Regulation of specific DNA binding by p53: evidence for a role for O-glycosylation and charged residues at the carboxy-terminus.
The carboxy-terminus of p53 contains a basic region which represses DNA binding, and this repression can be relieved by PAb421, an antibody against the basic region. The EB-1 human cell line contains wild type p53 protein which fails to express the PAb421 epitope and is highly active both in biological assays and in DNA binding assays. We show by wheat germ agglutinin chromatography and galactosyl-transferase labelling that this p53 is O-glycosylated, and that at least one of the sugar residues masks the PAb421 epitope, as demonstrated by recovery of reactivity with PAb421 after digestion of Western blots of EB-1 cell extract with hexosaminidase. A minor population of p53 molecules in EB-1 cells lacks the modification, and there is a correlation between the ability to bind DNA with high affinity and masking of the PAb421 epitope. We also show that strongly positively charged peptides, including short peptides from the basic region of p53, can derepress DNA binding, probably by disruption of an intramolecular interaction involving the basic region. We propose that any intervention which prevents this intramolecular interaction, including addition of bulky residues such as sugar groups, can activate DNA binding by p53.
Shaw
P
P
Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland.
Freeman
J
J
Bovey
R
R
Iggo
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D000939:Epitopes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA