Proteolysis by calpains: a possible contribution to degradation of p53.
p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.
Pariat
M
M
Institut de Génétique Moléculaire, UMR 9942, CNRS, Montpellier, France.
Carillo
S
S
Molinari
M
M
Salvat
C
C
Debüssche
L
L
Bracco
L
L
Milner
J
J
Piechaczyk
M
M
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics; D002154:Calpain / Q000378:metabolism*; D002417:Cattle; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D016296:Mutagenesis; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D014158:Transcription, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D009097:Multienzyme Complexes; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; D000001:Calcimycin; C033668:calpastatin; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex