Phorbol ester stimulates the activity of a protein tyrosine phosphatase containing SH2 domains (PTP1C) in HL-60 leukemia cells by increasing gene expression.

The affinity-purified antibody to a protein tyrosine phosphatase (PTP) containing two src homology 2 domains (PTP1C) was generated. The antibody recognized two types of PTP1C (PTP1C-alpha and -beta) of which the molecular sizes were 66 (alpha) and 62 kDa (beta), respectively, and these two types were expressed differentially in various cell types. The immune complex phosphatase assay using the antibody demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) and a vitamin D metabolite increased the PTP activity of immunoprecipitated PTP1C to 230 and 150% of control, respectively. By contrast, neither dimethyl sulfoxide nor retinoic acid significantly affected the PTP activity of PTP1C in HL-60 cells. The time course increment by TPA of PTP1C activity was closely correlated with that of the acquisition by HL-60 cells of a macrophage-like phenotype. In addition, TPA increased the amount of PTP1C detected by immunoblotting and immunoprecipitation and raised the level of expression of PTP1C mRNA in HL-60 cells. The increase of PTP1C mRNA induced by TPA treatment was inhibited by cycloheximide, suggesting that new protein synthesis is required for the increase by TPA of PTP1C mRNA expression. Furthermore, TPA increased the rate of transcription of the PTP1C gene without affecting the stability of PTP1C mRNA. These results suggest that (i) two subtypes of PTP1C may exist and function in various cell types, and (ii) TPA stimulates the PTP activity of PTP1C by increasing the transcription rate of PTP1C gene expression. The possible role of PTP1C in the macrophage differentiation will be also discussed.

The affinity-purified antibody to a protein tyrosine phosphatase (PTP) containing two arc homology 2 domains (PTPlC) was generated. The antibody recognized two types of PTPlC (PTPlC-a and -B) of which the molecular sizes were 66 (a) and 62 kDa (B), respectively, and these two types were expressed differentially in various cell types. The immune complex phosphatase assay using the antibody demonstrated that 12-0-tetradecanoylphorbol-13-acetate (TPA) and a vitamin D metabolite increased the PTP activity of immunoprecipitated PTPlC to 230 and 150% of control, respectively. By contrast, neither dimethyl sulfoxide nor retinoic acid significantly affected the PTP activity of PTPlC in HL-60 cells. The time course increment by TPA of PTPlC activity was closely correlated with that of the acquisition by HL-60 cells of a macrophage-like phenotype. In addition, TPA increased the amount of PTPlC detected by immunoblotting and immunoprecipitation and raised the level of expression of PTPlC mRNA in HL-60 cells. The increase of PTPlC mRNA induced by TPA treatment was inhibited by cycloheximide, suggesting that new protein synthesis is required for the increase by TPA of PTPlC mRNA expression. Furthermore, TPA increased the rate of transcription of the PTPlC gene without affecting the stability of PTPlC mRNA. These results suggest that (i) two subtypes of PTPlC may exist and function in various cell types, and (ii) TPA stimulates the PTP activity of PTPlC by increasing the transcription rate of PTPlC gene expression. The possible role of PTPlC in the macrophage differentiation will be also discussed.
