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.

No abstract

The reaction cycle of HECT domain ubiquitin ligases consists of three steps: 1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the ؊4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the ؊4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the ؊4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine ⑀-amino group of the substrate.The best characterized function of protein ubiquitination is as a recognition signal for the 26 S proteasome (1). Ubiquitin is covalently linked to substrate proteins via isopeptide bond linkages formed between the terminal carboxyl group of ubiquitin and ⑀-amino groups of lysine side chains of the substrate, or in some cases to the N-terminal ␣-amino group. Additional ubiquitin molecules can be conjugated sequentially at specific lysine residues of ubiquitin to form multi-ubiquitinated proteins, which are the principal substrate of the 26 S proteasome.Three groups of proteins cooperate in catalyzing ubiquitination: the E1 ubiquitin-activating enzyme, the E2 ubiquitinconjugating proteins (Ubc 1 proteins), and the E3 ubiquitin ligases (1). The E1 enzyme uses ATP to form a ubiquitinadenylate intermediate at the terminal carboxyl group of ubiquitin. A ubiquitin-thioester is then formed at the active site cysteine of the E1 enzyme, with release of AMP. The E1 enzyme transfers ubiquitin to the active site cysteine of the highly conserved family of E2 proteins, maintaining a ubiquitin-thioester linkage. The E3 ubiquitin-ligases interact with both the E2 proteins as well as with substrate proteins. Whereas several classes of E3 enzymes are now recognized (SCF, APC, CBC, RING, CHIP, and HECT E3s), they fall into two groups with respect to overall mechanism. The first group, which includes all classes except for the HECT E3s, are thought to function essentially as docking proteins, orienting the E2 and substrate so that ⑀-amino groups of substrate lysines can attack the E2-ubiquitin thioester, forming a ubiquitin-isopeptide bond. HECT E3s participate more directly in catalysis and are defined by a conserved C-terminal domain of ϳ350 amino aci...

No abstract