAA DVAS STQSICAS GMKV VSHSWCH

. .

m-I11 LSPP TNLH LEANPD'IGVLTVS WER STTPDITGYRIT TPPTN GQQGNSLEEVVBADQ SSCTFDN LSPGL EYNV SVYTVKD DKES VPISDTIIP

CONCENSENSUS P W G L P G Y SV S

FIG. 2. Sequence alignment of SAP-1. A , catalytic domain sequences of the following PTPs are aligned SAP-1, HPTPO (30), PTPlB (26), LAR (31), LCA (48), DPTPlOD (34,35). For those PTPs with two catalytic domains, only the N-terminal domain is used. Numbers in parentheses indicate the first residue shown. The amino acids that are identical in all PTPs are in boldface. B, sequences of the eight FN type 111 repeats of SAP-1 are aligned to domain 111-7 of human FN (49). The residue numbers of the initial amino acids of each repeat are indicated at the Left in parentheses. Residues shared by FN 111-7 and more than seven of the eight repeat sequences are indicated on the Consensus line at the bottom. Residues shared by more than six of eight repeats are indicated by asterisks at the top.

4.2 - * - SAP-I

FIG. 3. Expression of SAP-1 in human tissues. SAP-1 expression was determined by Northern hybridization using 3.0-kb human SAP-1 cDNA labeled with 32P as the probe. Two pg of poly(A)+ RNA isolated from different tissues was used (upper panel). The 1.2-kb mouse @-actin probe was used to examine the same blot (lowerpanel).

Southern blotting of genomic DNA extracted from these cancer cells showed that the SAP-1 gene was not amplified in these cells; suggesting that the amplification of the SAP-1 gene may not be involved in their elevated levels of SAP-1 transcripts. Thus, alterations in the regulation of transcrip-

T. Matozaki, unpublished observation.

tion or degradation rate of mRNA of SAP-1 gene may occur in these cells. It has been demonstrated that the p53 gene, a suppressor oncogene, is mutated and deleted in KATO-I11 cells (13) and most colon cancer cells (36). The p53 has also been shown to suppress the promoter-enhancer activity of numbers of genes (37, 38) so that the SAP-1 gene expression may possibly be suppressed by p53. Thus, inactivation of p53 in KATO-I11 cells and colon cancer cell lines may result in overexpression of SAP-1 gene; this possible mechanism for amplified expression of the SAP-1 gene in cancer cells is worth exploring next.

The increased expression of SAP-1 in WiDr cells and other colon and pancreatic cancer cells also suggests the possible involvement of SAP-1 in the oncogenesis of these cells. It has been demonstrated that the tyrosine-specific protein kinase activity of pp60""" obtained from human colon carcinomas and tumor-derived cell lines such as WiDr is consistently higher than that from normal colon tissues and cultures of normal colon mucosal cells (39). The protein-tyrosine kinase activity of pp6OC-" is augmented by dephosphorylation of Tyr527 at the carboxyl-terminal region of pp60""" (40). In addition, it has been shown that elevated pp6OC'"" kinase activity in colon tumor cells is associated with an apparent increase in the turnover rate of tyrosine phosphates within the carboxyl-terminal portion of the pp60""" from these tumor cell lines (41). Furthermore, it has recently been shown that overexpression of HPTPa, a transmembrane-type PTP, induces cell transformation through the dephosphorylation of the carboxyl-terminal portion of pp60'."" (lo), suggesting that

2080 Cloning of a PTP Expressed i n Gastrointestinal Cancers

Mr 1 2 3 4

200- cis, 4-

116- <

U c

4.2 - - II) k- II - SAP-1

FIG. 6. Immunoblotting and transient expression of SAP-1. WiDr cell ( l a n e 1 ) and HL-60 cell (lam 2 ) lysates (50 pg) were immunoblotted with anti-SAP-1 Ab as described under “Materials and Methods.” COS-7 cells were transfected with the pSRa vector alone (lam 3 ) or the vector containing a full length of SAP-1 cDNA ( l a n e 4 ) by the calcium phosphate precipitation method as described under “Materials and Methods.” Cells were lysed 3 days after transfection and immunoblotted with anti-SAP-1 Ab. Molecular weights are indicated. Arrows indicate the position of SAP-1 protein.

