IMMUNOCHEMICAL ANALYSIS OF THE INTERACTION OF P53 WITH MDM2 - FINE MAPPING OF THE MDM2 BINDING-SITE ON P53 USING SYNTHETIC PEPTIDES

The function of p53 is modulated by binding to a number of cellular an d viral proteins, such as MDM2 and SV40 large T antigen. An initial im munochemical characterization of the p53-MDM2 complex in a rat fibrobl ast cell line (Clone 6) suggested that the anti-p53 monoclonal antibod y Bp53-19 failed to immunoprecipitate the complex, and only recognized a fraction of the available p53 protein. Following the recent identif ication of the Bp53-19 epitope at the N-terminal end of p53, in the vi cinity of where MDM2 protein was known to bind, we investigated the po ssibility that Bp53-19 might identify a region of p53 that interacts w ith MDM2 protein. MDM2 was found to bind with great specificity to sho rt synthetic peptides derived from the N-terminus of p53. Several p53 synthetic peptides libraries, and an alanine substitution series at th e optimal binding site, were used to establish the MDM2 binding site, in fine detail, to the sequence TFSGLW (aa 18-23) in mouse and TFSDLW in man (aa 18-23). The key residues required for MDM2 binding are almo st identical to those required for the monoclonal antibody Bp53-19 to bind and this region of p53 is recognised by many other anti-p53 antib odies.