Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Cell Cycle
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Cell Cycle Proteins*
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Cell Division
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Cyclin D1 / metabolism
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DNA Mutational Analysis
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DNA-Binding Proteins*
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Down-Regulation
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E2F Transcription Factors
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E2F1 Transcription Factor
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Epitopes
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Flow Cytometry
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Glutathione Transferase / metabolism
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HeLa Cells
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Histone Deacetylase 2
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Histone Deacetylases / metabolism
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Humans
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Immunoblotting
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Lysine / chemistry
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Lysine / metabolism
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Models, Molecular
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Molecular Sequence Data
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Mutation
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Peptides / metabolism
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Plasmids / metabolism
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Precipitin Tests
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Promoter Regions, Genetic
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Protein Binding
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Protein Structure, Tertiary
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Proto-Oncogene Proteins c-myc / metabolism
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Recombinant Fusion Proteins / metabolism
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Repressor Proteins*
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Retinoblastoma Protein / chemistry*
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Retinoblastoma Protein / metabolism
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Sequence Homology, Amino Acid
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Transcription Factors / metabolism
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Transcription, Genetic
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Transfection
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Tumor Cells, Cultured
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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E2F Transcription Factors
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E2F1 Transcription Factor
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E2F1 protein, human
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Epitopes
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Peptides
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Proto-Oncogene Proteins c-myc
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Recombinant Fusion Proteins
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Repressor Proteins
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Retinoblastoma Protein
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Transcription Factors
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Cyclin D1
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Glutathione Transferase
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Histone Deacetylase 2
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Histone Deacetylases
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Lysine