Fig. 1

Schematic summary of the constructed p130 mutants based on the p130wt. a Schematic of the 1139 amino acid human p130 protein, showing the A and B domains of the pocket. Also indicated is the region of the B domain conserved between RB and p130 which was mutated in p130mE7. In p130mE7, amino acids Y1009 and N1010 were changed to alanines. b p130wt has two pocket domains which are A and B for viral oncoproteins binding and cyclin dependent kinase phosphorylation. The p130mE7 mutant was designed based on the work of Dick and Dyson (2002), who showed that a surface of the pRB B pocket was critical for binding proteins containing the L-X-C-X-E motif. This region of the pRB B pocket is partially conserved in p130, and two critical conserved amino acids (leucine and cysteine) in p130 were replaced with alanine by in vitro mutagenesis. The p130PM22 mutant has been mutated at the 22 CDK phosphorylation sites by replacing the phosphorylated serine and threonine with alanine (Farkas et al., 2002). The p130mE7/PM22 protein was mutated at both the HPV E7 binding and phosphorylation sites. All p130 mutants and a control wild-type p130 were tagged with HA to differentiate between endogenous and ectopically expressed p130