Isolation and Characterization of Murine Orthologue of PTP-BK

Biochemical and Biophysical Research Communications 276, 974 –981 (2000) doi:10.1006/bbrc.2000.3584, available online at http://www.idealibrary.com on

Isolation and Characterization of Murine Orthologue of PTP-BK Takuya Tomemori,* ,† Naohiko Seki,‡ Yo-ichi Suzuki,* Takahiko Shimizu,* Hiroshi Nagata,† Akiyoshi Konno,† and Takuji Shirasawa* ,1 *Department of Molecular Genetics, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan; ‡Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan; and †Department of Otorhinolaryngology, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan

Received August 23, 2000

We isolated and characterized a murine orthologue of PTP-BK, a receptor-type protein tyrosine phosphatase expressed in brain and kidney. The deduced amino acid sequences showed 89, 95, and 92% identities with human, rabbit, and chicken homologues of PTP-BK. Mouse PTP-BK encodes a polypeptide of 1,226 amino acids with calculated molecular weight of 138,598 Da. The mature form of PTP-BK constitutes of 3 domain structures including extracellular, transmembrane, and a single intracellular PTPase domain. Western blot analysis indicated that anti-PTP-BK antibody specifically immunoreacted to 2 distinct molecules of 200 kDa and 220 kDa in COS cells, possibly due to differential glycosylation. The recombinant PTP-BK showed the phosphatase activity specific for the phosphotyrosine, but not for the phosphoserine residue of phosphopeptides in vitro. Radiation hybrid panel assigned mouse PTP-BK gene to 5.02cR distal to from the marker D6Mit339 on chromosome 6. Mouse PTP-BK was classified in PTPRO family in novel nomenclature. We discuss here the diversity and physiological functions of PTPRO family of PTP. © 2000

diversities of PTPs and the novel and important functions of PTPs in phosphotyrosyl homeostasis. PTPs comprise at least 75 identified genes, about half of which belong to the receptor-like PTP subfamily (RPTP), predicting the existence of about 500 genes (1). In the previous study, we isolated a novel PTP gene of specifically expressed in the neonatal brains and kidneys and designated it as PTP-BK (2). The cDNA encodes receptor-type PTP with molecular mass of 138 kDa (3). Based on its structure, PTP-BK belongs to the type III class of RPTPs, whose extracellular region consists of multiple FN-III repeats. RNA blot analysis and in situ hybridization revealed that the expression in the brain is developmentally regulated in the postmitotic maturing neuronal cells (3). In order to clarify the role of PTP-BK in developing neuronal systems in mammals, in the present study, we isolated and characterized murine homologues of PTP-BK. We also assigned and localized the PTP-BK genomic loci to mouse chromosome 6. MATERIALS AND METHODS

Academic Press

Tyrosine phosphorylation is a crucial control mechanism for numerous physiological processes, including various signal transductions of nervous system, immune system, and hematopoietic system. The regulation of tyrosyl phosphorylation is controlled by coordinated actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Originally PTKs were considered to be the key enzymes for tyrosine phosphorylation while little was known for PTPs. However, recent studies revealed the unexpected structural To whom correspondence should be addressed. Fax: ⫹813-35794776. E-mail: [email protected]. 1

0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Molecular cloning of mouse PTP-BK cDNA. mRNA was isolated from 3-day-old mouse brain using oligo-dT cellulose Type 7 (Amersham Pharmacia Biotech, Buckinghamshire, UK). cDNA pools were created with Superscript II RNase H ⫺ reverse transcriptase (Life Technology, Grand Island, NY) followed by amplification with PTP-BK specific primers based on the flanking sequence of start codon (5⬘-ATT TGC GGC CGC CCA CGA TGG GGC ACC TGC C-3⬘) and stop codon (5⬘-CCG CTC GAG GGA CTT GCT GAC GTT CTC GTA-3⬘) of the rat PTP-BK (3). Three PCR fragments were isolated and subcloned into pBluescript II SK (⫺). Sequence reaction was done in both directions using a ABI PRISM 377 DNA Sequencer (Applied Biosystems, Foster City, CA). To verify the franking sequence around start and stop codon, several genomic clones containing 5⬘ UTR and 3⬘ UTR were further cloned from mouse diaphragm muscle genome libraries and sequenced using a ABI PRISM 377 DNA Sequencer (Applied Biosystems). The nucleotide and deduced amino acid sequences were compared with the sequences in the GenBank (Release 117.0) and SWISS-PROT (Release 39.0) databases respectively. Multiple alignments for PTP genes were adjusted

