Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase δ (p125)
Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase δ (p125)
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DOI：
10.1038/sj.onc.1203930
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发表时间：
2000-11-16
期刊：
影响因子：
8
通讯作者：
Lee, MYWT
中科院分区：
文献类型：
--
作者：
Krucher, NA;Zygmunt, A;Lee, MYWT
视网膜母细胞瘤基因产物（retinoblastoma gene product，pRb）与许多细胞蛋白相互作用，在细胞分裂、分化和凋亡的控制中起作用。几种pRb结合蛋白通过称为LXCXE基序的氨基酸序列与pRb复合，DNA聚合酶δ（p125）的催化亚基含有LXCXE基序，为了进一步研究该聚合酶的生化功能，我们试图确定p125是否与pRb相互作用。使用GST-pRb融合蛋白的实验表明，来自乳腺上皮（MCF 10A）细胞提取物的p125与pRb缔合。此外，GST-p125融合蛋白结合pRb从相同的细胞提取物。GST-p125结合的pRb大部分未被磷酸化。用细胞周期同步化的细胞进行的免疫共沉淀实验表明，p125和pRb主要在G（1）期形成复合物，在此期间，pRb大部分未被磷酸化。体外细胞周期蛋白依赖性激酶对GST-pRb的磷酸化降低了p125与GST-pRb结合的能力，将含有蛋白SV 40大T抗原的LXCXE加入GST-pRb中阻断了p125与pRb结合的能力，这表明p125可能通过LXCXE序列与pRb相互作用。体外聚合酶测定证明GST-pRb融合蛋白刺激DNA聚合酶δ活性。
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 delta (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 G(1) 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 delta activity.