Ubiquitination of p53 at multiple sites in the DNA-binding domain.
The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.
Chan
Wan Mui
WM
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
Mak
Man Chi
MC
Fung
Tsz Kan
TK
Lau
Anita
A
Siu
Wai Yi
WY
Poon
Randy Y C
RY
D000086042:3C Viral Proteases; D001665:Binding Sites; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006801:Humans; D008239:Lysine; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D015533:Transcriptional Activation / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; D014764:Viral Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; D008239:Lysine