Plant Molecular Biology

, Volume 23, Issue 6, pp 1177–1185 | Cite as

Identification and molecular cloning of two homologues of protein phosphatase X from Arabidopsis thaliana

  • Encarna Pérez-Callejón
  • Antonio Casamayor
  • Gemma Pujol
  • Elisabet Clua
  • Albert Ferrer
  • Joaquín Ariño
Research Article


In a recent paper [Ariño et al., Plant Mol Biol 21: 475–485 (1993)] we reported the amplification of a DNA fragment (AP-2) from the genome of Arabidopsis thaliana encoding an amino acid sequence corresponding to a Ser/Thr protein phosphatase distantly related to type 2A protein phosphatases. In this paper we report the use of the AP-2 fragment to isolate several cDNA clones from a leaf cDNA library. Two of these (EP 124 and Ep 129) largely overlap and contain the AP-2 sequence, whereas a third clone (EP 128) is different although very related in sequence (86% of identity). Clones EP 124/EP 129 and EP 128 were found to encode two highly related polypeptides (93% identity) of 305 residues, showing a very high identity (83%) to the catalytic subunit of protein phosphatase X (PPX) from rabbit. Therefore, they have been named PPX-1 (EP 124/EP 129) and PPX-2 (EP 128). Southern blot analysis of genomic DNA indicates that only these two genes encoding phosphatases closely related to PPX are present in the genome of A. thaliana. Both PPX-1 and PPX-2 are expressed at very low levels in A. thaliana flowers, leaves, stems and roots. The expression levels of four previously identified type 2A phosphatases are higher than those of PPX genes. PP2A-1 appears to be the major mRNA species detected in all the tissues analyzed.

Key words

A. thaliana protein phosphorylation Ser/Thr protein phosphatase cDNA cloning isoforms 


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  1. 1.
    Ariño, J, Woon, CW, Brautigan, DL, Miller, TB, Johnson, GL: Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunits isotypes. Proc Natl Acad Sci USA 85: 4252–4256 (1988).Google Scholar
  2. 2.
    Ariño, J, Pérez-Callejón, E, Cunillera, N, Camps, M, Posas, F, Ferrer, A: Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana. Plant Mol Biol 21: 475–485 (1993).Google Scholar
  3. 3.
    Arndt, KT, Styles, CA, Fink, GR: A suppressor of a HIS4 transcriptional defect encodes a protein with homology to the catalytic subunit of protein phosphatase. Cell 56: 527–537 (1989).Google Scholar
  4. 4.
    Berndt, N, Campbell, DG, Caudwell, B, Cohen, P, da Cruz e Silva, EF, da Cruz e Silva, OB, Cohen, PTW: Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: homology with protein phosphatase 2A. FEBS Lett. 223: 340–346 (1987).Google Scholar
  5. 5.
    Brewis, ND, Cohen, PTW: Protein phosphatase X has been highly conserved during mammalian evolution. Biochim Biophys Acta 1171: 231–233 (1992).Google Scholar
  6. 6.
    Brewis, ND, Street, AJ, Prescott, AR, Cohen, PTW: PPX, a novel proteine serine/threonine phosphatase localized to centrosomes. EMBO J. 12: 987–996 (1993).Google Scholar
  7. 7.
    Budde, RJA, Chollet, R: Regulation of enzyme activity in plants by reversible phosphorylation. Physiol Plant 72: 435–439 (1988).Google Scholar
  8. 8.
    Cohen, P: The structure and regulation of protein phosphatases. Annu Rev Biochem 58: 453–508 (1989).Google Scholar
  9. 9.
    Cohen, P: The structure and regulation of protein phosphatases. In: Nishizuka, Y et al. (eds) The Biology and Medicine of Signal Transduction, pp. 230–235. Raven Press, New York (1990).Google Scholar
  10. 10.
    Cohen, PTW, Brewis, ND, Hughes, V, Mann, DJ: Protein serine/threonine phosphatases: an expanding family. FEBS Lett 268: 355–359 (1990).Google Scholar
  11. 11.
    da Cruz e Silva, OB, da Cruz e Silva, EF, Cohen, PTW: Identification of a novel phosphatase catalytic subunit by cDNA cloning. FEBS Lett 242: 106–110 (1988).Google Scholar
  12. 12.
    Dean, L, Elzen, B, Tamaki, S, Dunsmuir, P, Bedbrook, J: Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small subunit multigene family. EMBO J 5: 3055–3061 (1985).Google Scholar
  13. 13.
    Dellaporta, SL, Wood, J, Hicks, JB: Maize DNA miniprep. In: Molecular Biology of Plants: A Laboratory Manual, pp. 36–37. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984).Google Scholar
  14. 14.
    Dombrádi, V, Axton, JM, Glover, DM, Cohen, PTW: Molecular cloning and chromosomal localization of a novel Drosophila protein phosphatase. FEBS Lett 247: 391–395 (1989).Google Scholar
  15. 15.
    Gerlach, WL, Bedbrook, JR: Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucl Acids Res 7: 1869–1885 (1979).Google Scholar
  16. 16.
    Hinnebusch, A: Evidence for translational regulation of the activator of general amino acid control in yeast. Proc Natl Acad Sci USA 81: 6442–6446 (1984).Google Scholar
  17. 17.
    Ingebritsen, TS, Cohen, P: Protein phosphatases: properties and role in cellular regulation. Science 221: 331–338 (1983).Google Scholar
  18. 18.
    Jagiello, I, Donella-Deana, A, Szczegielniak, J, Pinna, LA, Muszyńska, G: Identification of protein phosphatase activities in maize seedlings. Biochim Biophys Acta 1134: 129–136 (1992).Google Scholar
  19. 19.
    Kinoshita, N, Ohkura, H, Yanagida, M: Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle. Cell 63: 405–415 (1990).Google Scholar
  20. 20.
    Khew-Goodall, Y, Hemmings, BA: Tissue-specific expression of mRNAs-encoding α and β-catalytic subunits of protein phosphatases 2A. FEBS Lett. 238: 265–268 (1988).Google Scholar
  21. 21.
    Lohmer, S, Maddaloni, M, Motto, M, Salamini, F, Thompson, RD: Translation of the mRNA of the maize transcriptional activator Opaque-2 is inhibited by upstream open reading frames present in the leader sequence. Plant Cell 5: 65–73 (1993).Google Scholar
  22. 22.
    Lütcke, HA, Chow, KC, Mickel, FS, Moss, KA, Kern, HF, Scheele, GA: Selection of AUG initiation codons differs in plant and animals. EMBO J 6: 43–48 (1987).Google Scholar
  23. 23.
    Mackintosh, C, Cohen, P: Identification of high levels of type 1 and 2A protein phosphatases in higher plants. Biochem J 262: 335–339 (1989).Google Scholar
  24. 24.
    Mackintosh, C, Goggins, J, Cohen, P: Subcellular disktribution, detection of protein phosphatase 2C and identification of protein phosphatase 2A as the major quinate dehydrogenase phosphatase. Biochem J 273: 733–738 (1990).Google Scholar
  25. 25.
    Mackintosh, RW, Haycox, G, Hardie, DG, Cohen, PTW: Identification by molecular cloning of two cDNA sequences from the plant Brassica napus which are very similar to mammalian protein phosphatase-1 and-2A. FEBS Lett 276: 156–160 (1990).Google Scholar
  26. 26.
    Nitschke, K, Fleig, U, Schell, J, Palme, K: Complementation of the cs dis2–11 cell cycle mutant of Schizosaccharomyces pombe by a protein phosphatase 1. EMBO J 11: 1327–1333 (1992).Google Scholar
  27. 27.
    Orgad, S, Brewis, ND, Alphey, L, Axton, JM, Dudai, Y, Cohen, PTW: The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1. FEBS Lett 275: 44–48 (1990).Google Scholar
  28. 28.
    Posas, F, Casamayor, A, Morral, N, Ariño, J: Molecular cloning and analysis of a yeast protein phosphatase with an unusual amino-terminal region. J Biol Chem 267: 11734–11740 (1992).Google Scholar
  29. 29.
    Posas, F, Clotet, J, Muns, MT, Corominas, J, Casamayor, A, Ariño, J: The gene PPG encodes a novel protein phosphatase involved in glycogen accumulation. J Biol Chem 268: 1349–1354 (1993).Google Scholar
  30. 30.
    Ronne, H, Carlberg, M, Hu, G-Z, Nehlin, JO: Protein phosphatase 2A in Saccharomyces cerevisiae: Effects on cell growth and bud morphogenesis. Mol Cell Biol 11: 4876–4883 (1991).Google Scholar
  31. 31.
    Ruberti, I, Sessa, G, Luchetti, S, Morelli, G: A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif. EMBO J 10: 1787–1791 (1991).Google Scholar
  32. 32.
    Rundle, SJ, Nasrallah, JB: Molecular characterization of type 1 serine/threonine phosphatases from Brassica oleracea. Plant Mol Biol 20: 367–375 (1992).Google Scholar
  33. 33.
    Sanger, F, Nicklen, S, Coulson, AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).Google Scholar
  34. 34.
    Smith, RD, Walker, JC: Isolation and expression of a maize type 1 protein phosphatase. Plant Physiol 97: 677–683 (1991).Google Scholar
  35. 35.
    Smith, RD, Walker, JC: Expression of multiple type 1 phosphoprotein phosphatases in Arabidopsis thaliana. Plant Mol Biol 21: 307–316 (1993).Google Scholar
  36. 36.
    Sneddon, AA, Cohen, PTW, Stark, MJR: Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes. EMBO J 9: 4339–4346 (1990).Google Scholar
  37. 37.
    Somerville, CR, Ogreen, WL: Isolation of photorespiratory mutants of Arabidopsis. In: Edelman, M, Hallick, R, Chua, NH (eds) Methods in Chloroplast Molecular Biology, pp. 129–138. Elsevier, New York (1982).Google Scholar
  38. 38.
    Stein, J, Howlett, B, Boyes, D, Nasrallah, M, Nasrallah, J: Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc Natl Acad Sci USA 88: 8816–8820 (1991).Google Scholar
  39. 39.
    Swofford DL: Phylogenetic Analysis Using Parsimony, Version 3.0, Computer program distributed by the Illinois Natural History Survey, Champaign, Illinois (1991).Google Scholar
  40. 40.
    Trewavas, A, Gilroy, S: Signal transduction in plant cells. Trends Genet 7: 356–361 (1991).Google Scholar
  41. 41.
    Zhang, K, Tsukitani, Y, John, PLC: Mitotic arrest in tobacco caused by the phosphoprotein phosphatase inhibitor okadaic acid. Plant Cell Physiol 33: 677–688 (1992).Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Encarna Pérez-Callejón
    • 1
  • Antonio Casamayor
    • 2
  • Gemma Pujol
    • 1
  • Elisabet Clua
    • 2
  • Albert Ferrer
    • 1
  • Joaquín Ariño
    • 2
  1. 1.Unitat de Bioquímica, Facultat de FarmàciaUniversitat de BarcelonaBarcelonaSpain
  2. 2.Departament de Bioquímica i Biologia Molecular, Facultat de VeterinàriaUniversitat Autònoma de BarcelonaBarcelonaSpain

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