4.2 - - SAP-I

* ~ * ~ * * ~ .Irl) -Pactin

FIG. 4. Expression of SAP-1 in various human cell lines. Two pg of poly(A)+ RNA extracted from various cell lines as indicated was used for Northern blotting as described under “Materials and Methods.” The cell lines include a series of gastric cancer cells (KATO-ZZZ, KWS, TMK-I, MKNI, MKN28, MKN45, MKN74, AGS, JR-I) , a pancreatic cancer cell line (Pam-I, MIA PuCu-e), colon cancer cell lines (COLO230, COL0320, SW480, S W837, WiDr), glioblastoma cell lines (A172, T97G), a neuroblastoma cell line (ZMR- 32), a hepatoma cell line (Huh”/), myeloid cell lines (HL-60, SKM, SKNO-I), a B-lymphoid cell line (Ruji), an epidermoid cancer cell line (A431), a cervical carcinoma cell line (Hela) , and a breast cancer cell line (ZR-75-1). The mouse @-actin probe was used to examine the same blots.

A. B.

Mr

97 - 66-

1 2

2.0

a -GSTiSAP-I

45 - 0 v g 1.0

32 P-Ravtide pNPP

T T

FIG. 7. Fluorescence in situ hybridization of the human SAP-1 gene. Fluorescence in situ hybridization was performed as described under “Materials and Methods.” Double fluorescence spots were detected on R-banded chromosome 19 at the q13.4 region.

colon cancers and pancreatic cancers (44), is also mapped to 19q13.2 (45). In addition, CEA-related genes are also clustered near this region (46). CEA as well as SAP-1 has been shown to be a highly glycosylated protein potentially acting as an adhesion molecule (44); these structural and genetic similar- ities between the CEA family and SAP-1 seem to be interesting. On the other hand, DCC (47), a gene which encodes a putative adhesion molecule, has been shown to be often deleted in human colon cancers. The significance of amplification and deletion of genes encoding adhesion molecules in human colon cancer cells needs further elucidation.

In summary, we have isolated a novel transmembrane type PTP having a FN 111-like structure in the extracellular region and a single PTP catalytic domain in the cytoplasmic region. The high level of expression of this PTP in both pancreatic and colon cancers suggests the possibility that the elevated PTP activity of SAP-1 may induce dephosphorylation of src family protein-tyrosine kinases and subsequent cellular transformation of gastrointestinal cells, in concert with the activation of other oncogenes and inactivation of tumor suppressor genes.

Acknowledgments-We are grateful to Drs. M. Terada and Y. Hattori, Division of Genetics, National Cancer Research Institute, for the KATO-I11 cDNA library and their extensive technical advice.

*%,

FIG. 5. PTP activity of the GST-SAP-1 fusion protein. A, GST-SAP-1 fusion protein or GST alone was expressed and purified as described under “Materials and Methods.” Proteins were then electrophoresed on 10% polyacrylamide gel and visualized by Coo- massie Blue staining. Molecular sizes are indicated in kDa. Lane l , GST alone; lane 2, GST-SAP-1. B, PTP activities of GST alone and GST-SAP-I fusion protein were assayed by usingpNPP or 32P-labeled Raytide as a substrate as described under “Materials and Methods.” The results are the mean S.E. from three separate experiments.

unregulated elevation of PTP activity may contribute to malignant transformation by activation of pp6OC-””. Other src family PTKs such as ~ 5 6 “ ~ have also been shown to be activated by the dephosphorylation of their carboxyl-terminal region (42). Taken together, the elevated SAP-1 activity might result in the dephosphorylation of pp60“”“ or src family PTKs, thereby inducing malignant transformation of cells.

Chromosomal localization of the SAP-1 gene was assigned to 19q13.4. Although SAP-1 expression was not observed in most gastric cancer cell lines, deletion of 19q13 has not been reported in gastric cancer samples so far (43). On the other hand, CEA, a high level of which is usually associated with


We thank Drs. H. Saitoh and Y. Fujioka, Suma Red Cross Hospital, for providing human tissues and Drs. T. Nakagawa and S. Matozaki for providing hematopoietic cell lines. We thank Y. Kobayashi for technical assistance.

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