scholarly journals Hic-5 Communicates between Focal Adhesions and the Nucleus through Oxidant-Sensitive Nuclear Export Signal

2003 ◽  
Vol 14 (3) ◽  
pp. 1158-1171 ◽  
Author(s):  
Motoko Shibanuma ◽  
Joo-ri Kim-Kaneyama ◽  
Keiko Ishino ◽  
Nobuko Sakamoto ◽  
Tomoko Hishiki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Focal Adhesion ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Focal Adhesions ◽  
Dominant Negative ◽  
Nuclear Accumulation ◽  
Adhesion Protein ◽  
Focal Adhesion Protein ◽  
Export Signal

hic-5 was originally isolated as an H2O2-inducible cDNA clone whose product was normally found at focal adhesions. In this study, we found that Hic-5 accumulated in the nucleus in response to oxidants such as H2O2. Other focal adhesion proteins including paxillin, the most homologous to Hic-5, remained in the cytoplasm. Mutation analyses revealed that the C- and N-terminal halves of Hic-5 contributed to its nuclear localization in a positive and negative manner, respectively. After the finding that leptomycin B (LMB), an inhibitor of nuclear export signal (NES), caused Hic-5 to be retained in the nucleus, Hic-5 was demonstrated to harbor NES in the N-terminal, which was sensitive to oxidants, thereby regulating the nuclear accumulation of Hic-5. NES consisted of a leucine-rich stretch and two cysteines with a limited similarity to Yap/Pap-type NES. In the nucleus, Hic-5 was suggested to participate in the gene expression of c-fos. Using dominant negative mutants, we found that Hic-5 was actually involved in endogenous c-fos gene expression upon H2O2 treatment. Hic-5 was thus proposed as a focal adhesion protein with the novel aspect of shuttling between focal adhesions and the nucleus through an oxidant-sensitive NES, mediating the redox signaling directly to the nucleus.

scholarly journals LPP, an Actin Cytoskeleton Protein Related to Zyxin, Harbors a Nuclear Export Signal and Transcriptional Activation Capacity

2000 ◽  
Vol 11 (1) ◽  
pp. 117-129 ◽  
Author(s):  
Marleen M. R. Petit ◽  
Julie Fradelizi ◽  
Roy M. Golsteyn ◽  
Torik A.Y. Ayoubi ◽  
Bernadette Menichi ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Export ◽  
Protein Complexes ◽  
Nuclear Export Signal ◽  
Focal Adhesions ◽  
Mesenchymal Tumors ◽  
Adhesion Protein ◽  
Actin Organization ◽  
Focal Adhesion Protein ◽  
Activation Capacity

The LPP gene is the preferred translocation partner of the HMGIC gene in a subclass of human benign mesenchymal tumors known as lipomas. Here we have characterized theLPP gene product that shares 41% of sequence identity with the focal adhesion protein zyxin. LPP localizes in focal adhesions as well as in cell-to-cell contacts, and it binds VASP, a protein implicated in the control of actin organization. In addition, LPP accumulates in the nucleus of cells upon treatment with leptomycin B, an inhibitor of the export factor CRM1. The nuclear export of LPP depends on an N-terminally located leucine-rich sequence that shares sequence homology with well-defined nuclear export signals. Moreover, LPP displays transcriptional activation capacity, as measured by GAL4-based assays. Altogether, these results show that the LPP protein has multifunctional domains and may serve as a scaffold upon which distinct protein complexes are assembled in the cytoplasm and in the nucleus.

scholarly journals Identification of the Nuclear Export Signal and STAT-Binding Domains of the Nipah Virus V Protein Reveals Mechanisms Underlying Interferon Evasion

2004 ◽  
Vol 78 (10) ◽  
pp. 5358-5367 ◽  
Author(s):  
Jason J. Rodriguez ◽  
Cristian D. Cruz ◽  
Curt M. Horvath
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Nuclear Export ◽  
Nipah Virus ◽  
Nuclear Export Signal ◽  
Hendra Virus ◽  
Nuclear Accumulation ◽  
V Protein ◽  
Binding Domains ◽  
Export Signal

ABSTRACT The V proteins of Nipah virus and Hendra virus have been demonstrated to bind to cellular STAT1 and STAT2 proteins to form high-molecular-weight complexes that inhibit interferon (IFN)-induced antiviral transcription by preventing STAT nuclear accumulation. Analysis of the Nipah virus V protein has revealed a region between amino acids 174 and 192 that functions as a CRM1-dependent nuclear export signal (NES). This peptide is sufficient to complement an export-defective human immunodeficiency virus Rev protein, and deletion and substitution mutagenesis revealed that this peptide is necessary for both V protein shuttling and cytoplasmic retention of STAT1 and STAT2 proteins. However, the NES is not required for V-dependent IFN signaling inhibition. IFN signaling is blocked primarily by interaction between Nipah virus V residues 100 to 160 and STAT1 residues 509 to 712. Interaction with STAT2 requires a larger Nipah virus V segment between amino acids 100 and 300, but deletion of residues 230 to 237 greatly reduced STAT2 coprecipitation. Further, V protein interactions with cellular STAT1 is a prerequisite for STAT2 binding, and sequential immunoprecipitations demonstrate that V, STAT1, and STAT2 can form a tripartite complex. These findings characterize essential regions for Henipavirus V proteins that represent potential targets for therapeutic intervention.

scholarly journals RANTES induces tyrosine kinase activity of stably complexed p125FAK and ZAP-70 in human T cells.

