Phosphorylated retinoblastoma protein stimulates DNA polymerase α

Abstract

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10 – 100-fold the activity of DNA polymerase α from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated 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 α more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase α. 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 α antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase α. This result indicates that DNA polymerase α exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase α specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase α trapped to anti-DNA polymerase α antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication.