A Novel Cytoplasmic Dual Specificity Protein Tyrosine Phosphatase Implicated in Muscle and Neuronal Differentiation
1996; Elsevier BV; Volume: 271; Issue: 7 Linguagem: Inglês
10.1074/jbc.271.7.3795
ISSN1083-351X
AutoresRobert J. Mourey, Quinn Vega, Jean S. Campbell, Mary Pat Wenderoth, Stephen D. Hauschka, Edwin G. Krebs, Jack E. Dixon,
Tópico(s)Muscle Physiology and Disorders
ResumoDual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of mitogen-activated protein kinase (MAPK). In addition to hydrolyzing phosphotyrosine, dsPTPs can hydrolyze phosphoserine/threonine-containing substrates and have been shown to dephosphorylate activated MAPK. We have identified a novel dsPTP, rVH6, from rat hippocampus. rVH6 contains the conserved dsPTP active site sequence, VXVHCX2GX2RSX5AY(L/I)M, and exhibits phosphatase activity against activated MAPK. In PC12 cells, rVH6 mRNA is induced during nerve growth factor-mediated differentiation but not during insulin or epidermal growth factor mitogenic stimulation. In MM14 muscle cells, rVH6 mRNA is highly expressed in proliferating cells and declines rapidly during differentiation. rVH6 expression correlates with the inability of fibroblast growth factor to stimulate MAPK activity in proliferating but not in differentiating MM14 cells. rVH6 protein localizes to the cytoplasm and is the first dsPTP to be localized outside the nucleus. This novel subcellular localization may expose rVH6 to potential substrates that differ from nuclear dsPTPs substrates. Dual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of mitogen-activated protein kinase (MAPK). In addition to hydrolyzing phosphotyrosine, dsPTPs can hydrolyze phosphoserine/threonine-containing substrates and have been shown to dephosphorylate activated MAPK. We have identified a novel dsPTP, rVH6, from rat hippocampus. rVH6 contains the conserved dsPTP active site sequence, VXVHCX2GX2RSX5AY(L/I)M, and exhibits phosphatase activity against activated MAPK. In PC12 cells, rVH6 mRNA is induced during nerve growth factor-mediated differentiation but not during insulin or epidermal growth factor mitogenic stimulation. In MM14 muscle cells, rVH6 mRNA is highly expressed in proliferating cells and declines rapidly during differentiation. rVH6 expression correlates with the inability of fibroblast growth factor to stimulate MAPK activity in proliferating but not in differentiating MM14 cells. rVH6 protein localizes to the cytoplasm and is the first dsPTP to be localized outside the nucleus. This novel subcellular localization may expose rVH6 to potential substrates that differ from nuclear dsPTPs substrates. INTRODUCTIONExtracellular signals, such as mitogenic growth factors, bind to specific cell surface receptors that, in turn, initiate intracellular signaling through activation of a series of protein kinases (reviewed in (1.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4219) Google Scholar)). One such pathway involves the activation of mitogen-activated protein kinase (MAPK)( 1The abbreviations used are: MAPK mitogen-activated protein kinase MEK MAPK/ERK kinase PTP protein tyrosine phosphatase dsPTP dual specificity protein tyrosine phosphatase pNPP p-nitrophenyl phosphate NGF nerve growth factor EGF epidermal growth factor bFGF basic fibroblast growth factor PCR polymerase chain reaction GST glutathione S-transferase HA hemagglutinin bp base pair(s) kb kilobase(s) DMEM Dulbecco's modified Eagle's medium PBS phosphate-buffered saline CH2 domain cdc25 homology domain 2.) ( 2MAPK is also known as extracellular signal-regulated kinase (ERK) but will be referred to as MAPK in this paper.) (2.Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1476) Google Scholar). MAPK is regulated by an upstream kinase, MAPK/ERK kinase (MEK)( 3MEK is also known as MAP kinase kinase (MAPKK) but will be referred to as MEK in this paper.) (3.Ahn N.G. Seger R. Bratlien R.L. Diltz C.D. Tonks N.K. Krebs E.G. J. Biol. Chem. 1991; 266: 4220-4227Abstract Full Text PDF PubMed Google Scholar, 4.Wu J. Harrison J.K. Vincent L.A. Haystead C. Haystead T.A. Michel H. Hunt D.F. Lynch K.R. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 173-177Crossref PubMed Scopus (117) Google Scholar, 5.Zheng C.-F. Guan K.-L. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar), which phosphorylates MAPK on both Tyr and Thr residues within a TXY phosphorylation motif (reviewed in (1.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4219) Google Scholar)). This diphosphorylation activates MAPK and allows it to phosphorylate nuclear transcription factors, protein kinases, cytoskeletal proteins, and other cell growth-dependent substrates (see recent reviews in (1.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4219) Google Scholar) and (6.Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3185) Google Scholar)). Recently, a family of MAPKs, including the MAPK-related proteins c-Jun N-terminal kinase/stress-activated protein kinase and p38, has been identified and implicated in kinase cascades that respond to mitogenic, differentiation, and stress-induced signals (reviewed in (7.Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (996) Google Scholar)).MAPK and MAPK-related proteins can be inactivated by dephosphorylation of either Tyr or Thr residues (reviewed in (6.Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3185) Google Scholar)). Two protein phosphatases, MAP kinase phosphatase (CL100) and PAC1, have been shown to dephosphorylate and inactivate MAPK both in vitro(8.Alessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020PubMed Google Scholar, 9.Charles C.H. Sun H. Lau L.F. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5292-5296Crossref PubMed Scopus (183) Google Scholar, 10.Ward Y. Gupta S. Jensen P. Wartmann M. Davis R.J. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar) and in vivo(10.Ward Y. Gupta S. Jensen P. Wartmann M. Davis R.J. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar, 11.Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1021) Google Scholar). These phosphatases belong to the protein tyrosine phosphatase (PTP) class of proteins, as defined by their conserved active site sequence, HCXAGXXR(S/T) (reviewed in (12.Stone R.L. Dixon J.E. J. Biol. Chem. 1994; 269: 31323-31326Abstract Full Text PDF PubMed Google Scholar)). More specifically, MAPK phosphatase-1 and PAC1 are members of a subclass of PTPs commonly referred to as dual specificity protein tyrosine phosphatases (dsPTP) (reviewed in (13.Keyse S.M. Biochim Biophys Acta. 1995; 1265: 152-160Crossref PubMed Scopus (233) Google Scholar)), which hydrolyze phosphate from Ser/Thr residues as well as from Tyr. Transcription of dsPTPs are induced by a variety of extracellular stimuli, where they may function in such processes as tissue regeneration(14.Nathens D. Law L.F. Christy B. Hartzell S. Nakabeoou Y. Ryder K. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 893-900Crossref PubMed Google Scholar), serum and growth factor stimulation(9.Charles C.H. Sun H. Lau L.F. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5292-5296Crossref PubMed Scopus (183) Google Scholar, 15.Ishibashi T. Bottaro D.P. Michieli P. Kelley C.A. Aaronson S.A. J. Biol. Chem. 1994; 269: 29897-29902Abstract Full Text PDF PubMed Google Scholar, 16.Kwak S.P. Dixon J.E. J. Biol. Chem. 1995; 270: 1156-1160Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), oxidative stress and heat shock response(15.Ishibashi T. Bottaro D.P. Michieli P. Kelley C.A. Aaronson S.A. J. Biol. Chem. 1994; 269: 29897-29902Abstract Full Text PDF PubMed Google Scholar, 17.Keyse E.M. Emslie S.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (568) Google Scholar), nitrogen starvation(18.Guan K. Hakes D.J. Wang Y. Park H.-D. Cooper T.G. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12175-12179Crossref PubMed Scopus (75) Google Scholar), and mitogen stimulation(19.Rohan P.J. Davis P. Moskaluk C.A. Kearns M. Krutzsch H. Siebenlist U. Kelly K. Science. 1993; 259: 1763-1766Crossref PubMed Scopus (263) Google Scholar). dsPTPs may also regulate cell cycle events, e.g. controlling entry into mitosis (20.Millar J.B.A. Russell P. Cell. 1992; 68: 407-410Abstract Full Text PDF PubMed Scopus (192) Google Scholar) and interacting with cyclin-dependent kinases (21.