Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase δ (p125)

Nancy A. Krucher, Adam Zygmunt, Nayef Mazloum, Sama Tamrakar, John W. Ludlow, Marietta Y.W.T. Lee

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8 Citations (Scopus)

Abstract

The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, differentiation, and apoptosis. Several pRb binding proteins complex with pRb through an amino acid sequence called the LXCXE motif. The catalytic subunit of DNA polymerase δ (p125) contains a LXCXE motif. To further study the biochemical function of this polymerase, we sought to determine if p125 interacts with pRb. Experiments using GST-pRb fusion proteins showed that p125 from breast epithelial (MCF10A) cell extracts associates with pRb. In addition, GST-p125 fusion proteins bound pRb from the same cell extracts. The pRb that associated with GST-p125 was largely unphosphorylated. Coimmunoprecipitation experiments using cell cycle synchronized cells revealed that p125 and pRb form a complex predominantly during G1 phase, the phase during which pRb is mostly unphosphorylated. In vitro phosphorylation of GST-pRb by the cyclin dependent kinases reduced the ability of p125 to associate with GST-pRb. Addition of the LXCXE containing protein SV40 large T antigen to GST-pRb blocks the ability of p125 to associate with pRb, suggesting that it may be through a LXCXE sequence by which p125 interacts with pRb. Finally, in vitro polymerase assays demonstrate that GST-pRb fusion protein stimulates DNA polymerase δ activity.

Original languageEnglish
Pages (from-to)5464-5470
Number of pages7
JournalOncogene
Volume19
Issue number48
DOIs
Publication statusPublished - 16 Nov 2000

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Keywords

  • Cell cycle
  • Cyclin dependent kinase
  • DNA polymerase δ
  • Retinoblastoma

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cancer Research

Cite this

Krucher, N. A., Zygmunt, A., Mazloum, N., Tamrakar, S., Ludlow, J. W., & Lee, M. Y. W. T. (2000). Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase δ (p125). Oncogene, 19(48), 5464-5470. https://doi.org/10.1038/sj.onc.1203930