The Minimal Essential Core of a Cysteine-based Protein-tyrosine Phosphatase Revealed by a Novel 16-kDa VH1-like Phosphatase, VHZ

Abstract

The smallest active protein-tyrosine phosphatase yet (only 16 kDa) is described here and given the name VHZ for VH1-like member Z because it belongs to the group of small Vaccinia virus VH1-related dual specific phosphatases exemplified by VHR, VHX, and VHY. Human VHZ is remarkably well conserved through evolution as it has species orthologs in frogs, fish, fly, and Archaea. The gene for VHZ, which we designate as DUSP25, is located on human chromosome 1q23.1 and consists of only two coding exons. VHZ is broadly expressed in tissues and cells, including resting blood lymphocytes, Jurkat T cells, HL-60, and RAMOS. In transfected cells, VHZ was located in the cytosol and in other cells also in the nucleoli. Endogenous VHZ showed a similar but more granular distribution. We show that VHZ is an active phosphatase and analyze its structure by computer modeling, which shows that in comparison with the 185-amino acid residue VHR, the 150-residue VHZ is a shortened version of VHR and contains the minimal set of secondary structure elements conserved in all known phosphatases from this class. The surface charge distribution of VHZ differs from that of VHR and is therefore unlikely to dephosphorylate mitogen-activated protein kinases. The remarkably high degree of conservation of VHZ through evolution may indicate a role in some ancient and fundamental physiological process. The smallest active protein-tyrosine phosphatase yet (only 16 kDa) is described here and given the name VHZ for VH1-like member Z because it belongs to the group of small Vaccinia virus VH1-related dual specific phosphatases exemplified by VHR, VHX, and VHY. Human VHZ is remarkably well conserved through evolution as it has species orthologs in frogs, fish, fly, and Archaea. The gene for VHZ, which we designate as DUSP25, is located on human chromosome 1q23.1 and consists of only two coding exons. VHZ is broadly expressed in tissues and cells, including resting blood lymphocytes, Jurkat T cells, HL-60, and RAMOS. In transfected cells, VHZ was located in the cytosol and in other cells also in the nucleoli. Endogenous VHZ showed a similar but more granular distribution. We show that VHZ is an active phosphatase and analyze its structure by computer modeling, which shows that in comparison with the 185-amino acid residue VHR, the 150-residue VHZ is a shortened version of VHR and contains the minimal set of secondary structure elements conserved in all known phosphatases from this class. The surface charge distribution of VHZ differs from that of VHR and is therefore unlikely to dephosphorylate mitogen-activated protein kinases. The remarkably high degree of conservation of VHZ through evolution may indicate a role in some ancient and fundamental physiological process. The VH1-like phosphatases (1Fauman E.B. Saper M.A. Trends. Biochem. Sci. 1996; 21: 413-417Abstract Full Text PDF PubMed Scopus (319) Google Scholar) are members of the cysteine-based protein-tyrosine phosphatase (PTP) 1The abbreviations used are: PTP, protein-tyrosine phosphatase; MKP, mitogen-activated protein kinase phosphatase; DSP, dual specific protein phosphatase; GST, glutathione S-transferase; pNPP, p-nitrophenyl phosphate; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; VHR, VH1 related; VHZ, VH1-like member Z. family and contain the extended consensus signature motif DX27–30HCX2GX2R(S/T/A)X5A(Y/F)LM or slight variations thereof. The crystal structures of the catalytic domains of the VH1-like enzymes VHR (2Yuvaniyama J. Denu J.M. Dixon J.E. Saper M.A. Science. 1996; 272: 1328-1331Crossref PubMed Scopus (308) Google Scholar), mitogen-activated protein kinase phosphatase-3 (MKP-3) (3Stewart A.E. Dowd S. Keyse S.M. McDonald N.Q. Nat. Struct. Biol. 1999; 6: 174-181Crossref PubMed Scopus (148) Google Scholar), KAP (4Hanlon N. Barford D. Protein Sci. 1998; 7: 508-511Crossref PubMed Scopus (5) Google Scholar), PTEN (5Lee J.O. Yang H. Georgescu M.M. Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar), and MTMR2 (6Begley M.J. Taylor G.S. Kim S.