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1319) Google Scholar, 22.Hannon G.J. Casso D. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1731-1735Crossref PubMed Scopus (110) Google Scholar). Dephosphorylation of MAPK and other MAPK-like proteins by dsPTPs suggests a function for dsPTPs in the regulation of cellular mitogenesis and differentiation.In this study, we have identified a novel dsPTP from rat hippocampus, referred to as rVH6. rVH6 mRNA is detected in all tissues examined by Northern blot analysis. Recombinant rVH6 protein exhibits enzymatic activity against the artificial substrate, p-nitrophenyl phosphate (pNPP), and is able to dephosphorylate and inactivate MAPK in vitro. In COS-1 cells, expressed rVH6 protein localizes to the cytoplasm, and is thus the first dsPTP to be localized outside the nucleus. rVH6 mRNA is induced in PC12 cells following nerve growth factor (NGF)-mediated differentiation but is not induced following insulin or epidermal growth factor (EGF) mitogenic stimulation. The intermediate time course of rVH6 induction is distinct from other immediate-early gene dsPTPs and is closely correlated with the sustained activation and deactivation pattern of MAPK activity observed in NGF-mediated neuronal differentiation(23.Traverse S. Gomez N. Paterson H. Marshall C. Cohen P. Biochem. J. 1992; 288: 351-355Crossref PubMed Scopus (802) Google Scholar). Interestingly, in proliferating MM14 myoblasts, rVH6 mRNA levels are expressed at high levels and then decline rapidly following commitment to muscle differentiation. Moreover, the loss of rVH6 message correlates with the ability of basic fibroblast growth factor (bFGF) to stimulate MAPK activity in MM14 muscle cells. The widespread expression of rVH6, as well as its induction by NGF and bFGF, delineates a potential role for rVH6 in regulating proliferation and differentiation.EXPERIMENTAL PROCEDURESIsolation of dsPTP Clones from Rat Olfactory TissueTwo sets of degenerate oligonucleotide primers were designed to the conserved amino acid regions within the catalytic domain of dsPTPs and used to amplify dsPTPs from rat olfactory tissue by polymerase chain reaction (PCR). The 5′ primer, corresponding to amino acid sequence V(L/F)VHC(Q/L)A (5′-GT(C/G)(A/C/T)T(C/G/T)GT(C/G)CA(C/T)TG(C/T)C(A/T)GGC-3′; 288-fold degeneracy) was paired with a 3′ primer corresponding to amino acids GQLL(Q/D/E)FE (5′-CT(C/T)(A/G)(A/T)(A/G)(C/G)T(C/G)C(A/C)(A/G)CAGCTG(C/G)CC-3′; 512-fold degeneracy). The template for PCR was synthesized from rat olfactory epithelial poly(A)+ RNA using oligo(dT) primers and the cDNA cycle kit (Invitrogen) according to manufacturer's instructions. The PCR reactions were performed with the GeneAmp kit (Perkin-Elmer) and included 0.1 volume of the cDNA synthesis reaction and 500 ng of each primer for 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min. PCR products were isolated by agarose gel electrophoresis and products of expected size (approximately 177 bp) were subcloned into pCR(™) II using the TA Cloning kit (Invitrogen). Approximately 150 independent subclones were sequenced by dideoxy chain termination using Sequenase v. 2.0 polymerase (U. S. Biochemical Corp.).Library ScreeningA unique 177-bp PCR subclone (rVH6-pCR II), which had significant similarity to dsPTPs, was used to screen a rat hippocampus λZAPII cDNA library (Stratagene). The rVH6 insert was labeled by random priming and used as a hybridization probe to screen approximately one million library plaques by established procedures(24.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1987Google Scholar). Nitrocellulose filters were washed under high stringency with 0.2 × SSC (1 × SSC = 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) at 65°C. Positive library plaques were purified and cDNA rescued into pBluescript II (pBS II) (Stratagene) according to manufacturer's instructions. Several clones of 2.1 and one 1-kb clone were isolated. The sequences of both cDNA strands were verified for all subclones.Bacterial Expression and Purification of Recombinant rVH6Recombinant rVH6 was expressed as a glutathione S-transferase (GST)-fusion protein containing a carboxyl-terminal polyhistidine tag. Full-length rVH6 (1.14 kb) PCR product containing 5′ BamHI and 3′ EcoRI restriction sites was generated using oligonucleotides (5′-GATCGGATCCATGATAGATACGCTCA-3′) and (5′-GATCGAATTCCGTAGATTGCAGG GAGT-3′) and subcloned in-frame into BamHI/EcoRI-digested pGEX-KT (25.Hakes D.J. Dixon J.E. Anal. Biochem. 