Molecular Cloning and Characterization of a Novel Dual Specificity Phosphatase, LMW-DSP2, That Lacks the Cdc25 Homology Domain
2001; Elsevier BV; Volume: 276; Issue: 29 Linguagem: Inglês
10.1074/jbc.m100408200
ISSN1083-351X
AutoresKoji Aoyama, Miyuki Nagata, Kenji Oshima, Tsukasa Matsuda, Naohito Aoki,
Tópico(s)ATP Synthase and ATPases Research
ResumoA novel dual specificity phosphatase (DSP) designated LMW-DSP2 was cloned with a combination of reverse transcription-polymerase chain reaction and cDNA library screening strategies. The LMW-DSP2 open reading frame of 194 amino acids contained a single DSP catalytic domain but lacked the cdc25 homology domain, which is conserved in most known DSPs. Northern blot and reverse transcription-polymerase chain reaction analyses revealed that LMW-DSP2 was specifically expressed in testis. Recombinant LMW-DSP2 protein exhibited phosphatase activity toward an artificial low molecular weight substrate para-nitrophenyl phosphate, and the activity was inhibited completely by sodium orthovanadate but not sodium fluoride, pyrophosphate, and okadaic acid. The substitution of critical amino acid residues, aspartic acid and cysteine, resulted in a dramatic reduction of phosphatase activity. Transient transfection of LMW-DSP2 in COS7 cells resulted in the expression of a 21-kDa protein, and the phosphatase was shown to be distributed in both the cytosol and the nucleus. LMW-DSP2 dephosphorylated and deactivated p38, to a higher extent, and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases, in transfected COS7 cells andin vitro. Interestingly, mutation in a conserved docking motif of p38 and SAPK/JNK as well as in a cluster of aspartic acids of LMW-DSP2 did not affect the deactivation of the mitogen-activated protein kinases by LMW-DSP2. Furthermore, the binding between LMW-DSP2 and p38 and SAPK/JNK was also not disrupted by such mutations. Among the DSPs lacking the cdc25 homology domain, LMW-DSP2 is the first one that dephosphorylates and deactivates p38 and SAPK/JNK. A novel dual specificity phosphatase (DSP) designated LMW-DSP2 was cloned with a combination of reverse transcription-polymerase chain reaction and cDNA library screening strategies. The LMW-DSP2 open reading frame of 194 amino acids contained a single DSP catalytic domain but lacked the cdc25 homology domain, which is conserved in most known DSPs. Northern blot and reverse transcription-polymerase chain reaction analyses revealed that LMW-DSP2 was specifically expressed in testis. Recombinant LMW-DSP2 protein exhibited phosphatase activity toward an artificial low molecular weight substrate para-nitrophenyl phosphate, and the activity was inhibited completely by sodium orthovanadate but not sodium fluoride, pyrophosphate, and okadaic acid. The substitution of critical amino acid residues, aspartic acid and cysteine, resulted in a dramatic reduction of phosphatase activity. Transient transfection of LMW-DSP2 in COS7 cells resulted in the expression of a 21-kDa protein, and the phosphatase was shown to be distributed in both the cytosol and the nucleus. LMW-DSP2 dephosphorylated and deactivated p38, to a higher extent, and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases, in transfected COS7 cells andin vitro. Interestingly, mutation in a conserved docking motif of p38 and SAPK/JNK as well as in a cluster of aspartic acids of LMW-DSP2 did not affect the deactivation of the mitogen-activated protein kinases by LMW-DSP2. Furthermore, the binding between LMW-DSP2 and p38 and SAPK/JNK was also not disrupted by such mutations. Among the DSPs lacking the cdc25 homology domain, LMW-DSP2 is the first one that dephosphorylates and deactivates p38 and SAPK/JNK. extracellular signal-regulated kinase stress-activated protein kinase c-Jun N-terminal kinase mitogen-activated protein dual specificity phosphatase MAP kinase phosphatase mitogen-activated protein kinase reverse transcription polymerase chain reaction hemagglutinin double negative glutathione S- transferase protein-tyrosine phosphate Extracellular signal-regulated kinase (ERK),1 stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38/RK/CSBP (p38) are distinct classes of mitogen-activated protein (MAP) kinases. ERK is activated mainly by a variety of growth factors and phorbol esters and is associated with cellular proliferation and differentiation. SAPK/JNK and p38 are activated by extracellular stresses such as UV irradiation, osmotic stress, and inflammatory cytokines but are poorly activated by growth factors and phorbol esters. Activation of these protein kinases leads to a variety of responses such as gene expression, cell proliferation, differentiation, cell cycle arrest, apoptosis, early development, etc., depending on the cell type (1Ahn N.G. Seger R. Krebs E.G. Curr. Opin. Cell Biol. 1991; 4: 992-999Crossref Scopus (228) Google Scholar, 2Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (328) Google Scholar, 3Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 4Davis R.J. Trends Biochem. Sci. 1994; 19: 470-473Abstract Full Text PDF PubMed Scopus (917) Google Scholar, 5Kyriakis J.M. Avruch J. Bioessays. 1996; 18: 567-577Crossref PubMed Scopus (660) Google Scholar, 6Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar, 7Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar, 8Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1160) Google Scholar).Full MAP kinase activation needs phosphorylation on both tyrosine and threonine residues by selective upstream dual specificity kinases (7Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar, 9Cahill M.A. Janknechit R. Nordheim A. Curr. Biol. 1996; 6: 16-19Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 10Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (996) Google Scholar). Because MAP kinase activation is a reversible process, protein phosphatases play critical regulatory roles. Recently, there has been an emerging family of dual specificity phosphatases (DSPs)/MAP kinase phosphatases (MKPs) acting on the MAP kinase superfamily. Ten members of this group of DSPs have been reported including VHR (12Ishibashi T. Bottaro D.P. Chan A. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 89: 12170-12174Crossref Scopus (179) Google Scholar), CL100 (MKP-1) (13Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-646Crossref PubMed Scopus (568) Google Scholar, 14Charles C.H. Abler A.S. Lau L.F. Oncogene. 1992; 7: 187-190PubMed Google Scholar, 15Alessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020PubMed Google Scholar, 16Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1022) Google Scholar), PAC1 (17Rohan 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, 18Ward Y. Gupta S. Jensen P. Wartmann M. Davis R.J. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar), MKP-2 (hVH2, TYP-1) (19Guan K.-L. Butch E. J. Biol. Chem. 1995; 270: 7197-7203Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 20Misra-Press A. Rim C.S. Yao H. Roberson M.S. Stork P.J.S. J. Biol. Chem. 1995; 270: 14587-14596Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 21King A.G. Ozanne B.W. Smythe C. Ashworth A. Oncogene. 1995; 11: 2553-2563PubMed Google Scholar), hVH3 (B23) (22Kwak S.P Dixon J.E. J. Biol. Chem. 1995; 270: 1156-1160Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23Ishibashi 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), hVH5 (M3/6) (24Martell 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,25Theodosiou A.M. Rodrigues N.R. Nesbit M.A. Ambrose H.J. Paterson H. McLellan-Arnold E. Boyd Y. Leversha M.A. Owen N. Blake D.J. Ashworth A. Davies K.E. Hum. Mol. Genet. 1996; 96: 675-684Crossref Scopus (49) Google Scholar), MKP-3 (Pyst1, rVH6) (26Muda M. Boschert U. Dickinson R. Martinou J.-C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 27Mourey R.J. Vega Q.C. Campbell J.S. Wenderoth M.P. Hauschka S.D. Krebs E.G. Dixon J.E. J. Biol. Chem. 1996; 271: 3795-3802Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 28Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (370) Google Scholar), Pyst2 (29Dowd S. Sneddon A.A. Keyse S.M. J. Cell Sci. 1997; 111: 3389-3399Google Scholar), MKP-4 (Pyst3) (29Dowd S. Sneddon A.A. Keyse S.M. J. Cell Sci. 1997; 111: 3389-3399Google Scholar, 30Muda M. Boschert U. Smith A. Antonsson B. Gillierson C. Chabert C. Camps M. Martinou I. Ashworth A. Arkinstall S. J. Biol. Chem. 1997; 272: 5141-5151Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and MKP-5 (31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). They share sequence homology, but each has distinct properties concerning substrate specificity, tissue distribution, subcellular localization, and inducibility by extracellular stimuli (32Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (710) Google Scholar).As a part of the studies on DSP diversity and cellular function, we have been trying to clone novel members of the DSP family. A combination of RT-PCR and cDNA library screening revealed a novel clone designated LMW-DSP2 containing a single catalytic domain but lacking the cdc25 homology domain. Despite the absence of a putative common docking site for MAP kinases, which is normally located in the cdc25 homology domain, LMW-DSP2 dephosphorylated and deactivated p38, to a higher extent, and SAPK/JNK, but not ERK1/2, in transfected COS7 cells and in