Skip to main content
SpringerLink
Log in

The yeast phosphotyrosyl phosphatase activator protein, yPtpa1/Rrd1, interacts with Sit4 phosphatase to mediate resistance to 4-nitroquinoline-1-oxide and UVA

  • Research Article
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

We previously reported the isolation of mutants hypersensitive to the genotoxic agent 4-nitroquinoline-1-oxide, a potent inducer of oxidative stress. One of the mutants was defective in a gene designated yPTPA1, encoding a protein related to the human phosphotyrosyl phosphatase activator hPTPA, which is believed to play a role in activating the serine/threonine phosphatase PP2A. Yeast yptpa1Δ mutants are also sensitive to the UVA component of sunlight known to produce reactive oxygen species, suggesting a role for yPtpa1 in oxidative stress response. We now report the characterization of another 4-nitroquinoline-1-oxide-sensitive mutant, EBY20. We show that this mutant is defective in the SIT4 gene encoding a catalytic subunit of the PP2A phosphatases and that sit4Δ mutants exhibit hypersensitivity to 4-nitroquinoline-1-oxide and UVA, but not to UVC at 254 nm. Like the yptpa1Δ mutants, sit4Δ mutants are also defective in the repair of 4-nitroquinoline-1-oxide-induced DNA lesions. Genetic analysis revealed that both yPtpa1 and Sit4 function in the same pathway to protect cells against the lethal effects of 4-nitroquinoline-1-oxide and UVA. Moreover, we demonstrate that yPtpa1-affinity columns specifically retain Sit4, confirming a previous report that these two proteins indeed belong to a complex. Cellular localization studies using GFP-tagged proteins reveals that yPtpa1 is localized to the cytoplasm and the nucleus, while the Sit4 protein shows an intense staining spot in the cytoplasm and diffused staining in this organelle. We suggest that the yPtpa1–Sit4 complex may participate in a novel mechanism that mediates repair of oxidative DNA damage caused by 4-nitroquinoline-1-oxide and UVA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Ariza RR, Keyse SM, Moggs JG, Wood RD (1996) Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts. Nucleic Acids Res 24:433–440

    Article  CAS  PubMed  Google Scholar 

  • Baharians Z, Schonthal AH (1998) Autoregulation of protein phosphatase type 2A expression. J Biol Chem 273:19019–19024

    Article  CAS  PubMed  Google Scholar 

  • Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132

    Article  CAS  PubMed  Google Scholar 

  • Cayla X, Van Hoof C, Bosch M, Waelkens E, Vandekerckhove J, Peeters B, Merlevede W, Goris J (1994) Molecular cloning, expression, and characterization of PTPA, a protein that activates the tyrosyl phosphatase activity of protein phosphatase 2A. J Biol Chem 269:15668–15675

    CAS  PubMed  Google Scholar 

  • Demple B, Amabile-Cuevas CF (1991) Redox redux: the control of oxidative stress responses. Cell 67:837–839

    CAS  PubMed  Google Scholar 

  • Drobetsky EA, Turcotte J, Chateauneuf A (1995) A role for ultraviolet A in solar mutagenesis. Proc Natl Acad Sci USA 92:2350–2354

    CAS  PubMed  Google Scholar 

  • Felkner IC, Kadlubar F (1968) Parallel between ultraviolet light and 4-nitroquinoline-1-oxide sensitivity in Bacillus subtilis. J Bacteriol 96:1448–1449

    CAS  PubMed  Google Scholar 

  • Fellner T, Lackner DH, Hombauer H, Piribauer P, Mudrak I, Zaragoza K, Juno C, Ogris E (2003) A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo. Genes Dev 17:2138–2150

    Article  CAS  PubMed  Google Scholar 

  • Fiscella M, Zhang H, Fan S, Sakaguchi K, Shen S, Mercer WE, Vande Woude GF, O’Connor PM, Appella E (1997) Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. Proc Natl Acad Sci USA 94:6048–6053