1992; 202: 293-298Crossref PubMed Scopus (222) Google Scholar) modified to contain a COOH-terminal polyhistidine (6×) cassette. GST-rVH6-His6 was produced as described previously (25.Hakes D.J. Dixon J.E. Anal. Biochem. 1992; 202: 293-298Crossref PubMed Scopus (222) Google Scholar) and purified on glutathione-agarose and nickel-agarose resins. Pure protein (>90%) could routinely be obtained in a single step purification using a nickel-agarose column (QIAGEN). A catalytically inactive rVH6 mutant (Cys293 to Ser) generated by site-directed mutagenesis (26.Zhou G. Denu J.M. Wu L. Dixon J.E. J. Biol. Chem. 1994; 269: 28084-28090Abstract Full Text PDF PubMed Google Scholar) was expressed and purified under conditions identical to that of wild-type rVH6.Enzyme Activity AssayrVH6 enzymatic activity was assayed against the artificial substrate, pNPP, as described previously(26.Zhou G. Denu J.M. Wu L. Dixon J.E. J. Biol. Chem. 1994; 269: 28084-28090Abstract Full Text PDF PubMed Google Scholar). Various quantities of GST-rVH6 were incubated with 50 mM pNPP in 50 mM succinate buffer, pH 6.0, at 30°C for 30 min. The reactions were terminated with 0.1 N sodium hydroxide, and the molar concentration of pNPP hydrolyzed was determined by absorbance at 405 nm. Analysis of the data was performed using KinetAsyst software (IntelliKinetics, State College, PA).GST-MEK2, MAPK (p44) and the kinase-deficient mutant, MAPK K71R, were generous gifts from Dr. K. L. Guan. MAPK was activated by incubation with GST-MEK2 as described previously(27.Guan K.L. Butch E. J. Biol. Chem. 1995; 270: 7197-7203Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The ability of GST-rVH6 to inactivate MAPK was determined by incubation of activated MAPK (0.2 μg) with increasing concentrations of GST-rVH6 in Buffer A (50 mM HEPES, pH 7.5, 0.1% 2-mercaptoethanol) for 10 min at 30°C. The reactions were terminated by the addition of sodium vanadate (final concentration, 2 mM), and the remaining MAPK activity was determined as described(28.Sturgill T.W. Ray L.B. Anderson N.G. Erikson A.K. Methods Enzymol. 1991; 200: 342-351Crossref PubMed Scopus (18) Google Scholar). Wild-type MAPK and mutant MAPK K71R proteins were 32P-labeled by incubation with GST-MEK2 in the presence of [γ-32P]ATP and purified as described(5.Zheng C.-F. Guan K.-L. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar). The ability of GST-rVH6 to dephosphorylate MAPK was determined by incubation of 32P-labeled MAPK (0.2 μg) with increasing concentrations of GST-rVH6 as described above. The reactions were stopped by the addition of 5 × Laemmli buffer, boiled, and resolved by SDS-polyacrylamide gel electrophoresis. The proteins were transferred to a polyvinylidene difluoride membrane (Schleicher & Schuell) and exposed to film. After film development, radiolabeled MAPK was excised from the membrane and subjected to phosphoamino acid analysis(5.Zheng C.-F. Guan K.-L. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar).Cell CultureAll cell culture reagents were purchased from Life Technologies, Inc. PC12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% horse serum and 5% fetal bovine serum. When cells reached 50-70% confluency, serum was withdrawn for 18 h prior to growth factor treatment. PC12 cells were then induced to proliferate with EGF (50 ng/ml) or insulin (100 nM) or to differentiate with NGF (100 ng/ml) in DMEM for the indicated times. For each time point, one 140 × 20-mm tissue culture plate was harvested for poly(A)+ RNA extraction.MM14 mouse muscle cells were grown either in Ann Arbor from a cell stock generously provided by Dr. Jeffrey Chamberlain or