-A. Veine D.M. Dixon J.E. Stuckey J.A. Mol. Cell. 2003; 12: 1391-1402Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) show that they have the same topology and catalytic machinery as other members of the Class I cysteine-based PTP family exemplified by PTP1B (7Barford D. Flint A.J. Tonks N.K. Science. 1994; 263: 1397-1404Crossref PubMed Scopus (685) Google Scholar) and RPTPα (8Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar). However, whereas the catalytic cleft of most VH1-like phosphatases is only 6 Å deep to allow access by the shorter phosphoamino acid side chains of phosphoserine (Ser(P)) and phosphothreonine (Thr(P)) to the catalytic Cys at the bottom of the pocket, the deeper (9 Å) pocket of classical phosphatases like PTP1B only permits dephosphorylation of phosphotyrosine. On the other hand, the catalytic pockets of PTEN (5Lee J.O. Yang H. Georgescu M.M. Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar) and MTMR2 (6Begley M.J. Taylor G.S. Kim S.-A. Veine D.M. Dixon J.E. Stuckey J.A. Mol. Cell. 2003; 12: 1391-1402Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) are considerably wider to accommodate their unique physiological substrate, the lipid-bound inositol ring with phosphate at the D position. VH1-like phosphatases are found in all main classes of organisms from bacteria to plants, yeast, insects, worms, and mammals. The human and mouse genomes each encode at least 61 VH1-like enzymes 2Alonso, A., Sasin, J., Bottini, N., Friedberg, I., Friedberg, I., Osterman, A. Godzik, A., Hunter, T., Dixon, J. E., and Mustelin, T. (2004) Cell117, 699–711., 11 of which are collectively referred to as the MKPs. In addition to a catalytic VH1-like domain, these MKPs contain a non-catalytic CDC25 homology (CH2) domain for docking with mitogen-activated protein kinases (10Alonso A. Rojas A. Godzik A. Mustelin T. Top. Curr. Genet. 2003; 5: 333-358Crossref Google Scholar), which subsequently are dephosphorylated by the catalytic domain. Another group of 19 small VH1-like phosphatases lack the CH2 domain (in both mice and humans), 2Alonso, A., Sasin, J., Bottini, N., Friedberg, I., Friedberg, I., Osterman, A. Godzik, A., Hunter, T., Dixon, J. E., and Mustelin, T. (2004) Cell117, 699–711. but some of the phosphatases still dephosphorylate the phosphotyrosine and phosphothreonine residues in the activation loop of mitogen-activated protein kinases in vitro and in cells. In vitro, all of these 30 phosphatases as well as many other VH1-like enzymes dephosphorylate peptides with phosphate on any of three hydroxyl amino acids (serine, threonine, and tyrosine), and they are therefore often referred to as dual specific protein phosphatases (DSPs) (10Alonso A. Rojas A. Godzik A. Mustelin T. Top. Curr. Genet. 2003; 5: 333-358Crossref Google Scholar). The CDC25 phosphatases also show dual specific activity against threonine- and tyrosine-phosphorylated cyclin-dependent protein kinases (11Honda R. Ohba Y. Nagata A. Okayama H. Yasuda H. FEBS Lett. 1993; 318: 331-334Crossref PubMed Scopus (87) Google Scholar), but they have a different structure and topology (12Fauman E.B. Cogswell J.P. Lovejoy B. Rocque W.J. Holmes W. Montana V.G. Piwnica-Worms H. Rink M.J. Saper M.A. Cell. 1998; 93: 617-625Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) and do not share sequence similarity with DSPs apart from the CX5R motif common to all Cys-based phosphatases. Thus, the three CDC25s are not members of the VH1-like family (as defined above) but apparently became dual specific phosphatases as the result of convergent evolution. In addition, several VH1-like phosphatases dephosphorylate Tyr(P), Ser(P), and Thr(P) in vitro but have non-protein substrates in vivo. For example, phosphatase and tensin homology deleted on chromosome 10, the related phosphatases TPTE and TPIP, and the myotubularins dephosphorylate the Asp-3 phosphate of phosphatidylinositol (13Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2601) Google Scholar), whereas PIR1 (phosphatase that interacts with RNA/RNP complex 1) (14Deshpande T. Takagi T. Hao L. Buratowski S. Charbonneau H. J. Biol. Chem. 1999; 274: 16590-16594Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and mRNA capping enzyme (RNGTT) dephosphorylate the 5′ end of RNA transcripts during the mRNA capping process (14Deshpande T. Takagi T. Hao L. Buratowski S. Charbonneau H. J. Biol. Chem. 1999; 274: 16590-16594Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 15Takagi T. Moore C.R. Diehn F. Buratowski S. Cell. 1997; 89: 867-873Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Although much of the early work on DSPs focused on the biochemical and structural aspects of these enzymes, more recent work has begun to unravel the physiological functions of DSPs particularly in invertebrates. For example, the puckered (16Martin-Blanco E. Gampel A. Ring J. Virdee K. Kirov N. Tolkovsky A.M. Martinez-Arias A. Genes Dev. 1998; 12: 557-570Crossref PubMed Scopus (519) Google Scholar) and slingshot (17Niwa R. Nagata-Ohashi K. Takeichi M. Mizuno K. Uemura T. Cell. 2002; 108: 233-246Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar) genes in Drosophila melanogaster and LIP-1 (18Berset T. Hoier E.F. Battu G. Canevascini S. Hajnal A. Science. 2001; 291: 1055-1058Crossref PubMed Scopus (220) Google Scholar) in Caenorhabditis elegans have non-redundant functions revealed by gene deletion. Our understanding of the physiological roles of DSPs in mammals is much more rudimentary. The two reported DSP knock-out mice, MKP-1-/- (19Dorfman K. Carrasco D. Gruda M. Ryan C. Lira S.A. Bravo R. Oncogene. 1996; 13: 925-931PubMed Google Scholar) and VHX-/- (20Chen A.J. Zhou G. Juan T. Colicos S.M. Cannon J.P. Cabriera-Hansen M. Meyer C.F. Jurecic R. Copeland N.G. Gilbert D.J. Jenkins N.A. Fletcher F. Tan T.H. Belmont J.W. J. Biol. Chem. 2002; 277: 36592-36601Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) animals, had no apparent phenotype. However, new functions in cell lines and new substrates have recently been found for DSPs. For example, the cytoskeletal protein ADF/cofilin was identified as the substrate for the slingshot DSPs in the fruit fly D. melanogaster (17Niwa R. Nagata-Ohashi K. Takeichi M. Mizuno K. Uemura T. Cell. 2002; 108: 233-246Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar), whereas the hYVH1 (DUSP12) DSP was shown to dephosphorylate glucokinase, an enzyme involved in glucose metabolism (21Munoz-Alonso M.J. Guillemain G. Kassis N. Girard J. Burnol A.F. Leturque A. J. Biol. Chem. 2000; 275: 32406-32412Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Another small DSP, laforin (EMP2A), associates via a glycogen-binding domain (22Wang J. Stuckey J.A. Wishart M.J. Dixon J.E. J. Biol. Chem. 2002; 277: 2377-2380Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) with glycogen, an interaction that is important for cellular health because mutations in laforin cause a severe human disease, progressive myoclonus epilepsy or Lafora disease (23Minassian B.A. Lee J.R. Herbrick J.A. Huizenga J. Soder S. Mungall A.J. Dunham I. Gardner R. Fong C.Y. Carpenter S. Jardim L. Satishchandra P. Andermann E. Snead III, O.C. Lopes-Cendes I. Tsui L.C. Delgado-Escueta A.V. Rouleau G.A. Scherer S.W. Nat. Genet. 1998; 20: 171-174Crossref PubMed Scopus (409) Google Scholar). A first step toward understanding the physiological roles and possible redundancies within the DSP group of PTPs is the elucidation of the full repertoire of these enzymes expressed in cells and the characterization of their expression patterns and biochemical properties. Here we begin the characterization of a novel VH1-like phosphatase, which we call VHZ for VH1-like member Z. Antibodies and Reagents—The 9E10 hybridoma producing the monoclonal antibody that recognizes the c-Myc epitope tag was from the American Type Culture Collection (Manassas, VA). Polyclonal rabbit antisera were raised against the recombinant GST fusion protein of full-length VHZ protein and were used at 1:1,000 dilution. Expression Plasmids for VHZ—The cDNA for VHZ was cloned into the 5′ EcoRI to 3′ XhoI sites of the pcDNA3.1myc-his vector leaving the epitope tags in the C terminus of the insert. p-Nitrophenyl Phosphate (pNPP) Dephosphorylation Assay—The hydrolysis of pNPP was assayed at 30 °C for 30 min with 20 μg of enzyme in 100 μl of 0.1 m bis-Tris, pH 6.0, 150 mm NaCl, 1 mm dithiothreitol. The reaction was initiated by the addition of various concentrations of pNPP (ranging from 0.2 to 10 Km) to the reaction mixtures and terminated by the addition of 200 μl of Biomol reagent. Nonenzymatic hydrolysis of the substrate was corrected by measuring the control without the addition of enzyme. After a 30-min incubation, the absorbance at 620 nm was determined using a PowerWaveX340 microplate spectrophotometer (Bio-Tek Instruments, Inc.) and converted to a molar amount of product using a standard curve. The Km and Kcat values were determined by fitting the data to the Michaelis-Menten equations using nonlinear regression and the software program GraphPad Prism® (version 4.0). Cells and Transfections—293T, COS-1, HeLa, and other cell lines were kept at logarithmic growth in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mm l-glutamine, 1 mm sodium pyruvate, nonessential amino acids, and 100 units/ml each penicillin G and streptomycin. Peripheral blood lymphocytes (∼80% T cells) were obtained from venous blood from healthy donors (Red Cross Blood Bank, San Diego, CA); Ficoll gradient centrifugation was performed, and monocytes were removed by adherence to plastic at 37 °C for 2 h. 293T and COS-1 cells were transfected with a total of 5–10 μg of DNA by lipofection. Empty vector was added to control samples to make a constant amount of DNA in each sample. Cells were used for experiments and were as M. S. K. Mustelin T. Nat. Biol. 1999; PubMed Scopus Google Scholar, A. S. S. M. L. Godzik A. Mustelin T. Nat. 2003; PubMed Scopus Google Scholar, A. N. S. S. S. Mustelin T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells were in 20 mm pH 150 mm NaCl, mm 1 mm 10 and 100 and 1 mm and by centrifugation at for 20 The were on protein and with antibody for 2 this was by the addition of protein were three in in with m NaCl, in and in by were to and were with 1:1,000 or of monoclonal by or and the were by the to the and of transfected or VHZ was as described A. S. S. M. L. Godzik A. Mustelin T. Nat. 2003; PubMed Scopus Google Scholar, A. N. S. S. S. Mustelin T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. S. A. M. P. S. J. Mustelin T. J. 2000; PubMed Scopus (87) Google Scholar, H. A. E. M. M. Mustelin T. J. 2002; PubMed Scopus Google Scholar). 293T cells were in in with in and in serum, in for 30 min at and secondary antibody were in the same and with the cells for 1 each at After three with the cells were and a DNA was with at were also VHZ was using a on the structure of the human phosphatase and the phosphatase domain of the human cell protein on a J.M. A. D. L. 2001; PubMed Scopus Google Scholar). was by a T. J. N. 2003; PubMed Scopus Google the same was used for The was by the program M.J. J. Mol. Biol. 1997; PubMed Scopus Google Scholar). The in was obtained by the J.M. M.A. J.M. 2003; PubMed Scopus Google Scholar) on several of protein structural were obtained with the I. Gilbert PubMed Google Scholar). to the human set of PTP genes we a of in the data including a acid protein with the PTP consensus sequence In the new this is in a the conserved and in this it VHR T. A. T. S.A. Sci. S. A. 89: PubMed Scopus Google Scholar), which has a at the position. revealed that the new had the degree of homology to residues with protein from the and the degree of homology with the cell phosphatases from human and many other homology with residues was also with an PTP of physiological the new is related to VHR and belongs to the group of small kDa) dual specific including the Vaccinia virus and the human VHR, A. S. N. S. J. Mustelin T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), and A., J., A., J., and Mustelin, T. (2004) J. Biol. we to name the new member for member name also the of VHZ in the of for which with the enzymes and to toward and enzymes, as C. Biochem. 