Phosphorylation regulates the ferritoid–ferritin interaction and nuclear transport

Ferritin is an iron‐sequestering protein that is generally cytoplasmic; however, our previous studies have shown that in avian corneal epithelial (CE) cells ferritin is nuclear. We have also observed that this nuclear localization involves a tissue‐specific nuclear transporter that we have termed ferritoid, and that nuclear ferritin protects DNA from oxidative damage. Recently we have determined that ferritoid functions not only as a nuclear transporter, but also, within the nucleus, it remains associated with ferritin as a heteropolymeric complex. This ferritoid–ferritin complex has unique properties such as being half the size of a typical ferritin molecule and showing preferential binding to DNA. It is likely that the association between ferritoid and ferritin is involved both in the nuclear transport of ferritin and in determining certain of the properties of the complex; therefore, we have been examining the mechanisms involved in regulating the association of these two components. As the ferritoid sequence contains six putative phosphorylation sites, we have examined here whether phosphorylation is one such mechanism. We have determined that ferritoid in the nuclear ferritoid–ferritin complexes is phosphorylated, and that inhibition of this phosphorylation, using inhibitors of PKC, prevents its interaction with ferritin. Furthermore, in an experimental model system in which the nuclear transport of ferritin normally occurs (i.e., the co‐transfection of COS‐1 cells with full length constructs for ferritin and ferritoid), when phosphorylation sites in ferritoid are mutated, the interaction between ferritoid and ferritin is inhibited, as is the nuclear transport of ferritin. J. Cell. Biochem. 107: 528–536, 2009. © 2009 Wiley‐Liss, Inc.     

Nuclear Ferritin Protects DNA From UV Damage in Corneal Epithelial Cells

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3′-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.     

Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F

Centromere protein F (CENP-F) is a component of the kinetochore and a regulator of cell cycle progression. CENP-F recruits the dynein transport machinery and orchestrates several cell cycle-specific transport events, including transport of the nucleus, mitochondria and chromosomes. A key regulatory step for several of these functions is likely the G2 phase-specific export of CENP-F from the nucleus to the cytosol, where the cytoplasmic dynein transport machinery resides; however, the molecular mechanism of this process is elusive. Here, we have identified 3 phosphorylation sites within the bipartite classical nuclear localization signal (cNLS) of CENP-F. These sites are specific for cyclin-dependent kinase 1 (Cdk1), which is active in G2 phase. Phosphomimetic mutations of these residues strongly diminish the interaction of the CENP-F cNLS with its nuclear transport receptor karyopherin α. These mutations also diminish nuclear localization of the CENP-F cNLS in cells. Notably, the cNLS is phosphorylated in the ?1 position, which is important to orient the adjacent major motif for binding into its pocket on karyopherin α. We propose that localization of CENP-F is regulated by a cNLS, and a nuclear export pathway, resulting in nuclear localization during most of interphase. In G2 phase, the cNLS is weakened by phosphorylation through Cdk1, likely resulting in nuclear export of CENP-F via the still active nuclear export pathway. Once CENP-F resides in the cytosol, it can engage in pathways that are important for cell cycle progression, kinetochore assembly and the faithful segregation of chromosomes into daughter cells.     

Syndecan-1 and FGF-2, but Not FGF Receptor-1, Share a Common Transport Route and Co-Localize with Heparanase in the Nuclei of Mesenchymal Tumor Cells

Syndecan-1 forms complexes with growth factors and their cognate receptors in the cell membrane. We have previously reported a tubulin-mediated translocation of syndecan-1 to the nucleus. The transport route and functional significance of nuclear syndecan-1 is still incompletely understood. Here we investigate the sub-cellular distribution of syndecan-1, FGF-2, FGFR-1 and heparanase in malignant mesenchymal tumor cells, and explore the possibility of their coordinated translocation to the nucleus. To elucidate a structural requirement for this nuclear transport, we have transfected cells with a syndecan-1/EGFP construct or with a short truncated version containing only the tubulin binding RMKKK sequence. The sub-cellular distribution of the EGFP fusion proteins was monitored by fluorescence microscopy. Our data indicate that syndecan-1, FGF-2 and heparanase co-localize in the nucleus, whereas FGFR-1 is enriched mainly in the perinuclear area. Overexpression of syndecan-1 results in increased nuclear accumulation of FGF-2, demonstrating the functional importance of syndecan-1 for this nuclear transport. Interestingly, exogenously added FGF-2 does not follow the route taken by endogenous FGF-2. Furthermore, we prove that the RMKKK sequence of syndecan-1 is necessary and sufficient for nuclear translocation, acting as a nuclear localization signal, and the Arginine residue is vital for this localization. We conclude that syndecan-1 and FGF-2, but not FGFR-1 share a common transport route and co-localize with heparanase in the nucleus, and this transport is mediated by the RMKKK motif in syndecan-1. Our study opens a new perspective in the proteoglycan field and provides more evidence of nuclear interactions of syndecan-1.     

cGMP-dependent protein kinase I gamma encodes a nuclear localization signal that regulates nuclear compartmentation and function

cGMP-dependent protein kinase I (PKGI) plays an important role in regulating how cGMP specifies vascular smooth muscle cell (SMC) phenotype. Although studies indicate that PKGI nuclear localization controls how cGMP regulates gene expression in SMC, information about the mechanisms that regulate PKGI nuclear compartmentation and its role in directly regulating cell phenotype is limited. Here we characterize a nuclear localization signal sequence (NLS) in PKGIγ, a proteolytically cleaved PKGI kinase fragment that translocates to the nucleus of SMC. Immuno-localization studies using cells expressing native and NLS-mutant PKGIγ, and treated with a small molecule nuclear transport inhibitor, indicated that PKGIγ encodes a constitutively active NLS that requires importin α and β for regulation of its compartmentation. Moreover, studies utilizing a genetically encoded nuclear phospho-CREB biosensor probe and fluorescence lifetime imaging microscopy demonstrated that this NLS controls PKGIγ nuclear function. In addition, although cytosolic PKGIγ-activity was observed to stimulate MAPK/ERK-mediated nuclear CREB signaling in SMC, NLS-mediated PKGIγ nuclear activity alone was determined to increase the expression of differentiation marker proteins in these cells. These results indicate that NLS-mediated nuclear PKGIγ localization plays an important role in how PKGI regulates vascular SMC phenotype.     

AKT can be activated in the nucleus

To investigate issues about AKT/PKB nuclear localization in cells, we examined endogenous or transiently transfected AKT localization in cancer cell lines by immunofluorescence. We found that AKT can be detected in both the nucleus and cytoplasm of HEK 293, HeLa and MCF7E cells. It was found that an active process mediates AKT nuclear translocation as shown by fusing AKT with GFP3 protein. The cellular distribution pattern of serial deletion mutants from GFP3-HA-AKT revealed that more than one segment of AKT is required for AKT nuclear translocation, while the individual segment does not have any apparent nuclear transport activity. These results implied that the signal mediating AKT nuclear translocation is conformation dependent, or more likely, is dependent upon association with other proteins. It was also found that AKT does not contain any apparent nuclear export signal. Furthermore, we found that nuclear AKT was activated in MCF7E cells upon stimulation. The possibility that nuclear activated AKT was translocated from the cytoplasm was excluded through the generation of a chimeric AKT protein, in which a strong nuclear localization signal was fused to the C-terminal of AKT.     

The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis.

We have previously described the fact that the individual expression of influenza virus PA protein induced a generalized proteolysis (J.J. Sanz-Ezquerro, S. de la Luna, Ortin, and A. Nieto, J. Virol. 69:2420-2426, 1995). In this study, we have further characterized this effect by mapping the regions of PA protein required and have found by deletion analysis that the first 247 amino acids are sufficient to bring about this activity. PA mutants that were able to decrease the accumulation levels of coexpressed proteins also presented lower steady-state levels due to a reduction in their half-lives. Furthermore, the PA wild type produced a decrease in the stationary levels of different PA versions, indicating that is itself a target for its induced proteolytic process. All of the PA proteins that induced proteolysis presented nuclear localization, being the sequences responsible for nuclear transport located inside the first 247 amino acids of the molecule. To distinguish between the regions involved in nuclear localization and those involved in induction of proteolysis, we fused the nuclear localization signal of the simian virus 40 T antigen to the carboxy terminus of the cytosolic versions of PA. None of the cytosolic PA versions affected in the first 247-amino-acid part of PA, which were now located in the nucleus, were able to induce proteolysis, suggesting that conservation of a particular conformation in this region of the molecule is required for the effect observed. The fact that all of the PA proteins able to induce proteolysis presented nuclear localization, together with the observation that this activity is shared by influenza virus PA proteins from two different type A viruses, suggests a physiological role for this PA protein activity in viral infection.     

