Nuclear binding of purified retinoblastoma gene product is determined by cell cycle-regulated phosphorylation.
The retinoblastoma tumor suppressor gene product (pRb) is a nuclear protein subject to cell cycle-regulated hyperphosphorylation. I constructed a recombinant vaccinia virus vector that expresses both the underphosphorylated and hyperphosphorylated forms of pRb and purified the recombinant protein by using immunoaffinity chromatography directed toward a synthetic carboxy-terminal epitope. To investigate the hypothesis that hyperphosphorylation of pRb is a means of controlling its growth-regulating activity, I tested purified pRb for the ability to be reincorporated into pRb-deficient nuclei in vitro. The underphosphorylated form of pRb efficiently reassociated with nuclei, but the hyperphosphorylated form remained soluble in this assay. Nuclear binding of pRb was enhanced by phosphatase treatment and reduced by phosphorylation of pRb effected by using a preparation of the cell cycle-regulatory kinase p34cdc2. Mutant-encoded proteins with altered E1A-binding domains failed to bind to nuclei. Pretreatment of target nuclei with nucleases and high-salt extraction did not alter the specificity of binding for underphosphorylated pRb. These observations demonstrate that hyperphosphorylation of pRb can regulate its interaction with nuclei, supporting the hypothesis that hyperphosphorylation controls the growth-regulatory activities of pRb. Further, at least one target of pRb binding appears to be an integral component of the nuclear envelope.
Templeton
D J
DJ
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D016147:Genes, Tumor Suppressor / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics
D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D016203:CDC2 Protein Kinase; D010744:Phosphoric Monoester Hydrolases