in Seattle from the original MM14 cell stock. In both cases, the cells were grown in 0.6% gelatin-coated 140 - 20-mm plates containing Ham's F-10C nutrient medium supplemented with 15% horse serum and 2 ng/ml bFGF (29.Olwin B.B. Hauschka S.D. J. Cell Biol. 1988; 107: 761-769Crossref PubMed Scopus (146) Google Scholar). For maximal cell proliferation, MM14 cell medium was changed every 12 h, and the cells were split before reaching 400,000 cells/plate. To initiate a differentiation time course experiment, 550,000 cells were plated and allowed to proliferate for 24 h. The medium was then removed, the plates were washed with phosphate-buffered saline (PBS), and differentiation medium (Ham's F-10C containing 2% horse serum, 1 μM insulin, without bFGF) was added. Differentiating cells were fed every 24 h. For poly(A)+ RNA isolation, 20-30 plates were harvested per differentiation time point.For detection of cellular MAPK activities, MM14 cells withdrawn from serum and bFGF for the indicated lengths of time were stimulated briefly (5 min) with either 10 ng/ml of bFGF or 1.5% horse serum in F10C medium (control). The cells were harvested and lysed, and the high speed supernatants (100,000 × g) were fractionated over DE-52 "mini columns" (Whatman) as described previously(30.Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar). MAPK activity was eluted from the columns with 0.2 M NaCl, normalized for protein, and assayed for activity using myelin basic protein (Sigma) as substrate(30.Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar).RNA Isolation and Northern Blot AnalysisRat and mouse multiple tissue Northern blots were purchased from Clontech. Northern blots of cultured PC12 and MM14 cells were prepared by extraction of total RNA with TRIzol Reagent (Life Technologies, Inc.), followed by poly(A)+ RNA purification using a Poly(A)Tract kit (Promega). RNA samples were resolved in a formaldehyde-agarose gel (24.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1987Google Scholar) and transferred to a Nytran membrane (Schleicher & Schuell). An rVH6 riboprobe construct (rVH6Δ) was generated by digesting the rVH6-pBS II 1-kb partial length clone with AccI, regenerating a vector construct containing an insert of 407 bp (nucleotides 1069-1476; see Fig. 1). A 440-bp [32P]UTP-labeled rVH6 riboprobe, generated by T7 RNA polymerase transcription of EcoRI-linearized rVH6Δ, was hybridized to blots overnight in 400 mM sodium phosphate, 5% SDS, 1 mM EDTA, 1 mg/ml bovine serum albumin, 50% formamide at 60°C, 1-2 million cpm/ml. The membranes were washed at high stringency in 0.1 × SSC, 0.1% SDS, 1 mM EDTA at 70°C and exposed to XAR 5 film (Kodak) at −80°C with intensifying screens. A probe to mouse muscle creatine kinase was used to determine the degree of muscle differentiation, whereas rat cyclophilin was used as a control for amount of RNA loaded. RNA molecular size markers were from Life Technologies, Inc. Northern blot quantitation of mRNA was determined using a PhosphorImager (Molecular Dynamics), and RNA concentrations per lane were normalized against cyclophilin.Cellular Localization of rVH6rVH6 protein was expressed in COS-1 cells using the eukaryotic expression vector, pcDNA1 (Invitrogen), engineered to produce a COOH-terminal triple hemagglutinin (HA) (3 × YPYDVPDYA) epitope tag fusion protein. Full-length rVH6 cDNA (wild type and C/S mutant) was subcloned by PCR using 5′ (5′-GACTAAAGCTTACCGCCATGATAGATACGC-3′) and 3′ (5′-GACTAGAATTCCGTAGATTGCAGGGAGT-3′) oligonucleotide primers. hVH3 was provided by Dr. S. Kwak and expressed as a COOH-terminal c-myc epitope tag-fusion protein in COS-1 cells(16.Kwak S.P. Dixon J.E. J. Biol. Chem. 1995; 270: 1156-1160Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). COS-1 cells were plated at 5 × 104 cells/well (diameter, 3 cm) on glass coverslips (2 cm2) in DMEM + 5% fetal calf serum. Cells were transfected with the appropriate DNA (1 μg) using lipofectamine (Life Technologies, Inc.) in serum-free DMEM for 6 h before removing transfection solution and returning the cells to DMEM + 5% serum. Cells were fixed 48 h post-transfection with 4% paraformaldehyde in PBS, followed by acetone:methanol (1:1). All immunofluorescence incubations were conducted at room temperature. Primary mouse monoclonal antibodies anti-HA (Boehringer Mannheim) or anti-myc (9E10) (31.Kolodziej P.A. Young R.A. Methods Enzymol. 1991; 194: 508-519Crossref PubMed Scopus (423) Google Scholar) diluted to 1 μg/ml in blocking buffer (PBS, 0.1% saponin, 2% bovine serum albumin) were incubated with cells for 1 h. Cells were then washed four times with PBS + 0.1% saponin and incubated for 1 h with fluorescein-conjugated goat anti-mouse secondary antibody (Vector Labs) diluted 1:500 in blocking buffer. Following secondary antibody incubation, the cells were washed four times with PBS + 0.1% saponin and twice with PBS. Coverslips were mounted with Vecta-Shield (Vector Labs), and fluorescence was viewed with a Zeiss fluorescent microscope. For negative primary antibody controls, COS-1 cells were transfected with pcDNA-1 vector alone (minus epitope tag) and treated for immunofluorescence as described above.RESULTSIdentification and Cloning of rVH6 cDNADegenerate oligonucleotides were used to isolate novel dsPTPs from rat olfactory epithelium cDNA by PCR. The 5′ oligonucleotide was designed to partially overlap the PTP active site motif (I/V)HCXAGXXR(S/T), whereas the 3′ oligonucleotide was made against a conserved group of amino acids GQLLXFE (Fig. 1B). PCR generated a pool of cDNAs of the approximate predicted size (177 bp), which were subcloned and sequenced. Sequencing of 150 individual clones revealed that approximately 50% of the products were identical to the human dsPTP, CL100(17.Keyse E.M. Emslie S.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (568) Google Scholar). Approximately 25% of the clones contained a novel cDNA sequence, which when translated had 38-58% amino acid identity to previously identified dsPTPs. This dsPTP sequence was named rVH6 based on it being the sixth dsPTP identified in the laboratory of the senior author (J. E. D.), the first being the vaccinia H-1 gene product (VH1)(32.Guan K.L. Broyles S.S. Dixon J.E. Nature. 1991; 350: 359-362Crossref PubMed Scopus (323) Google Scholar).Preliminary Northern blot analysis (data not shown) using the rVH6 PCR fragment indicated that rVH6 was abundantly expressed in brain. Based on the high levels of expression in brain, a rat hippocampus cDNA library was screened with the rVH6 PCR fragment to isolate full-length cDNAs. Five hybridizing clones were isolated, four of which contained identical 2104-bp inserts and one of which contained a partial length insert of 1034 bp. The 2.1-kb cDNA sequence had a 1143-nucleotide (3.Ahn N.G. Seger R. Bratlien R.L. Diltz C.D. Tonks N.K. Krebs E.G. J. Biol. Chem. 1991; 266: 4220-4227Abstract Full Text PDF PubMed Google Scholar) open reading frame (Fig. 1A) with a putative initiator methionine in a region that matched the Kozak sequence motif (nucleotides 355-363; Fig. 1A)(33.Kozak M. Nucleic Acids Res. 1984; 12: 857-872Crossref PubMed Scopus (2378) Google Scholar). No other methionines were observed 5′ to this methionine in any other reading frame. In addition, 5′ to the Kozak sequence and 3′ to the in-frame stop codon (nucleotides 1504-1506; Fig. 1A), stop codons were observed in all three reading frames. No polyadenylation signal or poly(A) tail was observed in any rVH6 cDNA clone isolated. Based on the 3.2-kb message size observed in Northern blot analysis (Fig. 2), up to 1.1 kb of 5′ and 3′ untranslated sequence was not present in the isolated cDNA clones. Northern blot analysis using the rVH6 riboprobe revealed that the mRNA is expressed as a 3.2-kb transcript in all the tissues examined (Fig. 2). Relatively higher levels of expression were seen in brain and spleen, with lower levels in heart, lung, liver, and skeletal muscle. Very low levels of rVH6 mRNA were also detected in kidney and testis. A similar size rVH6 RNA transcript was also observed in a mouse multiple-tissue Northern blot (data not shown). The rVH6 mRNA tissue distribution between rat and mouse was similar, with the exception