Dimerization of MLH1 and PMS2 limits nuclear localization of MutLalpha

DNA mismatch repair maintains genomic stability by detecting and correcting mispaired DNA sequences and by signaling cell death when DNA repair fails. The mechanism by which mismatch repair coordinates DNA damage and repair with cell survival or death is not understood, but it suggests the need for regulation. Since the functions of mismatch repair are initiated in the nucleus, we asked whether nuclear transport of MLH1 and PMS2 is limiting for the nuclear localization of MutLalpha (the MLH1-PMS2 dimer). We found that MLH1 and PMS2 have functional nuclear localization signals (NLS) and nuclear export sequences, yet nuclear import depended on their C-terminal dimerization to form MutLalpha. Our studies are consistent with the idea that dimerization of MLH1 and PMS2 regulates nuclear import by unmasking the NLS. Limited nuclear localization of MutLalpha may thus represent a novel mechanism by which cells fine-tune mismatch repair functions. This mechanism may have implications in the pathogenesis of hereditary non-polyposis colon cancer.     

The E1 replication protein of bovine papillomavirus type 1 contains an extended nuclear localization signal that includes a p34cdc2 phosphorylation site.

Bovine papillomavirus (BPV) DNA replication occurs in the nucleus of infected cells. Most enzymatic activities are carried out by host cell proteins, with the viral E1 and E2 proteins required for the assembly of an initiation complex at the replication origin. In latently infected cells, viral DNA replication occurs in synchrony with the host cell chromosomes, maintaining a constant average copy number of BPV genomes per infected cell. By analyzing a series of mutants of the amino-terminal region of the E1 protein, we have identified the signal for transport of this protein to the cell nucleus. The E1 nuclear transport motif is highly conserved in the animal and human papillomaviruses and is encoded in a similar region in the related E1 genes. The signal is extended relative to the simple nuclear localization signals and contains two short amino acid sequences which contribute to nuclear transport, located between amino acids 85 and 108 of the BPV-1 E1 protein. Mutations in either basic region reduce nuclear transport of E1 protein and interfere with viral DNA replication. Mutations in both sequences simultaneously prevent any observable accumulation of the protein and reduce replication in transient assays to barely detectable levels. Surprisingly, these mutations had no effect on the ability of viral genomes to morphologically transform cells, although the plasmid DNA in the transformed cells was maintained at a very low copy number. Between these two basic amino acid blocks in the nuclear transport signal, at threonine 102, is a putative site for phosphorylation by the cell cycle regulated kinase p34cdc2. Utilizing an E1 protein purified from either a baculovirus vector system or Escherichia coli, we have shown that the E1 protein is a substrate for this kinase. An E1 gene mutant at threonine 102 encodes for a protein which is no longer a substrate for the p34cdc2 kinase. Mutation of this threonine to isoleucine had no observable effect on either nuclear localization of E1 or DNA replication of the intact viral genome.     

Functional analysis of the yeast Ran exchange factor Prp20p: in vivo evidence for the RanGTP gradient model

Numerous cellular processes rely on the movement of macromolecules into and out of the nucleus. The primary regulator of this movement is the small GTPase Ran. Like other small GTPases, the nucleotide-bound state of Ran is regulated by effectors that enhance the rate of nucleotide exchange or hydrolysis. Current models for vectorial nuclear transport suggest that it is the strict compartmentalization of these Ran effector molecules that generates a gradient of RanGTP between the nucleus and the cytoplasm to impart directionality to the transport process. Here we investigate the mechanism by which the Ran exchange factor is targeted to the nucleus, and test the impact of disrupting this nuclear compartmentalization on nucleocytoplasmic transport in vivo. Our results indicate that in Saccharomycces cerevisiae the nucleotide exchange factor Prp20p can be targeted to the nucleus via a classical nuclear localization sequence. This transport mechanism is dependent both on Ran and the receptor that recognizes the nuclear localization sequence, importin alpha. Mutations in the evolutionarily conserved nuclear localization sequence only partially inhibit nuclear import of Prp20p, suggesting the existence of a secondary mechanism for this critical nuclear targeting. In an in vivo test of the RanGTP gradient model, we demonstrate that overexpression of a functional cytoplasmic exchange factor inhibits cell growth and blocks both protein import and RNA export in wild-type cells that contain the endogenous nuclear Prp20 protein. Taken together, our results provide in vivo evidence for the idea that the compartmentalization of the exchange factor serves as a mechanism for establishing directional nuclear transport.     