1996 ◽  
Vol 184 (3) ◽  
pp. 873-882 ◽  
Author(s):  
K B Bacon ◽  
M C Szabo ◽  
H Yssel ◽  
J B Bolen ◽  
T J Schall
Keyword(s):  
T Cells ◽  
Tyrosine Kinase ◽  
Focal Adhesion ◽  
Cell Activation ◽  
Focal Adhesions ◽  
Adhesion Protein ◽  
Human T Cells ◽  
Multiple Protein ◽  
Transduction Mechanisms ◽  
Focal Adhesion Protein

The chemokine RANTES is a chemoattractant and activating factor for T lymphocytes. Investigation of the signal transduction mechanisms induced by RANTES in T cells revealed tyrosine phosphorylation of multiple protein species with prominent bands at 70-85 and 120-130 kD. Immunoprecipitation and Western analyses revealed that a protein of 125 kD was identical to the focal adhesion kinase (FAK) pp125FAK. RANTES stimulated phosphorylation of FAK as early as 30 seconds and immunoblots using antiphosphotyrosine monoclonal antibodies revealed that there was consistent phosphorylation of a 68-70 kD species in the pp125FAK immunoprecipitates. Immunoblotting and kinase assays showed this to be two separate proteins, the tyrosine kinase zeta-associated protein (ZAP) 70, and the focal adhesion protein paxillin. These results indicate a potentially important role for RANTES in the generation of T cell focal adhesions and subsequent cell activation via a molecular complex containing FAK, ZAP-70, and paxillin.

scholarly journals The nuclear lamina is a hub for the nuclear function of Jacob

2020 ◽  
Author(s):  
Sebastian Samer ◽  
Rajeev Raman ◽  
Gregor Laube ◽  
Michael R. Kreutz ◽  
Anna Karpova
Keyword(s):  
Gene Expression ◽  
Nuclear Membrane ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nuclear Lamina ◽  
Immunogold Electron Microscopy ◽  
Core Component ◽  
Inner Nuclear Membrane ◽  
Export Signal

Abstract Jacob is a synapto-nuclear messenger protein that couples NMDAR activity to CREB-dependent gene expression. In this study, we investigated the nuclear distribution of Jacob and report a prominent targeting to the nuclear envelope that requires NMDAR activity and nuclear import. Immunogold electron microscopy revealed preferential association of Jacob with the inner nuclear membrane where it directly binds to LaminB1, an intermediate filament and core component of the inner nuclear membrane (INM). The association with INM is transient; it involves a functional nuclear export signal in Jacob and a canonical CRM1-/RanGTP-dependent export mechanism that defines the residing time of the protein at the INM. Taken together, the data suggest a stepwise redistribution of Jacob within the nucleus following nuclear import and prior to nuclear export.

scholarly journals Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Ryan T. Behrens ◽  
Mounavya Aligeti ◽  
Ginger M. Pocock ◽  
Christina A. Higgins ◽  
Nathan M. Sherer
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Rna Binding ◽  
Nuclear Export Signal ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Virion Production ◽  
Export Signal ◽  
Hiv 1 ◽  
Rev Protein

ABSTRACT HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. IMPORTANCE HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.

scholarly journals Shuttling Imbalance of MLF1 Results in p53 Instability and Increases Susceptibility to Oncogenic Transformation

2007 ◽  
Vol 28 (1) ◽  
pp. 422-434 ◽  
Author(s):  
Noriko Yoneda-Kato ◽  
Jun-ya Kato
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Chromosomal Translocation ◽  
Nuclear Accumulation ◽  
Tumor Suppressor P53 ◽  
Oncogenic Transformation ◽  
Early Passage ◽  
Leptomycin B ◽  
Nuclear Shuttling ◽  
Export Signal

ABSTRACT Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation.

scholarly journals Paxillin-dependent Paxillin Kinase Linker and p21-Activated Kinase Localization to Focal Adhesions Involves a Multistep Activation Pathway

