Abstract
The product of the retinoblastoma susceptibility gene, pRB, is a nuclear phosphoprotein that controls cell growth by binding to and suppressing the activities of transcription factors such as the E2F family. Transactivation activity is inhibited when E2F is bound to hypophosphorylated pRB and released when pRB is phosphorylated by cyclin-dependent kinases (CDKs). To determine which of 16 potential CDK phosphorylation sites regulated the pRB-E2F interaction, mutant pRB proteins produced by site-directed mutagenesis were tested for the ability to suppress E2F-mediated transcription in a reporter chloramphenicol acetyltransferase assay. Surprisingly, no one CDK site regulated the interaction of pRB with E2F when E2F was bound to DNA. Instead, disruption of transcriptional repression resulted from accumulation of phosphate groups on the RB molecule.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Binding Sites / genetics
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CDC2-CDC28 Kinases*
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COS Cells
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Carrier Proteins*
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Cell Cycle Proteins*
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Cyclin E / metabolism
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases / metabolism
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DNA-Binding Proteins / genetics
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E2F Transcription Factors
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Gene Expression Regulation / genetics
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Genes, Reporter / genetics
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Mice
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Mutagenesis, Site-Directed / genetics
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Mutation / genetics
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Phosphoproteins / genetics
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Phosphoproteins / metabolism
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Phosphorylation
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Protein Serine-Threonine Kinases / metabolism
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Repressor Proteins / genetics
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Retinoblastoma Protein / genetics*
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Retinoblastoma Protein / metabolism
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Retinoblastoma-Binding Protein 1
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Transcription Factor DP1
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Transcription Factors / genetics*
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Transcription Factors / metabolism
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Tumor Cells, Cultured
Substances
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Arid4a protein, mouse
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Carrier Proteins
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Cell Cycle Proteins
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Cyclin E
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DNA-Binding Proteins
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E2F Transcription Factors
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Phosphoproteins
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Repressor Proteins
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Retinoblastoma Protein
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Retinoblastoma-Binding Protein 1
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Transcription Factor DP1
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Transcription Factors
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Protein Serine-Threonine Kinases
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CDC2-CDC28 Kinases
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Cdk2 protein, mouse
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases