Development of an in vitro assay for the proteolytic processing of the CDP/Cux transcription factor.
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 posttranslational 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.
Hebert
Sherry
S
Molecular Oncology Group, McGill University Health Center, Montreal, Quebec H3A1A1, Canada.
Bérubé
Ginette
G
Nepvue
Alain
A
D000818:Animals; D002457:Cell Extracts; D016193:G1 Phase; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016196:S Phase
D002457:Cell Extracts; C000624101:Cux1 protein, mouse; D018398:Homeodomain Proteins; D007976:Leupeptins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D007930:Leucine; C108192:aloxistatin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde