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. 1992 Mar;3(3):287–298. doi: 10.1091/mbc.3.3.287

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

R E Mayer-Jaekel 1, S Baumgartner 1, G Bilbe 1, H Ohkura 1, D M Glover 1, B A Hemmings 1
PMCID: PMC275530  PMID: 1320961

Abstract

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Axton J. M., Dombrádi V., Cohen P. T., Glover D. M. One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell. 1990 Oct 5;63(1):33–46. doi: 10.1016/0092-8674(90)90286-n. [DOI] [PubMed] [Google Scholar]
  2. Baumgartner S., Bopp D., Burri M., Noll M. Structure of two genes at the gooseberry locus related to the paired gene and their spatial expression during Drosophila embryogenesis. Genes Dev. 1987 Dec;1(10):1247–1267. doi: 10.1101/gad.1.10.1247. [DOI] [PubMed] [Google Scholar]
  3. Bourouis M., Moore P., Ruel L., Grau Y., Heitzler P., Simpson P. An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily. EMBO J. 1990 Sep;9(9):2877–2884. doi: 10.1002/j.1460-2075.1990.tb07477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cavener D. R. Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 1987 Feb 25;15(4):1353–1361. doi: 10.1093/nar/15.4.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen S. C., Kramer G., Hardesty B. Isolation and partial characterization of an Mr 60,000 subunit of a type 2A phosphatase from rabbit reticulocytes. J Biol Chem. 1989 May 5;264(13):7267–7275. [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  8. Cyert M. S., Thorner J. Putting it on and taking it off: phosphoprotein phosphatase involvement in cell cycle regulation. Cell. 1989 Jun 16;57(6):891–893. doi: 10.1016/0092-8674(89)90325-5. [DOI] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dombrádi V., Axton J. M., Glover D. M., Cohen P. T. Cloning and chromosomal localization of Drosophila cDNA encoding the catalytic subunit of protein phosphatase 1 alpha. High conservation between mammalian and insect sequences. Eur J Biochem. 1989 Aug 15;183(3):603–610. doi: 10.1111/j.1432-1033.1989.tb21089.x. [DOI] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Félix M. A., Cohen P., Karsenti E. Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid. EMBO J. 1990 Mar;9(3):675–683. doi: 10.1002/j.1460-2075.1990.tb08159.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gautier J., Solomon M. J., Booher R. N., Bazan J. F., Kirschner M. W. cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell. 1991 Oct 4;67(1):197–211. doi: 10.1016/0092-8674(91)90583-k. [DOI] [PubMed] [Google Scholar]
  14. Glover D. M. Mitosis in Drosophila. J Cell Sci. 1989 Feb;92(Pt 2):137–146. doi: 10.1242/jcs.92.2.137. [DOI] [PubMed] [Google Scholar]
  15. Goris J., Hermann J., Hendrix P., Ozon R., Merlevede W. Okadaic acid, a specific protein phosphatase inhibitor, induces maturation and MPF formation in Xenopus laevis oocytes. FEBS Lett. 1989 Mar 13;245(1-2):91–94. doi: 10.1016/0014-5793(89)80198-x. [DOI] [PubMed] [Google Scholar]
  16. Hemmings B. A., Adams-Pearson C., Maurer F., Müller P., Goris J., Merlevede W., Hofsteenge J., Stone S. R. alpha- and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure. Biochemistry. 1990 Apr 3;29(13):3166–3173. doi: 10.1021/bi00465a002. [DOI] [PubMed] [Google Scholar]
  17. Imaoka T., Imazu M., Usui H., Kinohara N., Takeda M. Resolution and reassociation of three distinct components from pig heart phosphoprotein phosphatase. J Biol Chem. 1983 Feb 10;258(3):1526–1535. [PubMed] [Google Scholar]
  18. Jimenez J., Alphey L., Nurse P., Glover D. M. Complementation of fission yeast cdc2ts and cdc25ts mutants identifies two cell cycle genes from Drosophila: a cdc2 homologue and string. EMBO J. 1990 Nov;9(11):3565–3571. doi: 10.1002/j.1460-2075.1990.tb07567.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Joshi B., Rundell K. Association of simian virus 40 small-t antigen with the 61-kilodalton component of a cellular protein complex. J Virol. 1990 Nov;64(11):5649–5651. doi: 10.1128/jvi.64.11.5649-5651.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kalderon D., Rubin G. M. Isolation and characterization of Drosophila cAMP-dependent protein kinase genes. Genes Dev. 1988 Dec;2(12A):1539–1556. doi: 10.1101/gad.2.12a.1539. [DOI] [PubMed] [Google Scholar]
  21. Khew-Goodall Y., Hemmings B. A. Tissue-specific expression of mRNAs encoding alpha- and beta-catalytic subunits of protein phosphatase 2A. FEBS Lett. 1988 Oct 10;238(2):265–268. doi: 10.1016/0014-5793(88)80493-9. [DOI] [PubMed] [Google Scholar]
  22. 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. 1990 Oct 19;63(2):405–415. doi: 10.1016/0092-8674(90)90173-c. [DOI] [PubMed] [Google Scholar]
