Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
Mol Cell Biochem. 2011 Oct;356(1-2):191-200. doi: 10.1007/s11010-011-0944-9. Epub 2011 Jul 8.
The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [γ-(32)P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPKTKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27 kDa, which were identified as α and β subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation.
SV40 大 T 抗原的核定位信号序列（NLS）对于蛋白质的核转位是必需和充分的。磷酸化通常调节信号蛋白的细胞内分布。在这项研究中，我们研究了大 T 抗原的 NLS 肽对蛋白质磷酸化的影响。当粗细胞裂解物与 [γ-(32)P]ATP 孵育时，野生型 NLS 肽（CPKKKRKVEDP）的添加刺激了内源性激酶对几种内源性底物（分子量为 100、80、50 和 45 kDa）的磷酸化。NLS 肽（CPKTKRKVEDP）和反向 NLS 肽（PDEVKRKKKPC）的核定位活性较弱，它们仅弱刺激这些底物的磷酸化。100 kDa 磷酸蛋白的迁移率与内质网（ER）驻留分子伴侣葡萄糖调节蛋白 94（Grp94）相同，属于 Hsp90 家族，并且纯化的 Grp94 通过 NLS 依赖的方式在细胞裂解物中的激酶中被磷酸化。100 kDa 蛋白通过免疫沉淀和重建实验被鉴定为 Grp94。通过顺序生化柱层析步骤纯化 NLS 依赖性 Grp94 激酶导致分离出分子量为 42 和 27 kDa 的两个多肽，通过 Western 印迹分析和生化特征鉴定分别为蛋白激酶 CK2 的α和β亚基。此外，过量 GTP 的效果和 V8 肽作图表明，细胞裂解物中 NLS 依赖性 Grp94 激酶与 CK2 相同。令人惊讶的是，纯化的 CK2 甚至在没有 NLS 肽的情况下也能磷酸化 Grp94，这表明 CK2 催化的 NLS 依赖性 Grp94 磷酸化需要额外的抑制因子。我们提出 CK2 催化的磷酸化在调节 NLS 依赖性蛋白质核转位中的可能普遍作用。
