Fig. 7.
The p53-binding region of HdmX is not required for HdmX to stimulate Hdm2-mediated p53 ubiquitination. (A) Schematic representation of the structure of Hdm2 and HdmX. bd, binding domain. (B) Coomassie stain of the various GST fusion proteins expressed in bacteria. fl, full-length protein; D N101, an Hdm2 or HdmX mutant, of which the N-terminal 101 amino acid residues of Hdm2 or HdmX were deleted. C463A and C464A, the respective cysteine residues of HdmX and Hdm2, were replaced by alanine. (C and D) In vitro ubiquitination of p53 in the presence of different amounts of different forms of Hdm2 and HdmX as indicated. The running positions of the nonmodified form and of the ubiquitinated forms of p53 are indicated by an arrowhead and an asterisk, respectively. (E) In vitro ubiquitination of a p53 mutant (D N43; deletion of the N-terminal 43-aa residues, which contain the binding site for Hdm2 and HdmX) in the presence of different amounts of full-length Hdm2 and HdmX as indicated. The running positions of the nonmodified form and of a potentially monoubiquitinated form of D N43 are indicated by an arrowhead and an asterisk, respectively. Amounts of Hdm2 and HdmX used are indicated in 10?1 m g.