  23. Langer-Safer P. R., Levine M., Ward D. C. Immunological method for mapping genes on Drosophila polytene chromosomes. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4381–4385. doi: 10.1073/pnas.79.14.4381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee T. H., Solomon M. J., Mumby M. C., Kirschner M. W. INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell. 1991 Jan 25;64(2):415–423. doi: 10.1016/0092-8674(91)90649-j. [DOI] [PubMed] [Google Scholar]
  25. Lehner C. F., O'Farrell P. H. Drosophila cdc2 homologs: a functional homolog is coexpressed with a cognate variant. EMBO J. 1990 Nov;9(11):3573–3581. doi: 10.1002/j.1460-2075.1990.tb07568.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mayer R. E., Hendrix P., Cron P., Matthies R., Stone S. R., Goris J., Merlevede W., Hofsteenge J., Hemmings B. A. Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform. Biochemistry. 1991 Apr 16;30(15):3589–3597. doi: 10.1021/bi00229a001. [DOI] [PubMed] [Google Scholar]
  27. Mozer B., Marlor R., Parkhurst S., Corces V. Characterization and developmental expression of a Drosophila ras oncogene. Mol Cell Biol. 1985 Apr;5(4):885–889. doi: 10.1128/mcb.5.4.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ohkura H., Yanagida M. S. pombe gene sds22+ essential for a midmitotic transition encodes a leucine-rich repeat protein that positively modulates protein phosphatase-1. Cell. 1991 Jan 11;64(1):149–157. doi: 10.1016/0092-8674(91)90216-l. [DOI] [PubMed] [Google Scholar]
  29. Orgad S., Brewis N. D., Alphey L., Axton J. M., Dudai Y., Cohen P. T. The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1. FEBS Lett. 1990 Nov 26;275(1-2):44–48. doi: 10.1016/0014-5793(90)81435-q. [DOI] [PubMed] [Google Scholar]
  30. Orgad S., Dudai Y., Cohen P. The protein phosphatases of Drosophila melanogaster and their inhibitors. Eur J Biochem. 1987 Apr 1;164(1):31–38. doi: 10.1111/j.1432-1033.1987.tb10988.x. [DOI] [PubMed] [Google Scholar]
  31. Pallas D. C., Shahrik L. K., Martin B. L., Jaspers S., Miller T. B., Brautigan D. L., Roberts T. M. Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell. 1990 Jan 12;60(1):167–176. doi: 10.1016/0092-8674(90)90726-u. [DOI] [PubMed] [Google Scholar]
  32. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  33. Saitoh Y., Yamamoto H., Ushio Y., Miyamoto E. Characterization of polyclonal antibodies to brain protein phosphatase 2A and immunohistochemical localization of the enzyme in rat brain. Brain Res. 1989 Jun 12;489(2):291–301. doi: 10.1016/0006-8993(89)90862-7. [DOI] [PubMed] [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Saunders R. D., Glover D. M., Ashburner M., Siden-Kiamos I., Louis C., Monastirioti M., Savakis C., Kafatos F. PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning. Nucleic Acids Res. 1989 Nov 25;17(22):9027–9037. doi: 10.1093/nar/17.22.9027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Siegfried E., Ambrosio L., Perrimon N. Serine/threonine protein kinases in Drosophila. Trends Genet. 1990 Nov;6(11):357–362. doi: 10.1016/0168-9525(90)90277-d. [DOI] [PubMed] [Google Scholar]
  37. Siegfried E., Perkins L. A., Capaci T. M., Perrimon N. Putative protein kinase product of the Drosophila segment-polarity gene zeste-white3. Nature. 1990 Jun 28;345(6278):825–829. doi: 10.1038/345825a0. [DOI] [PubMed] [Google Scholar]
  38. Stone S. R., Mayer R., Wernet W., Maurer F., Hofsteenge J., Hemmings B. A. The nucleotide sequence of the cDNA encoding the human lung protein phosphatase 2A alpha catalytic subunit. Nucleic Acids Res. 1988 Dec 9;16(23):11365–11365. doi: 10.1093/nar/16.23.11365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tautz D., Pfeifle C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma. 1989 Aug;98(2):81–85. doi: 10.1007/BF00291041. [DOI] [PubMed] [Google Scholar]
  40. Waelkens E., Goris J., Merlevede W. Purification and properties of polycation-stimulated phosphorylase phosphatases from rabbit skeletal muscle. J Biol Chem. 1987 Jan 25;262(3):1049–1059. [PubMed] [Google Scholar]
  41. Walter G., Ferre F., Espiritu O., Carbone-Wiley A. Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8669–8672. doi: 10.1073/pnas.86.22.8669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Woodgett J. R. Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J. 1990 Aug;9(8):2431–2438. doi: 10.1002/j.1460-2075.1990.tb07419.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yang S. I., Lickteig R. L., Estes R., Rundell K., Walter G., Mumby M. C. Control of protein phosphatase 2A by simian virus 40 small-t antigen. Mol Cell Biol. 1991 Apr;11(4):1988–1995. doi: 10.1128/mcb.11.4.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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