Phosphorylated retinoblastoma protein stimulates DNA polymerase α

• Published in: Oncogene Vol. 15; no. 20; pp. 2483 - 2492
• Main Authors: TAKEMURA, M, KITAGAWA, T, IZUTA, S, WASA, J, TAKAI, A, AKIYAMA, T, YOSHIDA, S
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing 13.11.1997; Nature Publishing Group
• Subjects: Biological and medical sciences; Burkitt Lymphoma - pathology; Burkitt's lymphoma; Calf thymus; Cancer; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Chromatography, Affinity; Cyclin E; Cyclin E - metabolism; Cyclin-dependent kinase; Cyclin-dependent kinase 2; Cyclin-dependent kinases; Deoxyribonucleic acid; Dephosphorylation; DNA; DNA biosynthesis; DNA polymerase; DNA Polymerase I - immunology; DNA Polymerase I - metabolism; DNA primase; DNA Replication; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - metabolism; Enzyme Activation - drug effects; Fundamental and applied biological sciences. Psychology; Humans; Immunoprecipitation; Insect cells; Molecular and cellular biology; Neoplasm Proteins - metabolism; Phosphoprotein phosphatase; Phosphoprotein Phosphatases - metabolism; Phosphorylation; Primase; Protein phosphatase; Protein Phosphatase 2; Protein Processing, Post-Translational; Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - pharmacology; Retina; Retinoblastoma; Retinoblastoma protein; Retinoblastoma Protein - chemistry; Retinoblastoma Protein - pharmacology; Tumors; Human; Phosphorylation; Enzyme; Transferases; Retinoblastoma protein; Molecular interaction; DNA synthesis; Nucleotidyltransferases; Regulation(control); Cell line; Molecular complex; DNA-directed DNA polymerase; Tumor suppressor gene
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/sj.onc.1201431

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.