Molecular and Cellular Biochemistry

, Volume 338, Issue 1–2, pp 291–298 | Cite as

Exogenously expressed human Ku70 stabilizes Ku80 in Xenopus oocytes and induces heterologous DNA-PK catalytic activity

  • Jyotshnabala Kanungo


The Ku protein is a heterodimer composed of 70 kD (Ku70) and 80 kD (Ku80) subunits. Ku is the regulatory component of the DNA-dependent protein kinase (DNA-PK) that has a catalytic subunit of ~460 kD (DNA-PKcs). In this study, the two polypeptides (Ku80/Ku70) of the human Ku were expressed in Xenopus oocytes in order to investigate their over-expression, sub-cellular localization, and functional interaction with the Xenopus DNA-PKcs. In vitro-transcribed mRNAs for Ku70 and Ku80 were obtained from the respective plasmid constructs. The exogenously expressed proteins from the injected mRNAs were immunoprecipitated using a specific anti-T7 Tag antibody. The T7 Tag epitope is present in the vector at the amino-terminus and is in-frame with the Ku cDNA sequences. While injected Ku70 mRNA translated to a full-length Ku70 polypeptide that translocated to the nucleus, injected Ku80 mRNA resulted in the expression of a truncated product that was retained in the cytoplasm. Although Ku80 mRNA was stable for a period of 18 h in the oocytes post-microinjection, the protein was only stabilized when co-expressed with Ku70, suggesting that Ku80 is susceptible to proteolytic degradation when not dimerized with Ku70. Furthermore, the immunocomplex was capable of phosphorylating the DNA-PK-specific substrate thereby indicating that the holoenzyme could functionally reconstitute in vivo in the oocytes by heterologous subunits thus demonstrating evolutionary conservation of the enzyme subunit structure and function among diverse species.


Ku DNA-PK Oocyte Xenopus Protein–protein interaction 



This study was supported by funds from the Institute of Molecular Medicine and Genetics and The Mason Trust (Medical College of Georgia).


  1. 1.
    de Vries E, van Driel W, Bergsma WG, Arnberg AC, van der Vliet PC (1989) HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA-multimeric protein complex. J Mol Biol 208:65–78CrossRefPubMedGoogle Scholar
  2. 2.
    Mimori T, Hardin JA (1986) Mechanism of interaction between Ku protein and DNA. J Biol Chem 261:10375–10379PubMedGoogle Scholar
  3. 3.
    Carter T, Vancurova I, Sun I, Lou W, DeLeon S (1990) A DNA-activated protein kinase from HeLa cell nuclei. Mol Cell Biol 10:6460–6471PubMedGoogle Scholar
  4. 4.
    Jackson SP, MacDonald JJ, Lees-Miller S, Tjian R (1990) GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell 63:155–165CrossRefPubMedGoogle Scholar
  5. 5.
    Satoh MS, Lindahl T (1992) Role of poly(ADP-ribose) formation in DNA repair. Nature 356:356–358CrossRefPubMedGoogle Scholar
  6. 6.
    Anderson CW, Lees-Miller SP (1992) The nuclear serine/threonine protein kinase DNA-PK. Crit Rev Eukaryot Gene Expr 2:283–314PubMedGoogle Scholar
  7. 7.
    Gottlieb TM, Jackson SP (1993) The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72:131–142CrossRefPubMedGoogle Scholar
  8. 8.
    Kanungo J, Calhoun B, Takeda Y, Hardin JA, Rasmussen H (1996) Gradual loss of a DNA-inducible protein kinase activity from the cytoplasmic extracts of Arbacia embryos. Biol Bull 191:281–282Google Scholar
  9. 9.
    Kanungo J, Cameron RS, Takeda Y, Hardin JA (1997) DNA-dependent protein phosphorylation activity in Xenopus is coupled to a Ku-like protein. Biol Bull 193:147–152CrossRefPubMedGoogle Scholar
  10. 10.
    Kanungo J, Walker JA, Woodrum D, Hardin JA, Rasmussen H (1996) A DNA-inducible kinase activity undergoes a change in cellular localization during development in Arbacia. Biol Bull 191:283–284Google Scholar
  11. 11.
    Brush GS, Anderson CW, Kelly TJ (1994) The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication. Proc Natl Acad Sci USA 91:12520–12524CrossRefPubMedGoogle Scholar
  12. 12.
    Tuteja N, Tuteja R, Ochem A, Taneja P, Huang NW, Simoncsits A, Susic S, Rahman K, Marusic L, Chen J et al (1994) Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen. EMBO J 13:4991–5001PubMedGoogle Scholar
  13. 13.
    Cao QP, Pitt S, Leszyk J, Baril EF (1994) DNA-dependent ATPase from HeLa cells is related to human Ku autoantigen. Biochemistry 33:8548–8557CrossRefPubMedGoogle Scholar
  14. 14.
    Vishwanatha JK, Baril EF (1990) Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase. Biochemistry 29:8753–8759CrossRefPubMedGoogle Scholar
  15. 15.
    Yagura T, Sumi K (1999) Molecular cloning and sequencing of cDNAs encoding homologues of human Ku70 and Ku80 autoantigen from Xenopus and their expression in various Xenopus tissues. Biochim Biophys Acta 1445:160–164PubMedGoogle Scholar
  16. 16.
    Labhart P (1997) mRNA encoding the catalytic subunit of DNA-dependent protein kinase is widely expressed in Xenopus cells. Gene 203:235–240CrossRefPubMedGoogle Scholar
  17. 17.
    Dumont JN (1972) Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol 136:153–179CrossRefPubMedGoogle Scholar
  18. 18.
    Gurdon JB (1976) Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal. J Embryol Exp Morphol 36:523–540PubMedGoogle Scholar
  19. 19.
    Mimori T, Ohosone Y, Hama N, Suwa A, Akizuki M, Homma M, Griffith AJ, Hardin JA (1990) Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome. Proc Natl Acad Sci USA 87:1777–1781CrossRefPubMedGoogle Scholar
  20. 20.
    Reeves WH, Sthoeger ZM (1989) Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. J Biol Chem 264:5047–5052PubMedGoogle Scholar
  21. 21.
    Suwa A, Hirakata M, Takeda Y, Jesch SA, Mimori T, Hardin JA (1994) DNA-dependent protein kinase (Ku protein–p350 complex) assembles on double-stranded DNA. Proc Natl Acad Sci USA 91:6904–6908CrossRefPubMedGoogle Scholar
  22. 22.
    Reeves WH (1985) Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera. J Exp Med 161:18–39CrossRefPubMedGoogle Scholar
  23. 23.
    Kanungo J (2008) DNase I-resistant DNA-dependent protein kinase activity in Xenopus oocytes. Mol Cell Biochem 309:33–40CrossRefPubMedGoogle Scholar
  24. 24.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  25. 25.
    Koike M, Ikuta T, Miyasaka T, Shiomi T (1999) Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal. Oncogene 18:7495–7505CrossRefPubMedGoogle Scholar
  26. 26.
    Paillard S, Strauss F (1993) Site-specific proteolytic cleavage of Ku protein bound to DNA. Proteins 15:330–337CrossRefPubMedGoogle Scholar
  27. 27.
    Wang J, Satoh M, Pierani A, Schmitt J, Chou CH, Stunnenberg HG, Roeder RG, Reeves WH (1994) Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers. J Cell Sci 107(Pt 11):3223–3233PubMedGoogle Scholar
  28. 28.
    Koike M, Shiomi T, Koike A (2000) Ku70 can translocate to the nucleus independent of Ku80 translocation and DNA-PK autophosphorylation. Biochem Biophys Res Commun 276:1105–1111CrossRefPubMedGoogle Scholar
  29. 29.
    Durant S, Karran P (2003) Vanillins—a novel family of DNA-PK inhibitors. Nucleic Acids Res 31:5501–5512CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  1. 1.Institute of Molecular Medicine and GeneticsMedical College of GeorgiaAugustaUSA
  2. 2.National Center for Toxicological Research, Food and Drug AdministrationJeffersonUSA

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