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• W2051438897 abstract "A tyrosine phosphatase, i.e. PTPase HA2, was previously isolated from 3T3-L1 cells and characterized using O-phospho Tyrosine19-422/aP2 protein (a target of the insulin receptor tyrosine kinase) as substrate. The nucleotide sequence of a PTPase HA2 cDNA showed it to be a homologue of PTPase 1B.When induced to differentiate into adipocytes, confluent 3T3-L1 preadipocytes undergo mitotic clonal expansion followed by growth arrest and then coordinate expression of adipocyte genes. During clonal expansion, expression of PTPase HA2 increases abruptly and then decreases concomitant with the transcriptional activation of adipocyte genes. Constitutive expression of the PTPase by 3T3-L1 preadipocytes using a PTPase HA2 expression vector prevents adipocyte gene expression and differentiation into adipocytes. Appropriately timed exposure of transfected preadipocytes to vanadate (a PTPase inhibitor), just as clonal expansion ceases restores their capacity to differentiate. Treatment of transfected preadipocytes with vanadate prior to or during clonal expansion fails to reverse PTPase HA2-blocked differentiation, whereas treatment of untransfected preadipocytes during mitotic clonal expansion blocks differentiation. Vanadate added following clonal expansion has no effect on differentiation. Thus, a critical tyrosine phosphorylation event(s) occurs between termination of clonal expansion and initiation of adipocyte gene expression while a critical tyrosine dephosphorylation event(s) occurs during clonal expansion. A tyrosine phosphatase, i.e. PTPase HA2, was previously isolated from 3T3-L1 cells and characterized using O-phospho Tyrosine19-422/aP2 protein (a target of the insulin receptor tyrosine kinase) as substrate. The nucleotide sequence of a PTPase HA2 cDNA showed it to be a homologue of PTPase 1B. When induced to differentiate into adipocytes, confluent 3T3-L1 preadipocytes undergo mitotic clonal expansion followed by growth arrest and then coordinate expression of adipocyte genes. During clonal expansion, expression of PTPase HA2 increases abruptly and then decreases concomitant with the transcriptional activation of adipocyte genes. Constitutive expression of the PTPase by 3T3-L1 preadipocytes using a PTPase HA2 expression vector prevents adipocyte gene expression and differentiation into adipocytes. Appropriately timed exposure of transfected preadipocytes to vanadate (a PTPase inhibitor), just as clonal expansion ceases restores their capacity to differentiate. Treatment of transfected preadipocytes with vanadate prior to or during clonal expansion fails to reverse PTPase HA2-blocked differentiation, whereas treatment of untransfected preadipocytes during mitotic clonal expansion blocks differentiation. Vanadate added following clonal expansion has no effect on differentiation. Thus, a critical tyrosine phosphorylation event(s) occurs between termination of clonal expansion and initiation of adipocyte gene expression while a critical tyrosine dephosphorylation event(s) occurs during clonal expansion. During the course of studies on insulin-stimulated glucose uptake by 3T3-L1 adipocytes, we discovered a cellular protein substrate of the insulin receptor tyrosine kinase (1Bernier M. Laird D.M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 844-1848Crossref Scopus (121) Google Scholar, 2Bernier M. Laird D.M. Lane M.D. J. Biol. Chem. 1988; 263: 13626-13634Abstract Full Text PDF PubMed Google Scholar, 3Frost S.C. Kohanski R.A. Lane M.D. J. Biol. Chem. 1987; 262: 9872-9876Abstract Full Text PDF PubMed Google Scholar, 4Hresko R.C. Bernier M. Hoffman R.D. Flores-Riveros J.R. Liao K. Laird D.M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 88: 8835-8839Crossref Scopus (69) Google