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. 2000 Sep;156(1):21–29. doi: 10.1093/genetics/156.1.21

Important role for phylogenetically invariant PP2Acalpha active site and C-terminal residues revealed by mutational analysis in Saccharomyces cerevisiae.

D R Evans 1, B A Hemmings 1
PMCID: PMC1461227  PMID: 10978272

Abstract

PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acalpha functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acalpha Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acalpha catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acalpha C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acalpha catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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Selected References

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  1. Barton G. J., Cohen P. T., Barford D. Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases. Eur J Biochem. 1994 Feb 15;220(1):225–237. doi: 10.1111/j.1432-1033.1994.tb18618.x. [DOI] [PubMed] [Google Scholar]
  2. Brunelli J. P., Pall M. L. A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences. Yeast. 1993 Dec;9(12):1299–1308. doi: 10.1002/yea.320091203. [DOI] [PubMed] [Google Scholar]
  3. Bryant J. C., Westphal R. S., Wadzinski B. E. Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Balpha subunit. Biochem J. 1999 Apr 15;339(Pt 2):241–246. [PMC free article] [PubMed] [Google Scholar]
  4. Chen J., Martin B. L., Brautigan D. L. Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science. 1992 Aug 28;257(5074):1261–1264. doi: 10.1126/science.1325671. [DOI] [PubMed] [Google Scholar]
  5. Chen J., Parsons S., Brautigan D. L. Tyrosine phosphorylation of protein phosphatase 2A in response to growth stimulation and v-src transformation of fibroblasts. J Biol Chem. 1994 Mar 18;269(11):7957–7962. [PubMed] [Google Scholar]
  6. Chung H., Brautigan D. L. Protein phosphatase 2A suppresses MAP kinase signalling and ectopic protein expression. Cell Signal. 1999 Aug;11(8):575–580. doi: 10.1016/s0898-6568(99)00033-9. [DOI] [PubMed] [Google Scholar]
  7. Chung H., Nairn A. C., Murata K., Brautigan D. L. Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2. Biochemistry. 1999 Aug 10;38(32):10371–10376. doi: 10.1021/bi990902g. [DOI] [PubMed] [Google Scholar]
  8. De Baere I., Derua R., Janssens V., Van Hoof C., Waelkens E., Merlevede W., Goris J. Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue. Biochemistry. 1999 Dec 14;38(50):16539–16547. doi: 10.1021/bi991646a. [DOI] [PubMed] [Google Scholar]
  9. Di Como C. J., Arndt K. T. Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Dev. 1996 Aug 1;10(15):1904–1916. doi: 10.1101/gad.10.15.1904. [DOI] [PubMed] [Google Scholar]
  10. Egloff M. P., Cohen P. T., Reinemer P., Barford D. Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate. J Mol Biol. 1995 Dec 15;254(5):942–959. doi: 10.1006/jmbi.1995.0667. [DOI] [PubMed] [Google Scholar]
  11. Evangelista C. C., Jr, Rodriguez Torres A. M., Limbach M. P., Zitomer R. S. Rox3 and Rts1 function in the global stress response pathway in baker's yeast. Genetics. 1996 Apr;142(4):1083–1093. doi: 10.1093/genetics/142.4.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Evans D. R., Hemmings B. A. Signal transduction. What goes up must come down. Nature. 1998 Jul 2;394(6688):23–24. doi: 10.1038/27782. [DOI] [PubMed] [Google Scholar]
  13. Evans D. R., Myles T., Hofsteenge J., Hemmings B. A. Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms. J Biol Chem. 1999 Aug 20;274(34):24038–24046. doi: 10.1074/jbc.274.34.24038. [DOI] [PubMed] [Google Scholar]
  14. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  15. Groves M. R., Hanlon N., Turowski P., Hemmings B. A., Barford D. The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell. 1999 Jan 8;96(1):99–110. doi: 10.1016/s0092-8674(00)80963-0. [DOI] [PubMed] [Google Scholar]
  16. Guo H., Damuni Z. Autophosphorylation-activated protein kinase phosphorylates and inactivates protein phosphatase 2A. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2500–2504. doi: 10.1073/pnas.90.6.2500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Healy A. M., Zolnierowicz S., Stapleton A. E., Goebl M., DePaoli-Roach A. A., Pringle J. R. CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase. Mol Cell Biol. 1991 Nov;11(11):5767–5780. doi: 10.1128/mcb.11.11.5767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huang H. B., Horiuchi A., Goldberg J., Greengard P., Nairn A. C. Site-directed mutagenesis of amino acid residues of protein phosphatase 1 involved in catalysis and inhibitor binding. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3530–3535. doi: 10.1073/pnas.94.8.3530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jiang Y., Broach J. R. Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J. 1999 May 17;18(10):2782–2792. doi: 10.1093/emboj/18.10.2782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee J., Stock J. Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase. J Biol Chem. 1993 Sep 15;268(26):19192–19195. [PubMed] [Google Scholar]
  21. Lizotte D. L., McManus D. D., Cohen H. R., DeLong A. Functional expression of human and Arabidopsis protein phosphatase 2A in Saccharomyces cerevisiae and isolation of dominant-defective mutants. Gene. 1999 Jun 24;234(1):35–44. doi: 10.1016/s0378-1119(99)00188-2. [DOI] [PubMed] [Google Scholar]
  22. Mayer-Jaekel R. E., Hemmings B. A. Protein phosphatase 2A--a 'ménage à trois'. Trends Cell Biol. 1994 Aug;4(8):287–291. doi: 10.1016/0962-8924(94)90219-4. [DOI] [PubMed] [Google Scholar]
  23. Millward T. A., Zolnierowicz S., Hemmings B. A. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci. 1999 May;24(5):186–191. doi: 10.1016/s0968-0004(99)01375-4. [DOI] [PubMed] [Google Scholar]
  24. Murata K., Wu J., Brautigan D. L. B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10624–10629. doi: 10.1073/pnas.94.20.10624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ogris E., Du X., Nelson K. C., Mak E. K., Yu X. X., Lane W. S., Pallas D. C. A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A. J Biol Chem. 1999 May 14;274(20):14382–14391. doi: 10.1074/jbc.274.20.14382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ogris E., Gibson D. M., Pallas D. C. Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen. Oncogene. 1997 Aug 18;15(8):911–917. doi: 10.1038/sj.onc.1201259. [DOI] [PubMed] [Google Scholar]
  27. Ogris E., Mudrak I., Mak E., Gibson D., Pallas D. C. Catalytically inactive protein phosphatase 2A can bind to polyomavirus middle tumor antigen and support complex formation with pp60(c-src). J Virol. 1999 Sep;73(9):7390–7398. doi: 10.1128/jvi.73.9.7390-7398.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ronne H., Carlberg M., Hu G. Z., Nehlin J. O. Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis. Mol Cell Biol. 1991 Oct;11(10):4876–4884. doi: 10.1128/mcb.11.10.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ruediger R., Hentz M., Fait J., Mumby M., Walter G. Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens. J Virol. 1994 Jan;68(1):123–129. doi: 10.1128/jvi.68.1.123-129.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ruediger R., Roeckel D., Fait J., Bergqvist A., Magnusson G., Walter G. Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus. Mol Cell Biol. 1992 Nov;12(11):4872–4882. doi: 10.1128/mcb.12.11.4872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sneddon A. A., Cohen P. T., Stark M. J. Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes. EMBO J. 1990 Dec;9(13):4339–4346. doi: 10.1002/j.1460-2075.1990.tb07883.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wera S., Hemmings B. A. Serine/threonine protein phosphatases. Biochem J. 1995 Oct 1;311(Pt 1):17–29. doi: 10.1042/bj3110017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Xie H., Clarke S. An enzymatic activity in bovine brain that catalyzes the reversal of the C-terminal methyl esterification of protein phosphatase 2A. Biochem Biophys Res Commun. 1994 Sep 30;203(3):1710–1715. doi: 10.1006/bbrc.1994.2383. [DOI] [PubMed] [Google Scholar]
  34. Xie H., Clarke S. Protein phosphatase 2A is reversibly modified by methyl esterification at its C-terminal leucine residue in bovine brain. J Biol Chem. 1994 Jan 21;269(3):1981–1984. [PubMed] [Google Scholar]
  35. Zhang J., Zhang Z., Brew K., Lee E. Y. Mutational analysis of the catalytic subunit of muscle protein phosphatase-1. Biochemistry. 1996 May 21;35(20):6276–6282. doi: 10.1021/bi952954l. [DOI] [PubMed] [Google Scholar]
  36. Zhuo S., Clemens J. C., Stone R. L., Dixon J. E. Mutational analysis of a Ser/Thr phosphatase. Identification of residues important in phosphoesterase substrate binding and catalysis. J Biol Chem. 1994 Oct 21;269(42):26234–26238. [PubMed] [Google Scholar]
  37. van Zyl W., Huang W., Sneddon A. A., Stark M., Camier S., Werner M., Marck C., Sentenac A., Broach J. R. Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Nov;12(11):4946–4959. doi: 10.1128/mcb.12.11.4946. [DOI] [PMC free article] [PubMed] [Google Scholar]

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