Bipartite signal sequence mediates nuclear translocation of the plant potyviral NIa protein.

The NIa protein of certain plant potyviruses localizes to the nucleus of infected cells. Previous studies have shown that linkage of NIa to reporter protein beta-glucuronidase (GUS) is sufficient to direct GUS to the nucleus in transfected protoplasts and in cells of transgenic plants. In this study, we mapped sequences in NIa that confer karyophilic properties. A quantitative transport assay using transfected protoplasts, as well as in situ localization technique using epidermal cells from transgenic plants, were employed. Two domains within NIa, one between amino acid residues 1 to 11 (signal domain I) and the other between residues 43 to 72 (signal domain II), were found to function additively for efficient localization of fusion proteins to the nucleus, although either region independently could facilitate a low level of translocation. Like signals from animal cells, both nuclear transport domains of NIa contain a high concentration of basic (arginine and lysine) residues. Nuclear transport signal domain II overlaps or is very near Tyr62, which is the residue that mediates covalent attachment of a subset of NIa molecules to the 5' terminus of viral RNA within infected cells. The nature of the NIa nuclear transport signal and the possibility for regulation of NIa translocation are discussed.     

A high-content chemical screen identifies ellipticine as a modulator of p53 nuclear localization

p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFP-mutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified a molecule that shifts the localization of p53 and enhances its nuclear activity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. G. Wei Xu and Imtiaz A. Mawji have contributed equally to this work.     

针对核质运输的高通量小分子筛选方法

核质运输是真核细胞的重大基本生命活动。核质运输小分子抑制剂不仅可以广泛应用并促进核质运输相关的基础研究,同时也为相关疾病、尤其是病毒性疾病的药物开发提供有利线索。然而,目前针对核质运输的商业化小分子仅有Leptomycin B一种。建立一个针对整个核质运输通路的小分子筛选平台,将有利于筛选与获得多种干扰核质运输的小分子。文章利用NZGFP和CZGFP可以重组为具有荧光GFP的特性,构建NZGFP-NES和CZGFP-NLS,将NZGFP和CZGFP分别定位在细胞质与细胞核中;当核内运或核外运通路被干扰,NZGFP和CZGFP定位发生改变并聚集重组为具有荧光的GFP。该方法可以有效检测核外运小分子抑制剂LeptomycinB的作用,为针对整个核质运输通路的高通量小分子筛选提供了一个有效平台。     

Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein.

The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region,90RKKR93, is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC50values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence specificity. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insufficient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and specifically during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic trafficking and not neccessarily as mediators of nuclear import.     

Cytoplasmic domain signal sequences that mediate transport of varicella-zoster virus gB from the endoplasmic reticulum to the Golgi

Normal herpesvirus assembly and egress depend on the correct intracellular localization of viral glycoproteins. While several post-Golgi transport motifs have been characterized within the cytoplasmic domains of various viral glycoproteins, few specific endoplasmic reticulum (ER)-to-Golgi transport signals have been described. We report the identification of two regions within the 125-amino-acid cytoplasmic domain of Varicella-Zoster virus gB that are required for its ER-to-Golgi transport. Native gB or gB containing deletions and specific point mutations in its cytoplasmic domain was expressed in mammalian cells. ER-to-Golgi transport of gB was assessed by indirect immunofluorescence and by the acquisition of Golgi-dependent posttranslational modifications. These studies revealed that the ER-to-Golgi transport of gB requires a nine-amino-acid region (YMTLVSAAE) within its cytoplasmic domain. Mutations of individual amino acids within this region markedly impaired the transport of gB from the ER to the Golgi, indicating that this domain functions by a sequence-dependent mechanism. Deletion of the C-terminal 17 amino acids of the gB cytoplasmic domain was also shown to impair the transport of gB from the ER to the Golgi. However, internal mutations within this region did not disrupt the transport of gB, indicating that its function during gB transport is not sequence dependent. Native gB is also transported to the nuclear membrane of transfected cells. gB lacking as many as 67 amino acids from the C terminus of its cytoplasmic domain continued to be transported to the nuclear membrane at apparently normal levels, indicating that the cytoplasmic domain of gB is not required for nuclear membrane localization.