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p27Kip1 (p27), a CDK inhibitor, migrates into the nucleus, where it controls cyclin-CDK complex activity for proper cell cycle progression. We report here that the classical bipartite-type basic amino-acid cluster and the two downstream amino acids of the C-terminal region of p27 function as a nuclear localization signal (NLS) for its full nuclear import activity. Importin a3 and a5, but not a1, transported p27 into the nucleus in conjunction with importin b, as evidenced by an in vitro transport assay. It is known that Akt phosphorylates Thr 157 of p27 and this reduces the nuclear import activity of p27. Using a pull-down experiment, 14-3-3 was identified as the Thr157-phosphorylated p27NLS-binding protein. Although importin a5 bound to Thr157-phosphorylated p27NLS, 14-3-3 competed with importin a5 for binding to it. Thus, 14-3-3 sequestered phosphorylated p27NLS from importin a binding, resulting in cytoplasmic localization of NLS-phosphorylated p27. These findings indicate that 14-3-3 suppresses importin a/b-dependent nuclear localization of Thr157-phosphorylated p27, suggesting implications for cell cycle disorder in Akt-activated cancer cells.

Y.Miyamoto and M.Hieda contributed equally to this workA classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin a/b complex. In this study, we found that importin a migrated into the nucleus without the addition of importin b, Ran or any other soluble factors in an in vitro transport assay. A mutant importin a lacking the importin b-binding domain ef®ciently entered the nucleus. Competition experiments showed that this import pathway for importin a is distinct from that of importin b. These results indicate that importin a alone can enter the nucleus via a novel pathway in an importin b-and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin a differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin a was affected by the addition of ATP, but not ATPgS. These results suggest that the nuclear import machinery for importin a at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis. Keywords: ATP/importin a/nuclear pore complex/ nuclear transport/time-lapse experiment IntroductionMolecular migration between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC) present in the double membrane of the nuclear envelope. The NPC, a huge proteinaceous structure composed of 50±100 different species of proteins called nucleoporins, is estimated to have a total mass of~125 MDa in higher eukaryotes and 66 MDa in Saccharomyces cerevisiae. Whereas molecules <20±40 kDa are able to passively diffuse through the NPC into the nucleus, the nuclear import of larger molecules is generally dependent on the presence of a speci®c signal sequence, the nuclear localization signal (NLS), and is often associated with a requirement for metabolic energy (Go Èrlich and Kutay, 1999). The ®rst identi®ed NLS was that of the SV40 large T-antigen, which consists of a short stretch of basic amino acids, designated as the basic type NLS. This type of NLS is divided into two groups, monopartite and bipartite, based on the number of basic amino acid clusters (Dingwall and Laskey, 1991).The nuclear import of basic type NLS-containing proteins is mediated by speci®c soluble factors that form a stable complex, the nuclear pore-targeting complex, in the cytoplasm (Imamoto et al., 1995a). The complex is composed of two essential components that are referred to as importin a and b (Go Èrlich and Mattaj, 1996). In addition to these molecules, several factors participate in this transport system, including a small GTPase Ran (Moore and Blobel, 1993) and Ran-binding proteins (Paschal and Gerace, 1995). Ran has a low intrinsic activity with respect to GDP/GTP exchange and GTP hydrolysis. RCC1, a guanine nucleotide exchange factor of Ran, accelerates the dissociation of the guanine nucleotide from Ran, ther...

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Carbon fiber-reinforced plastic (CFRP) has a great potential application in aircraft fuselage due to its light weight, high specific stiffness and high specific strength. It is crucial to coat the CFRP surface with an electrically conductive material to avoid the damage from lightning strike. Cold spray process has been developed for metallic coating technique. In this study, aluminum coating was fabricated onto the CFRP substrate using interlayer was investigated. It was difficult to fabricate cold-sprayed aluminum coating directly on the CFRP substrate. Though smaller size aluminum particles could be deposited on the CFRP substrate, but the coating was peeled off when the thickness was around 30 mm. On the other hand, it was possible to deposit aluminum coating on the CFRP substrate by plasma spray process. Our proposed structure is using a thin plasma-sprayed aluminum interlayer on the CFRP substrate before doing the cold spray. The interlayer with larger contact area could retain on the substrate and able to facilitate the deformation of the next incoming cold-sprayed particles to build a thick coating. The volume resistivity of cold spray coating is lower than the plasma sprayed aluminum coating because of high process gas temperature in the latter case enhances the oxidation of sprayed particle. Therefore, lower process gas temperature should be used to fabricate lower volume resistivity coating on cold spray.

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Many kinds of both metallic and ceramic powder particles were plasma-sprayed onto the mirror-polished metallic substrate surface, and the effect of both substrate temperature and ambient pressure on the flattening behavior of the particle was systematically investigated. In the flattening behavior of the sprayed particle onto the substrate surface, critical conditions were recognized both in the substrate temperature and ambient pressure. That is, the flattening behavior changed transitionally on that critical temperature and pressure range, respectively. We defined and introduced a transition temperature, Tt, and transition pressure, Pt, respectively, for those critical conditions. The role of the related factors (such as solidification of the bottom surface of the splat, desorption of adsorbates on the substrate surface and wetting at interface) on the transition behavior in the flattening was clarified from several points of views. The fact that the dependence both of transition temperature and transition pressure on the sprayed particle material had a similar tendency indicated that the wetting of the substrate by the molten particles seemed to be a domination in the flattening. A three-dimensional transition map by combining both transition temperature and transition pressure dependence was proposed as a controlling principle of the thermal spray process.