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by the Clustal method in Lasergene software packages (DNASTAR, London, UK). Transfection and Western blot analysis. The entire coding sequences of mouse and rat PTP-BK were amplified by PCR and cloned into the pFLAG-CMV-5a vector (Sigma, St. Louis, MO) to generate expression constructs with FLAG epitope in their C-terminus. COS-7 cells and NIH3T3 cells were transfected with expression constructs using Lipofectamine Plus Reagent (Life Technology). Transfectants were harvested at 48 h after transfection, washed twice with ice-cold phosphate-buffered saline (PBS) and resuspended in 0.5 ml of Lysis buffer consisting of 50 mM HEPES, pH 7.0, 2 mM EDTA, 2 mM EGTA, 10 mM 2-mercaptoethanol, 1 mM phenylmethyl-sulfonyl fluoride, 10 ␮g/ml leupeptin, and 2% Triton X-100. The lysates were incubated with occasional mixing at 4°C for 1 h, and centrifuged (10,000g for 10 min at 4°C) to obtain the solubilized fractions. Ten microliters of cellular lysates with SDS sample buffer were incubated at 100°C for 5 min. Samples were separated on 7.5% polyacrylamide gel under denaturing conditions, transferred to PVDF membranes (Millipore, Bedford, MA), and immunoblotted with M2 antiFLAG mouse monoclonal antibody (Sigma) and anti-PTP-BK antibody (kindly provided by Dr. H. Seimiya) (4) using ECL Western blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK). The signals were detected and analyzed by LAS1000 Lumino Image Analyzer (Fuji Photo Film, Tokyo, Japan). Tyrosine phosphatase assay. Cellular lysates were prepared as described above. Portions of the lysates were gently stirred with 50 ␮l of pre-equilibrated anti-FLAG M2 affinity gel (Sigma) at 4°C for 1 h. The immunocomplex was extensively washed three times with the TBS (20 mM Tris–HCl, pH 7.4, 150 mM NaCl) and twice with the PTP assay buffer (25 mM HEPES, pH 6.0, 5 mM EDTA, 10 mM 2,3-dihydroxybutane-1,4-dithiol). PTP activity in the immunocomplex was measured against tyrosine phosphopeptides, END(pY) INASL and serine phosphopeptides, VNA(pS)PDLPVAK by using the Tyrosine Phosphatase Assay System (Promega, Madison, WI), according to the instruction manual. Chromosome mapping of the mouse PTP-BK gene. Chromosomal assignment of mouse PTP-BK was performed using a radiation hybrid panel (T31 Mouse Radiation Hybrid Panel, Research Genetics Inc., Huntsville, AL) in the same manner as previous reports (5, 6). Primers used for PCR amplification correspond to the coding region of the gene (5⬘-GTC AAT GGT ACA GAC AGA GGA-3⬘ and 5⬘-GAT TTG CTG ACG TTC TCG TAG-3⬘, the PCR product size 120 bp). The radiation hybrid mapping data was processed using the RHMAPPER software package (http://www.genome.wi.mit.edu/ mouse_rh/index.html). The data vector for the gene was 010000000 0000000001 0010000000 1110001000 0000000000 0110100001 0000010000 0010000011 0110000010 110.

