|Budget Amount *help
¥12,800,000 (Direct Cost : ¥12,800,000)
Fiscal Year 1999 : ¥5,800,000 (Direct Cost : ¥5,800,000)
Fiscal Year 1998 : ¥7,000,000 (Direct Cost : ¥7,000,000)
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.