Scholar, 5Hresko R.C. Hoffman R.D. Flores-Riveros J.R. Lane M.D. J. Biol. Chem. 1990; 265: 21075-21085Abstract Full Text PDF PubMed Google Scholar). This 15-kDa phosphorylated protein, pp15, was subsequently identified as O-phosphotryosyl19-422(aP2) protein (4Hresko R.C. Bernier M. Hoffman R.D. Flores-Riveros J.R. Liao K. Laird D.M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 88: 8835-8839Crossref Scopus (69) Google Scholar). It was found that the phosphoryl group of pp15 turned over rapidly (t < 1 min) and that this turnover was prevented by the trivalent arsenical, phenylarsine oxide (PAO)1 1The abbreviations used are: PAOphenylarsine oxidePTPaseprotein tyrosine phosphataseC/EBPCCAAT/enhancer binding protein422/aP2adipocyte fatty acid binding proteinRT-PCRreverse-transcribed polymerase chain reactionDMEMDulbecco's modified Eagle's mediumMIX3-isobutyl-1-methylxanthineDEXdexamethasonePAGEpolyacrylamide gel electrophoresiskbkilobase(s)HPLChigh performance liquid chromatographyPBSphosphate-buffered salineIGFinsulin-like growth factor. (1Bernier M. Laird D.M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 844-1848Crossref Scopus (121) Google Scholar, 2Bernier M. Laird D.M. Lane M.D. J. Biol. Chem. 1988; 263: 13626-13634Abstract Full Text PDF PubMed Google Scholar). Trivalent arsenicals are known to bind to vicinal or neighboring -SH groups, and thereby, to block biological activity if such -SH groups are required for function (3Frost S.C. Kohanski R.A. Lane M.D. J. Biol. Chem. 1987; 262: 9872-9876Abstract Full Text PDF PubMed Google Scholar, 6Hoffman R.D. Lane M.D. J. Biol. Chem. 1992; 267: 14005-14011Abstract Full Text PDF PubMed Google Scholar, 7Liao K. Hoffman R.D. Lane M.D. J. Biol. Chem. 1991; 266: 6544-6553Abstract Full Text PDF PubMed Google Scholar). Thus, the enzymatic activity responsible for turnover of the phosphoryl group of pp15 would be expected to possess functional neighboring thiol groups. phenylarsine oxide protein tyrosine phosphatase CCAAT/enhancer binding protein adipocyte fatty acid binding protein reverse-transcribed polymerase chain reaction Dulbecco's modified Eagle's medium 3-isobutyl-1-methylxanthine dexamethasone polyacrylamide gel electrophoresis kilobase(s) high performance liquid chromatography phosphate-buffered saline insulin-like growth factor. By using [32P]pp15 as authentic substrate to assay phosphoryl group removing activity, two membrane-associated PAO-sensitive enzymes, i.e. HA1 and HA2, were purified ~10,000-fold from 3T3-L1 adipocytes (7Liao K. Hoffman R.D. Lane M.D. J. Biol. Chem. 1991; 266: 6544-6553Abstract Full Text PDF PubMed Google Scholar). Both HA1 and HA2 possessed the characteristics of protein phosphotyrosine-specific phosphatases (PTPases) and had molecular masses of ~60 and 38 kDa, respectively. HA2 is expressed by 3T3-L1 preadipocytes and to a lesser extent by adipocytes, whereas HA1 is expressed only by adipocytes. In the present paper we show that HA2 possesses extensive amino acid sequence similarity to PTPase 1B. PTPases are known to be sensitive to -SH reagents including trivalent arsenicals since a cysteinyl-SH group (Cys215) is known to function in the catalytic mechanism (8Guan K.L. Dixon J.E. J. Biol. Chem. 1991; 266: 17026-17030Abstract Full Text PDF PubMed Google Scholar). The recent three-dimensional x-ray structure of PTPase 1B (9Barford D. Flint A.J. Tonks N.K. Science. 