scholarly journals Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export

1997 ◽  
Vol 137 (3) ◽  
pp. 523-538 ◽  
Author(s):  
Jun Yang ◽  
Jimin Liu ◽  
Donald B. DeFranco
Keyword(s):  
Heat Shock ◽  
Nuclear Export ◽  
Glucocorticoid Receptors ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Sodium Molybdate ◽  
Hnrnp A1 ◽  
Permeabilized Cells ◽  
Subnuclear Trafficking

We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

scholarly journals Chromatin Recycling of Glucocorticoid Receptors: Implications for Multiple Roles of Heat Shock Protein 90

1999 ◽  
Vol 13 (3) ◽  
pp. 355-365 ◽  
Author(s):  
Jimin Liu ◽  
Donald B. DeFranco
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Nuclear Export ◽  
Glucocorticoid Receptors ◽  
Binding Capacity ◽  
Heat Shock Protein 90 ◽  
Multiple Roles ◽  
Chromatin Binding ◽  
Hormone Binding ◽  
Permeabilized Cells

Abstract Unliganded glucocorticoid receptors (GRs) released from chromatin after hormone withdrawal remain associated with the nucleus within a novel subnuclear compartment that serves as a nuclear export staging area. We set out to examine whether unliganded nuclear receptors cycle between distinct subnuclear compartments or require cytoplasmic transit to regain hormone and chromatin-binding capacity. Hormone-withdrawn rat GrH2 hepatoma cells were permeabilized with digitonin to deplete cytoplasmic factors, and then hormone-binding and chromatin-binding properties of the recycled nuclear GRs were measured. We found that recycled nuclear GRs do not require cytosolic factors or ATP to rebind hormone. Nuclear GRs that rebind hormone in permeabilized cells target to high-affinity chromatin-binding sites at 30 C, but not 0 C, in the presence of ATP. Since geldanamycin, a heat shock protein-90 (hsp90)-binding drug, inhibits hormone binding to recycled nuclear GRs, hsp90 may be required to reassemble the receptor into a form capable of productive interactions with hormone. Geldanamycin also inhibits GR release from chromatin during hormone withdrawal, suggesting that hsp90 chaperone function may play multiple roles to facilitate chromatin recycling of GR.

scholarly journals A Role for RanBP1 in the Release of CRM1 from the Nuclear Pore Complex in a Terminal Step of Nuclear Export

1999 ◽  
Vol 145 (4) ◽  
pp. 645-657 ◽  
Author(s):  
Ralph H. Kehlenbach ◽  
Achim Dickmanns ◽  
Angelika Kehlenbach ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Activated T Cells ◽  
Permeabilized Cells ◽  
Binding Domains ◽  
Biochemical Fractionation ◽  
Nuclear Export Receptor ◽  
Gtpase Ran

We recently developed an assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport.

scholarly journals Interaction of Vault Particles with Estrogen Receptor in the MCF-7 Breast Cancer Cell

1998 ◽  
Vol 141 (6) ◽  
pp. 1301-1310 ◽  
Author(s):  
Ciro Abbondanza ◽  
Valentina Rossi ◽  
Annarita Roscigno ◽  
Luigi Gallo ◽  
Angela Belsito ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Glucocorticoid Receptors ◽  
Sodium Molybdate ◽  
Nuclear Extract ◽  
Supernatant Fraction ◽  
Cdna Clones ◽  
Major Vault Protein ◽  
Estradiol Treatment ◽  
Mcf 7

A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.

scholarly journals The Dynamic Association of RCC1 with Chromatin Is Modulated by Ran-dependent Nuclear Transport

2004 ◽  
Vol 15 (1) ◽  
pp. 245-255 ◽  
Author(s):  
Ian Cushman ◽  
David Stenoien ◽  
Mary Shannon Moore
Keyword(s):  
Nuclear Export ◽  
Rna Polymerases ◽  
Mrna Transcription ◽  
Leptomycin B ◽  
Permeabilized Cells ◽  
Independent Process ◽  
Dependent Transport ◽  
Tsbn2 Cells

Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crm1/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or α-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo.

