Characterization of two peptide epitopes on Mdm2 oncoprotein that affect p53 degradation.
Phosphorylation of Mdm2, in response to DNA damage, resulted in prevention of p53 degradation in the cytoplasm as well as reduction of its binding with monoclonal antibody (mAb) 2A10. Using a 15-mer phage-peptide library, we identified two 2A10-epitopes on human Mdm2 (hdm2): at positions 255-266 (LDSEDYSLSEEG) and 389-400 (QESDDYSQPSTS). Synthetic peptides corresponding to the above sites, inhibit the binding of mAb2A10 to Mdm2 with high (4.5 x 10(-9)M) and moderate affinity (1.1 x 10(-7)M), respectively. Phospho-derivatives of these peptides, and of single human Mdm2 mutations S260D or S395D resulted in a considerable reduction in their binding with mAb2A10. These results provide a molecular explanation for the observation that reactivity of Mdm2 with mAb2A10 is inhibited by phosphorylation.
Balass
M
M
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. moshe.balass@weizmann.ac.il
Kalef
E
E
Maya
R
R
Wilder
S
S
Oren
M
M
Katchalski-Katzir
E
E
D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D018604:Epitope Mapping; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D014443:Tyrosine; D012694:Serine; D005982:Glutathione Transferase