1994; 263: 1397-1404Crossref PubMed Scopus (683) Google Scholar) shows that the -SH group involved in catalysis is juxtaposed near another -SH (Cys121) which accounts for the strong inhibitory action of vicinal-neighboring -SH group reagents, such as phenylarsine oxide. Two lines of evidence suggested that protein tyrosine phosphorylation-dephosphorylation plays a role in preadipocyte differentiation. First, the protocol used to induce differentiation includes treatment of preadipocytes with IGF-1 or a non-physiologically high level of insulin which acts through the IGF-1 receptor (10Smith P.J. Wise L.S. Berkowitz R. Wan C. Rubin C.S. J. Biol. Chem. 1988; 263: 9402-9408Abstract Full Text PDF PubMed Google Scholar). As the IGF-1 receptor is a mitogenic ligand-activated tyrosine kinase, it appears that tyrosine phosphorylation catalyzed by the receptor plays a role in the induction of differentiation. Second, mitosis which occurs during the clonal expansion phase of the differentiation program is thought to involve mitogen-activated protein kinase, an intermediate that undergoes tyrosine phosphorylation-dephosphorylation during mitogen-stimulated signal transduction (11Tamemoto H. Kadowaki T. Tobe K. Ueki K. Izumi T. Chatani Y. Kohno M. Kasuga M. Yazaki Y. Akanuma Y. J. Biol. Chem. 1992; 267: 20293-20297Abstract Full Text PDF PubMed Google Scholar). It is likely that these mitotic events and attendant DNA replication allow transacting factors to gain access to cis elements involved in the transcriptional activation of genes that control the differentiation program (12MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1994; 64: 345-373Crossref Scopus (934) Google Scholar). As mitotic clonal expansion ceases, expression of adipocyte-specific genes is initiated which ultimately gives rise to the terminally differentiated adipocyte phenotype (13Vasseur-Cognet M. Lane M.D. Curr. Opin. Genet. Dev. 1993; 3: 238-245Crossref PubMed Scopus (53) Google Scholar). In view of evidence that protein tyrosine phosphorylation-dephosphorylation is involved in the induction of differentiation of preadipocytes into adipocytes and our preliminary finding (14Liao K. Lane M.D. FASEB J. 1994; 82 (Abstr.): A1235Google Scholar) that expression of PTPase HA2 fluctuates during differentiation, we investigated the effect of constitutive vector-driven expression of PTPase HA2 on this process. It was found that 3T3-L1 preadipocytes transfected with a PTPase HA2 expression vector failed to differentiate and that treatment with vanadate (a potent PTPase inhibitor) at the appropriate point in the differentiation program restores their capacity to differentiate. Sequencing grade trypsin was purchased from Boehringer Mannheim. All restriction enzymes were from New England Biolabs. pBCMGneo vector (15Karasuyama H. Tohyama N. Tada T. J. Exp. Med. 1989; 169: 13-25Crossref PubMed Scopus (130) Google Scholar) was from Dr. Karasuyama (Basel, Switzerland). Sodium vanadate was purchased from Aldrich. Oil-Red-O was from Matheson Coleman & Bell (Norwood, OH). Antibody directed against PTPase1B was generously provided by Dr. Jack Dixon (University of Michigan). Peptide sequencing and oligonucleotide synthesis were performed in the Johns Hopkins University Protein/Peptide Facility. The Genebank data search was carried out through the Biomedical Supercomputing Center, National Cancer Institute, Frederick, MD. Purification of PTPase HA2 and Tryptic Peptide Sequencing-PTPase HA2 was purified ~20,000-fold from 800 10-cm monolayers of 3T3-L1 preadipocytes following previously described procedures (7Liao K. Hoffman R.D. Lane M.D. J. Biol. Chem. 1991; 266: 6544-6553Abstract Full Text PDF PubMed Google Scholar). The active PTPase HA2 fractions from the terminal glycerol gradient were concentrated using Centricon C-30 filters (from Amicon). The concentrated protein was subjected to SDS-PAGE (10% acrylamide, 16) (Fig. 1A) and then transferred to a nitrocellulose membrane in 25 mM Tris, 192 mM glycine, and 20% methanol, pH 8.3, at 250 mA for 2 h. Protein on the filters was stained with 0.1% Ponceau-S in 1% acetic acid solution, and the band corresponding to PTPase HA2 was cut out, destained in 200 mM NaOH, and the filter