vitro. Further analyses using various mutants suggested that LMW-DSP2 was a member of a distinct class of DSPs and that the mode of its dephosphorylation and deactivation action toward MAP kinases was different from that of other DSPs reported.DISCUSSIONWe have isolated a cDNA clone encoding a novel dual specificity phosphatase, LMW-DSP2. Unlike most of other DSPs/MKPs hitherto identified, LMW-DSP2 was unique in that it lacked the cdc25 homology domain that is conserved in the N-terminal region of DSPs (32Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (710) Google Scholar) and accordingly had a smaller molecular mass. VHR and recently cloned TMDP did not have the cdc25 homology domain as well (12Ishibashi T. Bottaro D.P. Chan A. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 89: 12170-12174Crossref Scopus (179) Google Scholar, 40Nakamura K. Shima H. Watanabe M. Haneji T. Kikuchi K. Biochem. J. 1999; 344: 819-825Crossref PubMed Google Scholar). Furthermore, we recently have cloned three additional members of this family by RT-PCR strategy, 3K. Aoyama, M. Nagata, K. Oshima, T. Matsuda, and N. Aoki, unpublished observation. and putative DSPs with a low molecular mass of various species have been registered in the GenBank and Swissprot data bases. It is now conceivable that a distinct subfamily of DSPs is actually present.Each DSP has its own substrate specificity for MAP kinases. MKP-1/CL100 deactivates MAPK/ERK, p38, and SAPK/JNK; PAC1 and Pyst2/B59 deactivate MAPK/ERK and p38; hVH2/MKP-2 deactivates MAPK/ERK and SAPK/JNK; hVH3/B23, Pyst1/MKP-3, and MKP4 deactivate MAPK/ERK; and hVH5/M3/6 and MKP-5 deactivate p38 and SAPK/JNK (22Kwak S.P Dixon J.E. J. Biol. Chem. 1995; 270: 1156-1160Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24Martell 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, 26Muda M. Boschert U. Dickinson R. Martinou J.-C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 28Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (370) Google Scholar, 31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 41Chu Y. Solski P.A. Khosravi-Far R. Der C.J. Kelly K. J. Biol. Chem. 1996; 271: 6497-6501Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). We determined the substrate specificity of LMW-DSP2. Upon co-expression in COS7 cells, LMW-DSP2 dephosphorylated and deactivated p38 and SAPK/JNK but not MAPK/ERK. The dephosphorylation and deactivation of p38 was greater than that of SAPK/JNK. Among the reported DSPs, only hVH5/M3/6 and MKP-5 exhibited the same or similar substrate specificity (24Martell 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,31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar).Although LMW-DSP2 does not contain the cdc25 homology domain that functions in other DSPs as a regulatory and essential domain in dephosphorylation toward MAP kinases (33Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (667) Google Scholar, 42Muda M. Theodosiou A. Gillieson C. Smith A. Chabert C. Camps M. Boschert U. Rodrigues N. Davies K. Ashworth A. Arkinstall S. J. Biol. Chem. 1998; 273: 9323-9329Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 43Camps M. Nichols A. Gillieron G. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (432) Google Scholar), it could dephosphorylate and deactivate p38 and SAPK/JNK MAP kinases in cultured cells (Fig. 4) and in vitro (Fig. 5). Further experiments with a variety of mutants of the phosphatase and MAP kinases revealed that static interaction between LMW-DSP2 and p38 and SAPK/JNK might not be required for dephosphorylation and deactivation of the MAP kinases by LMW-DSP2 in cultured cells (Fig. 6) and that specific binding between LMW-DSP2 and MAP kinases was not disrupted by mutating putative common docking sites (Fig. 7). It has also been reported that VHR lacking the cdc25 homology domain could dephosphorylate activated MAPK/ERK kinase in cultured cells and in vitro (44Todd J.L. Tanner K.G. Denu J.M. J. Biol. Chem. 1999; 274: 13271-13280Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This and our present study suggest the presence of a distinct dephosphorylation mechanism of MAP kinases by DSPs with a low molecular weight. Elucidation of this putative novel dephosphorylation mechanism is currently in progress using a series of mutants in our laboratory.LMW-DSP2 was shown here to localize evenly in the cytosol and the nucleus (Fig. 3). This subcellular localization of LMW-DSP2 is very similar to that of MKP-5 (31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The subcellular localization of LMW-DSP2 was unchanged after stimulation with anisomycin and NaCl (data not shown). p38 and SAPK/JNK also localized