PROTEOLYSIS BY CALPAINS - A POSSIBLE CONTRIBUTION TO DEGRADATION OF P53

p53 is a short-lived transcription factor that is frequently mutated i n 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 micro calpain, which are abundant and ubiquitous cytoplasmic proteases. Degr adation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility t o calpains, some of them being largely resistant to degradation and ot hers being more sensitive. This result suggests that the different mut ants tested here adopt slightly different conformations to which calpa ins are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by usin g the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, ac tivation 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 calpast atin expression vectors resulted in an increase in p53-dependent trans cription activity. Taken together, these data support the idea that ca lpains may also contribute to the regulation of wild-type p53 protein levels in vivo.