A Genetic Approach to Mapping the p53 Binding Site in the MDM2 Protein

Table 2 Summary of mutagenesis and binding assays with MDM2

Mutant	Derivation	Ab-reactivity	p53 Binding	In Vivo Regulation
wt		+	++	+
C2A	S.D. mutagenesis	ND	++	ND
G58D	Yeast screen	+	−	ND
G58A	S.D. mutagenesis	ND	−	−
D68A	S.D. mutagenesis	ND	−	—
E69A	S.D. mutagenesis	ND	+	ND
V75A	S.D. mutagenesis	ND	−	−
C77Y	Yeast screen	+	−	ND
C77A	S.D. mutagenesis	ND	—	ND

Eight MDM2 mutant proteins were made by site-directed mutagenesis and tested for the ability to bind to GST::p53 (1–82) (Figs. 2A and 4 and data not shown). Two retained full or partial binding ability (C2A and E69A), while the other six resulted in proteins that could not detectably interact with p53. Ab-reactivity is shown in Fig. 2B and in vivo regulation in Fig. 5. S.D., site-directed; ND, not determined.

ISSN: 1528-3658