Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition

Abstract

The e ect of a calpain-selective cell permeant inhibitor, benzyloxycarbonyl Leu-Leu-Tyr diazomethylketone (ZLLY-CHN 2 ), on the serum-stimulated growth of WI-38 human ®broblasts has been investigated. Only cell permeant protease inhibitors with activity against calpains prevented progression into S-phase. Protein blotting experiments indicated that p53 immunoreactivity increased in late G 1 cells treated with ZLLY-CHN 2 . The content of p21 Waf1/Cip1 CDK inhibitor also increased, providing a mechanism for the observed failure to enter S-phase. Further studies indicated that p53 could be degraded by a ZLLY-CHN 2 -sensitive protease immediately prior to S-phase, but that proteolysis did not occur after this critical time point. Chelation of extracellular Ca 2+ by addition of EGTA inhibited the p53 degradation. Consistent with proteolysis of p53 in late G 1 phase, m-calpain immunoreactivity transiently accumulated in cell nuclei at this time. ZLLY-CHN 2 did not appear to increase p53 mRNA in WI-38 cells. Puri®ed mcalpain required only 1 to 3 mM Ca 2+ to proteolyze p53 in WI-38 cell lysates. These results indicate that ZLLY-CHN 2 inhibits progression of WI-38 cells into S-phase by inactivating a calpain-like protease that is responsible for proteolysis of constitutively expressed p53 in late G 1 .

Regulation of BRCA1 by protein degradation

“…Induction of wild type p53 in MCF7 is a positive control for PS-341 activity resistant to ALLN (Figures 2a and 3c). Accumulation of wild type p53, previously shown to be sensitive to calpain as well as proteasome inhibition (Gonen et al, 1997;Kubbutat and Vousden, 1997;Pariat et al, 1997;Zhang et al, 1997), was demonstrated in MCF10 cells at the concentrations/incubation times used of the calpain inhibitors calpastatin peptide and PD150606 (Figure 4b). …”

A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development

“…Involvement of a calpain-like activity in G 1 3 S transitions has been suggested in other systems on the basis of indirect evidence (14,15). An S-phase delay has also been observed in P. berghei parasites lacking 1 of 2 eEF1a genes, but in that case, the total cell cycle was prolonged (16).…”

Activation and activities of the p53 tumour suppressor protein

“…Interference with the p53-JNK association reduced p53 ubiquitination and increased p53 half-life, suggesting that JNK might target p53 to ubiquitination and degradation (Fuchs et al, 1998). In addition to degradation by the proteasome, p53 has been shown to be cleaved by another protease, calpain, which may also serve to regulate p53 Pariat et al, 1997;Zhang et al, 1997;Atencio et al, 2000).…”