Fig. 2.

The phosphoacceptor sites serine-807 and serine-811 in the COOH-terminal region of Rb are required for cyclin D-mediated inhibition of Rb transcriptional repressor activity. To assay for active repression, Rb was fused to the DNA binding domain of Gal4 and coexpressed in Rb-null C33a cells, along with the pSVEC-G reporter containing Gal4 binding sites upstream of the SV40 enhancer. CAT activity from the reporter was measured with a phosphorimager. Rb, constructs containing wild-type Rb sequence (amino acids 379–928); RbΔ2, serine-807 and serine-811 have been converted to alanine to prevent phosphorylation of these sites. Cyclin D was coexpressed where indicated. Transfection of 0.5 μg of each expression vector (or vector control) and 0.2 μg of reporter was performed using the calcium phosphate method. Note that cyclin D blocks most of the transcriptional repression by Rb, but this effect is lost with mutation of serine-807/811.