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PubMed2004

SKAR is a specific target of S6 kinase 1 in cell growth control.

BACKGROUND: The mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) signaling pathways promote cell growth and cell cycle progression in response to nutritional, energy, and mitogenic cues. In mammalian cells, the ribosomal protein S6 kinases, S6K1 and S6K2, lie downstream of mTOR and PI3K, suggesting that translational control through the phosphorylation of S6 regulates cell growth. Interestingly, genetic experiments predict that a substrate that is specific to S6K1 but not S6K2 regulates cell growth. RESULTS: Here we identify SKAR as a novel and specific binding partner and substrate of S6K1 but not S6K2. We find that serines 383 and 385 of human SKAR are insulin-stimulated and rapamycin-sensitive S6K1 phosphorylation sites. Quantitative mass spectrometry reveals that serine 383/385 phosphorylation is sensitive to RNA interference (RNAi)-mediated S6K1 reduction, but not S6K2 reduction. Furthermore, RNAi-mediated reduction of SKAR decreases cell size. SKAR is nuclear protein with homology to the Aly/REF family of RNA binding proteins, which has been proposed to couple transcription with pre-mRNA splicing and mRNA export. CONCLUSIONS: We have identified a novel and specific target of S6K1, SKAR, which regulates cell growth. The homology of SKAR to the Aly/REF family links S6K1 with mRNA biogenesis in the control of cell growth.

Amino Acid Sequence↗
PubMed2003

The effects of ITIM-bearing FcgammaRIIB on the nuclear shuttling of MAP kinase in RBL-2H3 cells.

We have studied the effects of ITIM-bearing FcgammaRIIB2 on the FcepsilonRI-dependent nuclear shuttling of mitogen-activated protein (MAP) kinase (ERK2) in rat basophilic leukemia (RBL-2H3) cells. The cross-linking of FcepsilonRI elicited the sustained increase of the intracellular calcium ion concentration ([Ca(2+)](i)) and the translocation of ERK2 from the cytoplasm to the nucleus. The import of ERK2 to the nucleus reached the maximum at 6-7 min, thereafter ERK2 was exported within 30 min. The co-clustering of FcepsilonRI and FcgammaRIIB2 increased the [Ca(2+)](i) and induced the import of ERK2. However, the calcium increase was transient and ERK2 was rapidly exported to the cytoplasm. In addition, the phosphorylation of ERK2 and the production of TNF-alpha were decreased in case of co-clustering of FcepsilonRI and FcgammaRIIB2. This suggested that the co-clustering negatively control the production of pro-inflammatory cytokines through the suppression of nuclear shuttling of ERK2.

Animals↗
PubMed2001

Phosphorylation regulates the nucleocytoplasmic distribution of kinase suppressor of Ras.

KSR (kinase suppressor of Ras) has been proposed as a molecular scaffold regulating the Raf/MEK/ERK kinase cascade. KSR is phosphorylated on multiple phosphorylation sites by associated kinases. To identify potential mechanisms used by KSR to regulate ERK activation, green fluorescent protein was fused to intact and mutated KSR constructs lacking specific phosphorylation sites, and the subcellular distribution of each construct was observed in live cells. Mutation of a subset of KSR phosphorylation sites caused the redistribution of KSR to the nucleus. To determine whether intact KSR is normally imported to the nucleus, REF-52 fibroblasts expressing KSR were treated with 10 nm leptomycin B, which inhibits Crm1-dependent nuclear export. KSR accumulated in the nucleus within 2 h of treatment with leptomycin B, suggesting that KSR cycles continuously through the nucleus. Nuclear import of KSR was blocked by mutations that inhibit the interaction of KSR with MEK. Coexpression of fluorescent forms of KSR and MEK in cells revealed that each protein promoted the localization of the other in the cytoplasm. These data indicate that the subcellular distribution of KSR is dynamically regulated through phosphorylation and MEK interaction in a manner that may affect signaling through ERK.

Active Transport, Cell Nucleus↗
PubMed2002

Functional characterization of multiple domains involved in the subcellular localization of the hematopoietic Pbx interacting protein (HPIP).

We have previously reported the cloning of the Hematopoietic Pbx Interacting Protein (HPIP), a novel protein discovered through its interaction with Pbx1. HPIP is expressed in early hematopoietic precursors, can bind all members of the Pbx family and can inhibit the transcriptional activation of the oncogene E2A-Pbx. To further understand the function of HPIP, we have analysed its cellular localization and characterized its functional localization domains. Using fluorescence microscopy to follow the distribution of different HPIP sequences fused to GFP, we found that HPIP localizes predominantly to cytoskeletal fibers but has the potential ability to shuttle between the nucleus and the cytosol. The cytoskeletal localization of HPIP is mediated by an N-terminal leucine rich region (between aa 190-218) and can be disrupted by the microtubule destabilizing drug vincristine. The HPIP C-terminal domain (aa 443-731) bears a nuclear export activity that is blocked by the CRM1 inhibitor Leptomycin B. In addition, we found two basic amino acid regions located between aa 485-505 and aa 695-720 that contain nuclear import activities attenuated by nuclear export. These observations support a model in which the constitutive attachment of HPIP to the cytoskeleton could be modified by changes in functional domains implicated in nuclear export, import and cytoskeleton binding sequences, allowing the molecule to shuttle between the nucleus and the cytosol.

3T3 Cells↗
PubMed1999

L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons.

The molecular basis of learning and memory has been the object of several recent advances, which have focused attention on calcium-regulated pathways controlling transcription. One of the molecules implicated by pharmacological, biochemical and genetic approaches is the calcium/calmodulin-regulated phosphatase, calcineurin. In lymphocytes, calcineurin responds to specific calcium signals and regulates expression of several immediate early genes by controlling the nuclear import of the NF-ATc family of transcription factors. Here we show that NF-ATc4/NF-AT3 in hippocampal neurons can rapidly translocate from cytoplasm to nucleus and activate NF-AT-dependent transcription in response to electrical activity or potassium depolarization. The calcineurin-mediated translocation is critically dependent on calcium entry through L-type voltage-gated calcium channels. GSK-3 can phosphorylate NF-ATc4, promoting its export from the nucleus and antagonizing NF-ATc4-dependent transcription. Furthermore, we show that induction of the inositol 1,4,5-trisphosphate receptor type 1 is controlled by the calcium/calcineurin/NF-ATc pathway. This provides a new perspective on the function of calcineurin in the central nervous system and indicates that NF-AT-mediated gene expression may be involved in the induction of hippocampal synaptic plasticity and memory formation.

Biological Transport↗
PubMed2004

Inositol diphosphate signaling regulates telomere length.

Activation of phospholipase C-dependent inositol polyphosphate signaling pathways generates distinct messengers derived from inositol 1,4,5-trisphosphate that control gene expression and mRNA export. Here we report the regulation of telomere length by production of a diphosphorylinositol tetrakisphosphate, PP-IP4, synthesized by the KCS1 gene product. Loss of PP-IP4 production results in lengthening of telomeres, whereas overproduction leads to their shortening. This effect requires the presence of Tel1, the yeast homologue of ATM, the protein mutated in the human disease ataxia telangiectasia. Our data provide in vivo evidence of a regulatory link between inositol polyphosphate signaling and the checkpoint kinase family and describe a third nuclear process modulated by phospholipase C activation.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1999

The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human.

Human TAP is an orthologue of the yeast mRNA export factor Mex67p. In mammalian cells, TAP has a preferential intranuclear localization, but can also be detected at the nuclear pores and shuttles between the nucleus and the cytoplasm. TAP directly associates with mRNA in vivo, as it can be UV-crosslinked to poly(A)+ RNA in HeLa cells. Both the FG-repeat domain of nucleoporin CAN/Nup214 and a novel human 15 kDa protein (p15) with homology to NTF2 (a nuclear transport factor which associates with RanGDP), directly bind to TAP. When green fluorescent protein (GFP)-tagged TAP and p15 are expressed in yeast, they localize to the nuclear pores. Strikingly, co-expression of human TAP and p15 restores growth of the otherwise lethal mex67::HIS3/mtr2::HIS3 double knockout strain. Thus, the human TAP-p15 complex can functionally replace the Mex67p-Mtr2p complex in yeast and thus performs a conserved role in nuclear mRNA export.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by HIV-1-based lentivirus vectors expressing transdominant Rev.

Retrovirus vectors expressing transdominant-negative mutants of Rev (TdRev) inhibit human immunodeficiency virus type 1 (HIV-1) replication by preventing the nuclear export of unspliced viral transcripts, thus inhibiting the synthesis of Gag-Pol, Env, and genomic RNA. The use of HIV-1-based vectors to express TdRev would have the advantage of allowing access to nondividing hematopoietic cells. It would also provide additional levels of protection by sequestering the viral regulatory proteins Tat and Rev, competing for encapsidation into wild-type virions, and inhibiting reverse transcription. Here we describe HIV-1-based vectors that express TdRev. These vectors contain mutations in the splicing signals or replacement of the Rev-responsive element by the simian retrovirus type 1 constitutive transport element, making them less sensitive to the inhibitory effects of TdRev. In addition, overexpression of Rev and the use of an HIV-1 helper plasmid that drives high levels of Gag-Pol synthesis were used to transiently overcome the inhibition by TdRev of the synthesis of Gag-Pol during vector production. SupT1 cells transduced with these vectors were more resistant to HIV-1 replication than cells transduced with Moloney murine leukemia virus-based vectors expressing TdRev. Furthermore, we show that these vectors can be mobilized by the wild-type virus, reducing the infectivity of virions escaping inhibition and conferring protection against HIV-1 replication to previously untransduced cells.

Blotting, Southern↗
PubMed2004

Interaction of 14-3-3 protein with Chk1 affects localization and checkpoint function.

The protein kinase Chk1 is required for proper arrest of the cell cycle in response to DNA damage. We have previously shown in Schizosaccharomyces pombe, that upon DNA damage, phosphorylation of Chk1 correlates with checkpoint activation and that phosphorylated Chk1 is capable of interacting with the 14-3-3 proteins, Rad24 and Rad25. The interaction between Rad24 and Chk1 is stimulated tenfold after exposure to DNA damaging agents and we postulate that it is an important event in the DNA damage checkpoint response pathway in fission yeast. We identified a stretch of leucine residues as the domain in Chk1 that mediates the interaction with 14-3-3 proteins. Substitution of leucine residues with alanine disrupts the interaction with Rad24 and also prevents Chk1 from becoming phosphorylated in response to DNA damaging agents. Cells expressing the mutants are sensitive to UV radiation. In this study, we also show that Chk1 accumulates in the nucleus in response to DNA damage and this behavior is dependent on Rad24. Interestingly, the 14-3-3 binding domain mutants also fail to localize to the nucleus prompting a search for localization sequences within Chk1. Our investigations have identified the presence of both functional nuclear import and nuclear export sequences encoded in S. pombe Chk1 that, in conjunction with 14-3-3 proteins, may play a prominent role in regulating Chk1 localization and function.

14-3-3 Proteins↗
PubMed2005

Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor-induced Nur77 expression and apoptosis.

The molecular basis of thymocyte negative selection, a crucial mechanism in establishing central tolerance, is not yet resolved. Histone deacetylases (HDACs) have emerged as key transcriptional regulators in several major developmental programs. Recently, we showed that the class IIa member, HDAC7, regulates negative selection by repressing expression of Nur77, an orphan nuclear receptor involved in antigen-induced apoptosis of thymocytes. Engagement of the T cell receptor (TCR) alleviates this repression through phosphorylation-dependent nuclear exclusion of HDAC7. However, the identity of the TCR-activated kinase that phosphorylates and inactivates HDAC7 was still unknown. Here, we demonstrate that TCR-induced nuclear export of HDAC7 and Nur77 expression is mediated by activation of protein kinase D (PKD). Indeed, active PKD stimulates HDAC7 nuclear export and Nur77 expression. In contrast, inhibition of PKD prevents TCR-mediated nuclear exclusion of HDAC7 and associated Nur77 activation. Furthermore, we show that HDAC7 is an interaction partner and a substrate for PKD. We identify four serine residues in the NH(2) terminus of HDAC7 as targets for PKD. More importantly, a mutant of HDAC7 specifically deficient in phosphorylation by PKD, inhibits TCR-mediated apoptosis of T cell hybridomas. These findings indicate that PKD is likely to play a key role in the signaling pathways controlling negative selection.

Active Transport, Cell Nucleus↗
PubMed2000

The 5' RNA terminus of spleen necrosis virus stimulates translation of nonviral mRNA.

The RU5 region at the 5' RNA terminus of spleen necrosis virus (SNV) has been shown to facilitate expression of human immunodeficiency virus type 1 (HIV) unspliced RNA independently of the Rev-responsive element (RRE) and Rev. The SNV sequences act as a distinct posttranscriptional control element to stimulate gag RNA nuclear export and association with polyribosomes. Here we sought to determine whether RU5 functions to neutralize the cis-acting inhibitory sequences (INSs) in HIV RNA that confer RRE/Rev dependence or functions as an independent stimulatory sequence. Experiments with HIV gag reporter plasmids that contain inactivated INS-1 indicated that neutralization of INSs does not account for RU5 function. Results with luciferase reporter gene (luc) plasmids further indicated that RU5 stimulates expression of a nonretroviral RNA that lacks INSs. Northern blot and RT-PCR analyses indicated that RU5 does not increase the steady-state levels or nuclear export of the luc transcript but rather that the U5 region facilitates efficient polyribosomal association of the mRNA. RU5 does not function as an internal ribosome entry site in bicistronic reporter plasmids, and it requires the 5'-proximal position for efficient function. Our results indicate that RU5 contains stimulatory sequences that function in a 5'-proximal position to enhance initiation of translation of a nonretroviral reporter gene RNA. We speculate that RU5 evolved to overcome the translation-inhibitory effect of the highly structured encapsidation signal and other replication motifs in the 5' untranslated region of the retroviral RNA.

5' Untranslated Regions↗
PubMed2003

PKB/Akt phosphorylates the CDC25B phosphatase and regulates its intracellular localisation.

Regulation of the intracellular localisation of its actors is one of the key mechanisms underlying cell cycle control. CDC25 phosphatases are activators of Cyclin-Dependent Kinases (CDK) that undergo nucleo-cytoplasmic shuttling during the cell cycle and in response to checkpoint activation. Here we report that the protein kinase PKB/Akt phosphorylates CDC25B on serine 353, resulting in a nuclear export-dependent cytoplasmic accumulation of the phosphatase. Oxidative stress activates PKB/Akt and reproduces the effect on CDC25B phosphorylation and localisation. However, inhibition of PKB/Akt activity only partially reverted the effect of oxidative stress on CDC25B localisation and mutation of serine 353 abolishes phosphorylation but only delays nuclear exclusion. These results indicate that additional mechanisms are also involved in preventing nuclear import of CDC25B. Our findings identify CDC25B as a target of PKB/Akt and provide new insight into the regulation of its localisation in response to stress-activated signalling pathways.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2000

New steps toward the nucleocytoplasmic traffic of macromolecules.

The nucleocytoplasmic transport of functional molecules is mediated bidirectionally through the nuclear pore complex (NPC), which spans the double membranes of the nuclear envelope. It has recently been shown that signaling between the nucleus and the cytoplasm plays a key role in coordinating the cellular processes such as the cell cycle and cell differentiation (Yoneda, 2000). As the result of recent extensive analysis, significant progress has been made in our understanding of the fundamental mechanism of nuclear transport of proteins and RNAs and numerous transport factors have now been identified. In this special issue of review articles, we focus on our rapid growing knowledge of nucleocytoplasmic transport, especially the translocation of proteins through the NPC and mRNA export, and review this exciting field from various points of view including cell biology, structural biology and yeast genetics.

Active Transport, Cell Nucleus↗
PubMedDate not supplied

A symbiont-produced protein and bacterial symbiosis in Amoeba proteus.

Gram symbiotic X-bacteria present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29-kDa protein (S29x) into the host's cytoplasm across bacterial and symbiosome membranes. The S29x protein produced by E. coli transformed with the s29x gene is also rapidly secreted into the culture medium. Inside amoebae, S29x enters the host's nucleus as detected by confocal and immunoelectron microscopy, although it is not clear if S29x is selectively accumulated inside the nucleus. The deduced amino-acid sequence of S29x has a stretch of basic amino acids that could act as a nuclear localization signal, but there is no signal peptide at the N-terminus and the transport of S29x is energy independent. The functions of S29x are not known, but in view of its prominent presence inside the amoeba's nucleus, S29x is suspected to be involved in affecting the expression of amoeba's nuclear gene(s).

Amino Acid Sequence↗
PubMed2002

Nucleocytoplasmic transport: cargo trafficking across the border.

Transport of macromolecules between the cytoplasm and the nucleus is mediated by at least three different classes of soluble transport receptors, members of the importin-beta protein family, the Mex67/Tap family and the small nuclear transport factor 2 (NFT2). All nuclear transport factors can bidirectionally traverse the nuclear pore complex through specific interactions with phenylalanine/glycine-rich nuclear pore complex components. Recent kinetic and structural analyses revealed novel insight into the details of these interactions. In addition, new biochemical and genetic studies have dramatically improved our understanding of ribosomal and messenger RNA export, unveiling a tight coupling between RNA processing and transport.

Active Transport, Cell Nucleus↗
PubMed1998

Nuclear import and nucleolar accumulation of the human ribosomal protein S7 depends on both a minimal nuclear localization sequence and an adjacent basic region.

In the course of the eukaryotic ribosomal biogenesis, both the nuclear import and export are involved. We have studied the nuclear and nucleolar localization of the human ribosomal protein S7. We examined the subcellular distribution of the S7:beta-galactosidase fusion protein in SAOS-2 cells. We have identified two evolutionarily conserved domains, both of which are necessary for S7 nuclear and nucleolar targeting: amino acids 98 to 109 and 115 to 118. Out of the S7 protein context, a fragment 98...118, containing these domains, is sufficient for nuclear transport and nucleolar accumulation. Interestingly, a tetrapeptide 115KRPR118, which can act as an independent nuclear localization signal (NLS), is not sufficient for exclusively nuclear accumulation of the S7 protein if the adjacent region 98...109 is deleted. In addition, site-directed mutagenesis revealed that critical residues for nuclear targeting in this tetrapeptide and in the full-length S7 protein are different. While mutation of a Pro117 significantly impaired nuclear import of S7, similar substitution within the tetrapeptide-NLS had no effect on nuclear targeting. This suggests that to function perfectly, proper secondary structure of the S7 nuclear targeting domain is required.

Amino Acid Sequence↗
PubMed1997

An independent software system for the analysis of dynamic MR images.

PURPOSE: A computer system for the manual, semi-automatic, and automatic analysis of dynamic MR images was to be developed on UNIX and personal computer platforms. The system was to offer an integrated and standardized way of performing both image processing and analysis that was independent of the MR unit used. MATERIAL AND METHODS: The system consists of modules that are easily adaptable to special needs. Data from MR units or other diagnostic imaging equipment in techniques such as CT, ultrasonography, or nuclear medicine can be processed through the ACR-NEMA/DICOM standard file formats. A full set of functions is available, among them cine-loop visual analysis, and generation of time-intensity curves. Parameters such as cross-correlation coefficients, area under the curve, peak/maximum intensity, wash-in and wash-out slopes, time to peak, and relative signal intensity/contrast enhancement can be calculated. Other parameters can be extracted by fitting functions like the gamma-variate function. Region-of-interest data and parametric values can easily be exported. RESULTS AND CONCLUSION: The system has been successfully tested in animal and patient examinations.

Algorithms↗
PubMed2000

Aldosterone and nuclear volume cycling.

The response of target cells to the steroid hormone aldosterone has been divided into acute nongenomic (< 10 min) and sustained genomic (> 10 min) action. In the light of recent experiments this nomenclature does not hold anymore and should be abandoned. By applying atomic force microscopy (AFM) we observed in living endothelial cells that aldosterone induces cell volume increase in less than 10 minutes. The cell nucleus was identified as the swelling site. Hormone-induced nuclear swelling can reach 15 to 28% of total cell volume dissipating within 30 minutes. This phenomenon could have functional impact on flow resistance in small blood vessels. AFM-investigation of the intracellular signal pathway in nuclear envelope of aldosterone-injected Xenopus laevis oocytes visualizes putative intracellular receptors (40 kD granules) bound to nuclear pores 2 minutes after hormone injection, with subsequent macromolecule translocation into the nucleus. 15 minutes later macromolecules (800 kD plugs) appear in the central channels of the nuclear pores. The plugs resemble ribonucleoproteins that carry the aldosterone-induced mRNA to the ribosomes. We postulate that steroid-induced nuclear swelling is caused by a shift of receptors/transcription factors from cytoplasm into nucleoplasm followed by gene transcription. Nuclear volume returns to normal when mRNA export through the nuclear pores is finished. Thus, steroid-induced net-movements of macromolecules between intracellular compartments initiate shifts in cell volume compensated by volume regulatory transporters and ion channels in the plasma membrane.

Aldosterone↗