Abstract 283: Redox-mediated Regulation of Histone deacetylase4, a Class II HDAC, by Thioredoxin1 through Interaction with DnaJb5, a Heat Shock Protein 40

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Tetsuro Ago ◽  
Tong Liu ◽  
Hong Li ◽  
Jeffery Molkentin ◽  
Junichi Sadoshima
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cardiac Hypertrophy ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Pressure Overload ◽  
Class Ii ◽  
Luciferase Reporter

Thioredoxin1 (Trx1) reduces redox-sensitive proteins and regulates cell growth and death. We previously reported that cardiac hypertrophy induced by pressure-overload is suppressed in mice with cardiac specific overexpression of Trx1 (Tg-Trx1). To elucidate the mechanisms by which Trx1 suppresses cardiac hypertrophy, we performed DNA microarray analysis using Tg-Trx1 mouse hearts. We identified DnaJb5, a heat shock protein 40, as a gene significantly upregulated by Trx1. Immunostaining and immunoblot analyses indicated that Trx1 and DnaJb5 are co-localized in the nucleus of myocytes. Pull-down and immunoprecipitation assays showed that DnaJb5 interacts with TBP-2, a Trx1-binding protein. DnaJb5 did not disturb the interaction between Trx1 and TBP-2, and enhanced the Trx1 reducing activity. Both Trx1 and DnaJb5 attenuated phenylephrine (PE)-induced activation of NFAT and myocyte hypertrophy in vitro . Using transgenic mice harboring an NFAT luciferase reporter, we confirmed that Trx1 suppresses both NFAT activation and cardiac hypertrophy induced by PE in vivo . We also found that DnaJb5 binds directly to histone deacetylase 4 (HDAC4), a class II HDAC. An HDAC4 mutant lacking the minimal region responsible for the interaction with DnaJb5 (residues 628 – 881) was localized in the cytosol, in contrast to the nuclear localization of the wild-type HDAC4, suggesting the importance of the interaction for the nuclear localization of HDAC4. Overexpression of Trx1 suppressed PE-induced nuclear export of HDAC4 in myocytes. Using mass spectroscopy, we found that HDAC4 forms a disulfide bond between Cys-667 and -669, which was reduced by Trx1. The HDAC4 Cys667/669Ser mutant was localized in the cytosol, and its nuclear export was suppressed by leptomycin B, an inhibitor of exportin, suggesting that the redox modification induces nuclear export regardless of phosphorylation. Consistently, the Cys667/669Ser substitution abolished the suppressive effect of HDAC4 on NFAT activity and cardiac hypertrophy. Collectively, these results show that Trx1 upregulates DnaJb5, which recruits HDAC4 into the complex formed by Trx1-TBP-2-DnaJb5, thereby reducing HDAC4, retaining its nuclear localization, and suppressing NFAT activity and cardiac hypertrophy.

A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock

1999 ◽  
Vol 112 (10) ◽  
pp. 1465-1476 ◽  
Author(s):  
F. Weighardt ◽  
F. Cobianchi ◽  
L. Cartegni ◽  
I. Chiodi ◽  
A. Villa ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cellular Response ◽  
Rna Binding ◽  
Protein Composition ◽  
Heat Shock Factor 1 ◽  
Hnrnp A1 ◽  
Rich Region ◽  
Hnrnp Proteins ◽  
Hnrnp Protein

A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP). HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region. In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs. HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene. We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins. Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells. Heat-shock treatment at 42 degrees C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37 degrees C. The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes. Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.

scholarly journals LAM-003, a new drug for treatment of tyrosine kinase inhibitor–resistant FLT3-ITD–positive AML

2019 ◽  
Vol 3 (22) ◽  
pp. 3661-3673 ◽  
Author(s):  
Neil Beeharry ◽  
Sean Landrette ◽  
Sophia Gayle ◽  
Marylens Hernandez ◽  
Jeff E. Grotzke ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Heat Shock Protein 90 ◽  
Antileukemic Activity ◽  
Key Points

Key Points The heat shock protein 90 inhibitor LAM-003 displays potent in vitro and in vivo activity as a single agent and combined with venetoclax. LAM-003 retains antileukemic activity against AML cells rendered resistant to FLT3 kinase inhibitors by mutation or stromal signaling.

Evaluation of selective inhibitors of nuclear export (SINE) CRM1 inhibitors for the treatment of renal cell carcinoma (RCC).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 4634-4634 ◽  
Author(s):  
Hiromi Inoue ◽  
Michael Kauffman ◽  
Sharon Shacham ◽  
Yosef Landesman ◽  
Robert H Weiss
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Nuclear Export ◽  
Kinase Inhibitors ◽  
Chromosome Region ◽  
Human Kidney ◽  
Tumor Growth Inhibition ◽  
Novel Therapeutic

