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+>/钙调素依赖性蛋白激酶IV型（CaM激酶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，而在非神经元细胞（如甲状旁腺细胞）的细胞质中则存在。此外，CaM激酶IV的核定位信号尚未确定。将重组CaM激酶IV蛋白注入HeLa细胞（人宫颈癌细胞）或COS7细胞（非洲绿色猴子肾细胞）的细胞质中，它们迁移到COS7细胞的细胞核中，而不迁移到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转移到细胞核中。接下来，我们试图通过使用渗透性无细胞系统来确定CaM激酶IV核输入所需的因素。结果表明，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IBB（输入蛋白β-结合）结构域和输入蛋白β的n端NPC（核孔复合物）结合部分不抑制输入，这意味着CaM激酶IV的核迁移不是通过传统的输入蛋白α/β途径介导的。更有趣的是，研究发现，脑提取物介导的核输入未被小麦胚芽凝集素抑制，而埃利希腹水肿瘤细胞提取物则被抑制，这表明CaM激酶IV可能通过至少两种独立的途径转运到细胞核。在不久的将来，参与这些反应的因素应该被分离出来并加以表征。

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.