The phosphorylation of protein tyrosine residues is a crucial event in the regulation of normal cellular process such as proliferation and differentiation and is also involved in the malignant transformation of cells (1)(2)(3). The level of tyrosine phosphorylation is determined by the balance between the actions of protein tyrosine kinases and PTPs' (4,5). Recently, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviations used are: PTP(s), protein tyrosine phosphatase(s); TPA, 12-0-tetradecanoylphorbol-13-acetate; MeZSO, dimethyl sulfoxide; SHZ, src homology region 2; 1,25-(OH)*VD3, 1,25dihydroxyvitamin DB; kb, kilobase(s); MES, 4-morpholineethanesulfonic acid; pNPP, p-nitrophenyl phosphate; BSA, bovine serum albumin. much attention has been devoted to the role of PTP in the regulation of protein tyrosine phosphorylation, and it has been shown that there are numbers of PTPs which consist of two types, namely the transmembrane type and the nontransmembrane type (4,5). A PTP, PTPlC/HCP/SH-PTPl/SHP (6)(7)(8)(9), is a nontransmembrane type PTP that has been recently cloned. Of interest is that this PTP contains two SH, domains in its N-terminal region. The SH2 domain (10) has recently been found in various nonreceptor-type protein tyrosine kinases such as p~6 0 " -*~ (1,10) and other cytoplasmic signaling proteins such as phospholipase C-7 (ll), GTPactivating protein (1, lo), phophatidylinositol 3-kinase (12), and actin-binding protein (10). These signaling proteins bind phosphorylated tyrosine residues on activated growth factor receptors via SH, domains, and through the association they become substrates for the receptor tyrosine kinase (1, 10). Thus, SH2 domains of PTPlC may direct this unique PTP to tyrosine-phosphorylated protein, thereby modulating protein tyrosine kinase-related signal transduction (6). Subsequently, PTPlC has been shown to be expressed predominantly in hematopoietic cells (7)(8)(9). Thus, PTPlC may play an important role in a certain function that involves tyrosine phosphorylation and dephosphorylation in hematopoietic cells (7)(8)(9). Since PTPs have been suggested to function as a negative regulator of cellular proliferation (1, 13), PTPlC might suppress the proliferation of hematopoietic cells and induce cellular differentiation. It has been shown that promyelocytic leukemia cell line HL-60 is induced to differentiate by various compounds such as TPA (13)(14)(15) or MezSO (13,16). Therefore, we have determined whether these compounds stimulate the PTP activity of PTPlC in HL-60 cells. In the present study, we have generate an affinity-purified polyclonal antibody to PTPlC to determine the effects of TPA and MezSO on the specific activity of immunoprecipitated PTPlC. Results indicate that TPA, but not Me2S0, increased the specific PTP activity of immunoprecipitated PTPlC by increasing the expression of the PTPlC gene in differentiated HL-60 cells.
Affinity Purification of a Polyclonul Antibody-To generate a polyclonal antibody, a peptide containing 18 amino acids (CEKVKKQ RSADKEKSKGS, residues 577-593) of PTPlC (6) was chemically synthesized and conjugated to keyhole limpet hemocyanin by Peptide Institute Inc. (Osaka). Female rabbits were initially injected with 0.75 mg of the purified peptide in complete Freund's adjuvant and boosted every 2 weeks with 0.5 mg of antigen in incomplete Freund's adjuvant.
For affinity purification, 5 mg of peptide was rocked with 2 ml of Affi-Gel 10 at 4 "C overnight, and gels were washed with 30 ml of elution buffer containing 10 mM glycine (pH 7.5), 2 M NaCl, and 0.5 mM EGTA. Rabbit serum was then rocked with gels overnight at 4 "C followed by washing with 20 ml of 50 mM Hepes (pH 7.4) containing 0.5 M NaCl and 0.5 mM EGTA. The antibody bound to the gels was then eluted with 8 ml of elution buffer, and the eluate was combined with 15 ml of 1 M Hepes (pH 8.0) containing 0.5 mM EGTA, followed by concentration with Centriprep-10.
Phosphtase Assay-For the immune complex phosphatase assay, HL-60 cells treated with TPA or other compounds were lysed in 1 ml (approximately 2 mg/ml protein) of ice-cold lysis buffer (RIPA buffer: 50 mM Tris-HC1 (pH 7.5), 150 mM NaC1, 2 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 10% glycerol) containing 10 pg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride and 10 pg/ml leupeptin. The lysates were centrifuged at 10,000 X g for 15 min at 4 'C, and the resultant supernatants were used in the phosphatase assay. The protein concentration was determined by a modification of the method of Bradford (17) for application to cell lysates (18). Immunoprecipitation was performed by incubating 10-500 pg of HL-60 cell lysates with 5 pg of antibody prebound to Sepharose-protein A beads for 4 h at 4 "C. The beads with immunoprecipitated proteins were washed once with a buffer containing 50 mM Tris-HC1 (pH 7.6), 150 mM NaCl, and 0.1% Triton X-100, twice with 1 ml of WG buffer (50 mM Hepes (pH 7.6), 150 mM NaCl, 0.1% Triton X-loo), and twice with 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 the immune complex pellet were incubated at 30 "C for 30 min. The reaction was terminated by the addition of 200 ~1 of 1 N NaOH, and the absorbance at 410 nm was determined (19). The assay of the PTP activity of the immune complex was also performed using the "P-labeled synthetic peptide Raytide (20,21). For the radiolabeling of Raytide, 30 pg of Raytide dissolved in 30 pl of assay buffer (50 mM Hepes (pH 7.5), 0.1 mM EDTA, and 0.015% Briji 35) was incubated with 27 pl of kinase buffer (assay buffer with 0.1 mg/ml BSA and 0.2% 2-mercaptoethanol), 15 p1 of ATP mix (kinase buffer with 0.6 mM ATP and 60 mM MgCl,), 200 pCi of [r-'*P]ATP, and 1 pl of p60"' " tyrosine kinase as described previously (21). The reaction was incubated at 37 "C overnight and terminated by the addition of 0.5 ml of 20% trichloroacetic acid, 20 mM NaHzPO4 and 0.1 ml of 5 mg/ml acetylated BSA. After centrifugation, precipitates were washed five times with 20% trichloroacetic acid, 20 mM NaHzPOI and dissolved in 0.2 M Tris-HC1 (pH 8.0). In the PTP assay, the assay mixtures (100 pl) containing the immune complex pellet, 25 mM imidazole (pH 7.2), 0.1 mg/ml BSA, 10 mM dithiothreitol, and 1 pl of "P-labeled Raytide (approximately 10,000 cpm/pl) were incubated at 30 "C for 30 min. 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 NaH2P04, and 4% Norit A. After centrifugation in a microcentrifuge, the amount of radioactivity in 0.5 ml of supernatant was measured by a liquid scintillation counter.
