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 approximately 460 kD (DNA-PK(cs)). 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-PK(cs). 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.
Kanungo
Jyotshnabala
J
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA. jyotshnabala.kanungo@fda.hhs.gov
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D008845:Microinjections; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D051747:DNA-Activated Protein Kinase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen