Importin α transports CaMKIV to the nucleus without utilizing importin β
2005; Springer Nature; Volume: 24; Issue: 5 Linguagem: Inglês
10.1038/sj.emboj.7600587
ISSN1460-2075
AutoresIppei Kotera, Toshihiro Sekimoto, Yoichi Miyamoto, Takuya Saiwaki, Emi Nagoshi, Hiroyuki Sakagami, Hisatake Kondo, Yoshihiro Yoneda,
Tópico(s)Trace Elements in Health
ResumoArticle17 February 2005free access Importin α transports CaMKIV to the nucleus without utilizing importin β Ippei Kotera Ippei Kotera Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Toshihiro Sekimoto Toshihiro Sekimoto Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Yoichi Miyamoto Yoichi Miyamoto Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Takuya Saiwaki Takuya Saiwaki Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Emi Nagoshi Emi Nagoshi Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Hiroyuki Sakagami Hiroyuki Sakagami Department of Cell Biology, Graduate School of Medicine, Tohoku University, Aoba-ku, Sendai, Japan Search for more papers by this author Hisatake Kondo Hisatake Kondo Department of Cell Biology, Graduate School of Medicine, Tohoku University, Aoba-ku, Sendai, Japan Search for more papers by this author Yoshihiro Yoneda Corresponding Author Yoshihiro Yoneda Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Ippei Kotera Ippei Kotera Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Toshihiro Sekimoto Toshihiro Sekimoto Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Yoichi Miyamoto Yoichi Miyamoto Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Takuya Saiwaki Takuya Saiwaki Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Emi Nagoshi Emi Nagoshi Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Hiroyuki Sakagami Hiroyuki Sakagami Department of Cell Biology, Graduate School of Medicine, Tohoku University, Aoba-ku, Sendai, Japan Search for more papers by this author Hisatake Kondo Hisatake Kondo Department of Cell Biology, Graduate School of Medicine, Tohoku University, Aoba-ku, Sendai, Japan Search for more papers by this author Yoshihiro Yoneda Corresponding Author Yoshihiro Yoneda Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Author Information Ippei Kotera1, Toshihiro Sekimoto1, Yoichi Miyamoto2, Takuya Saiwaki1, Emi Nagoshi1, Hiroyuki Sakagami3, Hisatake Kondo3 and Yoshihiro Yoneda 1,2 1Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan 2Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan 3Department of Cell Biology, Graduate School of Medicine, Tohoku University, Aoba-ku, Sendai, Japan *Corresponding author. Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 4606; Fax: +81 6 6879 4609; E-mail: [email protected] The EMBO Journal (2005)24:942-951https://doi.org/10.1038/sj.emboj.7600587 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV) plays an essential role in the transcriptional activation of cAMP response element-binding protein-mediated signaling pathways. Although CaMKIV is localized predominantly in the nucleus, the molecular mechanism of the nuclear import of CaMKIV has not been elucidated. We report here that importin α is able to carry CaMKIV into the nucleus without the need for importin β or any other soluble proteins in digitonin-permeabilized cells. An importin β binding-deficient mutant (ΔIBB) of importin α also carried CaMKIV into the nucleus, which strongly suggests that CaMKIV is transported in an importin β-independent manner. While CaMKIV directly interacted with the C-terminal region of importin α, the CaMKIV/importin α complex did not form a ternary complex with importin β, which explains the nonrequirement of importin β for the nuclear transport of CaMKIV. The cytoplasmic microinjection of importin α-ΔIBB enhanced the rate of nuclear translocation of CaMKIV in vivo. This is the first report to demonstrate definitely that mammalian importin α solely carries a cargo protein into the nucleus without utilizing the classical importin β-dependent transport system. Introduction The import of macromolecules from the cytoplasmic compartment to the nucleus generally requires soluble transport receptors. The use of the classical basic type nuclear localization signal (cNLS) in conjunction with digitonin-permeabilized semi-intact cells enabled the molecular mechanisms of the classical nuclear protein import to be studied. Importin α (karyopherin α) recognizes a cargo protein containing cNLS, and the importin α is further recognized by importin β (karyopherin β) to form a nuclear pore-targeting complex (PTAC). This ternary complex interacts with the phenylalanine–glycine (FG) repeat domains of nucleoporins that are present in the nuclear pore complexes (NPCs), and the interaction is crucial for the passage of macromolecules. A small GTPase Ran that is predominantly GTP bound in the nucleus binds to importin β in the nucleoplasm to trigger the dissociation of the ternary complex. The nature of the dissociation of the cargo protein from importin α is unclear, but in yeast, it has been proposed that Nup2p, together with Cse1p (a yeast homolog of the cellular apoptosis susceptibility gene product, CAS) and RanGTP, assists the release of the cargo from importin α. The complexes of RanGTP/CAS/importin α and RanGTP/importin β are exported back to the cytoplasm where they are dissociated by the hydrolysis of GTP by Ran to complete the cycle of transport receptors (for reviews, see Görlich and Mattaj, 1996; Mattaj and Englmeier, 1998; Yoneda, 2000; Stewart et al, 2001). It has also been shown that multiple classes of importin β-like molecules that are present in a cell carry a variety of transport cargoes into the nucleus. Transportin, one of the importin β family members, mediates the nuclear import of a specific signal, termed M9, which was identified in hnRNP A1; the M9-containing cargoes are recognized by transportin without the aid of adaptor molecules such as importin α (Pollard et al, 1996). It has also been demonstrated that importin β by itself is able to transport cargoes including cyclin B1, sterol regulatory element-binding protein-2 (SREBP-2) and Smad3 through direct binding (Moore et al, 1999; Nagoshi et al, 1999; Kurisaki et al, 2001). It has also recently been reported in an X-ray crystallography study that importin β uses characteristic long helices, analogous to a pair of chopsticks, to bind to the active form of SREBP-2 (Lee et al, 2003). It is also well known that importin α functions as an adaptor molecule between the cNLS-containing cargo and importin β. Structural analyses of importin α revealed the existence of three functional domains: an importin β binding (IBB) domain in the N-terminus, a hydrophobic central domain known as the armadillo (arm) repeat domain and a short acidic domain in the C-terminus. A cNLS binds to two sites within the arm repeat domain, whereas the C-terminal acidic domain binds to CAS, which mediates the nuclear export of importin α. Furthermore, it has recently been shown that importin α is able to migrate into the nucleus in an importin β- and Ran-independent manner (Miyamoto et al, 2002). Although the issue of the physiological significance of the importin β-independent nuclear import of importin α is not yet resolved, it has very recently been reported that cellular stress, including UV irradiation, oxidative stress and heat shock, induces the nuclear accumulation of importin α, which results in the inhibition of conventional nuclear import (Miyamoto et al, 2004). However, the issue of whether importin α is able to carry cargoes into the nucleus in an importin β-independent manner in spite of the ability of importin α to translocate through the NPC by itself has not been resolved. Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV) is a multifunctional serine/threonine kinase and the phosphorylation of its substrates such as cAMP response element-binding protein (CREB) has been implicated in a number of cellular and physiological functions (for reviews, see Anderson and Kane, 1998; Krebs, 1998; Soderling, 1999). The phosphorylation of CREB at Ser133 by CaMKIV is crucial for the transcriptional activation of Ca2+-induced CREB-mediated signaling pathways (Enslen et al, 1994; Matthews et al, 1994). CaMKIV is particularly important among the Ca2+/calmodulin-dependent kinases because of its nuclear colocalization with CREB (Jensen et al, 1991; Matthews et al, 1994; Gao et al, 2004). The subcellular distribution of CaMKIV was observed to be rather dynamic; nuclear CaMKIV levels are increased in the neurons of the neocortex after the disruption of sensory input (Lalonde et al, 2004), whereas in granulosa cells of the ovary, CaMKIV migrates to the cytoplasm after a hormonal stimulus (Wu et al, 2000). Despite its importance in the nuclear localization and possible functional regulation by the dynamic movement within the cells, the nucleocytoplasmic transport mechanism of CaMKIV has not been elucidated. In this study, using a digitonin-permeabilized cell-free transport assay (Adam et al, 1990), we demonstrate that the addition of importin α alone reproduced the nuclear import of CaMKIV and that the nuclear migration of CaMKIV required neither importin β nor Ran. The cytoplasmic microinjection of importin α-ΔIBB was found to facilitate the nuclear translocation of CaMKIV in vivo. Thus, our data show that CaMKIV is transported to the nucleus by importin α without the need for importin β. This is the first example to demonstrate that importin α can act as a nuclear import carrier like importin β family molecule. In addition, the possibility that the solitary nuclear migration of importin α and the importin α-driven nuclear import of CaMKIV penetrate the NPC in similar manners is demonstrated. Results CaMKIV is transported to the nucleus by a facilitated mechanism The predicted molecular mass of CaMKIV is 53 kDa, suggesting that it could passively diffuse through the NPC. To determine empirically if CaMKIV migrates into the nucleus by diffusion or facilitated transport, in vivo microinjection assays with mammalian cell-expressed recombinant Flag-tagged CaMKIV were carried out. It has been established that wheat germ agglutinin (WGA) blocks facilitated transport, but not the free diffusion, of macromolecules through the NPC. To determine whether the nuclear translocation of CaMKIV is inhibited by WGA, CaMKIV and WGA were coinjected into the cytoplasm of NIH3T3 cells and the cells were incubated at 37°C for a period of up to 90 min. The nuclear migration of CaMKIV was inhibited by WGA, even after 90 min of incubation (Figure 1E), whereas the majority of the CaMKIV was translocated to the nucleus in the absence of WGA after 90 min of incubation (Figure 1D). These results indicate that CaMKIV is transported into the nucleus by facilitated transport, and not by passive diffusion. Figure 1.The slow and facilitated nuclear migration of CaMKIV. Flag-CaMKIV and GST-cNLS-CFP (10 μM each, left panels), along with GST-Venus (10 μM, right panels) as an injection marker, were cytoplasmically microinjected into NIH3T3 cells with or without WGA (2 mg/ml). The majority of Flag-CaMKIV remained in the cytoplasm during the first 15 min of incubation (A) and Flag-CaMKIV was gradually translocated to the nucleus with 30 (B), 60 (C) and 90 (D) min of incubation, whereas GST-cNLS-CFP was exclusively localized in the nucleus within 15 min after the injection (F). Coinjection of Flag-CaMKIV with WGA disrupted the transport of Flag-CaMKIV across the nuclear membrane even at 90 min after the injection (E). Download figure Download PowerPoint Although the subcellular localization of endogenous or exogenously injected CaMKIV was predominantly nuclear, a cytoplasmic signal was also apparent in both cases. We observed that, when microinjected into the nucleus, CaMKIV was exported to the cytoplasm (data not shown), which explains the fact that CaMKIV is distributed throughout the cell. Despite functioning as a shuttling molecule between the nucleus and cytoplasm, it is suspected that the rate of nuclear translocation of CaMKIV is considerably slower than that of the classical NLS (cNLS). In fact, the migration of GST-cNLS-GFP to the nucleus was completed within 15 min (Figure 1F), whereas the majority of CaMKIV remained in the cytoplasm after 15 min incubation (Figure 1A). Approximately 60 min was required for CaMKIV to reach its predominantly nuclear localization (Figure 1B and C). Thus, the nuclear translocation of CaMKIV occurs by facilitated transport, but the underlying mechanism of the transport might be different from that of the classical transport system. Importin α alone carries CaMKIV to the nucleus Using digitonin-permeabilized semi-intact cells with mammalian cell-expressed Flag-CaMKIV and baculo-virally expressed GFP-CaMKIV (Figure 2J), we attempted to determine the factors required for the nuclear transport of CaMKIV. A cytosolic extract prepared from Ehrlich ascites tumor cells was found to contain the necessary components for transport, as described previously (Imamoto et al, 1995); both Flag-CaMKIV (Figure 2A) and GFP-CaMKIV (Figure 2G) were efficiently transported to the nucleus when the extract and an ATP regeneration system were added, while the nuclear transport of Flag-CaMKIV (Figure 2B) and GFP-CaMKIV (Figure 2H) was dramatically diminished when the cytosolic extract was omitted, indicating that the nuclear import of CaMKIV requires soluble transport factor(s). Figure 2.Importin α mediates the nuclear transport of CaMKIV without utilizing importin β or Ran. In digitonin-permeabilized BHK cells, FLAG-CaMKIV-His (3 μM) was incubated at 30°C for 40 min along with various transport factors. After fixation with 4% paraformaldehyde, the cells were immunostained with anti-CaMKIV antibody. Along with an ATP regeneration system, CaMKIV was transported to the nucleus when an Ehrlich ascites tumor cell extract (14 mg/ml) was added (A) but not when the extract was omitted (B). GST-importin α (24 μM) solely transported CaMKIV to the nucleus (C). Despite the addition of importin α, importin β (6 μM) reduced the efficiency of the transport in the presence (D) or absence (E) of Ran (8 μM). ΔIBB mutant of importin α (24 μM) carried CaMKIV into the nucleus (F). GFP-CaMKIV-His was similarly transported to the nucleus when Ehrlich ascites tumor cell extract (G) or GST-importin α-ΔIBB (I) was added, but not when soluble proteinaceous factors were omitted (H). Mammalian cell-expressed Flag-CaMKIV-His and baculo-virally expressed GFP-CaMKIV-His used in this study were shown by Coomassie staining (J). Download figure Download PowerPoint In order to determine if CaMKIV is transported by classical transport factors, bacterially expressed importin α, importin β and Ran were used in semipermeable cells along with mammalian and insect cell-expressed CaMKIV and an ATP regeneration system. To our surprise, importin α alone had the highest activity for transporting CaMKIV to the nucleus without the addition of importin β or Ran (Figure 2C). In fact, the addition of importin β appeared to decrease the efficiency of transport, in both the presence and the absence of Ran (Figure 2D and E). To exclude the possibility that a residual amount of endogenous importin β in the permeabilized cells contributed to the nuclear migration of CaMKIV, the importin β binding-deficient mutant (ΔIBB) of importin α was used instead of the full-length importin α. The addition of only importin α-ΔIBB indeed reproduced the apparent nuclear import of Flag-CaMKIV (Figure 2F) and GFP-CaMKIV (Figure 2I) with the same efficiency as the full-length importin α. These results strongly suggest that CaMKIV is transported to the nucleus by importin α alone without the need for importin β or Ran. CaMKIV directly binds to importin α Classical transport requires the formation of a ternary complex of importin α, importin β and cNLS-cargo in the cytoplasm. To determine whether or not CaMKIV directly interacts with importin α, mammalian cell-expressed Flag-CaMKIV was incubated with bacterially expressed GST-importin α or mutants thereof that were immobilized on glutathione-Sepharose. CaMKIV directly interacted with immobilized importin α (Figure 3A, lane 2), consistent with a recent report (Lemrow et al, 2004). CaMKIV also bound to importin α-ΔIBB (Figure 3A, lane 3), in agreement with the above experiment in which importin α-ΔIBB was shown to transport CaMKIV to the nucleus. Figure 3.Interaction between importin α and CaMKIV. (A) GST-importin α (GST-imp α) and GST-importin α-ΔIBB (GST-imp α-ΔIBB) (15 pmol each) were immobilized on glutathione-Sepharose, and FLAG-CaMKIV (50 pmol) was applied to observe the interaction between importin α and CaMKIV. FLAG-CaMKIV interacted with both GST-importin α (lane 2) and GST-importin α-ΔIBB (lane 3), but not with GST alone (lane 1). The interaction between CaMKIV and importin α was disrupted by the addition of importin β (imp β). The amount of importin α-bound CaMKIV decreased as the amount of added importin β was increased from 100 pmol (lane 4) to 400 pmol (lane 6). Importin α-ΔIBB-bound CaMKIV was not affected by the addition of 400 pmol of importin β (lane 7). (B) Immobilized Flag-CaMKIV was used to examine the formation of a ternary complex with importin α and β. Importin α and/or β (100 pmol each) were added to immobilized Flag-CaMKIV (200 pmol) or GST-cNLS-GFP (100 pmol). A ternary complex of importin α, importin β and GST-cNLS-GFP was formed (lane 1). Importin β did not bind to importin α/CaMKIV complex (lane 3), nor to CaMKIV directly (lane 4). (C) Immobilized Flag-CaMKIV binds to importin α, but not to importin β, in a crude cytoplasmic extract of Ehrlich ascites tumor cells. The cytosolic extract was added to immobilized Flag-CaMKIV (6 nmol) or GST-cNLS-GFP (6 nmol). Importin β bound to a GST-cNLS-GFP/importin α complex (lane 1), but not to a CaMKIV/importin α complex (lane 3). Other importin β family molecules such as transportin or importin 4 did not bind to the CaMKIV/importin α complex (lower panels). Download figure Download PowerPoint It is generally known that the binding of importin β to the IBB domain of importin α enhances the interaction between importin α and the cNLS. The reason for this is because the IBB domain has autoinhibitory activity and the binding of importin β to the IBB domain eliminates this autoinhibitory effect, thus creating easier access for cNLS-bearing proteins (Moroianu et al, 1996; Kobe, 1999; Fanara et al, 2000). In contrast, in digitonin-permeabilized cells, the nuclear transport of CaMKIV was found to be somewhat decreased when importin β was added (Figure 2D). To elucidate the reason for why importin β is not only unnecessary, but also has an inhibitory effect on the import of CaMKIV, we examined the in vitro binding between CaMKIV and transport factors in more detail. Immobilized GST-importin α was incubated with CaMKIV, along with increasing amounts of importin β. An excess amount of importin β disrupted the interaction between importin α and CaMKIV (Figure 3A, lanes 4–6), but not between importin α-ΔIBB and CaMKIV (Figure 3A, lane 7), indicating that the binding of importin β to the IBB domain suppresses the binding of CaMKIV to importin α. Moreover, no CaMKIV-bound importin β signal was found on a Western blot assay (Figure 3B, lane 4), indicating that CaMKIV does not bind to importin β directly. In addition, even in the presence of importin α, CaMKIV was unable to form a ternary complex with importin α and β (Figure 3B, lane 3), whereas immobilized GST-cNLS-GFP efficiently formed a ternary transport complex with importin α and β (Figure 3B, lane 1). The result with a crude cytosolic extract of Ehrlich ascites carcinoma cells confirmed that Flag-CaMKIV interacted with endogenous importin α, but not with endogenous importin β (Figure 3C, lane 3), whereas GST-cNLS-GFP formed a ternary complex with endogenous importin α and β (Figure 3C, lane 1). Moreover, other importin β family molecules such as transportin or importin 4 did not bind to the importin α/CaMKIV complex. These results are in good agreement with the result of the digitonin-permeabilized transport assay, which showed that CaMKIV was transported by importin α alone, and the transport was inhibited somewhat by the presence of importin β. ATP is required for the nuclear translocation of CaMKIV but not as an energy source The nuclear transport of CaMKIV was completely inhibited without the addition of exogenous ATP in digitonin-permeabilized cells, even in the presence of cytosolic extract (Figure 4A) or importin α (Figure 4B). Although at present, how ATP is used for the nuclear transport is a controversial issue, one interpretation is that ATP is required for the de novo synthesis of cellular GTP, which, in turn, is required for the maintenance of RanGTP/GDP gradient across the nuclear membrane (Schwoebel et al, 2002). However, when we substituted ATP with ATPγS, a nonhydrolyzable analog of ATP, importin α-driven transport of CaMKIV continued to be active in digitonin-permeabilized cells (Figure 4C) and Ran was not required for the nuclear import of CaMKIV in vitro. These results and the report showing that a non-ATP-binding mutant of CaMKIV is nuclear translocation-incompetent (Lemrow et al, 2004) raise the possibility that the binding of ATP to CaMKIV is required for its nuclear translocation rather than its use as an energy source. It is unlikely, however, that ATP is a prerequisite for the interaction between CaMKIV and importin α, since it was shown that the non-ATP-binding mutant of CaMKIV retains the ability to bind importin α (Lemrow et al, 2004). Thus, the issue of how ATP is used for the nuclear translocation of CaMKIV is yet to be determined. Figure 4.CaMKIV is transported to the nucleus in an ATP-dependent manner. In digitonin-permeabilized BHK cells, the nuclear transport of CaMKIV by cytosolic extract was inhibited when no ATP was added (A). Importin α-driven nuclear import of CaMKIV was also inhibited without ATP (B). The importin α-driven nuclear import of CaMKIV was restored when nonhydrolyzable ATPγS (3 mM) was added instead of ATP (C). In digitonin-permeabilized BHK cells, cNLS peptide or rNLS peptide (125 μM each) was added along with GST-importin α, FLAG-CaMKIV and an ATP regeneration system. The nuclear translocation of CaMKIV was inhibited by the addition of cNLS peptide (D), whereas the addition of rNLS did not affect the nuclear transport of CaMKIV (E). (F) cNLS does not disrupt the interaction between CaMKIV and importin α. GST-importin α (15 pmol) was immobilized on glutathione-Sepharose, and FLAG-CaMKIV (50 pmol) (upper) or GFP-cNLS (50 pmol) (lower) was added along with an excess amount of cNLS or rNLS peptides (6 nmol each). The interaction between CaMKIV and importin α was not affected by the addition of either peptide. (G) CaMKIV and cNLS simultaneously bind to importin α in an excess amount of cNLS. Flag-CaMKIV (50 pmol) and GFP-cNLS (250 pmol) were added to immobilized GST-importin α (15 pmol). Importin α-bound Flag-CaMKIV and GFP-cNLS were detected by anti-CaMKIV and anti-GFP antibodies, respectively. Download figure Download PowerPoint Importin α-driven nuclear translocation of CaMKIV may utilize the same pathway as importin β-independent nuclear import of importin α Importin α has been reported to migrate to the nucleus without utilizing importin β and Ran (Miyamoto et al, 2002). This report also concluded that the presence of an excess amount of the cNLS peptide dramatically inhibited the nuclear import of importin α in the absence of importin β and Ran. In order to further examine the possibility that both the unassisted migration of importin α to the nucleus and the importin α-driven nuclear translocation of CaMKIV use the same pathway, a peptide corresponding to the cNLS was added to the digitonin-permeabilized cells along with importin α and CaMKIV. As expected, the addition of the cNLS peptide dramatically reduced the nuclear import of CaMKIV (Figure 4D), while reversed NLS (rNLS) did not (Figure 4E). We then attempted to determine if the inhibition was due to a blockage of the nuclear translocation of importin α rather than the disruption of the interaction between CaMKIV and importin α. Immobilized GST-importin α and GFP-cNLS were incubated with CaMKIV in the presence of an excess of the cNLS or rNLS peptide. Although GFP-cNLS was completely dissociated from importin α in an excess of cNLS peptide (Figure 4F, lower panel), the interaction between CaMKIV and importin α was not affected by cNLS peptide (Figure 4F, upper panel). In order to understand if cNLS and CaMKIV bind to importin α alongside, excess amounts of GFP-cNLS and Flag-CaMKIV were incubated with immobilized GST-importin α. As expected, both Flag-CaMKIV and GFP-cNLS were detected in the bound fraction of importin α (Figure 4G). These results indicate that both the importin β-independent nuclear translocation of importin α and importin α-driven nuclear translocation of CaMKIV are inhibited by the binding of cNLS to importin α in a similar fashion, suggesting that they may enter the nucleus using the same pathway. CaMKIV binds to the C-terminal region of importin α In spite of the fact that the binding of cNLS to importin α abolishes its ability to migrate to the nucleus independent of importin β, why does CaMKIV-bound importin α enter the nucleus? A possible explanation would be that the binding site for CaMKIV differs from that of the cNLS so that the binding of CaMKIV does not affect the ability of importin α to enter the nucleus. To test this hypothesis, the CaMKIV-binding region of importin α was determined using a variety of GST-tagged importin α deletion mutants. The immobilized GST-importin α mutants were incubated with Flag-CaMKIV or GFP-cNLS in an attempt to determine which domain of importin α is required for the interaction (Figure 5A). GST-importin α (1–459) showed efficient binding activity to CaMKIV (Figure 5B, lane 2), but the binding was dramatically reduced when GST-importin α (1–412) was used (Figure 5B, lane 3). GST-importin α (338–538) showed significant binding activity to CaMKIV (Figure 5B, lane 4), but a lower binding activity was observed for GST-importin α (425–538) (Figure 5B, lane 5). These data indicate that CaMKIV interacts with the C-terminal region of importin α, including armadillo repeats 9 and 10, which is clearly different from the binding site for cNLS. The result, at least in part, may explain the reason for why the importin β-independent nuclear migration of importin α is inhibited by cNLS, but not CaMKIV, although the issue of how the difference of binding sites affects the molecular state of importin α remains unknown. Figure 5.CaMKIV interacts with the C-terminal region of importin α. (A) Schematic representation of importin α and its mutants. (B) Flag-CaMKIV and GFP-cNLS were incubated with GST-importin α and its mutants immobilized on glutathione-Sepharose beads. GST-importin α (lane 1) and GST-importin α (1–459) (lane 2) showed binding activity to CaMKIV, whereas GST-importin α (1–412) (lane 3) showed dramatically reduced binding. GST-importin α (338–538) (lane 4) interacted with CaMKIV, and a somewhat reduced binding was observed with importin α (425–538) (lane 5). GFP-cNLS bound to all importin α mutants except the C-terminal deleted mutants (338–538) and (425–538) (lanes 4 and 5, lower). Download figure Download PowerPoint The in vivo nuclear transport rate of CaMKIV is enhanced by the cytoplasmic microinjection of importin α-ΔIBB In order to confirm the importin α-driven nuclear transport of CaMKIV in vivo, we attempted to inhibit the nuclear import of CaMKIV by microinjecting concentrated anti-importin α antibodies or C-terminal region-containing mutants of importin α, which might function dominant-negatively, but none of them succeeded (data not shown). This is probably due to the diversity and abundance of importin α family molecules in intact cells. Therefore, we attempted to determine whether the nuclear transport rate of CaMKIV could be accelerated by the cytoplasmic microinjection of recombinant importin α-ΔIBB. The IBB region of importin α has
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