J Biol Chem, Vol. 273, Issue 45, 29367-29372, November 6, 1998

Purification and Cloning of PZR, a Binding Protein and Putative Physiological Substrate of Tyrosine Phosphatase SHP-2^*

Zhizhuang Joe Zhao and Runxiang Zhao

From the Hematology Division, Department of Medicine, Vanderbilt Cancer Center, Vanderbilt University, Nashville, Tennessee 37232-6305

	ABSTRACT

Top Abstract Introduction Procedures Results Discussion References

Overexpression of a catalytically inactive mutant of tyrosine phosphatase SHP-2 in 293 cells resulted in hyperphosphorylation of a glycoprotein specifically associated with the enzyme. The protein has been purified to near homogeneity. Based on the amino acid sequences of peptides obtained from the protein, a full-length cDNA was isolated. The cDNA encodes a protein with a single transmembrane segment and a signal sequence. The extracellular portion of the protein contains a single immunoglobulin-like domain displaying 46% sequence identity to that of myelin P0, a major structural protein of peripheral myelin. The intracellular segment of the protein shows no significant sequence identity to any known protein except for two immunoreceptor tyrosine-based inhibitory motifs. We name the protein PZR for protein zero related. Transfection of the PZR cDNA in Jurkat cells gave rise to a protein of expected molecular size. Stimulation of cells with pervanadate resulted in tyrosine phosphorylation of PZR and a near-stoichiometric association of PZR with SHP-2. Northern blotting analyses revealed that PZR is widely expressed in human tissues and is particularly abundant in heart, placenta, kidney, and pancreas. As a binding protein and a putative substrate of SHP-2, PZR protein may have an important role in cell signaling.

	INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Protein tyrosine phosphatases (PTPs)¹ represent a highly diverse family of enzymes that have a pivotal role in cell proliferation, differentiation, and transformation (1-3). SHP-1 and SHP-2, representing a subfamily of PTPs containing Src homology 2 domains have been extensively studied in recent years (for review, see Refs. 4-11). These two enzymes share nearly 60% overall sequence identity and are regulated in similar manners. Nevertheless, in many systems, they have distinct physiological functions. SHP-1 has a negative role in proliferation of hematopoietic cells, whereas SHP-2 is a positive transducer of growth factor signal transduction. This distinction in functions is presumably attributable to different physiological targets. Recently, a number putative substrates of SHP-1 and SHP-2 have been identified (12-22), and one of them, designated SIRP or SHPS-1, has been cloned (23, 24). In our earlier studies, by overexpressing catalytically inactive mutants of SHP-1 and SHP-2, we have identified several hyperphosphorylated proteins associated with the inactive SHP-1 and/or SHP-2 (25, 26). Among these is a 43-kDa membrane protein that is specifically associated with SHP-2. Here we report the purification and molecular cloning of this protein. It represents a novel transmembrane protein with an extracellular segment homologous to myelin protein zero and an intracellular portion containing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). We name it PZR for protein zero related.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials-- Polyclonal anti-SHP-1 and anti-SHP-2 antibodies were raised in rabbits against full-length SHP-1 and an Src homology 2 domain-truncated form of SHP-2, respectively (27, 28). An anti-SHP-2 antibody column was made by immobilization of affinity-purified anti-SHP-2 antibody via NH₂ groups to CNBr-activated Sepharose resins (Sigma). Monoclonal anti-phosphotyrosine 4G10 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Endoglysosidase F-N-glycosidase F was from Sigma. The stably transfected 293 cells overexpressing the catalytic inactive mutant of SHP-1 or SHP-2 were obtained as previous described (25, 26). Pervanadate was made by mixing equal moles of sodium vanadate and H₂O₂ and incubating at room temperature for 20 min before addition to the cells (29).

Purification of PZR from 293 Cells Overexpressing the Catalytically Inactive Mutant of SHP-2-- The stably transfected 293 cells overexpressing the catalytic inactive mutant of SHP-2 were grown in Dulbecco's modified Eagle's medium/high containing 10% calf serum and 100 units/ml penicillin and streptomycin and 0.25 mg/ml G418 sulfate. After growing to confluency, the cells were treated with 0.1 mM pervanadate for 20 min before harvesting in ice-cold phosphate-buffered saline. The collected cells were broken up with a Dounce glass homogenizer in Buffer A containing 25 mM -glycerophosphate (pH 7.3), 10 mM EDTA, 2 mM EDTA, 0.2 mM Na₃VO₄, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µM pepstatin A, and 1 µg/ml aprotinin. Nuclear pellets were removed by centrifugation at 800 × g for 20 min, and the remaining postnuclear extract was further centrifuged at 100,000 × g for 45 min to give a clear cytosolic supernatant and a pelleted membrane fraction. The latter pellet, washed once with Buffer A and then dissolved in the same buffer supplemented with 1% Triton X-100, was referred to as the membrane extract. After centrifugation at 10,000 × g for 30 min, the clear membrane extract was loaded onto a fast-flow Q-Sepharose column (Amersham Pharmacia Biotech) equilibrated with Buffer B (25 mM -glycerolphosphate, pH 7.3, 1 mM EDTA, and 2 mM -mercaptoethanol), and the flow through was loaded directly onto a fast-flow SP-Sepharose column (Amersham Pharmacia Biotech). The proteins were then eluted with Buffer B supplemented with 0.3 M NaCl. This was followed by separation of proteins on a wheat germ agglutinin column, which was equilibrated with Buffer B and eluted with 0.3 M N-acetylglucosamine. The eluates were loaded onto an anti-SHP-2 antibody-Sepharose column, which was equilibrated with Buffer B, washed with 0.5 M NaCl, and eluted with 2.0 M NaSCN. The final purification step was achieved by using a 7.5% preparative SDS gel (Bio-Rad). Throughout the purification procedure, the proteins were followed by anti-phosphotyrosine Western blot analyses.

Isolation and Sequencing of Peptides-- The purified protein was digested with endoproteinase Lys-C. Resulting peptide fragments were isolated by reverse-phase high-performance liquid chromatography equipped with a C18 column. Several peptide peaks were chosen for peptide sequence analyses by using a gas phase sequenator at the Vanderbilt Cancer Center.

Molecular Cloning of PZR-- Peptide sequence analyses gave rise to four clean peptide sequences. Search of the expressed sequence tags (EST) database of The Institute for Genomic Research with two of the peptide sequences pulled out an EST that potentially codes for part of a protein. PCR primers were thus designed to amplify the full-length cDNA according to the rapid amplification of cDNA ends (RACE) strategy by using the Marathon cDNA amplification kit from CLONTECH (Palo Alto, CA). One RACE primer (AP1) was provided in the kit. Two gene-specific primers were designed according to the EST sequence. They were 5'-TCCGAGGAGCCTGCTTAACTGGTGAC-3' for 5' RACE and 5'-GTAGTGGTGGGCATAGTTACTGCTGT-3' for 3' RACE. The Advantage KlenTaq polymerase and the Advantage-GC cDNA polymerase, two Taq polymerase mixtures from CLONTECH, were used for PCR amplification according to the manufacturer's protocol. The PCR products were cloned into the pCR2.1 TA cloning vector (Invitrogen, San Diego, CA) and were then sequenced. Combining of the 5' and 3' RACE products, which had an overlapping sequence, gave rise to a complete cDNA encoding a protein containing all the four peptides sequenced. To isolate the full-length coding region, two gene-specific primers corresponding to the 5' and 3' coding regions of the cDNA were designed. They were 5'-GATGGCAGCGTCCGCCGGAGCCGG-3' and 5'-CCAGTTTGGTTTTGTTTCTTGCTGAGG-3'. PCR was performed by using the high-fidelity DNA polymerase Pfu and Turbo Pfu (Stratagene, La Jolla, CA) in addition to the Taq DNA polymerase mixes from CLONTECH as used above. HeLa cell and human kidney Marathon-ready cDNAs purchased from CLONTECH and 293 cell cDNAs prepared by using the reverse transcription-PCR amplification kit (CLONTECH) were used as templates. The PCR was run for 25 cycles at 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 4 min. The products obtained with the Pfu enzymes were subcloned into the pBluescript KS vector (Stratagene), which was opened by EcoRV digestion, whereas those obtained with the Taq polymerases were subcloned into pCR2.1 as described above. DNA sequencing was performed by using the automated DNA sequencer at the Vanderbilt Cancer Center.

Production of Anti-PZR Antibody-- For antibody production, the intracellular portion (corresponding to amino acid residues 192-269) of PZR was expressed in Escherichia coli as a glutathione S-transferase fusion protein by using the pGex-2T vector (Amersham Pharmacia Biotech) and purified by using a glutathione-Sepharose column. A rabbit was injected with the fusion protein to produce the antiserum.