Zmmunoblotting and Immunoprecipitation-For immunoblotting experiments, cellular lysates were prepared as described above. A 10-50 pg sample of each lysate was subjected to electrophoresis on a 10% SDS-polyacrylamide gel which was then electroblotted to Immobilon P. The blots were blocked for 60 min with 3% BSA in TBST (10 mM Tris (pH 7.6), 150 mM NaCl, 0.05% Tween 20). Blots were then incubated with the anti-PTPlC antibody (1:1,000 dilution) in TBST for 2 h followed by two washes (15 min each) in TBST and visualized with alkaline phosphatase-conjugated goat anti-mouse IgG (1:5,000) in the presence of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (22).
For %S labeling experiments, HL-60 cells were labeled overnight in methionine-free, cystein-free RPMI 1640 supplemented with 10% dialyzed serum using Tran3'S-label at 100 pCi/ml. Labeled cells were lysed in RIPA buffer as described above and centrifuged. The lysates were incubated with 5 p1 of the purified antibody prebound to Sepharose-protein A beads for 4 h at 4 "C. The immunoprecipitates were washed twice in buffer A (20 mM NazHP04 (pH 8.6), 0.5% Triton X-100, 0.1% SDS, 1 M NaC1, 0.1% BSA), twice in buffer B (20 mM Na2HP04 (pH 8.6), 0.5% Triton X-100, 0.1% SDS, 150 mM NaCl), and twice in WG buffer before SDS sample buffer was added. The samples were then boiled for 4 min, loaded onto a 10% SDS-acrylamide gel, and subjected to electrophoresis followed by autoradiography. The radioactivities in 66-kDa PTPlC bands were determined using a Fujix BAS 2000 Bio-image Analyzer.
Zsokztion of Human PTPIC Clones-To isolate the gene encoding PTPlC, a 720-base pair DNA fragment of PTPlC gene was amplified by reverse transcriptase polymerase chain reaction using total RNA extracted from the gastric cancer cell line MKN-45 as described previously (22). The sense and antisense primers used were 5'-GAAGGATCCCGGCCAGAGAACAAGGGCAAGAAC (nucleotides 1060-1083) and 5'-ATAGAATTCGAACTGGGCGATGGCCACGT-AGATG (nucleotides 1774-1806), respectively. The nucleotide numbers used here correspond to the numbering used by Shen et al. (6). The primers contained flanking sequences for BamHI and EcoRI fragment was labeled with [a-3ZP]dCTP (3,000 Ci/mmol) by the sites to facilitate subsequent cloning. This polymerase chain reaction random primer method (23) and used to screen a cDNA bacteriophage library constructed from the breast cancer cell line ZR-75-1. Two independent clones, 1.9 and 2.0 kb in length, were obtained, ligated, and subcloned into the EcoRI site of pBluescript I1 for subsequent sequencing in both directions by the chain termination method as described previously (23).
