scholarly journals Intersectin goes nuclear: secret life of an endocytic protein

2018 ◽  
Vol 475 (8) ◽  
pp. 1455-1472 ◽  
Author(s):  
Gualtiero Alvisi ◽  
Lucia Paolini ◽  
Andrea Contarini ◽  
Chiara Zambarda ◽  
Veronica Di Antonio ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Chromosome Region ◽  
Coiled Coil ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Trafficking ◽  
Multidomain Structure ◽  
Endocytic Protein ◽  
Localization Signal ◽  
Src Homology ◽  
Energy Dependent

Intersectin 1-short (ITSN1-s) is a 1220 amino acid ubiquitously expressed scaffold protein presenting a multidomain structure that allows to spatiotemporally regulate the functional interaction of a plethora of proteins. Besides its well-established role in endocytosis, ITSN1-s is involved in the regulation of cell signaling and is implicated in tumorigenesis processes, although the signaling pathways involved are still poorly understood. Here, we identify ITSN1-s as a nucleocytoplasmic trafficking protein. We show that, by binding to importin (IMP)α, a small fraction of ITSN1-s localizes in the cell nucleus at the steady state, where it preferentially associates with the nuclear envelope and interacts with lamin A/C. However, upon pharmacological ablation of chromosome region maintenance 1 (CRM-1)-dependent nuclear export pathway, the protein accumulates into the nucleus, thus revealing its moonlighting nature. Analysis of deletion mutants revealed that the coiled coil (CC) and Src homology (SH3) regions play the major role in its nucleocytoplasmic shuttling. While no evidence of nuclear localization signal (NLS) was detected in the CC region, a functional bipartite NLS was identified within the SH3D region of ITSN1-s (RKKNPGGWWEGELQARGKKRQIGW-1127), capable of conferring energy-dependent nuclear accumulation to reporter proteins and whose mutational ablation affects nuclear import of the whole SH3 region. Thus, ITSN1-s is an endocytic protein, which shuttles between the nucleus and the cytoplasm in a CRM-1- and IMPα-dependent fashion.

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.

Inhibition of CRM1-Mediated Nuclear Export of Transcription FACTORS by Leukemogenic NUP98 Fusion Proteins.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1959-1959
Author(s):  
Akiko Takeda ◽  
Anmaar M Abdul-Nabi ◽  
Nabeel R. Yaseen
Keyword(s):  
Transcription Factors ◽  
Nuclear Export ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Nuclear Accumulation ◽  
Human Leukemia ◽  
Dependent Manner ◽  
Nucleocytoplasmic Trafficking ◽  
Nuclear Retention

Abstract Abstract 1959 Poster Board I-982 NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin (NPMc) and HIV-1 Rev. In-vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG-repeat motif in a Ran-GTP dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of myeloid cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFΚB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFΚB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators. Disclosures: No relevant conflicts of interest to declare.

scholarly journals BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

2006 ◽  
Vol 26 (19) ◽  
pp. 7318-7330 ◽  
Author(s):  
Ilmin Kwon ◽  
Jiwon Lee ◽  
Seok Hoon Chang ◽  
Neon Cheol Jung ◽  
Byung Ju Lee ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Export ◽  
Clock Gene ◽  
Clock Genes ◽  
Nuclear Accumulation ◽  
Cry Proteins ◽  
Embryonic Mouse ◽  
E Box ◽  
Localization Signal ◽  
Functional Nuclear Localization Signal

ABSTRACT CLOCK and BMAL1 are bHLH-PAS-containing transcription factors that bind to E-box elements and are indispensable for expression of core circadian clock components such as the Per and Cry genes. A key step in expression is the heterodimerization of CLOCK and BMAL1 and their accumulation in the nucleus with an approximately 24-h periodicity. We show here that nucleocytoplasmic shuttling of BMAL1 is essential for transactivation and for degradation of the CLOCK/BMAL1 heterodimer. Using serial deletions and point mutants, we identified a functional nuclear localization signal and Crm1-dependent nuclear export signals in BMAL1. Transient-transfection experiments revealed that heterodimerization of CLOCK and BMAL1 accelerates their turnover, as well as E-box-dependent clock gene transcription. Moreover, in embryonic mouse fibroblasts, robust transcription of Per2 is tightly associated with massive degradation of the CLOCK/BMAL1 heterodimer. CRY proteins suppressed this process during the transcription-negative phase and led to nuclear accumulation of the CLOCK/BMAL1 heterodimer. Thus, these findings suggest that the decrease of BMAL1 abundance during the circadian cycle reflects robust transcriptional activation of clock genes rather than inhibition of BMAL1 synthesis.

scholarly journals Cell adhesion differentially regulates the nucleocytoplasmic distribution of active MAP kinases

2002 ◽  
Vol 115 (13) ◽  
pp. 2781-2790 ◽  
Author(s):  
Andrew E. Aplin ◽  
Brian P. Hogan ◽  
Jeannie Tomeu ◽  
R. L. Juliano
Keyword(s):  
Cell Adhesion ◽  
Map Kinase ◽  
Nuclear Export ◽  
Cellular Localization ◽  
Map Kinases ◽  
Nuclear Accumulation ◽  
Adherent Cells ◽  
Nucleocytoplasmic Trafficking ◽  
3T3 Fibroblasts ◽  
Nucleocytoplasmic Distribution

Cells decide whether to undergo processes, such as proliferation,differentiation and apoptosis, based upon the cues they receive from both circulating factors and integrin-mediated adhesion to the extracellular matrix. Integrins control the activation of the early signaling pathways. For example, growth factor activation of the ERK cascade is enhanced when cells are adherent. In addition, adhesion receptors oversee the cellular localization of critical signaling components. We have recently shown that ERK signaling to the nucleus is regulated by cell adhesion at the level of nucleocytoplasmic trafficking. Since the ERKs are only one class of MAP kinase, we extended these studies to include both JNK and p38 MAP kinases. We have rendered JNK and p38 activation in NIH 3T3 fibroblasts anchorage-independent either by treatment with anisomycin or by expression of upstream activators. Under conditions whereby JNK activation is anchorage-independent, we show that localization of JNK to the nucleus and JNK-mediated phosphorylation of c-Jun and Elk-1 is not altered by loss of adhesion. Likewise, the ability of activated p38 to accumulate in the nucleus was similar in suspended and adherent cells. Finally, we show that expression of a form of ERK, which is activated and resistant to nuclear export, reverses the adhesion-dependency of ERK phosphorylation of Elk-1. Thus, adhesion differentially regulates the nucleocytoplasmic distribution of MAP kinase members; ERK accumulation in the nucleus occurs more efficiently in adherent cells, whereas nuclear accumulation of active p38 and active JNK are unaffected by changes in adhesion.

A Putative NES Mediates Cytoplasmic Localization of Apoptin in Normal Cells

2004 ◽  
Vol 36 (12) ◽  
pp. 817-823 ◽  
Author(s):  
Qing-Ming Wang ◽  
Guo-Cai Fan ◽  
Ji-Zhong Chen ◽  
Hui-Peng Chen ◽  
Fu-Chu He
Keyword(s):  
Tumor Cells ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Chicken Anemia Virus ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Normal Cells ◽  
Localization Signal ◽  
Anemia Virus ◽  
Export Signal

Abstract Apoptin, a protein expressed by chicken anemia virus, is found predominantly in the cytoplasm in normal cells, whereas it localizes in the nucleus in transformed and malignant cells. However, the mechanisms that regulate the different subcellular localization of Apoptin in normal and tumor cells have not been fully clarified. In this work, a putative nuclear export signal (NES) in Apoptin was predicted. It was testified that the putative NES (pNES) of Apoptin was not a functional NES, but actually acted as a cytoplasmic retention signal. Deletion of the pNES led to the nuclear accumulation of Apoptin in normal cells. In addition, when a strong nuclear localization signal was introduced into Apoptin, it exclusively translocated to the nucleus in normal cells. These observations indicated that the cytoplasmic localization of Apoptin in normal cells results from the balance between cytoplasmic retention and nuclear import. On the other hand, the pNES was also proved to be necessary for Apoptin multimerization. Mutants lacking the pNES did not form obviously visible globular aggregates in normal or tumor cells.

scholarly journals Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705–SH2 through C-terminal tail modulation

2019 ◽  
Author(s):  
Junhao Yang ◽  
Hiroyuki Kunimoto ◽  
Bumpei Katayama ◽  
Hong Zhao ◽  
Takashi Shiromizu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Nuclear Export ◽  
Chromosome Region ◽  
Intramolecular Interactions ◽  
Nuclear Accumulation ◽  
Proper Function ◽  
Functional Studies ◽  
Reciprocal Interactions ◽  
The Stability ◽  
Transcription 3

Abstract Signal transducer and activator of transcription 3 (STAT3) is involved in many biological processes, including immunity and cancer. STAT3 becomes phosphorylated at Tyr705 and Ser727 on IL-6 stimulation. Phospho-Tyr705 (pY705) stabilizes the STAT3 dimer with reciprocal interactions between pY705 and the SH2 of the other molecule and phospho-Ser727 (pS727) accelerates pY705 dephosphorylation. We study how pS727 regulates STAT3 in both structural and biological perspectives. Using STAT3 reconstituted in HepG2-stat3-knockout cells, we show that pS727, together with a handshake N-terminal domain (NTD) interaction, causes rapid inactivation of STAT3 for pY705 dephosphorylation and a chromosome region maintenance 1 (CRM1)-independent nuclear export, which is critical for faithful STAT3 response to the cellular signals. The various N-terminal tags, GFP-related Ruby and FLAG, rendered the export CRM1-dependent and especially FLAG-tag caused nuclear accumulation of STAT3, indicating the presence of conformational changes in inactivation. Impaired reactivation of STAT3 by S727A or FLAG-tag delayed or inhibited the IL-6-induced saa1 mRNA expression, respectively. The detailed analysis of the pY705–SH2 structure identified the C-terminal tail (CTT) from L706 to P715 as a key regulator of the CTT–CTT intermolecular and the CTT–SH2 intramolecular interactions that support pY705–SH2 association. The functional studies using multiple STAT3 mutants indicated that the degree of the two interactions determines the stability of pY705–SH2 interaction. Importantly, Pro715 was critical for the pS727's destabilizing activity and the known phosphorylation and acetylation at the CTT structurally inhibited the pY705–SH2 interaction. Thus, pS727 triggers pY705–SH2 dissociation by weakening the supportive interactions likely through CTT modulation, inducing rapid cycles of STAT3 activation–inactivation for proper function of STAT3.

scholarly journals Nuclear-Cytoplasmic Shuttling of Chibby Controls β-Catenin Signaling

2010 ◽  
Vol 21 (2) ◽  
pp. 311-322 ◽  
Author(s):  
Feng-Qian Li ◽  
Adaobi Mofunanya ◽  
Victoria Fischer ◽  
Jason Hall ◽  
Ken-Ichi Takemaru
Keyword(s):  
Nuclear Export ◽  
Intracellular Localization ◽  
Gene Activation ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Cooperative Action ◽  
Importin Α ◽  
Localization Signal ◽  
Bona Fide ◽  
Nuclear Export Receptor

In the canonical Wnt pathway, β-catenin acts as a key coactivator that stimulates target gene expression through interaction with Tcf/Lef transcription factors. Its nuclear accumulation is the hallmark of active Wnt signaling and is frequently associated with cancers. Chibby (Cby) is an evolutionarily conserved molecule that represses β-catenin–dependent gene activation. Although Cby, in conjunction with 14-3-3 chaperones, controls β-catenin distribution, its molecular nature remains largely unclear. Here, we provide compelling evidence that Cby harbors bona fide nuclear localization signal (NLS) and nuclear export signal (NES) motifs, and constitutively shuttles between the nucleus and cytoplasm. Efficient nuclear export of Cby requires a cooperative action of the intrinsic NES, 14-3-3, and the CRM1 nuclear export receptor. Notably, 14-3-3 docking provokes Cby binding to CRM1 while inhibiting its interaction with the nuclear import receptor importin-α, thereby promoting cytoplasmic compartmentalization of Cby at steady state. Importantly, the NLS- and NES-dependent shuttling of Cby modulates the dynamic intracellular localization of β-catenin. In support of our model, short hairpin RNA–mediated knockdown of endogenous Cby results in nuclear accumulation of β-catenin. Taken together, these findings unravel the molecular basis through which a combinatorial action of Cby and 14-3-3 proteins controls the dynamic nuclear-cytoplasmic trafficking of β-catenin.

scholarly journals Kinetics of nuclear-cytoplasmic translocation of Foxo1 and Foxo3A in adult skeletal muscle fibers

2012 ◽  
Vol 303 (9) ◽  
pp. C977-C990 ◽  
Author(s):  
Tova Neustadt Schachter ◽  
Tiansheng Shen ◽  
Yewei Liu ◽  
Martin F. Schneider
Keyword(s):  
Skeletal Muscle ◽  
Nuclear Export ◽  
Time Course ◽  
Chromosome Region ◽  
Muscle Fibers ◽  
High Rate ◽  
Nuclear Accumulation ◽  
Experimental Conditions ◽  
Adult Skeletal Muscle ◽  
Skeletal Muscle Fibers

In skeletal muscle, the transcription factors Foxo1 and Foxo3A control expression of proteins that mediate muscle atrophy, making the nuclear concentration and nuclear-cytoplasmic movements of Foxo1 and Foxo3A of therapeutic interest in conditions of muscle wasting. Here, we use Foxo-GFP fusion proteins adenovirally expressed in cultured adult mouse skeletal muscle fibers to characterize the time course of nuclear efflux of Foxo1-GFP in response to activation of the insulin-like growth factor-1 (IGF-1)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway to determine the time course of nuclear influx of Foxo1-GFP during inhibition of this pathway and to show that Akt mediates the efflux of nuclear Foxo1-GFP induced by IGF-1. Localization of endogenous Foxo1 in muscle fibers, as determined via immunocytochemistry, is consistent with that of Foxo1-GFP. Inhibition of the nuclear export carrier chromosome region maintenance 1 by leptomycin B (LMB) traps Foxo1 in the nucleus and results in a relatively rapid rate of Foxo1 nuclear accumulation, consistent with a high rate of nuclear-cytoplasmic shuttling of Foxo1 under control conditions before LMB application, with near balance of unidirectional influx and efflux. Expressed Foxo3A-GFP shuttles ∼20-fold more slowly than Foxo1-GFP. Our approach allows quantitative kinetic characterization of Foxo1 and Foxo3A nuclear-cytoplasmic movements in living muscle fibers under various experimental conditions.

scholarly journals Unique Motif for Nucleolar Retention and Nuclear Export Regulated by Phosphorylation

2002 ◽  
Vol 22 (4) ◽  
pp. 1126-1139 ◽  
Author(s):  
Frédéric Catez ◽  
Monique Erard ◽  
Nathalie Schaerer-Uthurralt ◽  
Karine Kindbeiter ◽  
Jean-Jacques Madjar ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Temperature Dependent ◽  
Localization Signal ◽  
New Type ◽  
Simplex Virus ◽  
Energy Dependent ◽  
Us11 Protein

ABSTRACT By microinjecting purified glutathione S-transferase linked to all or parts of herpes simplex virus type 1 US11 protein into either the nucleus or the cytoplasm, we have demonstrated that this nucleolar protein exhibits a new type of localization signal controlling both retention in nucleoli and export to the cytoplasm. Saturated mutagenesis combined with computer modeling allowed us to draw the fine-structure map of this domain, revealing a new proline-rich motif harboring both activities, which are temperature dependent and regulated by phosphorylation. Finally, crossing the nuclear pore complex from the cytoplasm to the nucleus is an energy-dependent process for US11 protein, while getting to nucleoli through the nucleoplasm is energy independent.

scholarly journals Characterization of localization and export signals of bovine torovirus nucleocapsid protein responsible for extensive nuclear and nucleolar accumulation and their importance for virus growth

2020 ◽  
Author(s):  
Makoto Ujike ◽  
Yukako Kawachi ◽  
Yui Matsunaga ◽  
Yuka Etho ◽  
Hideki Asanuma ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Rna Viruses ◽  
Nuclear Export Signal ◽  
Economic Loss ◽  
Nuclear Accumulation ◽  
Structural Protein ◽  
Nucleocytoplasmic Trafficking ◽  
Virus Growth ◽  
N Proteins

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N-proteins, but little is known about ToV N-proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N-protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with EGFP revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN-accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth. IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN predominantly accumulated in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. Understanding its unique features, BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.

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