Three regions of the pRB pocket domain affect its inactivation by human papillomavirus E7 proteins.
A critical event in papillomavirus transformation of human cells is the inactivation of pRB by the E7 protein. E7, like many other viral oncoproteins, possesses a well-characterized LXCXE peptide motif that interacts with the pocket domain of pRB. Disruption of the LXCXE-binding cleft on pRB renders it resistant to E7 binding and inactivation. Such binding cleft mutants of pRB are capable of inducing a G(1) arrest in the human papillomavirus 18-transformed HeLa cell line. We show here that the efficient inactivation of pRB in HeLa cells does not simply depend on the integrity of the LXCXE-binding cleft. Multiple site-directed mutants that alter conserved surfaces of the pRB pocket domain cause HeLa cells to accumulate in G(1). We divide these mutants into two classes: those that can be bound by E7 and those that cannot. The E7 interacting mutants include changes in conserved residues that lie in a groove between the A and B halves of the pocket. Surprisingly, none of these mutants show a clear defect in any of the known mechanisms for pRB inactivation by E7. Analysis of mutants that are compromised for E7 binding reveals that this interaction depends on both the LXCXE-binding cleft and on a conserved group of lysines adjacent to the cleft. These basic amino acids on pRB define a discrete interaction point with E7. These residues most likely form ionic interactions with conserved acidic amino acids on E7 since a stable pRB/E7 interaction was restored when the lysine residues on pRB and the acidic residues on E7 were interchanged.
Dick
Frederick A
FA
Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA.
Dyson
Nicholas J
NJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016193:G1 Phase; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D027383:Papillomaviridae / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D009856:Oncogene Proteins, Viral; D016160:Retinoblastoma Protein