Interaction of the retinoblastoma protein (pRb) with the catalytic subunitof DNA polymerase delta (p125)

The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, differentiation, and apoptosi s, Several pRb binding proteins complex with pRb through an amino acid sequ ence called the LXCXE motif, The catalytic subunit of DNA polymerase delta (p125) contains a LXCXE motif, To further study the biochemical function of this polymerase, we sought to determine if p125 interacts with pRb, Experi ments using GST-pRb fusion proteins showed that p125 from breast epithelial (MCF10A) cell extracts associates with pRb. In addition, GST-p125 fusion p roteins bound pRb from the same cell extracts. The pRb that associated with GST-p125 was largely unphosphorylated, Coimmunoprecipitation experiments u sing cell cycle synchronized cells revealed that p125 and pRb form a comple x predominantly during G(1) phase, the phase during which pRb is mostly unp hosphorylated. 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 abi lity of p125 to associate with pRb, suggesting that it may be through a LXC XE sequence by which p125 interacts with pRb, Finally, in vitro polymerase assays demonstrate that GST-pRb fusion protein stimulates DNA polymerase de lta activity.