Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Phosphorylation within the bipartite NLS alters the localization and toxicity of the ER stress response factor DDIT3/CHOP.

ABSTRACT: Regulated nuclear-cytoplasmic trafficking is a well-established mechanism utilized by cells to regulate adaptive and maladaptive responses to acute oxidant stress. Commonly associated with endoplasmic reticulum stress, the bZIP transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP/DDIT3) mediates the cellular response to redox stress with effects on cellular growth, differentiation, and survival. We show through functional analyses that CHOP contains a conserved, compound pat4/bipartite nuclear localization signal within the basic DNA-binding domain. Using phylogenetic analyses and mass spectrometry, we now show that Ser107 located within the linker region of the bipartite NLS domain is a substrate for phosphorylation under standard culture conditions. Studies using the S107E phospho-mimic of CHOP indicate that changes in the charge properties at this residue regulate CHOP's nuclear-to-cytoplasmic ratio. And while co-stimulation with the SERCA inhibitor thapsigargin induced injury in cells expressing wild-type CHOP, the S107A point-mutant blocked this response. These findings indicate that phosphorylation within the bipartite NLS exerts regulatory effects on both the subcellular localization and toxic potential of DDIT3/CHOP. Future studies geared towards defining the relevant kinase/phosphatase networks that converge on the phosphorylation-regulated NLS (prNLS) phosphoepitope may provide an opportunity to constrain cellular damage in the context of acute ER stress.

SUBMITTER: Bartko JC

PROVIDER: S-EPMC7484389 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Chop/Ddit3 depletion in β cells alleviates ER stress and corrects hepatic steatosis in mice.

Project description:Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, hyperinsulinemia, and insulin resistance (IR). During the early phase of T2D, insulin synthesis and secretion by pancreatic β cells is enhanced, which can lead to proinsulin misfolding that aggravates endoplasmic reticulum (ER) protein homeostasis in β cells. Moreover, increased circulating insulin may contribute to fatty liver disease. Medical interventions aimed at alleviating ER stress in β cells while maintaining optimal insulin secretion are therefore an attractive therapeutic strategy for T2D. Previously, we demonstrated that germline Chop gene deletion preserved β cells in high-fat diet (HFD)-fed mice and in leptin receptor-deficient db/db mice. In the current study, we further investigated whether targeting Chop/Ddit3 specifically in murine β cells conferred therapeutic benefits. First, we showed that Chop deletion in β cells alleviated β cell ER stress and delayed glucose-stimulated insulin secretion (GSIS) in HFD-fed mice. Second, β cell-specific Chop deletion prevented liver steatosis and hepatomegaly in aged HFD-fed mice without affecting basal glucose homeostasis. Third, we provide mechanistic evidence that Chop depletion reduces ER Ca2+ buffering capacity and modulates glucose-induced islet Ca2+ oscillations, leading to transcriptional changes of ER chaperone profile ("ER remodeling"). Last, we demonstrated that a GLP1-conjugated Chop antisense oligonucleotide strategy recapitulated the reduction in liver triglycerides and pancreatic insulin content. In summary, our results demonstrate that Chop depletion in β cells provides a therapeutic strategy to alleviate dysregulated insulin secretion and consequent fatty liver disease in T2D.

| S-EPMC8557800 | biostudies-literature

Nuclear Localization of the DNA Repair Scaffold XRCC1: Uncovering the Functional Role of a Bipartite NLS.

Project description:We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior provides a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.

| S-EPMC4548243 | biostudies-literature

Distinct cytoplasmic and nuclear functions of the stress induced protein DDIT3/CHOP/GADD153.

Project description:DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders.

| S-EPMC3322118 | biostudies-literature

An actin-regulated importin ?/?-dependent extended bipartite NLS directs nuclear import of MRTF-A.

Project description:Myocardin-related transcription factors (MRTFs) are actin-regulated transcriptional coactivators, which bind G-actin through their N-terminal RPEL domains. In response to signal-induced actin polymerisation and concomitant G-actin depletion, MRTFs accumulate in the nucleus and activate target gene transcription through their partner protein SRF. Nuclear accumulation of MRTFs in response to signal is inhibited by increased G-actin level. Here, we study the mechanism by which MRTF-A enters the nucleus. We show that MRTF-A contains an unusually long bipartite nuclear localisation signal (NLS), comprising two basic elements separated by 30 residues, embedded within the RPEL domain. Using siRNA-mediated protein depletion in vivo, and nuclear import assays in vitro, we show that the MRTF-A extended bipartite NLS uses the importin (Imp)?/?-dependent import pathway, and that import is inhibited by G-actin. Interaction of the NLS with the Imp?-Imp? heterodimer requires both NLS basic elements, and is dependent on the Imp? major and minor binding pockets. Binding of the Imp?-Imp? heterodimer to the intact MRTF-A RPEL domain occurs competitively with G-actin. Thus, MRTF-A contains an actin-sensitive nuclear import signal.

| S-EPMC2964165 | biostudies-literature

ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif.

Project description:Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kap?2) for its transportation into the nucleus. We identify a potential PY-NLS motif ((774)gpgfgssppGaeaapslRyvPY(795)) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X(2-5)PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kap?2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kap?2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kap?2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kap?2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kap?2 interacts with ULK2 through ULK2's putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.

| S-EPMC4460075 | biostudies-literature

Structural characterisation of the nuclear import receptor importin alpha in complex with the bipartite NLS of Prp20.

Project description:The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-? (IMP?) adaptor protein, which in turn binds to IMP? to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMP? in complex with the NLS of Prp20. We observe 1,091 Å(2) of buried surface area mediated by an extensive array of contacts involving residues on armadillo repeats 2-7, utilising both the major and minor NLS binding sites of IMP? to contact bipartite NLS clusters (17)RAKKMSK(23) and (3)KR(4), respectively. One notable feature of the major site is the insertion of Prp20NLS Ala(18) between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMP? and cargo.

| S-EPMC3858281 | biostudies-literature

Affinity for DNA Contributes to NLS Independent Nuclear Localization of MeCP2.

Project description:MeCP2 is a nuclear protein that is mutated in the severe neurological disorder Rett syndrome (RTT). The ability to target ?-galactosidase to the nucleus was previously used to identify a conserved nuclear localization signal (NLS) in MeCP2 that interacts with the nuclear import factors KPNA3 and KPNA4. Here, we report that nuclear localization of MeCP2 does not depend on its NLS. Instead, our data reveal that an intact methyl-CpG binding domain (MBD) is sufficient for nuclear localization, suggesting that MeCP2 can be retained in the nucleus by its affinity for DNA. Consistent with these findings, we demonstrate that disease progression in a mouse model of RTT is unaffected by an inactivating mutation in the NLS of MeCP2. Taken together, our work reveals an unexpected redundancy between functional domains of MeCP2 in targeting this protein to the nucleus, potentially explaining why NLS-inactivating mutations are rarely associated with disease.

| S-EPMC6130050 | biostudies-literature

Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ.

Project description:HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington's, Parkinson's diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S).

| S-EPMC3406602 | biostudies-literature

ER stress time course (WT vs CHOP-/-)

Project description:Primart mouse embryonic fibroblasts (pass 3 to 4) were treated with tunicamycin (2 micrograms/ml) for specified times - genotypes were WILDTYPE and CHOP-/-. Each time point n=4 Keywords: ordered

2004-12-17 | GSE2082 | GEO

A minimal nuclear localization signal (NLS) in human phospholipid scramblase 4 that binds only the minor NLS-binding site of importin alpha1.

Project description:Importin α1 can bind classical nuclear localization signals (NLSs) in two NLS-binding sites, known as "major" and "minor." The major site is located between ARM repeats 2-4, whereas the minor site spans ARM 7-8. In this study, we have characterized the cellular localization of human phospholipid scramblase 4 (hPLSCR4), a member of the phospholipid scramblase protein family. We identified a minimal NLS in hPLSCR4 ((273)GSIIRKWN(280)) that contains only two basic amino acids. This NLS is both necessary for nuclear localization of hPLSCR4 in transfected HeLa cells and sufficient for nuclear import of a non-diffusible cargo in permeabilized cells. Mutation of only one of the two basic residues, Arg(277), correlates with loss of nuclear localization, suggesting this amino acid plays a key role in nuclear transport. Crystallographic analysis of mammalian importin α1 in complex with the hPLSCR4-NLS reveals this minimal NLS binds specifically and exclusively to the minor binding site of importin α. These data provide the first structural and functional evidence of a novel NLS-binding mode in importin α1 that uses only the minor groove as the exclusive site for nuclear import of nonclassical cargos.

| S-EPMC3151061 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data