Overexpression of PZR in Jurkat Cells-- PZR cDNA encoding the entire coding sequence of the protein was constructed into the pCDNA3 vector (Invitrogen), and the cDNA plasmid was used to transfect Jurkat cells by electroporation. The cells were grown to ~2 × 10⁶/ml in RPMI 1640 medium supplemented with 10% fetal calf serum and 50 µg/ml streptomycin and penicillin. Cells (1 × 10⁷) were collected by centrifugation, washed with plain medium without serum, and then resuspended in 300 µl of the same plain medium. The cDNA plasmid (20 µg) in 100 µl of water was added to the cells. The electroporation was performed under 950 microfarads, 250 V, and 72 ohms with 4-mm cuvettes by using the ECM 600 electroporation system (BTX Inc., San Diego, CA). After sitting on ice for 15 min, the cells were transferred to 5 ml of complete medium and continued in culture for 72 h before further treatment.

Cell Stimulation, Immunoprecipitation, and Western Blotting Analyses-- To investigate tyrosine phosphorylation of PZR, Jurkat cells transiently overexpressing PZR and wild-type 293 cells were treated with 100 mM pervanadate for 30 min. After washing with ice-cold phosphate-buffered saline, the cells were lysed in Buffer A supplemented with 1% Triton X-100. Extracts were cleared by centrifugation. For immunoprecipitation, cell extracts were incubated overnight with the anti-PZR antibodies prebound to protein A-Sepharose. The beads were washed three times with Buffer A supplemented with 0.3 M NaCl. For Western blot analyses, samples were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were probed with various primary antibodies and were detected by using the ECL system with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech).

Northern Blot Analysis-- To determine the level of expression of PZR in various tissues, a Human Multiple Tissue Northern blot system (CLONTECH) was used as described previously (30). This was performed according to the manufacturer's protocol. Briefly, the blot was prehybridized for 1 h and then hybridized for 1 h at 68 °C in the ExpressHyb hybridization solution provided in the kit. The probe (PZR fragment) was labeled with [-³²P]dCTP by using the T7 Quick Prime kit (Amersham Pharmacia Biotech). The blot was washed three times with 2 × SSC and 0.05% SDS at room temperature and three times with 0.1 × SSC and 0.1% SDS at 50 °C before exposure to x-ray film at 80 °C.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Identification and Purification of a 43-kDa Hyperphosphorylated Protein-- All PTPs contain a highly conserved cysteinyl residue within their catalytic centers, which is directly involved in the formation of a thiophosphate intermediate essential for the catalysis (for review, see Refs. 1 and 2). Mutation of this cysteinyl residue to serine impairs the phosphatase activity. The Cys-to-Ser mutants of SHP-1 and SHP-2 display dominant negative effects and cause hyperphosphorylation of specific cellular proteins on tyrosine as described previously (25, 26). In human embryonic kidney 293 cells, expression of the catalytically inactive Cys-to-Ser mutant form of SHP-2 resulted in tyrosine phosphorylation of 43- and 95-kDa proteins, which were associated with SHP-2, whereas overexpression of the mutant of SHP-1 led to tyrosine phosphorylation of 95- and 110-kDa proteins, which were associated with SHP-1 (Fig. 1A). Tyrosine phosphorylation of these proteins and their association with SHP-1 and/or SHP-2 were also observed in cells (including 293 and HeLa cells) treated with pervanadate, a potent inhibitor of PTPs (data not shown). Because hyperphosphorylation of these proteins correlated with the inactivation of SHP-1 and SHP-2, they are putative substrates of the enzymes. The selective interaction of the 43-kDa protein with SHP-2 suggests its specific role in cell signaling involving SHP-2. In this study, we focus on the purification of the 43-kDa protein (referred to as p43 or PZR hereafter).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   A, identification of the 43-kDa hyperphosphorylated protein designated p43 or PZR. Whole-cell extracts from vector control 293 cells or 293 cells expressing catalytically inactive Cys-to-Ser mutants of SHP-1 or SHP-2 were immunoprecipitated with anti-SHP-1 or SHP-2 antibodies. The immunoprecipitates (IP) were separated on SDS gels, transferred to polyvinylidene difluoride membranes, and detected by anti-phosphotyrosine Western blotting. Positions of SHP-1, SHP-2, and the heavy chain of IgG are indicated. p43, p95, and p110 denote the 43-, 95-, and 110-kDa tyrosine-phosphorylated proteins, respectively. B, deglycosylation of purified p43. Purified p43 (0.5 µg) was treated with 2 units of N-glycosidase F for the indicated periods. Proteins were detected by Coomassie Brilliant Blue R-250 staining. C, specific dephosphorylation of p43 by SHP-2. Purified p43 (0.25 µg) was incubated with 0.1 unit of SHP-1 or SHP-2 for 10 min in a buffer containing 25 mM -glycerolphosphate (pH 7.3), 1 mM EDTA, and 2 mM -mercaptoethanol. Tyrosine phosphorylation was detected by Western blotting analyses with anti-phosphotyrosine. Activity of SHP-1 and SHP-2 was determined by using para-nitrophenylphosphate as a substrates as described (27, 28).

For purification of p43, 293 cells expressing the catalytically inactive mutant of SHP-2 were treated with 0.1 mM pervanadate for 20 min to enhance tyrosine phosphorylation of the protein. After lysis of the cells in Buffer A, p43 was partitioned exclusively in the membrane extract. The protein did not bind to the anionic fast-flow Q-Sepharose column, but this step was necessary for efficient separation of p43 on the next cationic fast-flow SP-Sepharose column, which was eluted with 0.3 M NaCl. In the following step, p43 bound to the wheat germ agglutinin column, and it was eluted with 0.3 M N-acetylglucosamine. This suggests that p43 is a glycoprotein. Because p43 and SHP-2 formed a tight complex, the anti-SHP-2-Sepharose column was able to pull down both proteins. The 95-kDa protein was also found in the complex, but it was less abundant (Fig. 1A). After purification of the NaSCN eluate of the antibody column on a preparative SDS gel, ~50 µg of purified p43 was obtained from 300 plates (150 mm) of transfected 293 cells. On SDS gels, purified p43 ran as a broad band at ~43 kDa, but on deglycosylation by N-glycosidase F, it displayed a sharp band at ~30 kDa (Fig. 1B). This further confirms that p43 is a glycosylated protein and indicates that the glycosylation causes heterogenous migration of p43 on SDS gels. To demonstrate specific dephosphorylation of p43 by SHP-2, we incubated purified p43 with equal units of SHP-1 or SHP-2. As shown in Fig. 1C, SHP-2 caused complete dephosphorylation of p43, whereas SHP-1 only produced a partial dephosphorylation, indicating that p43 may be a physiological substrate of SHP-2.

Peptide Mapping and Amino Acid Analysis-- For this purpose, the gel-purified protein was digested with endoproteinase Lys-C. After separation of the peptides on a reverse-phase C18 column, 28 peaks were obtained. Sequencing of peptides corresponding to four of the peaks gave rise to four clean peptide sequences. These, in single-letter amino acid symbols, are peak 15, RDXTGCSTSESLSPVK; peak 17, SLPSGSHQGPVIYAQLDHSGGHHSDK; peak 19, DRISWAGDLDK; and peak 26, NPPDIVVQPGHIRLYVVEK. The X in the sequence of the peak 15 peptide corresponded to a cycle that gave no regular amino acid signal. Sequencing of peptides corresponding to several other peaks yielded mixed peptides. A search of the protein and nucleotide databases of the National Center for Biotechnology Information by using the BLAST program revealed that peptides corresponding to peaks 19 and 26 showed significant sequence homology to peptide segments of human peripheral myelin P0. A search of the EST database of The Institute for Genomic Research with peptide sequences from peaks 15 and 17 pulled out an EST with an identification number of THC211134. The EST spans 2892 bp, and it has multiple ambiguous bases. When the EST sequence is inverted, its 5' end potentially codes for part of a protein that contains the peptide sequences found in peaks 15 and 17.

cDNA Cloning of PZR-- ESTs are partial, single-base sequences from either end of a cDNA clone. The EST strategy was developed to allow rapid identification of expressed genes by sequence analysis. We thus synthesized two specific PCR primers derived from the EST sequence and used them to amplify a RACE-ready HeLa cell cDNA library. The 3' RACE gave rise to a 521-bp PCR product with a poly(A) tail. The non-poly(A) region essentially verified the EST sequence, which had two uncertain bases in this region. The 5' RACE yielded a 784-bp PCR product with a GC-rich 5' region and an initial codon. Combining of the 3' and 5' RACE products, which had an overlapping sequence, resulted in a cDNA of 1151 bp. The cDNA contained a 807-bp open reading frame encoding a 269-amino acid protein that contained all the peptides sequenced. To clone the coding region of the cDNA, we synthesized two specific PCR primers corresponding to the 5' and 3' coding regions. For PCR amplification, we used three cDNA libraries (from kidney, HeLa, and 293 cells) and four different thermo-DNA polymerases including two hot-start Taq polymerase mixtures (CLONTECH) and two high-fidelity Pfu enzymes (Stratagene). All gave rise to an identical PCR product matching that obtained from RACE. This not only confirmed the coding region but also essentially ruled out possible cloning artifacts caused by PCR.

DNA Sequence Analysis-- The nucleotide sequence of PZR cDNA and the amino acid sequence deduced from it are presented in Fig. 2. The open reading frame consists of 807 nucleotides encoding a protein of 269 amino acids with a calculated molecular mass of 29,081 Da, which is very close to the size of the deglycosylated protein on the SDS gel (Fig. 1B). The deduced amino acid sequence contains a signal sequence at the amino terminus, a membrane-spanning segment in the middle, and an 80-amino acid carboxyl-terminal intracellular portion. The primary structure predicts that PZR is a transmembrane protein, which is consistent with our previous observation that p43 co-localized with catalytically inactive SHP-2(C-S) on the plasma membrane (25). The 132-bp 5'-untranslated sequence has 75% G+C. There are a G at the +4 position and a purine A at the 3 position from the initiating ATG, which conforms with requirements for efficient translation as defined by Kozak (31). The 3' untranslated region stretches 184 bp before reaching the poly(A) tail.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Nucleotide sequence of the entire PZR cDNA and deduced amino acid sequence of the PZR protein. Amino acid residues are numbered on the left, and nucleotide positions are on the right. The putative signal sequence and transmembrane segment are underlined. The ITIM sequences in the cytosolic domain are bold face. Two putative N-linked glycosylation sites and two cysteinyl residues potentially involved in disulfide bond formation in an immunoglobulin-like domain in the extracellular domain are underlined and bold face. The putative tyrosine phosphorylation site Tyr200 is shown in italic and bold face.

Sequence analysis revealed that the extracellular portion of PZR forms an immunoglobulin-like domain with two cysteinyl residues and two potential N-linked glycosylation sites. It shares 45.8% sequence identity and 60.2% sequence similarity with the extracellular domain of myelin P0 (Fig. 3A), a major structural protein of peripheral myelin, which is mutated in type 1B Charcot-Marie-Tooth disease (32, 33). The intracellular segment of PZR displayed no significant sequence identity with any known protein except for two ITIMs, which have a (V/I)XYXXX(L/V) consensus sequence (Fig. 3B). The ITIM was initially defined in FCRB (34) and later in many other hematopoietic cell proteins, including KIR (35) and LAIR (36). Interestingly, this motif is also found in SIRP/SHPS-1, a putative SHP-2 substrate that has recently been cloned (23, 24). It should be noted that PZR shares no significant overall sequence identity with SIRP/SHPS-1. The ITIMs corresponding to Tyr²⁴¹ and Tyr²⁶³ of PZR resemble the consensus sequence for binding of SHP-2 Src homology 2 domains, suggesting that Tyr²⁴¹ and Tyr²⁶³ may provide docking sites for the enzyme. Tyr²⁰⁰ corresponding to a peptide sequencing cycle that gave no signal is probably fully phosphorylated in the purified protein. It has an acidic amino acid residue on the amino-terminal side, conferring the consensus phosphorylation sequence for many tyrosine kinases. Because the Src homology 2 domain of SHP-2 requires hydrophobic residues at the third position after the phosphotyrosine, for specific binding (37), Tyr²⁰⁰ is unlikely to serve as a docking site for SHP-2. It may participate in interactions with other proteins and may presumably act as a target of the catalytic domain of SHP-2. In this regard, PZR is a physiological substrate of SHP-2. Above all, the structural features of PZR make it an important player in cell signaling involving SHP-2.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   A, sequence alignment of PZR with myelin P0. Identical amino acid residues are shown in the middle. +, similar residues. B, sequence alignment of ITIMs. Note that all the proteins listed except for FcRIIB have two ITIMs.

Overexpression of PZR and Its Association with SHP-2-- Western blot analyses with anti-PZR antibody showed that Jurkat cells express essentially no endogenous PZR. Transfection of the cells with PZR cDNA resulted in expression of PZR with the expected molecular size (Fig. 4A). The heterogeneous distribution of the protein on the SDS gel can be attributed to the different degrees of glycosylation. To confirm the tyrosine phosphorylation of PZR and its association with SHP-2, the transfected cells were stimulated with pervanadate. As shown in Fig. 4B, PZR was heavily phosphorylated on tyrosine, and it formed a complex with SHP-2, which itself was phosphorylated on tyrosine. Similar results were observed in wild-type 293 cells treated with pervanadate (Fig. 4C). In this case, in addition to SHP-2, a number of tyrosine-phosphorylated proteins with molecular sizes ranging from 80 to 180 kDa were also co-immunoprecipitated with PZR. In both Jurkat and 293 cells, co-immunoprecipitation of SHP-1 with PZR was not detected, although both cell lines express a high level of SHP-1 (data not shown). We have also treated cells with epidermal growth factor, insulin, and platelet-derived growth factor. However, none of these growth factors could induce tyrosine phosphorylation of PZR (data provided for scrutiny by reviewers). This suggests that PZR might be involved in different signaling systems.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   A, overexpression of PZR in Jurkat cells. Jurkat cells were transfected with the pCDNA3-PZR plasmid as described under "Experimental Procedures." Cell extracts (20 µg) from control and PZR construct-transfected cells were analyzed for PZR expression by using the anti-PZR antibody. B-E, association of PZR with SHP-2. pCDNA3-PZR-transfected Jurkat cells (B) and wild-type 293 cells (C-E) were either left untreated ( lanes) or were treated with 0.1 mM pervanadate (+ lanes) for 30 min. Cells extracts were immunoprecipitated with anti-PZR or anti-SHP-2 antibodies, and the immunoprecipitates or the immunodepleted supernatants were subjected to Western blot analyses with anti-phosphotyrosine, anti-SHP-2, and anti-PZR as indicated. NSB, nonspecific bands, which essentially reflect equal loading of samples.

To characterize further the association of PZR with SHP-2, we performed immunodepletion of PZR and SHP-2. As shown in Fig. 4, D and E, both proteins were totally depleted from cell extracts by correspondent antibodies. In the nonstimulated cells, depletion of one protein had no effect on the presence of the other protein in the cell extracts. In the pervanadate-treated cells, however, depletion of SHP-2 resulted in a >90% loss of PZR in the extracts, whereas depletion of PZR caused ~50% removal of SHP-2. These data not only reveal a near-stoichiometric association of PZR with SHP-2 but also indicate that PZR may be a major anchor of SHP-2 on the plasma membrane.

Northern Blot Analyses of PZR Expression in Human Tissues-- Northern analyses showed that PZR is expressed in all human tissues investigated (Fig. 5). Expression in heart, placenta, kidney, and pancreas appeared to be particular high. The size of the major transcript from the PZR gene is ~4.0 kb. Two minor forms of 3.8 and 1.3 kb were also seen. The cDNA obtained from RACE may correspond to the 1.3-kb transcript, which happened to be predominantly amplified in the PCR reaction because of its short length. The 4.0- and 3.8-kb transcripts may result from alternate splicing or an extended 3' noncoding region. In fact, the EST sequence pulled out from the database spanned 2580 bp after the termination codon.

View larger version (75K): [in this window] [in a new window]   Fig. 5.   Expression of PZR in various human tissues. Each lane contains 2 µg of poly(A)+ RNA. The numbers on the right refer to the sizes of the RNAs given in kb.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

The present study reports purification, molecular cloning, and preliminary characterization of PZR, a binding protein and putative physiological substrate of SHP-2. PZR was purified based on its hyperphosphorylation caused by expression of a catalytically inactive mutant of SHP-2 and on its association with the enzyme, and it was cloned according to peptide sequences. PZR is a novel member of the immunoglobulin superfamily. Its extracellular segment has significant sequence homology to myelin P0, whereas its intracellular portion has two tyrosine phosphorylation sites resembling ITIMs. Myelin P0 is a major transmembrane glycoprotein in the myelin sheath, and it has strong pathological implications. It has been shown that mutation of myelin P0 is responsible for type 1B Charcot-Marie-Tooth disease, and homophilic interaction between P0 molecules mediates the apposition of two neighboring membrane layers of myelin (38, 39). With 60% sequence similarity to myelin P0, PZR might play a similar role in mediating cell-cell interactions in a variety of cells. The ITIM was initially identified in several inhibitory immunoglobulin superfamily members, including human KIR, FcRII, LAIR, gp49, and gp91 (34-36). In contrast to the immunoreceptor tyrosine-based activation motifs found in proteins associated with cell surface immunoglobulin receptors, T-cell antigen receptors, and certain Fc receptors, ITIMs play an important role in signal inhibition by recruiting terminating enzymes, including protein tyrosine phosphatases SHP-1 and SHP-2 and inositol phosphatase SHIP (40-43). The presence of ITIMs in PZR, which is widely distributed in nonhematopoietic cells, suggests a general importance of these motifs.

