Human
retinoblastoma (
Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10-100-fold the activity 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 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 alpha 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 alpha. 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 co-precipitated with
DNA polymerase alpha. This result indicates that
DNA polymerase alpha exists as a complex containing phosphorylated
Rb protein in cells.
DNA polymerase alpha specifically bound to a purified
Rb protein-immobilized
Sepharose column.
Rb protein also bound to
DNA polymerase alpha trapped to anti-
DNA polymerase alpha 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.