Figure 3.

Functional interaction between MDM2 and pRB. (A) pRB restores Sp1 activity in cells with amplified MDM2. NIH 3T3 cells and a hybrid with amplified MDM2 (MDM2+) were transfected with the synthetic Sp1-dependent promoter construct (Sp1)₃BCAT and increasing amounts of CMV-RB. CAT activity is expressed as cpm above background. Values represent the average of duplicate dishes, with <20% difference between duplicates. (B) MDM2 inhibits Sp1 activity in the absence of pRB. SAOS2 cells were transfected with the Sp1-dependent promoter construct (Sp1)₃BCAT, the Sp1 expression vector CMVSp1, and increasing amounts of either CMV-MDM2 or CMV-MDMX. CAT activity was measured as in Fig. 1 and is expressed as percentage of activated levels. (C) In vitro-translated MDM2 binds to GST-RB and GST-Sp1. Radiolabeled, full-length MDM2 and the two MDM2 deletion mutants 1–294 and 1–220 were incubated with GST-Sp1 (zinc + D domain) or GST-RB (pRB amino acids 379–928) and subjected to a GST-pulldown assay. (D) Schematic representation of additional MDM2 deletion mutants were analyzed using the GST pulldown assay. (E) In vitro-translated MDMX binds to GST-RB and to GST-Sp1. Radiolabeled MDMX was incubated with GST-RB and GST-Sp1 and subjected to a GST-pulldown assay.