Our previous studies have shown that hyperphosphorylation of PZR as a consequence of the overexpression of the catalytically inactive mutant of SHP-2 correlated with the inhibition of mitogen-activated protein kinase activation induced by growth factors (25). This suggests that PZR may play a role in down-regulation of growth factor signals. SIRP/SHPS-1, a putative SHP-2 substrate that also contains ITIMs, has been shown to inhibit signaling through tyrosine kinase receptors (23). This inhibitory effect is presumably mediated by ITIMs, which may serve as binding sites for SHP-2. Finally, because PZR specifically interacts with SHP-2 and not SHP-1, it may be responsible for the distinctly different functions of these two enzymes in cell signaling. Considering the crucial role of SHP-2 in cell signaling, as a binding protein and putative physiological substrate, PZR could be an important signaling molecule.

	ACKNOWLEDGEMENTS

We are grateful to Drs. Sanford B. Krantz, Edmond H. Fischer, and Stanley Cohen for critical review of the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant RO1 HL57393.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF087020.

To whom correspondence should be addressed: Hematology Division, 547 MRB II, Dept. of Medicine, Vanderbilt Cancer Center, Vanderbilt University, 2220 Pierce Ave., Nashville, TN 37232-6305. Tel.: 615-936-1797; Fax: 615-936-3853; E-mail: joe.zhao{at}mcmail.vanderbilt.edu.

The abbreviations used are: PTP, protein tyrosine phosphatase; ITIM, immunoreceptor tyrosine-based inhibitory motif; EST, expressed sequence tags; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction.

	REFERENCES

Top Abstract Introduction Procedures Results Discussion References

1. Fischer, E. H., Charbonneau, H., and Tonks, N. K. (1991) Science 253, 401-406[Abstract/Free Full Text]
2. Walton, K. M., and Dixon, J. E. (1993) Annu. Rev. Biochem. 62, 101-120[CrossRef][Medline] [Order article via Infotrieve]
3. Hunter, T. (1995) Cell 80, 225-236[CrossRef][Medline] [Order article via Infotrieve]
4. Zhao, Z., Shen, S. H., and Fischer, E. H. (1995) Adv. Protein Phosphatases 9, 297-317
5. Streuli, M. (1996) Curr. Opin. Cell Biol. 183, 182-188
6. Scharenberg, A. M., and Kinet, J. P. (1996) Cell 87, 961-964[CrossRef][Medline] [Order article via Infotrieve]
7. Tonks, N. K., and Neel, B. G. (1996) Cell 87, 365-368[CrossRef][Medline] [Order article via Infotrieve]
8. Frearson, J. A., and Alexander, D. R. (1997) Bioessays 19, 417-427[CrossRef][Medline] [Order article via Infotrieve]
9. Ulyanova, T., Blasioli, J., and Thomas, M. L. (1997) Immunol. Res. 16, 101-113[Medline] [Order article via Infotrieve]
10. Byon, J. C., Kenner, K. A., Kusari, A. B., and Kusari, J. (1997) Proc. Soc. Exp. Biol. Med. 216, 1-20[Abstract]
11. Neel, B. G., and Tonks, N. K. (1997) Curr. Opin. Cell Biol. 9, 193-204[CrossRef][Medline] [Order article via Infotrieve]
12. Xiao, S., Rose, D. W., Sasaoka, T., Maegawa, H., Burke, T. R., Jr., Roller, P. P., Shoelson, S. E., and Olefsky, J. M. (1994) J. Biol. Chem. 269, 21244-21248[Abstract/Free Full Text]
13. Milarski, K. L., and Saltiel, A. L. (1994) J. Biol. Chem. 269, 21239-21243[Abstract/Free Full Text]
14. Noguchi, T., Matozaki, T., Horita, K., Fujioka, Y., and Kasuga, M. (1994) Mol. Cell. Biol. 14, 6674-6682[Abstract/Free Full Text]
15. Yamauchi, K., Milarski, K. L., Saltiel, A. R., and Pessin, J. E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 664-668[Abstract/Free Full Text]
16. Yamauchi, K., Ribon, V., Saltiel, A. R., and Pessin, J. E. (1995) J. Biol. Chem. 270, 17716-17722[Abstract/Free Full Text]
17. Frearson, J. A., Yi, T., and Alexander, D. R. (1996) Eur. J. Immunol. 26, 1539-1543[Medline] [Order article via Infotrieve]
18. Valiante, N. M., Phillips, J. H., Lanier, L. L., and Parham, P. (1996) J. Exp. Med. 184, 2243-2250[Abstract/Free Full Text]
19. Carlberg, K., and Rohrschneider, L. R. (1997) J. Biol. Chem. 272, 15943-15950[Abstract/Free Full Text]
20. Ruff, S. J., Chen, K., and Cohen, S. (1997) J. Biol. Chem. 272, 1263-1267[Abstract/Free Full Text]
21. Gu, H., Griffin, J. D., and Neel, B. G. (1997) J. Biol. Chem. 272, 16421-16430[Abstract/Free Full Text]
22. Jiao, H., Yang, W., Berrada, K., Tabrizi, M., Shultz, L., and Yi, T. (1997) Exp. Hematol. 25, 592-600[Medline] [Order article via Infotrieve]
23. Kharitonenkov, A., Chen, Z., Sures, I., Wang, H., Schilling, J., and Ullrich, A. (1997) Nature 386, 181-186[CrossRef][Medline] [Order article via Infotrieve]
24. Fujioka, Y., Matozaki, T., Noguchi, T., Iwamatsu, A., Yamao, T., Takahashi, N., Tsuda, M., Takada, T., and Kasuga, M. (1996) Mol. Cell. Biol. 16, 6887-6899[Abstract]
25. Zhao, Z., Tan, T., Wright, J. H., Diltz, C. D., Shen, S.-H., Krebs, E. G., and Fischer, E. H. (1995) J. Biol. Chem. 270, 11765-17769[Abstract/Free Full Text]
26. Su, L., Zhao, Z., Bouchard, P., Banville, D., Fischer, E. H., Krebs, E. G., and Shen, S.-H. (1996) J. Biol. Chem. 271, 10385-10390[Abstract/Free Full Text]
27. Zhao, Z., Larocque, R., Ho, W.-T., Fischer, E. H., and Shen, S.-H. (1994) J. Biol. Chem. 269, 8780-8785[Abstract/Free Full Text]
28. Zhao, Z., Bouchard, P., Diltz, C. D., Shen, S. H., and Fischer, E. H. (1993) J. Biol. Chem. 268, 2816-2820[Abstract/Free Full Text]
29. Zhao, Z., Tan, T., Diltz, C. D., You, M., and Fischer, E. H. (1996) J. Biol. Chem. 271, 22251-22255[Abstract/Free Full Text]
30. Ahmad, S., Banville, D., Zhao, Z., Fischer, E. H., and Shen, S.-H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2197-2201[Abstract/Free Full Text]
31. Kozak, M. (1989) J. Cell Biol. 108, 229-241[Abstract/Free Full Text]
32. Filbin, M. T., and Tennekoon, G. I. (1992) Bioessays 14, 541-547[CrossRef][Medline] [Order article via Infotrieve]
33. Harding, A. E. (1995) Brain 118, 809-818[Abstract/Free Full Text]
34. Muta, T., Kurosaki, T., Misulovin, Z., Sanchez, M., Nussenzweig, M. C., and Ravetch, J. V. (1994) Nature 368, 70-73[CrossRef][Medline] [Order article via Infotrieve]
35. Colonna, M., and Samaridis, J. (1995) Science 268, 405-408[Abstract/Free Full Text]
36. Meyaard, L., Adema, G. J., Chang, C., Woollatt, E., Sutherland, G. R., Lanier, L. L., and Phillips, J. H. (1997) Immunity 7, 283-290[CrossRef][Medline] [Order article via Infotrieve]
37. Lee, C. H., Kominos, D., Jacques, S., Margolis, B., Schlessinger, J., Shoelson, S. E., and Kuriyan, J. (1994) Structure 2, 423-438[Abstract/Free Full Text]
38. Shapiro, L., Doyle, J. P., Hensley, P., Colman, D. R., and Hendrickson, W. A. (1996) Neuron 17, 435-449[CrossRef][Medline] [Order article via Infotrieve]
39. Choe, S. (1996) Neuron 17, 363-365[CrossRef][Medline] [Order article via Infotrieve]
40. Unkeless, J. C., and Jin, J. (1997) Curr. Opin. Immunol. 9, 338-343[CrossRef][Medline] [Order article via Infotrieve]
41. Vely, F., Olivero, S., Olcese, L., Moretta, A., Damen, J. E., Liu, L., Krystal, G., Cambier, J. C., Daeron, M., and Vivier, E. (1997) Eur. J. Immunol. 27, 1994-2000[Medline] [Order article via Infotrieve]
42. Vely, F., and Vivier, E. (1997) J. Immunol. 159, 2075-2077[Abstract/Free Full Text]
43. Isakov, N. (1997) Immunol. Res. 16, 85-100[Medline] [Order article via Infotrieve]