Gao Yan, Zhou Yajing, Xie Bin, Zhang Sufang, Rahmeh Amal, Huang Hua-Shan, Lee Marietta Y W T, Lee Ernest Y C
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Biochemistry. 2008 Oct 28;47(43):11367-76. doi: 10.1021/bi801122t. Epub 2008 Oct 1.
Protein phosphatase-1 (PP1) is a Ser/Thr protein phosphatase that participates in the phosphorylation/dephosphorylation regulation of a diverse range of cellular processes. The PP1 catalytic subunit (PP1) achieves this by its ability to interact with many targeting subunits such that PP1 activity is thereby specified against phosphoprotein substrates in the microvicinity of its targeting subunit. DNA polymerase delta (Pol delta) is a key enzyme in mammalian chromosomal replication. It consists of four subunits, p125, p50, p68, and p12. We identify p68 as a novel PP1 targeting subunit. PP1 was shown to associate with human DNA polymerase delta by affinity chromatography and coimmunoprecipitation assays from mammalian cell lysates and in vitro by pull-down assays. The binding domain for PP1 was identified as the sequence KRVAL, a variant of the canonical RVxF PP1 binding motif. These studies provide the first evidence for the targeting of PP1 to DNA polymerase delta. We also show that CK2 phosphorylates the Pol delta p125, p68, and p12 subunits and that these phosphorylated subunits are substrates for PP1. These findings identify a new role for p68 as a PP1 targeting subunit that implicates PP1 in the dephosphorylation of Pol delta. Our findings also show that CK2 is a strong candidate for the protein kinase involved in the in vivo phosphorylation of p68.
蛋白磷酸酶-1（PP1）是一种丝氨酸/苏氨酸蛋白磷酸酶，参与多种细胞过程的磷酸化/去磷酸化调节。PP1催化亚基（PP1）通过与许多靶向亚基相互作用来实现这一点，从而使PP1活性针对其靶向亚基微环境中的磷蛋白底物而得以确定。DNA聚合酶δ（Polδ）是哺乳动物染色体复制中的关键酶。它由四个亚基组成，即p125、p50、p68和p12。我们将p68鉴定为一种新的PP1靶向亚基。通过亲和色谱法以及从哺乳动物细胞裂解物中进行的共免疫沉淀试验，并且在体外通过下拉试验表明，PP1与人DNA聚合酶δ相关联。PP1的结合结构域被鉴定为序列KRVAL，它是典型的RVxF PP1结合基序的变体。这些研究为PP1靶向DNA聚合酶δ提供了首个证据。我们还表明，CK2使Polδ的p125、p68和p12亚基磷酸化，并且这些磷酸化的亚基是PP1的底物。这些发现确定了p68作为PP1靶向亚基的新作用，这意味着PP1参与Polδ的去磷酸化过程。我们的发现还表明，CK2是参与p68体内磷酸化的蛋白激酶的有力候选者。