    Article  CAS  PubMed  Google Scholar 

  • Galiegue-Zouitina S, Bailleul B, Ginot YM, Perly B, Vigny P, Loucheux-Lefebvre MH (1986) N2-guanyl and N6-adenyl arylation of chicken erythrocyte DNA by the ultimate carcinogen of 4-nitroquinoline-1-oxide. Cancer Res 46:1858–1863

    CAS  PubMed  Google Scholar 

  • Gershoni JM, Palade GE (1983) Protein blotting: principles and applications. Anal Biochem 131:1–15

    CAS  PubMed  Google Scholar 

  • Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425

    CAS  PubMed  Google Scholar 

  • Goldstein AL, McCusker JH, Knop M, Siegers K, Pereira G, Zachariae W, Winsor B, Nasmyth K, Schiebel E, Pan X, Wach A, Brachat A, Pohlmann R, Philippsen P (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 15:1541–1553

    CAS  PubMed  Google Scholar 

  • Guthrie C, Fink GR (1991) Guide to yeast genetics and molecular biology. Methods Enzymol 194:3–37

    CAS  PubMed  Google Scholar 

  • Hoeijmakers JH (1993) Nucleotide excision repair. II. From yeast to mammals. Trends Genet 9:211–217

    Article  CAS  PubMed  Google Scholar 

  • Keyse SM, Emslie EA (1992) Oxidative stress and heat shock induce a human gene encoding a protein–tyrosine phosphatase. Nature 359:644–647

    Article  CAS  PubMed  Google Scholar 

  • Kohda K, Tada M, Kasai H, Nishimura S, Kawazoe Y (1986) Formation of 8-hydroxyguanine residues in cellular DNA exposed to the carcinogen 4-nitroquinoline 1-oxide. Biochem Biophys Res Commun 139:626–632

    CAS  PubMed  Google Scholar 

  • Leduc A, He CH, Ramotar D (2003) Disruption of the Saccharomyces cerevisiae cell-wall pathway gene SLG1 causes hypersensitivity to the antitumor drug bleomycin. Mol Genet Genomics 269:78–89

    CAS  PubMed  Google Scholar 

  • Luke MM, Della Seta F, Di Como CJ, Sugimoto H, Kobayashi R, Arndt KT (1996) The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase. Mol Cell Biol 16:2744–2755

    CAS  PubMed  Google Scholar 

  • Mitchell DA, Sprague GF Jr (2001) The phosphotyrosyl phosphatase activator, Ncs1p (Rrd1p), functions with Cla4p to regulate the G(2)/M transition in Saccharomyces cerevisiae. Mol Cell Biol 21:488–500

    Article  CAS  PubMed  Google Scholar 

  • Moan J, Dahlback A, Setlow RB (1999) Epidemiological support for an hypothesis for melanoma induction indicating a role for UVA radiation. Photochem Photobiol 70:243–247

    Article  CAS  PubMed  Google Scholar 

  • Nunoshiba T, Demple B (1993) Potent intracellular oxidative stress exerted by the carcinogen 4-nitroquinoline-N-oxide. Cancer Res 53:3250–3252

    CAS  PubMed  Google Scholar 

  • Prakash S, Sung P, Prakash L (1993) DNA repair genes and proteins of Saccharomyces cerevisiae. Annu Rev Genet 27:33–70

    CAS  PubMed  Google Scholar 

  • Ramotar D, Belanger E, Brodeur I, Masson JY, Drobetsky EA (1998) A yeast homologue of the human phosphotyrosyl phosphatase activator PTPA is implicated in protection against oxidative DNA damage induced by the model carcinogen 4-nitroquinoline-1-oxide. J Biol Chem 273:21489–21496

    Article  CAS  PubMed  Google Scholar 

  • Ruiz-Laguna J, Ariza RR, Prieto-Alamo MJ, Boiteux S, Pueyo C (1994) Fpg protein protects Escherichia coli K-12 from mutation induction by the carcinogen 4-nitroquinoline-1-oxide. Carcinogenesis 15:425–429

    CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    PubMed  Google Scholar 

  • Sherman F, Fink G, Hicks J (1983) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

  • Shu Y, Yang H, Hallberg E, Hallberg R (1997) Molecular genetic analysis of Rts1p, a B′ regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A. Mol Cell Biol 17:3242–3253

    CAS  PubMed  Google Scholar 

  • Stark MJ (1996) Yeast protein serine/threonine phosphatases: multiple roles and diverse regulation. Yeast 12:1647–1675

    Article  CAS  PubMed  Google Scholar 

  • Sugimura T, Otake H, Matsushima T (1968) Single strand scissions of DNA caused by a carcinogen, 4-hydroxylaminoquinoline-1-oxide. Nature 218:392

    CAS  PubMed  Google Scholar 

  • Tada M (1976) Main binding sites of the carcinogen, 4-nitroquinoline-1-oxide in nucleic acids. Biochim Biophys Acta 454:558–566

    Article  CAS  PubMed  Google Scholar 

  • Tong AH, Lesage G, Bader GD, Ding H, Xu H, et al (2004) Global mapping of the yeast genetic interaction network. Science 303:808–813

    Article  CAS  PubMed  Google Scholar 

  • Vongsamphanh R, Fortier PK, Ramotar D (2001) Pir1p mediates translocation of the yeast Apn1p endonuclease into the mitochondria to maintain genomic stability. Mol Cell Biol 21:1647–1655

    CAS  PubMed  Google Scholar 

  • Wang H, Ramotar D (2002) Cellular resistance to bleomycin in Saccharomyces cerevisiae is not affected by changes in bleomycin hydrolase levels. Biochem Cell Biol 80:789–796

    Article  CAS  PubMed  Google Scholar 

  • Wera S, Hemmings BA (1995) Serine/threonine protein phosphatases. Biochem J 311:17–29

    CAS  PubMed  Google Scholar 

  • Yacoub A, Kelley MR, Deutsch WA (1997) The DNA repair activity of human redox/repair protein APE/Ref-1 is inactivated by phosphorylation. Cancer Res 57:5457–5459

    CAS  PubMed  Google Scholar 

  • Yamamoto K, Inoue S, Kawanishi S (1993) Site-specific DNA damage and 8-hydroxydeoxyguanosine formation by hydroxylamine and 4-hydroxyaminoquinoline-1-oxide in the presence of Cu(II): role of active oxygen species. Carcinogenesis 14:1397–1401

    CAS  PubMed  Google Scholar 

  • Zhao Y, Boguslawski G, Zitomer RS, DePaoli-Roach AA (1997) Saccharomyces cerevisiae homologs of mammalian B and B′ subunits of protein phosphatase 2A direct the enzyme to distinct cellular functions. J Biol Chem 272:8256–8262

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Anick Leduc for assistance with the microscope and Cyril Sabbah for reading the manuscript. This study was supported by a grant to D.R. from the Canadian Institutes of Health Research. During the tenure of this work, D.R. was supported by a career scientist award from the NCIC, and currently by a senior fellowship from the Fonds de la Recherche en Sante du Quebec.

Author information

Authors and Affiliations

  1. Centre de Recherche, Hopital Maisonneuve-Rosemont, Université de Montréal, 5415 Boulevard de l’ Assomption, Montréal, Quebec, H1T 2M4, Canada

    J. Douville, J. David, P.-K. Fortier & Dindial Ramotar

Authors
  1. J. Douville
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. David
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P.-K. Fortier
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dindial Ramotar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dindial Ramotar.

Additional information

Communicated by S. Hohmann

Rights and permissions

Reprints and permissions

About this article

Cite this article

Douville, J., David, J., Fortier, PK. et al. The yeast phosphotyrosyl phosphatase activator protein, yPtpa1/Rrd1, interacts with Sit4 phosphatase to mediate resistance to 4-nitroquinoline-1-oxide and UVA. Curr Genet 46, 72–81 (2004). https://doi.org/10.1007/s00294-004-0513-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-004-0513-9

Keywords

Search

Navigation