RESULTS AND DISCUSSIONS Isolation and characterization of mouse PTP-BK cDNA. To isolate murine orthologue of PTP-BK gene, we amplified cDNA from mouse neonatal brain mRNA by RT-PCR using specific primers based on the flanking sequences around start and stop codon of rat PTP-BK (3). Three independent PCR products encoding mouse PTP-BK were isolated and sequenced in both directions. In order to determine the sequence around start codon, a genomic fragment containing 5⬘ UTR and start codon was further isolated and sequenced. The nucleotide sequence of mouse PTP-BK contained an open reading frame of 3,678 bp, which encoded a polypeptide of 1,226 amino acid residues with a molecular weight of 138,598 Da and an isoelec-

tric point of 5.86. The sequence around a putative initiator methionine codon, CCGCGATGG, was consistent with the Kozak consensus sequence (7) with G at position ⫺3 and G at ⫹4. The nucleotide and the deduced amino acid sequences of mouse PTP-BK are shown in Fig. 1. The deduced amino acid sequence of mouse PTP-BK showed a putative signal peptide sequence (residues 1–29), seven repeats of FN-III-like domain (FNI, residues 141–191; FNII, residues 261–336; FNIII, residues 341– 409; FNIV, residues 441–519; FNV, residues 531– 619; FNVI, residues 640 –714; FNVII, residues 731– 804), a transmembrane portion (residues 830 – 854) and a unique phosphatase catalytic domain (residues 927–1202) containing the consensus motif for PTP active site, IHCSAGVGRTG. The mouse PTP-BK showed 15 possible N-linked glycosylation sites in its extracellular domain (See annotation of GenBank AF295638). The nucleotide and amino acid sequences were subjected for a homology search against GenBank (Release 117.0) and SWISS-PROT (Release 39.0) databases, respectively. The result indicated that mouse PTP-BK showed a significant homology to human PTP-U2 with 82.6% identity at the nucleotide level and 89.0% identity at the amino acid level. In the alignment, the mouse PTP-BK has 10 additional amino acid residues in the extracellular domain compared with human PTP-U2. One additional proline is inserted at residue 256 and the sequence of 9 amino acid residues, SPPSNVSSA, is inserted at residues 284 –292. The survey also showed the highest overall homology to rat PTP-BK with 89.8% identity at the nucleotide and 95.2% identity at the amino acid level. The PTP domain was then searched for its homology with other members of PTP-BK family. PTP catalytic domain is highly homologous to mouse PTP-phi (99% identity at amino acid level), rabbit GLEPP1 (95% identity), human PTP-U2 (95% identity), chicken CRYP-2 (92% identity), and to Drosophila DPTP10D (46% identity), indicating that PTP domain is well-conserved compared to the structure of extracellular domain (Fig. 2A). As shown in Fig. 2A, the consensus motif for PTP active site was highly conserved among these PTPs (Fig. 2A, asterisks), indicating that PTP/substrate relationship may also be conserved in PTP-BK family. Interestingly, PTP-BK showed a significant overall homology to Drosophila DPTP10D that is essential for the axon guidance in the development of Drosophila nervous system (Fig. 2A). To analyze the molecular evolution of PTP-BK, we calculated the genetic distances on the aligned sequences of the entire primary structure, the extracellular domain, and the catalytic domain among PTP-BK family members; mouse PTP-BK, human PTP-U2, rabbit GLEPP-1, chicken CRYP-2, and Drosophila PTP10D (Fig. 2B). Base on the phylogenetic trees, these members are suggested to have evolved

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FIG. 1. Nucleotide sequence and deduced amino acid sequence of mouse PTP-BK. The deduced amino acid sequence is shown under the nucleotide sequence. The predicted signal peptide is boxed. The FN-III-like repeats are underlined with a solid line. The transmembrane region is underlined with a dotted line. Putative PTP domain is indicated by filled boxes. The sequence is deposited in GenBank under Accession No. AF295638.

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FIG. 2. (A) Alignment of catalytic domains among mouse PTP-BK and related PTPs. Putative PTP domains of mouse PTP-BK, human PTP-U2 (Z48541), rabbit GLEPP-1 (U09490), chicken CRYP-2 (U65891), and Drosophila DPTP10D (M80465) were aligned by the Clustal method. Amino acid residues that showed at least three identical residues among these PTP members are indicated by filled boxes. Asterisks (*) show the active site consensus motifs. (B) Phylogenic trees of PTPs. Genetic distance was calculated on the aligned sequences of entire primary structures, PTP catalytic domains, and extracellular domains examined in A. The scale shows the relative distance between sequences.

from a common ancestral gene with the entire structure being evolutionarily conserved, although Drosophila DPTP10D was less conserved than chicken and

mammalian PTP-BKs (Fig. 2B, middle panel). The extracellular domain of PTP-BK is less conserved among the species examined, suggesting that the

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FIG. 3. (A) Western blot analysis of PTP-BK. COS-7 cells transfected with mouse and rat PTP-BK/FLAG expression vector were immunoprecipitated with anti-FLAG antibody. Both anti-PTP-BK and anti-FLAG antibodies detected two bands at 220 kDa and 200 kDa. No band was detected in mock transfectants. (B) PTP assay revealed protein tyrosine phosphatase activity of PTP-BK. Note inhibitory effect of sodium orthovanadate, a nonspecific PTP inhibitor. pY, Tyrosine phosphopeptide; pS, Serine phosphopeptide; Vanadate, Sodium orthovanadate (5 mM). ***P ⬍ 0.001 (Student’s t test).

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FIG. 4. Chromosomal placement of the mouse RPTP-BK gene on the WICGR radiation hybrid map of the mouse genome (http:// www.genome.wi.mit.edu/mouse_rh/index.html). Human/mouse homologous region around the RPTP-BK gene is indicated. Distances are in centimorgans (cM) from the top of chromosome 6 linkage group.

physiological function(s) of the extracellular domain such as binding to ligand molecule(s) may be evolutionarily diverged while the substrate molecule(s) for PTP-BK may be conserved among the species of animals. Characterization of mouse PTP-BK protein by Western blot analysis. In order to characterize mouse PTP-BK protein biochemically, we transiently expressed FLAG-tagged PTP-BK in COS-7 cells or NIH3T3 cells. An anti-PTP-BK C-terminal antibody or an anti-FLAG antibody (M2, Sigma) specifically detected the molecules of 200 kDa and 220 kDa in the immunodetection of mouse PTP-BK as well as rat PTP-BK expressed in COS-7 cells (Fig. 3) or NIH3T3 (data not shown). The shorter form of PTP-BK (200 kDa) presents as the major form while the longer form (220 kDa) presents weak but distinct band, although the physiological significance of these forms still remains to be defined. Since the calculated molecular weights of mouse and rat PTP-BK are both around 138 kDa, 2 forms of PTP-BK shown in Fig. 3A may be posttranslationally modified in COS cells. In addition, the primary structure of PTP-BK deduced 15 potential N-glycosylation sites in the extracellular domain, suggesting that 2 forms of PTP-BK may both be extensively glycosylated in COS cells as previously suggested in the biochemical study on GLEPP1 (8). Phosphatase activity of mouse PTP-BK. In order to confirm that mouse PTP-BK possesses an intrinsic PTPase activity, we investigated PTP enzymatic activities using FLAG-tagged PTP-BK expressed in COS-7

cells. To exclude the influences of other endogenous PTPase, the transfectants were immunoprecipitated with anti-FLAG M2 affinity gel (Sigma). In these immunoprecipitates (IP), we assayed PTPase activities using Tyrosine Phosphatase Assay Kit (Promega). As shown in Fig. 3B, IP fraction containing the recombinant mouse PTP-BK showed a significant phosphatase activity against phosphotyrosine, but not against phosphoserine (release of free phosphate, 1,356 ⫾ 315 vs 156 ⫾ 42 pmol/h/well against 100 ␮M of phosphopeptide; P ⬍ 0.001, unpaired Student’s t test). This phosphatase activity was specifically inhibited by 5 mM of sodium orthovanadate (306 ⫾ 19 pmol/h/well; P ⬍ 0.001, unpaired Student’s t test). IP fraction containing rat PTP-BK also showed a comparable level of phosphatase activity against phosphotyrosine, but not against phosphoserine (release of free phosphate, 925 ⫾ 253 vs 172 ⫾ 35 pmol/h/well; P ⬍ 0.001, unpaired Student’s t test). The phosphatase activity of rat PTP-BK was also specifically inhibited by 5 mM of sodium orthovanadate (289 ⫾ 10 pmol/h/well; P ⬍ 0.001, unpaired Student’s t test). These activities were not detected when the control vector was transfected or only reagent was assayed (Fig. 3B). Chromosome mapping of the mouse PTP-BK gene. We determined the precise chromosomal location of mouse PTP-BK using a radiation hybrid panel. The consequent statistical report indicated the gene was mapped to 5.02 cR distal to from the marker D6Mit339 on chromosome 6 (Fig. 4). This region contains homologous organization to the human chromosome 12p12-

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FIG. 5. Comparison of domain structure of PTP-BK and its related PTPs. Known species and new nomenclatures are listed for each PTPs. Abbreviations for species are as follows: M, Mouse; R, Rat; H, Human; Rb, Rabbit; C, Chicken; D, Drosophila. Protein distributions of PTPRO/PTPROt family are also listed.

p13 region (www.ncbi.nlm.nih.gov/Homology) (Fig. 4). According to the previous report, human PTP-U2 gene was mapped to chromosome 12p13 region by fluorescence in situ hybridization (FISH) method (4). Thus, it was confirmed that the mouse and human genes encoding PTP-BK are localized in a region with conserved linkage homology between these species. The classification of PTP-BK in novel nomenclature. Recently, the Human Gene Nomenclature Committee of the Human Genome Organization classified human PTPs (Fig. 5). According to new nomenclature, PTP-BK belongs to PTPRO family including CRYP-2 (9), PTP-U2 (4) and GLEPP-1 (8, 10). In this nomenclature, PTPRB, PTPRJ and PTPRH were classified as HPTPbeta (11)/DPTP10D (12), DEP-1 (13)/Byp (14)/ PTPbeta2 (15) and SAP-1 (16), respectively. In this study, we isolated and characterized a murine orthologue of PTP-BK. As shown in Fig. 5, PTP-BK is a member of PTPRO family, which is characterized by its various tissue-specific isoforms generated by alternative splicings. PTPROt is a truncated form of PTPRO with a short truncated extracellular domain of 8 amino acid residues. PTP-phi was identified as a member of PTPROt from a murine macrophage cell line with three

isoforms, two membrane-spanning and one cytoplasmic form (17). Interestingly, the insertion of 28 amino acid residues in the juxtamembrane region of membrane-spanning PTP-phi was also found in PTPBK, CRYP-2 and PTP-U2, but not in GLEPP1. Tissue blot analysis of human, rabbit, and mouse indicated that alternatively spliced PTPRO transcripts are expressed in a tissue-specific manner as summarized in Fig. 5. Thus it has been shown that at least two different alternatively splicing plays a key role in producing various expressions and functions of PTPRO family. Suggested functions of PTP-BK. The functional role of PTP-BK remains to be determined while recent studies suggest PTPRO family has a variety of functions. For example, the transfection of PTP-U2 induced the apoptosis in U937 monocytic leukemia cell line after the terminal differentiation (18). In immune cells, PTPROt was expressed in naive quiescent B cell subpopulation and showed an enhancing effects on G0/G1 arrest (19). In chick embryos, CRYP-2 is strongly expressed in axons and growth cones of the developing avian retinotectal system, suggesting that it functions as an adhesion molecule in axon guidance in coordination with other PTPs (20).

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More recently, increasing evidence implicate that PTPs control the neural development. In Drosophila, 4 CNS-specific PTPs; DLAR, DPTP69D, DPTP99A and DPTP10D, have been localized in CNS axons (21–23). In the DPTP69D mutant embryos, the growth cones of motorneuron showed the defective recognition of muscle targets (21). These observations support a critical role for PTPs in the regulation of adhesions that control axonal path finding. Since PTP-BK is highly similar to DPTP10D and its expression is restricted to brain and kidney, PTP-BK may play an important role in axon guidance in the mammalian CNS as well as CRYP-2. Based on these speculations, the identification of in vivo substrates and associating molecules or the phenotype of PTP-BK deficient mouse will be of interest.

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