segment blocked with 0.5% PVP-40 (Sigma) and 100 mM acetic acid solution for 30 min at 37°C, followed by extensive washing in deionized water. After cutting the filter segment into ~1-mm2 pieces, trypsin (~1/20 the amount of PTPase HA2 protein) in 50 ml of 95% 100 mM NH4HCO3, pH 8.2, and 5% acetonitrile was added and digestion carried out for 48 h at 37°C. After centrifugation, the pellet was washed once with 50 ml of 95% 100 mM NH4HCO3, pH 8.2 and 5% acetonitrile solution. The two supernatants were combined and lyophilized three times to remove all traces of salt. The pellet was dissolved in 100 ml of 0.1% trifluoroacetic acid and injected into a pre-equilibrated microbore C-18 reverse-phase HPLC column (Vydac). After washing with 0.06% trifluoroacetic acid solution (buffer A) at a flow rate of 0.15 ml/min for 10 min, peptides were eluted with a two-step linear gradient from 100% of Buffer A to 62.5% of Buffer A and 37.5% of 0.052% trifluoroacetic acid and 80% acetonitrile solution (Buffer B) in 60 min followed with gradient from 62.5% of Buffer A and 37.5% of buffer B to 25% of Buffer A and 75% of Buffer B in 30 min at 0.15 ml/min flow rate. The column effluent was monitored at 215 nm, and fractions were collected manually. Selected fractions were concentrated and subjected to gas-phase amino acid sequencing using an Applied Biosystems peptide microsequenator. A pair of peptide sequences of PTPase HA2, i.e. n-LHQEDNDYINAS-c and n-FIMGDSSVQDQ-c, were selected as PCR primer sites. A Genebank search showed identical matches of these peptides to those of PTPase 1B. Based on this information, the corresponding oligonucleotides, i.e. 5’-GGAATTCGCACCAGGAAGATAATGACTATATCAATGCCAGC-3’ and 5’-GGAATTCACTGATCCTGCACTGACGAGTCGCCCATGATG-3’ with EcoRI restriction enzyme sites were synthesized. Reverse-transcription was carried out with total cellular RNA from day 0 3T3-L1 preadipocytes and day 5 3T3-L1 adipocytes. The RNA template was heated to 80°C for 2 min, held at 65°C for 40 min, and then slowly cooled to 37°C. The reverse-transcription reaction contained 5 μg of heat-treated RNA, 50 mM KCl, 10 mM Tris-HCl, pH 8.4, 4 mM MgCl2, 1 mM dNTPs, 5 mM primer 2 as initial primer for reverse-transcription, 20 units of placental RNase inhibitor, and 50 units of murine reverse transcriptase in 20 ml. After incubating at 37°C for 30 min, the reaction mixture was heated to 95°C for 5 min to inactivate the reverse transcriptase. Ten μl of the RT reaction mixture were used for DNA amplification in 50 ml containing 50 mM KCl, 10 mM Tris-HCl, pH 8.4, 1.5 mM MgCl2, 200 mM dNTPs, 1 mM each primer, and 2.5 units of Taq DNA polymerase. The PCR reaction was carried out for 30 cycles (94°C for 30 s, 50°C for 1.5 min, and 72°C for 2 min), and half of the product was analyzed by electrophoresis in 1% agarose gel. The DNA band (~694 bp) was isolated by glass wool filtration and then reamplified. The reamplified band was isolated by electrophoresis, digested with EcoRI, inserted into the pBluescript KS(-) vector (Stratagene) and sequenced (17Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52511) Google Scholar). The nucleotide sequences of the RT-PCR products of both the day 0 and day 5 DNA samples were identical. The RT-PCR fragment was labeled with [α-32P]dATP by the random-priming method (18Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16646) Google Scholar) and used as probe to screen 1.2 million phage plaques of a day 5 mouse 3T3-L1 cell cDNA library (19Kaestner K.H. Ntambi J.M. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1989; 264: 14755-14761Abstract Full Text PDF PubMed Google Scholar) in [lamda]ZAP (Stratagene) using low stringency conditions (i.e. hybridization in 20% formamide, 4 × SSC (standard saline citrate), 1 × Denhardt's solution, 1% SDS, 50 mg/ml yeast tRNA, 0.5 mg/ml sodium pyrophosphate, and 50 mM sodium phosphate, pH 7.0, at 42°C overnight and washing with 2 × SSC and 0.1% SDS 2 × 30 min at 42°C). Ten positive clones were identified and purified. The inserts of these clones were characterized, and a full-length cDNA clone was constructed. Total RNA was isolated from proliferating 3T3-L1 cells, confluent cells, 2-day post-confluent cells, and cells at various differentiation stages by the guanidine isothiocyanate method (20Chirgwin J.M. Przybyla A.E. MacDougald R.J. Rutter W.J. Biochemistry. 1979; 24: 5294-5299Crossref Scopus (16648) Google Scholar). For Northern blot analysis, 20 mg of total RNA was denatured with glyoxal and dimethyl sulfoxide and resolved by electrophoresis on 1% agarose gels as described (21McMaster G.K. Carmichael G.G. Proc. Natl. Acad. Sci. U. S. A. 1977; 77: 5201-5205Google Scholar, 22Thomas P.S. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4835-4838Crossref Scopus (5857) Google Scholar). After transfer to Hybond-N membrane (Amersham Corp.) and UV cross-linking, the RNA was stained with methlene blue to locate 28 S and 18 S rRNAs and verify equal loading. The blot was then hybridized with DNA probes in 50% formamide, 4 × SSC, 1 × Denhardt's solution, 50 mM sodium phosphate, pH 7.0, 1% SDS, 50 μg/ml yeast tRNA, and 0.5 mg/ml sodium pyrophosphate at 42°C overnight and washed with 1 × SSC and 0.1% SDS twice at 50°C and 0.1 × SSC and 0.1% SDS twice at 65°C. Chromosomal DNA from one 10-cm dish of confluent 3T3-L1 preadipocytes was isolated by the method of Wigler et al.(23Wigler M. Sweet R. Sim G.K. Wold B. Pellicer A. Lacy E. Maniatis T. Silverstein S. Axel R. Cell. 1979; 16: 777-785Abstract Full Text PDF PubMed Scopus (860) Google Scholar). DNA samples (20 μg each) were double-digested overnight with XhoI and BamHI and resolved by electrophoresis on 0.9% agarose gel. After denaturation, neutralization, and transfer to Hybond-N membranes (24Southern E.M. J. Mol. Biol. 1975; 98: 503-517Crossref PubMed Scopus (21443) Google Scholar), hybridization was carried out as described for Northern blotting (above). The copy number of the PTPase HA2 transgene in chromosomal DNA was quantitated by densitometry normalized to the signal a known quantity of plasmid PTPase HA2 DNA insert. Total cellular protein was extracted from proliferating 3T3-L1 cells and cells at each different stage of the differentiation program. Each 10-cm monolayer of cultured cells was washed twice with ice-cold phosphate-buffered saline (PBS), pH 7.5. The cells were then lysed directly on the plate with boiling 1 × Laemmli SDS sample buffer containing 20 mM dithiolthretol (16Laemmli V.D. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206658) Google Scholar). The cellular lysate was boiled for 5 min and protein determined using the BCA protein method (Pierce). Protein (100 μg) was subjected to 10% SDS-PAGE and transferred to Immobilon-P membranes (Millipore). After Ponceau-S staining, to ensure equal loading of protein, membranes were incubated with primary antibody to the targeted protein, followed with horseradish peroxidase-conjugated secondary antibody. The immunoreactive protein was visualized by ECL (enhanced chemiluminescence, Amersham). Full-length PTPase HA2 cDNA, excised from pBluescript with EcoRI, was blunt end-inserted into the XhoI site of the pBCMGneo expression vector (15Karasuyama H. Tohyama N. Tada T. J. Exp. Med. 1989; 169: 13-25Crossref PubMed Scopus (130) Google Scholar). The sense insert orientation plasmid, pBCMGFS, was identified by restriction analysis and used to transfect 3T3-L1 cells. For transfection, 3 × 105 low passage 3T3-L1 preadipocytes were plated onto 10-cm culture dishes the day before and transfected with 20 μg of pBCMGFS or pBCMGneo control plasmid DNA by the calcium phosphate precipitation method (25Graham F.L. Van der Eb A.J. Virology. 1973; 52: 456-457Crossref PubMed Scopus (6485) Google Scholar). Briefly, 0.5 ml of DNA-CaCl2 (0.25 M) solution was added dropwise to 0.5 ml NaCl-HEPES-Na2HPO4 solution (0.28 M NaCl, 50 mM HEPES, pH 7.12, 1.5 mM Na2HPO4) to form the DNA-calcium phosphate precipitate. After standing for 30 min, the mixture was added directly to culture medium of the cell monolayers. After 4 h at 37°C in the CO2 incubator, cells were shocked with 10% dimethyl sulfoxide-PBS for 3 min and then cultured for 24 h in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum. G418 (350 μg/ml) was added for selection, and resistant foci were isolated and cultured. The 3T3-L1 preadipocytes were cultured in DMEM with 10% calf serum and allowed to achieve confluence (designated as day −2). Differentiation was induced by adding 1 μg/ml insulin, 1 mM dexamethasone (DEX), and 0.5 mM 3-isobutyl-1-methylxanthine (MIX) to 2-day post-confluent cells (designated as day 0) in DMEM with 10% fetal bovine serum (26Reed B.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 285-289Crossref PubMed Scopus (161) Google Scholar, 27Student A.K. Hsu R.Y. Lane M.D. J. Biol. Chem. 1980; 255: 4745-4750Abstract Full Text PDF PubMed Google Scholar). After 48 h (i.e. on day 2), the medium was replaced with DMEM containing 10% fetal bovine serum and 1 μg/ml insulin, and the cells were then fed every other day with DMEM containing 10% fetal bovine serum. By day 3, expression of adipocyte mRNAs (e.g. the 422/aP2 message) occurs and by day 4 small cytoplasmic triglyceride droplets are evident. By day 8-9, the cells are fully differentiated (28Bernlohr D.A. Bolanowski M.A. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1985; 260: 5563-5567Abstract Full Text PDF PubMed Google Scholar). In vanadate reversal experiments, the cell differentiation was induced in the usual manner as above. After 48 h, 35 μM sodium vanadate was added to the normal feeding medium. Vanadate treatment lasted for 2 days with a second addition of 35 μM sodium vanadate made after the first day. 3T3-L1 adipocytes were washed with PBS and fixed for 2 min with 3.7% formaldehyde in PBS. A 0.5% Oil-Red-O isopropanol solution was diluted with 1.5 volume of water, filtered, and added to the fixed adipocyte monolayers for 1 h. Cells were then washed and stained triglyceride droplets visualized and/or photographed (29Mackall J.C. Student A.K. Polakis S.E. Lane M.D. J. Biol. Chem. 1976; 251: 6462-6464Abstract Full Text PDF PubMed Google Scholar). PTPase HA2 was purified ~10,000-fold to near homogeneity from 800 10-cm monolayers of 3T3-L1 preadipocytes by procedures described previously (7Liao K. Hoffman R.D. Lane M.D. J. Biol. Chem. 1991; 266: 6544-6553Abstract Full Text PDF PubMed Google Scholar). As a final step in the purification the protein was subjected to SDS-PAGE (Fig. 1A) and then transferred to a nitrocellulose membrane. The segment of the membrane containing PTPase HA2 was cut out and digested with trypsin, and the tryptic peptides were subjected to HPLC on a microbore C-18 reverse-phase column. The elution profile revealed several major peaks, three of which, i.e. peaks 1, 2 and 3 in Fig. 1B, were subjected to gas-phase amino acid microsequencing. Peaks 1 and 3 were each found to be comprised of two distinct peptides (peptides 3 and 4 in peak 1 and peptides 1 and 2 in peak 3, see Fig. 1C). Owing to the large differences (~3-fold) in the amounts of the two peptides in peaks 1 and 3 at each cycle of Edman degradation, the amino acid sequences of both peptides could be readily deduced. The ordering of amino acids in peptides 1 and 2 was verified by the sequence of the peptide in peak 2 which contained the first 5 amino acids in the sequence of peptide 2. A computer search for possible sequence similarities of these peptides (Fig. 1C) to those in the SwissProt protein data base revealed matches to sequences in different regions of human PTPase 1B (Fig. 2B) (30Tonks N.K. Diltz C.D. Fischer E.H. J. Biol. Chem. 1988; 263: 6722-6730Abstract Full Text PDF PubMed Google Scholar, 31Tonks N.K. Diltz C.D. Fischer E.H. J. Biol. Chem. 1988; 263: 6731-6737Abstract Full Text PDF PubMed Google Scholar). The fact that the first amino acid in three of the four peptide sequences followed an Arg or Lys in the PTPase 1B sequence verified their tryptic cleavage sites in the protein (Fig. 2B). A blank in the first cycle of Edman degradation of peptide 3 is consistent with a Cys (which is normally destroyed during gas-phase sequencing) following a Lys in the sequence of human PTPase 1B (Fig. 2B). These results indicated that PTPase HA2 was a homologue of human PTPase 1B. Oligonucleotide primers were prepared which corresponded to the amino acid sequences of peptide 1 (5’→ 3’) and peptide 2 (3’→ 5’) taking into consideration the nucleotide sequences at comparable sites in PTPase 1B cDNAs found in the GenEMBL data base. RT-PCR using these primers and RNA isolated from confluent 3T3-L1 preadipocytes or day 5 3T3-L1 adipocytes gave rise to the same ~694-bp fragment whose identity was verified by sequencing. This 694-bp RT-PCR fragment was then used as probe to screen a day 5 3T3-L1 cell λZap cDNA library (19Kaestner K.H. Ntambi J.M. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1989; 264: 14755-14761Abstract Full Text PDF PubMed Google Scholar). Ten positive clones were isolated and shown by sequencing to represent the same mRNA. As shown in Fig. 2A the full-length 1969-bp cDNA had an open reading frame encoding 432 amino acids with a calculated molecular mass of 49.5 kDa. The apparent discrepancy between the molecular mass derived from the cDNA and that of purified PTPase HA2 (38 kDa by SDS-PAGE) suggests that the purified protein may have been post-translationally modified or undergone proteolysis during purification. The amino acid sequence of PTPase HA2 shares 83% identity with that of human PTPase 1B (Fig. 2B). The full-length cDNA constructed from two overlapping cDNA clones corresponds to a 1.9-kb mRNA which is in good agreement with the size of an mRNA detected by Northern blot analysis (Fig. 5A) of RNA isolated from 3T3-L1 cells. In addition to the 1.9-kb transcript, a transcript similar in size to 28 S rRNA (~4.8 kb) was detected using the PTPase cDNA as probe. The amount of this transcript, however, varied in different blots with no consistent pattern of expression; thus, it is not clear whether this RNA represents an intermediate in mRNA processing or a nonspecific hybridizing RNA. When exposed to the appropriate complement of external inducers, 3T3-L1 preadipocytes differentiate into adipocytes. To ascertain whether PTPase HA2 is differentially expressed during this process, total cellular RNA and protein extracted from cells at various stages of differentiation were analyzed. Northern blot analysis revealed that expression of PTPase HA2 mRNA is regulated during differentiation, the level of expression being highest during mitotic clonal expansion and lowest during growth arrest (Fig. 3B). Thus, following induction of differentiation, the expression of PTPase HA2 message and protein increases dramatically (day 0 to day 2, Fig. 3, B and C) concomitant with clonal expansion, and then falls as the cells undergo growth arrest and begin to coordinately express adipocyte genes, including the C/EBPα, insulin receptor, 422/aP2, SCD1, GLUT4, as well as others genes (28Bernlohr D.A. Bolanowski M.A. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1985; 260: 5563-5567Abstract Full Text PDF PubMed Google Scholar, 32Bernlohr D.A. Angus C.W. Lane M.D. Bolanowski M.A. Kelly Jr., T.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5468-5472Crossref PubMed Scopus (241) Google Scholar, 33Christy R.J. Yang V.W. Ntambi J.M. Geiman D.E. Landschulz W.H. Friedman A.D. Nakabeppu Y. Kelly Jr., T.J. Lane M.D. Genes & Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (465) Google Scholar, 34Kaestner K.H. Christy R.J. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 251-255Crossref PubMed Scopus (226) Google Scholar, 35Lin F.-T. Lane M.D. Genes & Dev. 1992; 6: 533-544Crossref PubMed Scopus (274) Google Scholar, 36Ntambi J.M. Buhrow S.A. Kaestner K.H. Christy R.J. Sibley E. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1988; 263: 17291-17300Abstract Full Text PDF PubMed Google Scholar, 37Spiegelman B.M. Frank M. Green M. J. Biol. Chem. 1983; 258: 10083-10098Abstract Full Text PDF PubMed Google Scholar, 38Yang V.W. Christy R.J. Cook J.S. Kelly T.J. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3629-3633Crossref PubMed Scopus (68) Google Scholar) that give rise to adipocyte characteristics. To determine whether the inappropriate timing of expression of PTPase HA2 might alter the course of the differentiation program, the effect of constitutive expression of the phosphatase on the program was investigated. A bovine papilloma virus-based PTPase HA2 expression vector, pBCMGFS, was constructed (Fig. 4A) in which the full-length PTPase HA2 cDNA including the 5’-untranslated region was inserted in the sense orientation just 3’ of a human cytomegalovirus gene promoter (15Karasuyama H. Tohyama N. Tada T. J. Exp. Med. 1989; 169: 13-25Crossref PubMed Scopus (130) Google Scholar, 35Lin F.-T. Lane M.D. Genes & Dev. 1992; 6: 533-544Crossref PubMed Scopus (274) Google Scholar). In addition to a neomycin resistance gene, this vector contains bovine papilloma virus sequences that allow extrachromosomal replication at high copy number, as well as integration into the cellular genome. The PTPase expression vector and the parental vector lacking an insert (pBCMGneo) were separately transfected into 3T3-L1 preadipocytes. Selection with G418 generated several PTPase positive and control cell lines. Quantitative Southern blot analysis indicated copy numbers of 2, 1, 8, and 1, respectively, for four representative cell lines, i.e. pBCMGFS-1, −2, −3, and −4 (Fig. 4B), that were used for the studies described below. Northern analysis verified that the pBCMGFS-transfected cell lines exhibited a high level of expression of the exogenous PTPase HA2 mRNA in both the proliferating and growth-arrested confluent states (Fig. 5A). Expression (albeit at a lower level than the exogenous PTPase message), of the endogenous PTPase HA2 message occurred in proliferating, but not growth-arrested preadipocytes (Fig. 5A). The FS-3 cell line, which possessed the highest copy number of the transgene, exhibited the highest level of expression of exogenous PTPase HA2 message (which as indicated below did not lead to a comparable high level of expression of the protein). 3T3-L1 cells that either had not been transfected or had been transfected with the insertless control vector (pBCMGneo) expressed low levels of endogenous PTPase message in the proliferating state and lower levels in the growth-arrested state. Western" @default.
• W2051438897 created "2016-06-24" @default.
• W2051438897 creator A5012475457 @default.
• W2051438897 creator A5043469056 @default.
• W2051438897 date "1995-05-01" @default.
• W2051438897 modified "2023-09-28" @default.
• W2051438897 title "The Blockade of Preadipocyte Differentiation by Protein-tyrosine Phosphatase HA2 Is Reversed by Vanadate" @default.
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• W2051438897 doi "https://doi.org/10.1074/jbc.270.20.12123" @default.
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