2002 ◽  
Vol 13 (5) ◽  
pp. 1550-1565 ◽  
Author(s):  
Michael C. Brown ◽  
Kip A. West ◽  
Christopher E. Turner
Keyword(s):  
Focal Adhesion ◽  
Focal Adhesions ◽  
Identical Result ◽  
Cytoskeletal Reorganization ◽  
Serine Threonine Kinase ◽  
Adhesion Protein ◽  
Spatial Regulation ◽  
P21 Activated Kinase ◽  
Focal Adhesion Protein ◽  
Activation Cascade

The precise temporal-spatial regulation of the p21-activated serine-threonine kinase PAK at the plasma membrane is required for proper cytoskeletal reorganization and cell motility. However, the mechanism by which PAK localizes to focal adhesions has not yet been elucidated. Indirect binding of PAK to the focal adhesion protein paxillin via the Arf-GAP protein paxillin kinase linker (PKL) and PIX/Cool suggested a mechanism. In this report, we demonstrate an essential role for a paxillin–PKL interaction in the recruitment of activated PAK to focal adhesions. Similar to PAK, expression of activated Cdc42 and Rac1, but not RhoA, stimulated the translocation of PKL from a generally diffuse localization to focal adhesions. Expression of the PAK regulatory domain (PAK1–329) or the autoinhibitory domain (AID 83–149) induced PKL, PIX, and PAK localization to focal adhesions, indicating a role for PAK scaffold activation. We show PIX, but not NCK, binding to PAK is necessary for efficient focal adhesion localization of PAK and PKL, consistent with a PAK–PIX–PKL linkage. Although PAK activation is required, it is not sufficient for localization. The PKL amino terminus, containing the PIX-binding site, but lacking paxillin-binding subdomain 2 (PBS2), was unable to localize to focal adhesions and also abrogated PAK localization. An identical result was obtained after PKLΔPBS2 expression. Finally, neither PAK nor PKL was capable of localizing to focal adhesions in cells overexpressing paxillinΔLD4, confirming a requirement for this motif in recruitment of the PAK–PIX–PKL complex to focal adhesions. These results suggest a GTP-Cdc42/GTP-Rac triggered multistep activation cascade leading to the stimulation of the adaptor function of PAK, which through interaction with PIX provokes a functional PKL PBS2–paxillin LD4 association and consequent recruitment to focal adhesions. This mechanism is probably critical for the correct subcellular positioning of PAK, thereby influencing the ability of PAK to coordinate cytoskeletal reorganization associated with changes in cell shape and motility.

scholarly journals TORC1 signaling exerts spatial control over microtubule dynamics by promoting nuclear export of Stu2

2017 ◽  
Vol 216 (11) ◽  
pp. 3471-3484 ◽  
Author(s):  
Babet van der Vaart ◽  
Josef Fischböck ◽  
Christine Mieck ◽  
Peter Pichler ◽  
Karl Mechtler ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Cell Cycle Progression ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cycle Progression ◽  
Cytoskeletal Organization ◽  
Spindle Positioning ◽  
Cellular Processes ◽  
Export Signal

The target of rapamycin complex 1 (TORC1) is a highly conserved multiprotein complex that functions in many cellular processes, including cell growth and cell cycle progression. In this study, we define a novel role for TORC1 as a critical regulator of nuclear microtubule (MT) dynamics in the budding yeast Saccharomyces cerevisiae. This activity requires interactions between EB1 and CLIP-170 plus end–tracking protein (+TIP) family members with the TORC1 subunit Kog1/Raptor, which in turn allow the TORC1 proximal kinase Sch9/S6K1 to regulate the MT polymerase Stu2/XMAP215. Sch9-dependent phosphorylation of Stu2 adjacent to a nuclear export signal prevents nuclear accumulation of Stu2 before cells enter mitosis. Mutants impaired in +TIP–TORC1 interactions or Stu2 nuclear export show increased nuclear but not cytoplasmic MT length and display nuclear fusion, spindle positioning, and elongation kinetics defects. Our results reveal key mechanisms by which TORC1 signaling controls Stu2 localization and thereby contributes to proper MT cytoskeletal organization in interphase and mitosis.

scholarly journals CDK-dependent phosphorylation of Alp7–Alp14 (TACC–TOG) promotes its nuclear accumulation and spindle microtubule assembly

2014 ◽  
Vol 25 (13) ◽  
pp. 1969-1982 ◽  
Author(s):  
Naoyuki Okada ◽  
Takashi Toda ◽  
Masayuki Yamamoto ◽  
Masamitsu Sato
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Coiled Coil ◽  
Microtubule Assembly ◽  
Nuclear Accumulation ◽  
Cyclin Dependent Kinase ◽  
Mitotic Entry ◽  
Localization Signal ◽  
Export Signal ◽  
Overexpressed Gene

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7–Alp14 (transforming acidic coiled-coil–tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7–Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7–Alp14 complex to localize it to the nucleus.

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