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