Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins.
The pocket domain of pRB is required for pRB to arrest the cell cycle. This domain was originally defined as the region of the protein that is necessary and sufficient for pRB's interaction with adenovirus E1A and simian virus s40 large T antigen. These oncoproteins, and other pRB-binding proteins that are encoded by a variety of plant and animal viruses, use a conserved LXCXE motif to interact with pRB. Similar sequences have been identified in multiple cellular pRB-binding proteins, suggesting that the viruses have evolved to target a highly conserved binding site of pRB that is critical for its function. Here we have constructed a panel of pRB mutants in which conserved amino acids that are predicted to make close contacts with an LXCXE peptide were altered. Despite the conservation of the LXCXE binding site throughout evolution, pRB mutants that lack this site are able to induce a cell cycle arrest in a pRB-deficient tumor cell line. This G(1) arrest is overcome by cyclin D-cdk4 complexes but is resistant to inactivation by E7. Consequently, mutants lacking the LXCXE binding site were able to induce a G(1) arrest in HeLa cells despite the expression of HPV-18 E7. pRB mutants lacking the LXCXE binding site are defective in binding to adenovirus E1A and human papillomavirus type 16 E7 protein but exhibit wild-type binding to E2F or DP, and they retain the ability to interact with CtIP and HDAC1, two transcriptional corepressors that contain LXCXE-like sequences. Consistent with these observations, the pRB mutants are able to actively repress transcription. These observations suggest that viral oncoproteins depend on the LXCXE-binding site of pRB for interaction to a far greater extent than cellular proteins that are critical for cell cycle arrest or transcriptional repression. Mutation of this binding site allows pRB to function as a cell cycle regulator while being resistant to inactivation by viral oncoproteins.
Dick
F A
FA
MGH Cancer Center, Charlestown, Massachusetts 02129, USA.
Sailhamer
E
E
Dyson
N J
NJ
D017500:Adenovirus E1A Proteins; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D017124:Conserved Sequence; D056741:Cyclin D; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D017500:Adenovirus E1A Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056741:Cyclin D; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; C059731:oncogene protein E7, Human papillomavirus type 16; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases