Analysis of neuron-specific nuclear protein transport by using CaM kinase IV as a substrate.

为了了解神经元特异性核蛋白转运的分子机制，我们使用Ca^<2+>/钙调蛋白依赖性蛋白激酶IV型（CaM kinase IV）作为模型底物。已知CaM激酶IV定位于神经元细胞的细胞核中，而遍及非神经元细胞如甲状旁腺细胞的细胞质。此外，尚未鉴定CaM激酶IV的核定位信号。当将重组CaM激酶IV蛋白注射到HeLa细胞（人宫颈癌细胞）或COS 7细胞（非洲绿色猴肾细胞）的细胞质中时，它们迁移到COS 7细胞的细胞核中，而不是HeLa细胞的细胞核中，这表明CaM激酶IV以细胞类型特异性的方式从细胞质易位到细胞核。接下来，我们试图通过使用透化的无细胞系统来确定CaM激酶IV的核输入所需的因子。已经证明，脑提取物比埃利希腹水肿瘤细胞提取物更有效地支持CaM激酶IV的核输入。此外，输入蛋白α的伊布（importin β-binding）结构域和输入蛋白β的N-末端NPC（nuclear pore complex）结合部分的加入并不抑制输入，这意味着CaM激酶IV的核迁移不是由传统的输入蛋白α/β途径介导的。更有趣的是，发现脑提取物介导的核输入不被麦胚凝集素抑制，而被埃利希腹水瘤细胞提取物抑制，这表明CaM激酶IV可能通过至少两个独立的途径被转运到核中。在不久的将来，参与这些反应的因素应该被分离和表征。

In order to know the molecular mechanism of neuron-specific nuclear protein transport, we used Ca^<2+>/calmodulin-dependent protein kinase type IV (CaM kinase IV) as a model substrate. CaM kinase IV is known to be localized in the nucleus of neuronal cells, while throughout the cytoplasm of non-neuronal cells such as parathyroid cells. Further, the nuclear localization signal of CaM kinase IV has not yet been identified. When the recombinant CaM kinase IV proteins were injected into the cytoplasm of HeLa cells (human cervical cancer cells) or COS7 cells (African green monkey kidney cells), they migrated into the nuclei of COS7 cells but not those of HeLa cells, suggesting that CaM kinase IV is translocated from cytoplasm to the nucleus in a cell-type specific manner. Next, we tried to identify factors required for the nuclear import of CaM kinase IV by using a permeabilized cell-free system. It was demonstrated that brain extracts support the nuclear import of CaM kinase IV more efficiently than Ehrlich ascites tumor cell extracts. Moreover, the import was not inhibited by the addition of IBB (importin β-binding) domain of importin α and the N-terminal NPC (nuclear pore complex)-binding portion of importin β, meaning that the nuclear migration of CaM kinase IV is not mediated by conventional importin α/β pathway. More interestingly, it was found that the nuclear import mediated by brain extracts was not inhibited by the treatment with wheat germ agglutinin, whereas was that by Ehrlich ascites tumor cell extracts, suggesting that CaM kinase IV may be transported into the nucleus through at least two independent pathways. In the near future, factors involved in these reactions should be isolated and characterized.