Northern Blot Analysis-For the analysis by Northern blotting, total RNA (20 pg) was extracted from HL-60 cells and was subjected to electrophoresis on a 1.2% agarose/formaldehyde gel and transferred to a nylon filter as described previously (24). A PTPlC cDNA probe was prepared from plasmid pBluescript 11-PTPlC, constructed as described above, by purifying a 2.4-kilobase pair human PTPlC cDNA fragment. The blot was hybridized with the 32P-labeled PTPlC cDNA probe 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 at 42 'C. The blots were then washed three times at room temperature in 2 X SSC and 0.1% SDS, followed by washing twice for 30 min at 50 "C in 0.1 X SSC and 0.1% SDS. The filter was then rehybridized with 32P-labeled human c-myc or with 32P-labeled mouse @-actin probe.
Assay for Nuclear Transcription-Transcription of nuclear mRNA was compared in cells cultured for 24 or 48 h in the medium without TPA and cells induced to differentiate with 10 nM TPA. The assay was performed as described previously (25,26). Briefly, 3-5 x lo7 nuclei were prepared by using the lysis buffer containing 30% sucrose, 40 mM Tris-HC1 (pH 7.5), 37 mM KCl, 12 mM MgC12, and 0.5% Nonidet P-40. Nuclei resuspended in 100 pl of 50 mM Tris-HC1 (pH 8.4), 40% glycerol, 5 mM MgC12, and 0.1 mM EDTA were incubated with 100 pCi of [32P]UTP for 30 min at 30 "C. The RNA was purified free of DNA, protein, and unincorporated isotope and precipitated with 10% trichloroacetic acid; labeled RNA was recovered on nitrocellulose filters. The radiolabeled RNA was then dissolved in 1 ml of hybridization buffer containing 50% formamide, 5 X SSPE, 0.5% SDS, 5 X Denhardt's solution, and 100 pg/ml yeast tRNA and hybridized at 42 "C overnight with 2 pg of the pBluescript plasmid immobilized to a nylon filter or with immobilized plasmid containing PTPlC cDNA or @-actin cDNA. The filter was washed with a solution containing 0.5 X SSPE and 0.1% SDS at 60 "C and then treated with 10 pg/ml RNase A and 1 pg/ml RNase T1 at 37 "C for 30 min, followed by autoradiography. The radioactivity in each band was determined by a Fujix BAS 2000 Bio-image Analyzer, and transcription rates of PTPlC gene are expressed as the percentage of 0-actin transcription rate.
Other Methods-Monocyte-macrophage differentiation was determined by the percentage of cells with nonspecific esterase activity as described previously (27). Myelocytic differentiation was monitored by the ability of cells to reduce nitro blue tetrazolium (28).
The results presented are the mean & S.E. of three or more experiments unless otherwise stated.

RESULTS
Characterization of the Affinity-purified Polyclonul Anti-PTPl C Antibody-We generated a rabbit polyclonal antibody against a synthetic peptide corresponding to 18 amino acids of the C terminus of PTPlC as described under "Experimental Procedures." This antibody immunoprecipitated a 66-kDa protein from HL-60 cells metabolically labeled with [35S] methionine (Fig. lA, lane 2), whereas the preimmune serum from the same rabbit did not (Fig. lA, lane 1). In addition, when the immunoprecipitation was performed in the presence of excess amounts of the peptide used to raise the antibody, a 66-kDa protein was specifically eliminated (Fig. lA, lune 3 ) .
The molecular size (66 kDa) of the radiolabeled protein specifically recognized by the antibody closely corresponded to the molecular size predicted from the amino acid sequence of cloned P T P l C (6-9). As shown in Fig. lB, lane 1, Western blotting using the anti-peptide antibody showed a single immunoreactive protein band in the HL-60 cell lysate. In the HeLa cell lysate (Fig. lB, lane 2) this antibody also recognized a single protein whose relative molecular size was smaller than that of the protein detected in HL-60 cells and was estimated to be 62 kDa. Thus, we tentatively named these two different P T P l C proteins PTPlC-a (HL-60 cell type) and PTPlC-P (HeLa cell type), respectively. Furthermore, in two gastric cancer cell lines MKN-45 and KATO-111, both PTPlC-a and -P were simultaneously expressed (Fig. lB,   lanes 3 and 4 ) . In the breast cancer cell line ZR-75-1, from which the PTPlC cDNA was originally isolated (6), PTPlCa was predominantly expressed (Fig. lB, lane 5 ) .
Immunoprecipitation was carried out with increasing amounts of HL-60 cell lysate, and the PTP activity of the precipitated was then assayed using pNPP as a PTP substrate. PTP activity was recovered in the immune complex, and the assay was linear in the range of 10 pg up to 500 pg of HL-60 cell lysate (Fig. 2 A ) . When radiolabeled Raytide was used as a PTP substrate, PTP activity was also observed in the immunoprecipitate prepared from HL-60 cell lysate (Fig.   2B). Furthermore, the PTP activity in the immune complex was completely inhibited by 1 mM vanadate, a inhibitor of PTP (Fig. 2, A and B ) . These results indicate that the purified polyclonal antibody specifically recognizes PTPlC and allows us to measure the PTP activity of P T P l C immunoprecipitated from HL-60 cells.
Effects of TPA on P T P l C Activity in HL-60 Cells-We next determined the changes in the PTP activity of P T P l C when HL-60 cells were treated with various compounds that induce HL-60 cell differentiation. When HL-60 cells were treated with 10 nM TPA for 72 h, TPA increased P T P l C activity in HL-60 cells to 230 & 18% of control ( n = 6) (Fig. 3). In addition, a vitamin DJ metabolite, 1,25-(OH)2D3, also increased P T P l C activity to 150 f 8% of control ( n = 3) after a 72-h exposure. In contrast, when HL-60 cells were treated with 1.2% MezSO or 100 nM retinoic acid, no significant effect on P T P l C activity was detected, even after a 120-h exposure (Fig. 3). After a 120-h exposure of HL-60 cells to 1.2% Me2S0, cells attained the characteristics of granulocytes, as measured by their capacity to reduce nitro blue tetrazolium to insoluble formazan granules (67 f 8% of cells positive, n = 3). TPA increased the PTPlC activity in a time-dependent manner; the detectable increase in P T P l C activity was observed after a 24-h exposure of HL-60 cells to TPA, and the maximal increase of P T P l C activity stimulated by TPA was observed after a 72-h exposure (Fig. 4A). The time course of the stimulation by TPA of P T P l C activity was closely related to the acquisition of nonspecific esterase activity, a marker of the monocyte-macrophage phenotype (25) (Fig. 4A). Furthermore, TPA stimulated P T P l C activity in a concentrationdependent fashion with a detectable increase observed at 1 nM TPA and a maximal stimulation at 10 nM TPA (Fig. 4B). Effects of TPA on the Protein Level of P T P l C in HL-60 Cells-To explore the mechanism by which TPA increases P T P l C activity in macrophage-like differentiated HL-60 cells, we next examined TPA-induced changes in the PTPlC protein level as detected by immunoblotting. As shown in Fig.  5A, both 10 nM TPA and 100 nM 1,25-(OH)*VD3 significantly increased the intensity of the PTPlC band, whereas neither Me2S0 nor retinoic acid had any effect. When HL-60 cells were treated with 10 nM TPA for 6-72 h and the lysates were immunoblotted, the intensity of the PTPlC band was increased in a time-dependent fashion (Fig. 5B). Furthermore, when HL-60 cells were treated with or without 10 nM TPA for 48 h followed by incubation with "S-labeled amino acids for another 16 h, TPA increased the radioactivity of the P T P l C 66-kDa band to 255 * 24% of control ( n = 3) (Fig.   5 C ) . Thus, these results suggest that TPA may increase the level of P T P l C protein, thereby elevating the PTP activity of P T P l C in macrophage-like differentiated HL-60 cells. It was constantly observed that a 25-kDa protein coimmunoprecipitated with P T P l C (Fig. 5C, lanes 1 and 2), suggesting that this protein may specifically form a complex with PTPlC in HL-60 cells.
Effects of TPA on P T P l C mRNA Expression in HL-60 Cells-Since TPA increased the level of P T P l C protein, it seemed possible that TPA might stimulate the synthesis of P T P l C protein by increasing P T P l C gene expression in differentiated HL-60 cells. To determine the level of PTPlC gene expression we isolated P T P l C cDNA from a breast cancer cell (ZR-75-1) cDNA library. T o isolate the PTPlC gene, we generated a 750-base pair DNA fragment corresponding to the nucleotide sequence of the PTP domain of PTPlC by reverse transcriptase polymerase chain reaction and used it as a probe for library screening. The sequence of the cloned P T P l C cDNA was identical to the nucleotide sequence published by Shen et al. (6) except that our PTPlC cDNA contained 32 additional nucleotides (GGAGAAGAGCA-AGGGTTCCCTCAAGAGGAAGT) in the 5"noncoding region. This suggests that two different PTPlC cDNAs may utilize different 5' exons.
