Multiple Lysine Mutations in the C-Terminal Domain of p53 Interfere with MDM2-Dependent Protein Degradation and Ubiquitination

Abstract

To investigate the effect of mutations in the p53 C-terminal domain on MDM2-mediated degradation, we introduced single and multiple point mutations into a human p53 cDNA at four putative acetylation sites (amino acid residues 372, 373, 381, and 382). Substitution of all four lysine residues by alanines (the A4 mutant) and single lysine-to-alanine substitutions were functional in sequence-specific DNA binding and transactivation; however, the A4 mutant protein was resistant to MDM2-mediated degradation, whereas the single lysine substitutions were not. Although the A4 mutant protein and the single lysine substitutions both bound MDM2 reasonably well, the single lysine substitutions underwent normal MDM2-dependent ubiquitination, whereas the A4 protein was inefficiently ubiquitinated. In addition, the A4 mutant protein was found in the cytoplasm as well as in the nucleus of a subpopulation of cells, unlike wild-type p53, which is mostly nuclear. The partially cytoplasmic distribution of A4 mutant protein was not due to a defect in nuclear import because inhibition of nuclear export by leptomycin B resulted in nuclear accumulation of the protein. Taken together, the data suggest that mutations in the putative acetylation sites of the p53 C-terminal domain interfere with ubiquitination, thereby regulating p53 degradation.

Hyperubiquitylation of wild-type p53 contributes to cytoplasmic sequestration in neuroblastoma

“…16,17 In line with this, we previously reported a covalent modification of the p53 C terminus in NB cell lines, as indicated by NB-specific masking of the modification-sensitive epitope of monoclonal p53 antibody PAb421 (residues 372-382) and a dramatic shift in immune-isoelectrofocussing in the p53 isoform profile to a ladder of hyperacidic forms (Supplementary Figure 2). 10 Given the current data, these isoforms can now be identified as the hyperubiquitylated p53 species described here.…”

“…Hdm2, the major E3 ligase which itself is a target gene of p53, ubiquitylates p53, thus constituting a negative-feedback loop. 16,17 Importantly, this ubiquitylation of p53 is required for its nuclear export via the export receptor CRM 1. [18][19][20] Furthermore, the fate of p53 depends on the type of ubiquitylation.…”

Multiple lysine mutations in the C-terminus of p53 make it resistant to degradation mediated by MDM2 but not by human papillomavirus E6 and induce growth inhibition in MDM2-overexpressing cells

“…It has been published that multiple C-terminal lysine residues target p53 its degradation (Rodriguez et al, 2000). We have previously shown that A4 is less ubiquitinated than is wild-type p53 and is therefore not degraded e ciently by MDM2 mediated degradation (Nakamura et al, 2000). The possibility exists that the four lysine residues that have been changed to alanine residues in A4 are potential sites for ubiquitination and are required for proteosome-mediated degradation.…”

“…Actin was used as a loading control. (b) Nuclear extractions and electromobility shift assays were performed as previously described (Nakamura et al, 2000) using the p53 consensus DNA-binding sequence (p53CON) GGACATGCC-CGGGCA TGTCC and either wild-type p53 or A4. The following antibodies were used for supershift assay: Ab-1, PAb 421, Ab-2, Pab-1801, and Ab-6, DO1 (Oncogene Sciences).…”

Proteomic techniques to probe the ubiquitin landscape

“…These can have different signaling properties, as has been shown for IKKγ [64], or can act simply as alternative sites that trigger the same event [65][66][67].…”