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Development of an in Vitro Assay for the Proteolytic Processing of the CDP/Cux Transcription Factor

  • Hebert, Sherry (Molecular Oncology Group, McGill University Health Center) ;
  • Berube, Ginette (Molecular Oncology Group, McGill University Health Center) ;
  • Nepveu, Alain (Molecular Oncology Group, McGill University Health Center)
  • Received : 2003.01.07
  • Accepted : 2003.02.27
  • Published : 2003.07.31

Abstract

The CDP/Cux transcription factor was previously shown to be proteolytically processed at the G1/S transition. In view of characterizing and eventually identifying the protease responsible for CDP/Cux processing, we have established an in vitro proteolytic processing assay. CDP/Cux recombinant proteins expressed in mammalian or bacterial cells were efficiently processed in vitro using as a source of protease either whole cell extracts, the nuclear or the cytoplasmic fraction. Processing was found to take place optimally at a lower pH, to be insensitive to variations in salt concentration, and to be inhibited by the protease inhibitors MG132 and E64D. Interestingly, the bacterially-produced substrate was more efficiently processed than the substrate purified from mammalian cells. Moreover, processing in vitro was more efficient when CDP/Cux substrates were purified from populations of cells enriched in the S phase than in the G1 phase of the cell cycle. Altogether, these results suggest that post-translational modifications of CDP/Cux in mammalian cells inhibits processing and contributes to the cell cycle-dependent regulation of processing. The in vitro processing assay described in this study will provide a useful tool for the purification and identification of the protease responsible for the processing of CDP/Cux.

Keywords

CDP/Cux;Proteolytic processing;In vitro assay;Cell cycle

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