4634 Background: For the ~30% of patients who present with RCC at the metastatic stage, multi-kinase inhibitors have been used with moderate success: progression-free survival remains at only one to two years, and thus it is imperative to discover novel therapeutic approaches for metastatic disease. We asked whether (1) SINE inhibitors of chromosome region maintenance protein 1 (CRM1) attenuate key cell cycle regulatory and apoptotic molecules and whether these compounds exert salutary effects in a human RCC xenograft mouse model. Methods: Four RCC cell lines (ACHN, Caki-1, 786-O, and A498) with distinct genotypes, and primary normal human kidney (NHK) cell lines, were used in this study. The cells were treated with the chemically related SINE CRM1 inhibitors KPT-185 or 251 and MTT assays were performed. In addition, cell cycle analyses, immunofluorescence for p53 and p21, and immunoblotting for CRM1, p53, p21, p27, and p-MDM2 were performed for all cell lines. RCC mice with Caki-1 xenografts were treated with vehicle, the orally-available CRM1 inhibitor KPT-251, or sorafenib for 26 days. Tumor volume was measured over several days. Results: Both KPT185 and 251 specifically reduced CRM1 protein levels in RCC cells. KPT-185 caused dose-dependent cytotoxicity in RCC cells, which was greater than sorafenib in RCC cell lines but less in NHK cells, suggesting a possible clinical advantage of KPT-185 over sorafenib. By FACS analysis, we showed that KPT-185 arrests the cell cycle in both G2/M and G1, and increased the sub-G0 cell population. KPT-185 and 251 both increased p53 and p21 in RCC cells, and KPT-185 confined these proteins to the nucleus. In vivo, KPT-251 inhibited Caki-1 xenografts in mice compared to both vehicle and sorafenib without obvious systemic adverse effects. Conclusions: We introduce a completely novel therapeutic approach to the treatment of RCC based on inhibition of the nuclear export of key cell cycle regulatory proteins. Inhibition of CRM1 leads to forced nuclear retention, and thereby activation, of several key p53-pathway proteins, leading to cell cycle arrest and apoptosis in RCC cell lines in vitro and tumor growth inhibition in vivo.

scholarly journals The 70-kD heat shock cognate protein (hsc70) facilitates the nuclear export of the import receptors

2005 ◽  
Vol 171 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Shingo Kose ◽  
Maiko Furuta ◽  
Makiko Koike ◽  
Yoshihiro Yoneda ◽  
Naoko Imamoto
Keyword(s):  
Heat Shock ◽  
Nuclear Export ◽  
Nucleocytoplasmic Transport ◽  
Living Cells ◽  
Transport Systems ◽  
Nuclear Accumulation ◽  
Import Receptors ◽  
Heat Shock Cognate Protein ◽  
Cognate Protein

Transport receptors of the importin β family continuously shuttle between the nucleus and cytoplasm. We previously reported that the nuclear export of importin β involves energy-requiring step(s) in living cells. Here, we show that the in vitro nuclear export of importin β also requires energy input. Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin β. Further purification revealed that the active component in the absorbed fraction was a 70-kD heat shock cognate protein (hsc70). The addition of recombinant hsc70, but not an ATPase-deficient hsc70 mutant, to the depleted cytosol restored the export activity. In living cells, depletion of hsc70 caused the significant nuclear accumulation of importin β. These effects of hsc70 were observed in the nuclear export of importin β, but also for other import receptors, transportin and importin α. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins.

A novel function for the 90 kDa heat-shock protein (Hsp90): facilitating nuclear export of 60 S ribosomal subunits

2002 ◽  
Vol 362 (3) ◽  
pp. 675-684 ◽  
Author(s):  
Harald SCHLATTER ◽  
Thomas LANGER ◽  
Susann ROSMUS ◽  
Marie-Luise ONNEKEN ◽  
Hugo FASOLD
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Nuclear Export ◽  
Vitro Assay ◽  
Ribosomal Subunits ◽  
Cytoplasmic Proteins ◽  
Energy Dependent

Ribosomal subunits are assembled in the nucleus, and mature 40S and 60S subunits are exported stoichiometrically into the cytoplasm. The nuclear export of ribosomal subunits is a unidirectional, saturable and energy-dependent process. An in vitro assay for the nuclear export of 60S ribosomal subunits involves the use of resealed nuclear envelopes. The export of ribosomal subunits from resealed nuclear envelopes is enhanced by cytoplasmic proteins. Here we present evidence that the export-promoting activity was due to the cytoplasmic 90kDa heat-shock protein (Hsp90). Isolated, purified Hsp90 vastly enhanced the export of 60S ribosomal subunits from resealed nuclear envelopes, while inhibition of Hsp90 function, either with the Hsp90-binding drug geldanamycin or with anti-Hsp90 antibodies, resulted in reduced release of 60S ribosomal subunits. To confirm these findings under in vivo conditions, corresponding experiments were performed with Xenopus oocytes using microinjection techniques; the results obtained confirmed the findings obtained with resealed nuclear envelopes. These findings suggest that Hsp90 facilitates the nuclear export of 60S ribosomal subunits, probably by chaperoning protein interactions during the export process.

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