Role of LXCXE motif-dependent interactions in the activity of the retinoblastoma protein.
Cell cycle control by pRb requires the integrity of the pocket domain, which is a region necessary for interactions with a variety of proteins, including E2F and LXCXE-motif containing proteins. Through knowledge of the crystal structure of pRb we have prepared a panel of pRb mutant derivatives in which a cluster of lysine residues that demark the LXCXE peptide binding domain were systematically mutated. One of the mutant derivatives, Rb6A, exhibits significantly reduced LXCXE-dependent interactions with HPV E7, cyclinD1 and HDAC2, but retained LXCXE-independent binding to E2F. Consistent with these results, Rb6A could down-regulate E2F-1-dependent activation of different E2F responsive promoters, but was compromised in Rb-dependent repression. Most importantly, Rb6A retained wild-type growth arrest activity, and colony forming activity similar to wild-type pRb. It is compatible with these results that directly targeting HDAC2 to E2F responsive promoters as an E2F/HDAC hybrid protein failed to effect cell cycle arrest. These results suggest that LXCXE-dependent interactions are not essential for pRb to exert growth arrest.
Chan
H M
HM
Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, UK.
Smith
L
L
La Thangue
N B
NB
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002455:Cell Division; D019938:Cyclin D1 / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D000939:Epitopes; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D000939:Epitopes; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D019938:Cyclin D1; D005982:Glutathione Transferase; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases; D008239:Lysine