2002; PubMed Scopus Google Scholar) and A. S. N. S. J. Mustelin T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). We have given the gene that VHZ the species orthologs of VHZ were found in a of organisms from mammals to and a possible species in the which is and similar indicate that VHZ is a well conserved and ancient The human gene that VHZ is located at 1q23.1 and consists of a 1 by two coding both of which are by expressed sequence that this is a expressed we with the full-length VHZ as a and a mRNA in all tissues were in and whereas the were in and we cloned the VHZ cDNA into the pcDNA3.1myc-his vector and expressed it in cells We also the and which were expressed at similar as the enzyme and three at on as from the of 16 with an 2 from the tag amino For a A., J., A., J., and Mustelin, T. (2004) J. Biol. was expressed at to the VHZ we an by with a GST fusion protein full-length The antisera well with the expressed VHZ and with a protein of 16 in blood lymphocytes, Jurkat T cells, cells, cells, and the cell lines and of 293T cells transfected with VHZ and with the monoclonal antibody against its epitope tag showed that VHZ had a of In most cells, VHZ was the whereas in some cells it was found also in the and of A of cells had VHZ only in the in cell VHZ was found in the the were Endogenous VHZ was also located in the cytosol but in a more granular with a of the only in some cells this distribution of VHZ had any to the cell as the homology with we cells for VHZ and DNA and focused on cells in various of In cells, VHZ showed a granular distribution the The only in the of VHZ during was its into the of the of the In this VHZ was more but not for to the showed that VHZ was in the same but not on the that a VHZ protein in cells, we to this remarkably small DSP protein is a active The smallest VH1-like phosphatase is the 185-amino acid residue VHR T. A. T. S.A. Sci. S. A. 89: PubMed Scopus Google Scholar), which has been (2Yuvaniyama J. Denu J.M. Dixon J.E. Saper M.A. Science. 1996; 272: 1328-1331Crossref PubMed Scopus (308) Google Scholar) and found to a catalytic domain with only a residues in its and C VHZ contains only 150 amino acid residues and is therefore the catalytic domain of A GST fusion protein full-length VHZ as well as its and were expressed in and A standard phosphatase with p-nitrophenyl phosphate showed that VHZ dephosphorylated this substrate with a Km of mm and a Kcat of Although the Km is similar to the values for the small DSPs VHX, and the is VHZ is an active activity of VHZ and related small values the from values the from in a new the structural elements and catalytic of a phosphatase of only 150 amino acid residues we a computer of showed that VHZ consists of the same of by and three as all other Class I Cys-based The main the VHZ and VHR structure to the of several and as well as of the in the surface topology and surface charge distribution of VHZ differs from of VHR and A. S. N. S. J. Mustelin T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). VHZ has two on each side of the catalytic but it a of charge to the catalytic pocket found in this has been to important for docking of with VHR M.A. A.E. Denu J.M. 2002; PubMed Scopus Google Scholar), that VHZ is unlikely to this We to that an of VHZ, of the in is by a the of the protein similar to that of the human is also possible and not on A more comparison of the structure of VHZ with other phosphatases showed that the which is of by and three on each side is found in all Cys-based many elements have been added to this structure during evolution. added elements are shown in as for and for each to in the For example, the classical PTPs and contain with the both and to the common also have and to the deeper catalytic pocket of these classical In VHR and VHZ only of the common and are to the structure of an an sequence of in which an Cys-based of the minimal of or by only or two on each side was used as a for a first phosphatase, which to more protein phosphatases and PTPs through the addition of more is by the of enzymes in all of including and whereas PTPs are found in VHZ to a well conserved enzyme with a in the T. Thus, it is possible that VHZ an ancient in the evolution of phosphatases. in the human and expression in tissues and cell may indicate that VHZ has some important and in cell On the amino acid sequence the of VHZ are the which are of cell R. K. S. M. A. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the sequence with is for any of VHZ have to in the