in both the cytosol and nucleus, and their subcellular localizations were not dramatically affected by stress stimulation, whereas MAPK/ERK translocated into the nucleus upon stimulation (data not shown). The correspondence of the subcellular localization between LMW-DSP2 and its target substrates, p38 and SAPK/JNK, might reflect some physiological relevance.LMW-DSP2 was shown to be specifically expressed in testis (Fig. 8,A and B), and p38 and SAPK/JNK were also shown to be expressed in testis (Fig. 8 C), suggesting that p38 and SAPK/JNK are physiological substrates of LMW-DSP2. However, we cannot exclude the possibility that LMW-DSP2 might target other unknown molecules specifically expressed in testis. Substrate-trapping mutants of LMW-DSP2 (D57A and C88S) might be useful in the identification of the other putative substrates.Several protein phosphatases are reported to be expressed specifically in the testis. Serine/threonine protein phosphatase PP12 is abundant in the rat testis and localized in the nuclei of late spermatocytes and early spermatids (45Shima H. Haneji T. Hatano Y. Kasugai I. Sugimura T. Nagao M. Biochem. Biophys. Res. Commun. 1993; 194: 930-937Crossref PubMed Scopus (61) Google Scholar). The PTP Typ was also shown to be expressed specifically in testicular germ cells (46Ohsugi M. Kuramochi S. Matsuda S. Yamamoto T. J. Biol. Chem. 1997; 272: 33092-33099Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). We also observed that cytosolic PTP20 is expressed abundantly in mouse and rat testis, 4K. Aoyama, M. Nagata, K. Oshima, T. Matsuda, and N. Aoki, unpublished data. and we showed that one of the splice variants of PTP36 (PTP36-B) is specifically expressed in testis (11Aoyama K. Matsuda T. Aoki N. Biochem. Biophys. Res. Commun. 1999; 266: 523-531Crossref PubMed Scopus (8) Google Scholar). One of the DSPs, TMDP, was also shown to be predominantly expressed in the testis and skeletal muscle (40Nakamura K. Shima H. Watanabe M. Haneji T. Kikuchi K. Biochem. J. 1999; 344: 819-825Crossref PubMed Google Scholar). Moreover, we have observed that three novel DSP clones are also specifically expressed in testis.4 Testis-specific or predominant expression of many protein phosphatases might suggest involvement in a testis-specific cellular event, particularly in spermatogenesis.In summary, we have cloned and characterized a novel DSP with low molecular weight designated LMW-DSP2. LMW-DSP2 does not seem to belong to known subfamilies of DSPs with respect to structural features and substrate specificity. LMW-DSP2 is specific for p38 and SAPK/JNK, but the dephosphorylation and deactivation mechanism does not require static interaction between the phosphatase and MAP kinases. Thus, LMW-DSP2 may be a novel member of the DSP family acting on the MAP kinase family. Extracellular signal-regulated kinase (ERK),1 stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38/RK/CSBP (p38) are distinct classes of mitogen-activated protein (MAP) kinases. ERK is activated mainly by a variety of growth factors and phorbol esters and is associated with cellular proliferation and differentiation. SAPK/JNK and p38 are activated by extracellular stresses such as UV irradiation, osmotic stress, and inflammatory cytokines but are poorly activated by growth factors and phorbol esters. Activation of these protein kinases leads to a variety of responses such as gene expression, cell proliferation, differentiation, cell cycle arrest, apoptosis, early development, etc., depending on the cell type (1Ahn N.G. Seger R. Krebs E.G. Curr. Opin. Cell Biol. 1991; 4: 992-999Crossref Scopus (228) Google Scholar, 2Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (328) Google Scholar, 3Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 4Davis R.J. Trends Biochem. Sci. 1994; 19: 470-473Abstract Full Text PDF PubMed Scopus (917) Google Scholar, 5Kyriakis J.M. Avruch J. Bioessays. 1996; 18: 567-577Crossref PubMed Scopus (660) Google Scholar, 6Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar, 7Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar, 8Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1160) Google Scholar). Full MAP kinase activation needs phosphorylation on both tyrosine and threonine residues by selective upstream dual specificity kinases (7Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar, 9Cahill M.A. Janknechit R. Nordheim A. Curr. Biol. 1996; 6: 16-19Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 10Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (996) Google Scholar). Because MAP kinase activation is a reversible process, protein phosphatases play critical regulatory roles. Recently, there has been an emerging family of dual specificity phosphatases (DSPs)/MAP kinase phosphatases (MKPs) acting on the MAP kinase superfamily. Ten members of this group of DSPs have been reported including VHR (12Ishibashi T. Bottaro D.P. Chan A. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 89: 12170-12174Crossref Scopus (179) Google Scholar), CL100 (MKP-1) (13Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-646Crossref PubMed Scopus (568) Google Scholar, 14Charles C.H. Abler A.S. Lau L.F. Oncogene. 1992; 7: 187-190PubMed Google Scholar, 15Alessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020PubMed Google Scholar, 16Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1022) Google Scholar), PAC1 (17Rohan 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, 18Ward Y. Gupta S. Jensen P. Wartmann M. Davis R.J. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar), MKP-2 (hVH2, TYP-1) (19Guan K.-L. Butch E. J. Biol. Chem. 1995; 270: 7197-7203Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 20Misra-Press A. Rim C.S. Yao H. Roberson M.S. Stork P.J.S. J. Biol. Chem. 1995; 270: 14587-14596Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 21King A.G. Ozanne B.W. Smythe C. Ashworth A. Oncogene. 1995; 11: 2553-2563PubMed Google Scholar), hVH3 (B23) (22Kwak S.P Dixon J.E. J. Biol. Chem. 1995; 270: 1156-1160Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23Ishibashi 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), hVH5 (M3/6) (24Martell 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,25Theodosiou A.M. Rodrigues N.R. Nesbit M.A. Ambrose H.J. Paterson H. McLellan-Arnold E. Boyd Y. Leversha M.A. Owen N. Blake D.J. Ashworth A. Davies K.E. Hum. Mol. Genet. 1996; 96: 675-684Crossref Scopus (49) Google Scholar), MKP-3 (Pyst1, rVH6) (26Muda M. Boschert U. Dickinson R. Martinou J.-C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 27Mourey R.J. Vega Q.C. Campbell J.S. Wenderoth M.P. Hauschka S.D. Krebs E.G. Dixon J.E. J. Biol. Chem. 1996; 271: 3795-3802Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 28Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (370) Google Scholar), Pyst2 (29Dowd S. Sneddon A.A. Keyse S.M. J. Cell Sci. 1997; 111: 3389-3399Google Scholar), MKP-4 (Pyst3) (29Dowd S. Sneddon A.A. Keyse S.M. J. Cell Sci. 1997; 111: 3389-3399Google Scholar, 30Muda M. Boschert U. Smith A. Antonsson B. Gillierson C. Chabert C. Camps M. Martinou I. Ashworth A. Arkinstall S. J. Biol. Chem. 1997; 272: 5141-5151Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and MKP-5 (31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). They share sequence homology, but each has distinct properties concerning substrate specificity, tissue distribution, subcellular localization, and inducibility by extracellular stimuli (32Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (710) Google Scholar). As a part of the studies on DSP diversity and cellular function, we have been trying to clone novel members of the DSP family. A combination of RT-PCR and cDNA library screening revealed a novel clone designated LMW-DSP2 containing a single catalytic domain but lacking the cdc25 homology domain. Despite the absence of a putative common docking site for MAP kinases, which is normally located in the cdc25 homology domain, LMW-DSP2 dephosphorylated and deactivated p38, to a higher extent, and SAPK/JNK, but not ERK1/2, in transfected COS7 cells and in vitro. Further analyses using various mutants suggested that LMW-DSP2 was a member of a distinct class of DSPs and that the mode of its dephosphorylation and deactivation action toward MAP kinases was different from that of other DSPs reported. DISCUSSIONWe have isolated a cDNA clone encoding a novel dual specificity phosphatase, LMW-DSP2. Unlike most of other DSPs/MKPs hitherto identified, LMW-DSP2 was unique in that it lacked the cdc25 homology domain that is conserved in the N-terminal region of DSPs (32Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (710) Google Scholar) and accordingly had a smaller molecular mass. VHR and recently cloned TMDP did not have the cdc25 homology domain as well (12Ishibashi T. Bottaro D.P. Chan A. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 89: 12170-12174Crossref Scopus (179) Google Scholar, 40Nakamura K. Shima H. Watanabe M. Haneji T. Kikuchi K. Biochem. J. 1999; 344: 819-825Crossref PubMed Google Scholar). Furthermore, we recently have cloned three additional members of this family by RT-PCR strategy, 3K. Aoyama, M. Nagata, K. Oshima, T. Matsuda, and N. Aoki, unpublished observation. and putative DSPs with a low molecular mass of various species have been registered in the GenBank and Swissprot data bases. It is now conceivable that a distinct subfamily of DSPs is actually present.Each DSP has its own substrate specificity for MAP kinases. MKP-1/CL100 deactivates MAPK/ERK, p38, and SAPK/JNK; PAC1 and Pyst2/B59 deactivate MAPK/ERK and p38; hVH2/MKP-2 deactivates MAPK/ERK and SAPK/JNK; hVH3/B23, Pyst1/MKP-3, and MKP4 deactivate MAPK/ERK; and hVH5/M3/6 and MKP-5 deactivate p38 and SAPK/JNK (22Kwak S.P Dixon J.E. J. Biol. Chem. 1995; 270: 1156-1160Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24Martell 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, 26Muda M. Boschert U. Dickinson R. Martinou J.-C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 28Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (370) Google Scholar, 31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 41Chu Y. Solski P.A. Khosravi-Far R. Der C.J. Kelly K. J. Biol. Chem. 1996; 271: 6497-6501Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). We determined the substrate specificity of LMW-DSP2. Upon co-expression in COS7 cells, LMW-DSP2 dephosphorylated and deactivated p38 and SAPK/JNK but not MAPK/ERK. The dephosphorylation and deactivation of p38 was greater than that of SAPK/JNK. Among the reported DSPs, only hVH5/M3/6 and MKP-5 exhibited the same or similar substrate specificity (24Martell 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,31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar).Although LMW-DSP2 does not contain the cdc25 homology domain that functions in other DSPs as a regulatory and essential domain in dephosphorylation toward MAP kinases (33Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (667) Google Scholar, 42Muda M. Theodosiou A. Gillieson C. Smith A. Chabert C. Camps M. Boschert U. Rodrigues N. Davies K. Ashworth A. Arkinstall S. J. Biol. Chem. 1998; 273: 9323-9329Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 43Camps M. Nichols A. Gillieron G. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (432) Google Scholar), it could dephosphorylate and deactivate p38 and SAPK/JNK MAP kinases in cultured cells (Fig. 4) and in vitro (Fig. 5). Further experiments with a variety of mutants of the phosphatase and MAP kinases revealed that static interaction between LMW-DSP2 and p38 and SAPK/JNK might not be required for dephosphorylation and deactivation of the MAP kinases by LMW-DSP2 in cultured cells (Fig. 6) and that specific binding between LMW-DSP2 and MAP kinases was not disrupted by mutating putative common docking sites (Fig. 7). It has also been reported that VHR lacking the cdc25 homology domain could dephosphorylate activated MAPK/ERK kinase in cultured cells and in vitro (44Todd J.L. Tanner K.G. Denu J.M. J. Biol. Chem. 1999; 274: 13271-13280Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This and our present study suggest the presence of a distinct dephosphorylation mechanism of MAP kinases by DSPs with a low molecular weight. Elucidation of this putative novel dephosphorylation mechanism is currently in progress using a series of mutants in our laboratory.LMW-DSP2 was shown here to localize evenly in the cytosol and the nucleus (Fig. 3). This subcellular localization of LMW-DSP2 is very similar to that of MKP-5 (31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The subcellular localization of LMW-DSP2 was unchanged after stimulation with anisomycin and NaCl (data not shown). p38 and SAPK/JNK also localized in both the cytosol and nucleus, and their subcellular localizations were not dramatically affected by stress stimulation, whereas MAPK/ERK translocated into the nucleus upon stimulation (data not shown). The correspondence of the subcellular localization between LMW-DSP2 and its target substrates, p38 and SAPK/JNK, might reflect some physiological relevance.LMW-DSP2 was shown to be specifically expressed in testis (Fig. 8,A and B), and p38 and SAPK/JNK were also shown to be expressed in testis (Fig. 8 C), suggesting that p38 and SAPK/JNK are physiological substrates of LMW-DSP2. However, we cannot exclude the possibility that LMW-DSP2 might target other unknown molecules specifically expressed in testis. Substrate-trapping mutants of LMW-DSP2 (D57A and C88S) might be useful in the identification of the other putative substrates.Several protein phosphatases are reported to be expressed specifically in the testis. Serine/threonine protein phosphatase PP12 is abundant in the rat testis and localized in the nuclei of late spermatocytes and early spermatids (45Shima H. Haneji T. Hatano Y. Kasugai I. Sugimura T. Nagao M. Biochem. Biophys. Res. Commun. 