DAP kinase-related apoptosis-inducing kinase 2 (DRAK2) is a serine/threonine kinase of the death-associated protein kinase family. DRAK2 mediates apoptosis induced by extracellular stimuli, including UV irradiation and interleukin-2, and also regulates T-cell receptor sensitivity in developing thymocytes. During these events, the subcellular localization of DRAK2 changes between the nucleus and cytoplasm. We found that DRAK2 has a putative nuclear-localization signal (NLS) sequence. Mutations in this sequence interfered with DRAK2 localization to the nucleus. Furthermore, green fluorescence protein fused to the putative NLS accumulated in the nucleus, indicating that the putative sequence functions as an NLS. We also found that the function of the NLS was regulated by phosphorylation. Phorbol myristate acetate (PMA) induced the accumulation of DRAK2 in the cytoplasm of NIH3T3 cells, whereas in the absence of PMA, DRAK2 was localized to the nucleus. Ectopic expression of PKC-gamma induced cytoplasmic localization of DRAK2 and PKC-gamma phosphorylated Ser350 flanking the NLS. DRAK2, but not the Ser350Asp mutant, accumulated in the nuclei of ACL-15 cells in response to UV-irradiation. These results suggest that phosphorylation of Ser350 plays an essential role in regulating translocation of DRAK2 to the nucleus from the cytoplasm, possibly by affecting the activity of the NLS.

Apoptosis, a type of programmed cell death, plays an essential role in the survival of multicellular organisms and is observed in a wide variety of physiological situations. 1)Apoptosis is important for biological self-defense, and defects in the ability to undergo apoptosis can lead to various diseases, including cancer, neurodegenerative diseases, and autoimmune diseases.2) Apoptosis is a well-defined cellular process characterized by morphological changes that include condensation of nuclear chromatin, cell shrinkage, and plasma membrane blebbing.3)The Fas family cytokine and tumor necrosis factor (TNF) cascades can mediate apoptosis, 4,5) but recently, many additional apoptotic components have been identified. Using a functional cloning strategy, Deiss et al. identified the deathassociated protein kinase (DAPK) as a positive mediator of apoptosis triggered by interferon-g.6) The kinase activity of DAPK, which shares homology with myosin light chain kinases, is regulated by calmodulin in a Ca 2+ -dependent manner in vitro.7) Overexpression of DAPK in various cell lines results in cell death, and this death-promoting property strictly depends on the intrinsic kinase activity. Additional isoforms of the DAPK family, including DRP-1, ZIP kinase, DAP kinase-related apoptosis-inducing kinase 1 (DRAK1), and DRAK2 have been identified. These proteins share homology in their primary structures, especially in the catalytic domain.8-11) All of these kinases have an apoptosis-promoting activity that is dependent on the kinase activity.We have studied the function of the small Ca 2ϩ -binding protein CHP1 (calcineurin B homologous protein1).12) CHP1 is a multifunctional regulator of various proteins, including Na ϩ /H ϩ antiporters, a kinesin-like motor protein, and calcineurin. [13][14][15] We identified DRAK2 as a CHP1 binding protein, and found that CHP1 inhibits the kinase activity of DRAK2 in a Ca 2ϩ -dependent manner.16) DRAK2 is found in thymus, testis, spleen, and brain, tissues in which apoptosis plays an important role.12) Overexpression of DRAK2 in NIH3T3 cells causes apoptosis like cell death through a mechanism that requires the kinase activity. 10) However, overexpression studies do not necessarily indicate whether DRAK2 causes apoptosis during normal physiological processes.Several components of apoptotic signal transduction pathways, such as DNases, Bcl family members, and caspases, are localized to specific areas of the cell.17) During UV-induced apoptosis, damaged double stranded DNA signals translocation of the mitochondrial components AIF and Endo-G to the nucleus. 18,19) UV irradiation also activates cytoplasmic targets, such as the transcriptional factors AP-1 and NF-kB.20,21) NF-kB migrates to the nucleus and binds to specific response elements of various genes. Thus, the intracellular localization and translocation of molecules are important elements of apoptosis, and understanding these dynamics will elucidate the regulatory mechanisms of apoptosis.The DAPK family members ZIP kinase and DAP kinase...

Calcineurin homologous protein (CHP) is an EF-hand Ca(2+)-binding protein capable of interacting with various cellular proteins including Na(+)/H(+) exchangers, kinesin-related proteins, and apoptosis-inducing protein kinase DRAK2. We investigated the role of CHP on the DRAK2 protein kinase in vitro. CHP significantly reduced (approximately 85% inhibition) the kinase activity of DRAK2 for both autophosphorylation and phosphorylation of exogenous substrate (myosin light chain). The inhibitory effect of CHP was dependent on the presence of Ca(2+), whereas the interaction between CHP and DRAK2 was not Ca(2+)-dependent. These observations suggest that CHP negatively regulates the apoptosis-inducing protein kinase DRAK2 in a manner that depends on intracellular Ca(2+)-concentration.

In this paper, we examined a new method for decontamination of soils by magnetic separation to reduce the volume of contaminated soils. In this method, we separate paramagnetic 2:1 type clay minerals with strong adsorbility of Cs ions from diamagnetic 1:1 type clay minerals with weak adsorbility of Cs ions. In addition, Cs ions desorbed from 1:1 types by washing are migrated to 2:1 type clay minerals. As a result, the doses of 1:1 type clay minerals can be reduced. First, it became apparent by the particle trajectory simulation that vermiculite, which is 2:1 type clay mineral, could be selectively separated by high gradient magnetic separation using superconducting magnet. Second, we confirmed the separation of vermiculite coexisting with kaolinite, which is 1:1 type clay mineral, by the experiments based on the simulation. Moreover, the experiments using the actual contaminated soils were conducted to examine the migration of Cs ions from 1:1 to 2:1 type clay minerals. As a result, it was observed that Cs ions desorbed from 1:1 types could be adsorbed to 2:1 type clay minerals when using 0.1 mol/L KI as wash solution. It showed the possibility of decreasing the dose of 1:1 type clay minerals.

In guided bone regeneration (GBR), a barrier membrane is applied to provide a space for bone regeneration to promote cell adhesion, cell proliferation, and bone regeneration by inducing cell migration to the surface of the bone-deficient site. We prepared a non-woven absorbable membrane for GBR comprised of a copolymer of L-lactic acid and glycolic acid (PLLGA) using the dry spinning method. A thin and dense membrane comprising unsintered hydroxyapatite (uHA) and poly-L-lactic acid (PLLA) was used for comparison. Osteoblast TMS-12 cells were seeded onto the membranes, and cell adhesion and proliferation rates were examined. Calcification levels were quantified by Alizarin Red S staining after cell culture in calcification induction medium. Osteoblastic gene expression was examined by RT-PCR. Cell adhesion and proliferation rates and calcification on PLLGA membranes were significantly higher than those on uHA/PLLA membranes. PLLGA membranes upregulated osteogenic gene expression compared to on uHA/PLLA membranes. Non-woven PLLGA membranes may be useful in GBR for promoting bone formation and maturation.