that mouse kidney and skeletal muscle expressed higher levels of rVH6 (data not shown).Figure 2:Tissue distribution of rVH6. Rat multiple tissue Northern blot (Clontech) of poly(A)+ RNA (2 μg/lane) was screened with a rVH6 antisense probe as described under "Experimental Procedures." RNA size markers are shown to the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Data base searches with the deduced amino acid sequence of rVH6 (Fig. 1B) indicate that rVH6 is similar to all known dsPTPs. The rVH6 cDNA predicted open reading frame codes for a protein of 381 amino acids (∽42,315 Daltons), which is similar in size and amino acid identity to CL100 (36%,(17.Keyse E.M. Emslie S.A. Nature. 1992; 359: 644-647Crossref PubMed Scopus (568) Google Scholar)), Pac-1 (35%,(19.Rohan P.J. Davis P. Moskaluk C.A. Kearns M. Krutzsch H. Siebenlist U. Kelly K. Science. 1993; 259: 1763-1766Crossref PubMed Scopus (263) Google Scholar)), hVH2 (35%, (27.Guan K.L. Butch E. J. Biol. Chem. 1995; 270: 7197-7203Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar)), and hVH3 (33%,(16.Kwak S.P. Dixon J.E. J. Biol. Chem. 1995; 270: 1156-1160Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar)). rVH6 contains the active site sequence, VXVHCXXGXXRSXXXXXAY(L/I)M (Fig. 1B), characteristic of virtually all dsPTPs(34.Martell K.J. Seasholtz A.F. Kwak S.P. Clemens K.K. Dixon J.E. J. Neurochem. 1995; 65: 1823-1833Crossref PubMed Scopus (92) Google Scholar). The structure of rVH6 can be divided based on its amino acid alignment with other dsPTPs (Fig. 1B) into a 181-amino acid COOH-terminal catalytic domain and a 200-residue NH2-terminal extension. The rVH6 catalytic domain possesses amino acid identities to hVH2 (45%), CL100 (44%), Pac-1 (43%) hVH5 (42%,(34.Martell K.J. Seasholtz A.F. Kwak S.P. Clemens K.K. Dixon J.E. J. Neurochem. 1995; 65: 1823-1833Crossref PubMed Scopus (92) Google Scholar)), hVH3 (38%) VHR (35%,(35.Ishibashi T. Bottaro D.P. Chan A. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12170-12174Crossref PubMed Scopus (179) Google Scholar)), and VH1 (27%).The NH2 terminus of rVH6 contains two regions of amino acid similarity to the cell cycle regulator phosphatase, cdc25(36.Keyse S.M. Ginsburg M. Trends. Biochem. Sci. 1993; 18: 377-378Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 37.Kwak S.P. Hakes D.J. Martell K.J. Dixon J.E. J. Biol. Chem. 1994; 269: 3596-3604Abstract Full Text PDF PubMed Google Scholar). These two regions (underlined in Fig. 1B) are referred to as cdc25 homology domains 2 (CH2 domains) (37.Kwak S.P. Hakes D.J. Martell K.J. Dixon J.E. J. Biol. Chem. 1994; 269: 3596-3604Abstract Full Text PDF PubMed Google Scholar) and have been observed in the NH2 terminus of several other dsPTPs (CL100, Pac-1, hVH2, hVH3, and hVH5). The function of these CH2 domains is unknown. Interposed between the NH2-terminal CH2-containing, and COOH-terminal catalytic domains, is a serine-rich (30%) amino acid region (amino acids 150-210; Fig. 1B) that also contains two putative proline-directed serine kinase substrate recognition motifs, XSP, (amino acids 162-164 and 200-202; Fig. 1B) (reviewed in (1.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4219) Google Scholar)). In addition, the NH2-terminal domain of rVH6 contains two putative nuclear export motifs, LXLXLXXL and LXXLXLXXL (Fig. 1A), implicated in the function of proteins that export specific proteins and RNAs from the nucleus(38.Fischer U. Huber J. Boelens W.C. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-484Abstract Full Text PDF PubMed Scopus (980) Google Scholar, 39.Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (994) Google Scholar). No other significant amino acid similarity to other Genbank protein sequences was observed in the NH2-terminal domain of rVH6.Catalytic Properties of rVH6To determine whether the rVH6 cDNA coded for a protein possessing phosphatase activity, recombinant rVH6 was expressed, purified, and assayed for enzymatic activity. The fusion protein, GST-rVH6, hydrolyzed the tyrosine phosphatase substrate, p
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