When HL-60 cells were treated with 10 nM TPA as a function of time, TPA significantly increased a 2.4-kb transcript of P T P l C after a 24-h exposure and maximally increased mRNA expression a t 48 h (Fig. 6A). Measurement of radioactivities in 2.4-kb bands showed that TPA increased

Regulation of PTPl C Activity in HL-60 Cells
A.  (Fig. 6A) as described previously (13,15). The time course of the increased expression of the PTPlC gene overlapped closely with that of an increase in the PTPlC activity in TPA-treated HL-60 cells (see Fig. 4A). Since TPA increased the levels of PTPlC mRNA only after a 24-h exposure, we next determined the effect of cycloheximide on TPA-induced PTPlC gene expression. Although the increased level of PTPlC mRNA was observed after treatment of HL-60 cells with TPA for 48 h, simultaneous treatment with cycloheximide inhibited the increase in the level of PTPlC mRNA induced by TPA (Fig.   6B). This result suggests that the increase of PTPlC mRNA induced by TPA may require new protein synthesis.
The increase of PTPlC mRNA in response to TPA may result from either alteration of transcription rate or degradation rate of mRNA. To examine these possibilities we next performed nuclear run-off transcription assays. The results of these experiments were normalized to the rate of transcription of the @-actin gene. As shown in Fig. 7A, a significant tracted from HL-60 cells. The yield of total RNA extracted from HL-60 cells was not changed by actinomycin D treatment up to 8 h. The autoradiogram of disappearance of P T P l C mRNA with time in HL-60 cells treated with or without TPA is shown in Fig. 7B. However, the calculated half-life of P T P l C mRNA in cells treated with TPA was comparable to that in control cells (TPA-treated, 3.3 k 0.3 h; control, 3.6 ? 0.5 h, n = 3). Thus, the results indicate that the stability of P T P l C mRNA may not be changed by TPA treatment.

DISCUSSION
In the present study, we generated an affinity-purified polyclonal antibody to a C-terminal peptide of PTPlC. Both immunoprecipitation and immunoblotting showed that the antibody specifically recognized PTPlC as a 66-kDa protein in the HL-60 cell lysate. Furthermore, in the HeLa cell lysate this antibody recognized another type of PTPlC (PTPlC-8) of which the estimated molecular size was 62 kDa. In the breast cancer cell line ZR-75-1, PTPlC-n was predominant, whereas both types of P T P l C were expressed in two gastric cancer cell lines. Furthermore, both types of P T P l C were also expressed in two pancreatic cancer cell lines (Panc-1 and MIA Paca-2) and three colon cancer cell lines (SW480, SW837, and WiDr).' Thus, these results indicate that two different types of P T P l C proteins were expressed differentially and may function in various cell types. The molecular size of PTPlC-n (66 kDa) closely corresponds to the molecular size estimated by P T P l C cDNA sequence published previously (6)(7)(8)(9). In addition, PTPlC-n is predominantly expressed in ZR-75-1 cell and cDNA of P T P l C has been originally isolated from a cDNA library of ZR-75-1 cell (6). Thus, PTPlC-a might be encoded by the PTPlC gene originally described (6). On the other hand, alternative splicing of PTPlC-n gene may generate both N and ( 3 forms, or the a and p forms may be encoded by distinct P T P l C genes. The isolation of a cDNA clone of PTPlC-0 from a HeLa cell cDNA library is currently being carried out in our laboratory. The affinity-purified antibody was found to immunoprecipitate PTPlC and allowed us to measure the changes in the PTP activity of P T P l C during the differentiation of HL-60 cells. By immune complex phosphatase assay, we have demonstrated that TPA increases the PTP activity of PTPlC in macrophage-like differentiated HL-60 cells. This stimulatory effect of TPA on PTPlC activity was found to be time-and TPA concentration-dependent. The concentrations of TPA required for the induction of an increase in PTPlC activity correspond well with those required to induce the macrophage-like differentiation of HL-60 cells. Furthermore, the time course of the TPA-induced differentiation of HL-60 cells and that of the stimulation of P T P l C activity by TPA closely overlapped. A derivative of vitamin D, 1,25-(OH)2VD3, which induces the monocyte-like differentiation of HL-60 cells (13), also increased the PTP activity of PTPlC. By contrast, neither Me2S0 nor retinoic acid, both of which induce the myelocyte-like differentiation of HL-60 cells (131, affected ' T. Matozaki and T. Uchida, unpublished observation. the PTPlC activity in HL-60 cells. It has been suggested that PTPs function as