Pharmacogenetics section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, U.S.A.
Mass Spectrometry Research and Support Group, Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, U.S.A.
Biosci Rep. 2020 Apr 30;40(4). doi: 10.1042/BSR20200498.
Vaccinia-related kinase 1 (VRK1) is a chromatin-associated Ser-Thr kinase that regulates numerous downstream factors including DNA repair as well as stress factors c-Jun and p53. Both c-Jun and p53 are phosphorylated at Ser63 and Thr18, respectively, in response to low glucose (40 mg/dl of medium) but not high glucose (140 mg/dl of medium) in human hepatoma-derived Huh-7 cells. Here, we have determined the molecular mechanism by which VRK1 phosphorylates these residues in response to glucose in Huh-7 cells. Human VRK1 auto-phosphorylates Ser376 and Thr386 in in vitro kinase assays. In Huh-7 cells, this auto-phosphorylation activity is regulated by glucose signaling; Thr386 is auto-phosphorylated only in low glucose medium, while Ser376 is not phosphorylated in either medium. A correlation of this low glucose response phosphorylation of Thr386 with the phosphorylation of c-Jun and p53 suggests that VRK1 phosphorylated at Thr386 catalyzes this phosphorylation. In fact, VRK1 knockdown by siRNA decreases and over-expression of VRK1 T386D increases phosphorylated c-Jun and p53 in Huh-7 cells. Phosphorylation by VRK1 of c-Jun but not p53 is regulated by cadherin Plakophilin-2 (PKP2). The PKP2 is purified from whole extracts of Huh-7 cells cultured in low glucose medium and is characterized to bind a C-terminal peptide of the VRK1 molecules to regulate its substrate specificity toward c-Jun. siRNA knockdowns show that PKP2 transduces low glucose signaling to VRK1 only to phosphorylate c-Jun, establishing the low glucose-PKP2-VRK1-c-Jun pathway as a glucose stress signaling pathway.
痘苗病毒相关激酶 1（VRK1）是一种与染色质相关的丝氨酸-苏氨酸激酶，可调节包括 DNA 修复以及应激因子 c-Jun 和 p53 在内的众多下游因子。在人肝癌衍生的 Huh-7 细胞中，c-Jun 和 p53 分别在低糖（培养基中 40mg/dl）而非高糖（培养基中 140mg/dl）条件下，其丝氨酸 63 位和苏氨酸 18 位被磷酸化。在此，我们确定了 VRK1 在 Huh-7 细胞中响应葡萄糖而磷酸化这些残基的分子机制。人 VRK1 在体外激酶测定中自身磷酸化丝氨酸 376 和苏氨酸 386。在 Huh-7 细胞中，这种自身磷酸化活性受葡萄糖信号调节；只有在低糖培养基中，Thr386 才会被自身磷酸化，而在两种培养基中，Ser376 均未被磷酸化。这种 Thr386 低葡萄糖响应磷酸化与 c-Jun 和 p53 磷酸化的相关性表明，磷酸化 Thr386 的 VRK1 催化了这种磷酸化。事实上，通过 siRNA 敲低 VRK1 会减少，而过表达 VRK1 T386D 会增加 Huh-7 细胞中磷酸化的 c-Jun 和 p53。VRK1 对 c-Jun 的磷酸化而非 p53 的磷酸化受钙黏蛋白 Plakophilin-2（PKP2）调节。PKP2 从在低糖培养基中培养的 Huh-7 细胞的全提取物中被纯化出来，并被鉴定为结合 VRK1 分子的 C 端肽，以调节其对 c-Jun 的底物特异性。siRNA 敲低表明，PKP2 将低葡萄糖信号转导至 VRK1 仅使其磷酸化 c-Jun，从而建立了低葡萄糖-PKP2-VRK1-c-Jun 途径作为葡萄糖应激信号途径。
