| Research Abstract |
The purpose of this project is to approach the mechanism of transition from G1 to S pahse in cell cycle from the viewpoint of protein-protein interaction. We have observed that human retinoblastoma (Rb) protein, a product of representative tumor suppressor gene, suppress the DNA polymerase alpha-stimulating effect of the simian virus 40 T-antigen, by a protein-protein interaction. This result may imply that an underphosphorylated Rb protein suppress the initiation of DNA replication in G1 phase. In the present study, we have focused our work on the possible role of the hyperphosphorylated form of Rb protein, which persists in cell nuclei throughout the S-phase. Rb protein, immunopurified from an extract of recombinant baculovirus-infected cells, stimulated 10-to 100-fold the avtivity of DNA polymerase alpha from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and underphosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase alpha more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I.By immunoprecipitation using anti-DNA polymerase alpha antibody, Rb protein in nuclear extract of Raji cells was coprecipitated with DNA polymerase alpha. This result indicates that DNA polymerase alpha exists as a complex containing phosphorylated Rb protein in cells. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication (Takemura et al.Oncogene 15,2843,1997)
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