a counterpart of protein tyrosine kinases; protein tyrosine kinases generally stimulates cellular proliferation, whereas PTPs negatively regulate cellular proliferation. The cellular differentiation that is part of the maturation process involves the programmed shutdown of the proliferation capacity of the cell (13). In addition, PTPlC has been demonstrated to be highly expressed in macrophage-derived cell lines (7). Taken together, the present results suggest the possibility that PTPlC might be involved in the process of the macrophage differentiation. To examine this possibility further it is required to investigate whether transfection of PTPlC cDNA or microinjection of PTPlC protein to HL-60 cells induces macrophage-like differentiation of HL-60 cells or not; these experiments are being performed at present in our laboratory. Although MezSO did not increase PTPlC activity in HL-60 cells, it has been demonstrated that both TPA and Me2S0 increase the total PTP activity in differentiated HL-60 cells (29). Therefore, another PTP, but not PTPlC, might be involved in the myelocyte-like differentiation of this leukemia cell line. In fact, it has recently been demonstrated that MezSO induces the gene expression of CD45, a transmembrane-type PTP, during the myelocyte-like differentiation of HL-60 cells (30).
In the present study, the mechanism underlying the increase of PTPlC activity induced by TPA has been also investigated. TPA increased both the synthesis of PTPlC and PTPlC gene expression in HL-60 cells. Thus, the results suggest that the TPA-induced increase of PTPlC activity may be, in part, caused by the stimulation of the synthesis of PTPlC protein by increasing the expression of the PTPlC gene. TPA and 1,25-(OH)zD3 have been demonstrated to induce the expression of several genes including c -j m (31) and c-fos (32) in HL-60 cells. By contrast, these compounds depress c-myc expression during the differentiation of HL-60 cells (13,15). The depression by TPA of c-myc expression occurs rapidly after a 6-h exposure of the cells to TPA, and c-myc expression is nearly undetectable after a 12-h exposure of HL-60 cells to TPA (15,31). On the other hand, c-fms expression (31) and increases in PTPlC gene expression are detectable after a 24-h exposure to TPA and become maximal following 48-72 h of induction. In the case that an increase of PTPlC activity could be involved in the macrophage-like differentiation of HL-60 cells, present data above suggest that the increase in PTPlC activity may not have a primarily function but may play a role in the later events of differentiation of HL-60 cells. This is also supported by the result showing that cycloheximide treatment inhibits the increased PTPlC gene expression induced by TPA. TPA-induced synthesis of a new protein seems to be necessary for the increase by TPA of PTPlC gene expression.
The mechanism for the elevation of PTPlC mRNA induced by TPA was further explored in our study, and results have shown that the increase of transcription rate of PTPlC gene but not the change of mRNA stability may be responsible for the increase of PTPlC mRNA detected by Northern blotting.
Similarly, Me2S0 has been demonstrated to increase CD45 mRNA expression through activation of gene transcription in HL-60 cells (29). Isolation of genomic DNA of PTPlC is required to investigate whether or not a TPA-responsive element exists in the enhancer-promoter region of PTPlC gene. Although present results have demonstrated that the TPA-induced increase in the level of PTPlC protein is a possible mechanism for the increase of PTPlC activity in-duced by TPA, it is also possible that TPA may induce the phosphorylation of PTPlC, thereby increasing its activity. In the preliminary study, we did not observed any increase in phosphorylation of PTPlC immunoprecipitated from 32Plabeled HL-60 cells treated with TPA.3 However, it is still possible that TPA may phosphorylate a certain protein which then interacts with PTPlC, thereby altering the activity of PTPlC in HL-60 cells.
The results of this study have led us to suggest a physiological role for PTPlC for the first time. However, the role of the SH2 domains of PTPlC is still unknown, and the target protein of PTPlC has not been identified. We identified a 25-kDa protein that was immunoprecipitated along with PTPlC (Fig. 5B). This 25-kDa protein band was observed even in the presence of excess amounts of the peptide used for immunization. Thus, this 25-kDa protein may be the putative target protein of PTPlC, and further characterization and purification of this protein are necessary to establish its identity.