1993; 194: 930-937Crossref PubMed Scopus (61) Google Scholar). The PTP Typ was also shown to be expressed specifically in testicular germ cells (46Ohsugi M. Kuramochi S. Matsuda S. Yamamoto T. J. Biol. Chem. 1997; 272: 33092-33099Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). We also observed that cytosolic PTP20 is expressed abundantly in mouse and rat testis, 4K. Aoyama, M. Nagata, K. Oshima, T. Matsuda, and N. Aoki, unpublished data. and we showed that one of the splice variants of PTP36 (PTP36-B) is specifically expressed in testis (11Aoyama K. Matsuda T. Aoki N. Biochem. Biophys. Res. Commun. 1999; 266: 523-531Crossref PubMed Scopus (8) Google Scholar). One of the DSPs, TMDP, was also shown to be predominantly expressed in the testis and skeletal muscle (40Nakamura K. Shima H. Watanabe M. Haneji T. Kikuchi K. Biochem. J. 1999; 344: 819-825Crossref PubMed Google Scholar). Moreover, we have observed that three novel DSP clones are also specifically expressed in testis.4 Testis-specific or predominant expression of many protein phosphatases might suggest involvement in a testis-specific cellular event, particularly in spermatogenesis.In summary, we have cloned and characterized a novel DSP with low molecular weight designated LMW-DSP2. LMW-DSP2 does not seem to belong to known subfamilies of DSPs with respect to structural features and substrate specificity. LMW-DSP2 is specific for p38 and SAPK/JNK, but the dephosphorylation and deactivation mechanism does not require static interaction between the phosphatase and MAP kinases. Thus, LMW-DSP2 may be a novel member of the DSP family acting on the MAP kinase family. We have isolated a cDNA clone encoding a novel dual specificity phosphatase, LMW-DSP2. Unlike most of other DSPs/MKPs hitherto identified, LMW-DSP2 was unique in that it lacked the cdc25 homology domain that is conserved in the N-terminal region of DSPs (32Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (710) Google Scholar) and accordingly had a smaller molecular mass. VHR and recently cloned TMDP did not have the cdc25 homology domain as well (12Ishibashi T. Bottaro D.P. Chan A. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 89: 12170-12174Crossref Scopus (179) Google Scholar, 40Nakamura K. Shima H. Watanabe M. Haneji T. Kikuchi K. Biochem. J. 1999; 344: 819-825Crossref PubMed Google Scholar). Furthermore, we recently have cloned three additional members of this family by RT-PCR strategy, 3K. Aoyama, M. Nagata, K. Oshima, T. Matsuda, and N. Aoki, unpublished observation. and putative DSPs with a low molecular mass of various species have been registered in the GenBank and Swissprot data bases. It is now conceivable that a distinct subfamily of DSPs is actually present. Each DSP has its own substrate specificity for MAP kinases. MKP-1/CL100 deactivates MAPK/ERK, p38, and SAPK/JNK; PAC1 and Pyst2/B59 deactivate MAPK/ERK and p38; hVH2/MKP-2 deactivates MAPK/ERK and SAPK/JNK; hVH3/B23, Pyst1/MKP-3, and MKP4 deactivate MAPK/ERK; and hVH5/M3/6 and MKP-5 deactivate p38 and SAPK/JNK (22Kwak S.P Dixon J.E. J. Biol. Chem. 1995; 270: 1156-1160Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24Martell 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, 26Muda M. Boschert U. Dickinson R. Martinou J.-C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 28Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (370) Google Scholar, 31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 41Chu Y. Solski P.A. Khosravi-Far R. Der C.J. Kelly K. J. Biol. Chem. 1996; 271: 6497-6501Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). We determined the substrate specificity of LMW-DSP2. Upon co-expression in COS7 cells, LMW-DSP2 dephosphorylated and deactivated p38 and SAPK/JNK but not MAPK/ERK. The dephosphorylation and deactivation of p38 was greater than that of SAPK/JNK. Among the reported DSPs, only hVH5/M3/6 and MKP-5 exhibited the same or similar substrate specificity (24Martell 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,31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Although LMW-DSP2 does not contain the cdc25 homology domain that functions in other DSPs as a regulatory and essential domain in dephosphorylation toward MAP kinases (33Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (667) Google Scholar, 42Muda M. Theodosiou A. Gillieson C. Smith A. Chabert C. Camps M. Boschert U. Rodrigues N. Davies K. Ashworth A. Arkinstall S. J. Biol. Chem. 1998; 273: 9323-9329Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 43Camps M. Nichols A. Gillieron G. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (432) Google Scholar), it could dephosphorylate and deactivate p38 and SAPK/JNK MAP kinases in cultured cells (Fig. 4) and in vitro (Fig. 5). Further experiments with a variety of mutants of the phosphatase and MAP kinases revealed that static interaction between LMW-DSP2 and p38 and SAPK/JNK might not be required for dephosphorylation and deactivation of the MAP kinases by LMW-DSP2 in cultured cells (Fig. 6) and that specific binding between LMW-DSP2 and MAP kinases was not disrupted by mutating putative common docking sites (Fig. 7). It has also been reported that VHR lacking the cdc25 homology domain could dephosphorylate activated MAPK/ERK kinase in cultured cells and in vitro (44Todd J.L. Tanner K.G. Denu J.M. J. Biol. Chem. 1999; 274: 13271-13280Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This and our present study suggest the presence of a distinct dephosphorylation mechanism of MAP kinases by DSPs with a low molecular weight. Elucidation of this putative novel dephosphorylation mechanism is currently in progress using a series of mutants in our laboratory. LMW-DSP2 was shown here to localize evenly in the cytosol and the nucleus (Fig. 3). This subcellular localization of LMW-DSP2 is very similar to that of MKP-5 (31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The subcellular localization of LMW-DSP2 was unchanged after stimulation with anisomycin and NaCl (data not shown). p38 and SAPK/JNK also localized in both the cytosol and nucleus, and their subcellular localizations were not dramatically affected by stress stimulation, whereas MAPK/ERK translocated into the nucleus upon stimulation (data not shown). The correspondence of the subcellular localization between LMW-DSP2 and its target substrates, p38 and SAPK/JNK, might reflect some physiological relevance. LMW-DSP2 was shown to be specifically expressed in testis (Fig. 8,A and B), and p38 and SAPK/JNK were also shown to be expressed in testis (Fig. 8 C), suggesting that p38 and SAPK/JNK are physiological substrates of LMW-DSP2. However, we cannot exclude the possibility that LMW-DSP2 might target other unknown molecules specifically expressed in testis. Substrate-trapping mutants of LMW-DSP2 (D57A and C88S) might be useful in the identification of the other putative substrates. Several protein phosphatases are reported to be expressed specifically in the testis. Serine/threonine protein phosphatase PP12 is abundant in the rat testis and localized in the nuclei of late spermatocytes and early spermatids (45Shima H. Haneji T. Hatano Y. Kasugai I. Sugimura T. Nagao M. Biochem. Biophys. Res. Commun. 1993; 194: 930-937Crossref PubMed Scopus (61) Google Scholar). The PTP Typ was also shown to be expressed specifically in testicular germ cells (46Ohsugi M. Kuramochi S. Matsuda S. Yamamoto T. J. Biol. Chem. 1997; 272: 33092-33099Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). We also observed that cytosolic PTP20 is expressed abundantly in mouse and rat testis, 4K. Aoyama, M. Nagata, K. Oshima, T. Matsuda, and N. Aoki, unpublished data. and we showed that one of the splice variants of PTP36 (PTP36-B) is specifically expressed in testis (11Aoyama K. Matsuda T. Aoki N. Biochem. Biophys. Res. Commun. 1999; 266: 523-531Crossref PubMed Scopus (8) Google Scholar). One of the DSPs, TMDP, was also shown to be predominantly expressed in the testis and skeletal muscle (40Nakamura K. Shima H. Watanabe M. Haneji T. Kikuchi K. Biochem. J. 1999; 344: 819-825Crossref PubMed Google Scholar). Moreover, we have observed that three novel DSP clones are also specifically expressed in testis.4 Testis-specific or predominant expression of many protein phosphatases might suggest involvement in a testis-specific cellular event, particularly in spermatogenesis. In summary, we have cloned and characterized a novel DSP with low molecular weight designated LMW-DSP2. LMW-DSP2 does not seem to belong to known subfamilies of DSPs with respect to structural features and substrate specificity. LMW-DSP2 is specific for p38 and SAPK/JNK, but the dephosphorylation and deactivation mechanism does not require static interaction between the phosphatase and MAP kinases. Thus, LMW-DSP2 may be a novel member of the DSP family acting on the MAP kinase family. We are grateful to Drs. J. Pouyssegur, J. S. Gutkind, J. Woodgett, and S. Arkinstall for providing pcDNA1-HA/p44 ERK1, pcDNA3-HA/p38 HOG, pMT2T-HA/p54 SAPKβ, and pMTSM-MycMKP-4, respectively. We also thank Dr. Hagiwara for providing a mouse testis cDNA library. Retraction: Molecular cloning and characterization of a novel dual specificity phosphatase, LMW-DSP2, that lacks the Cdc25 homology domain.Journal of Biological ChemistryVol. 288Issue 35PreviewVOLUME 276 (2001) PAGES 27575–27583 Full-Text PDF Open Access
Referência(s)