Model system to study classical nuclear export signals

Kanwal, Charu; Li, Henan; Lim, Carol S.

doi:10.1208/ps040318

Cited by 21 publications

(23 citation statements)

References 25 publications

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“…5, D and E). An NES was previously reported to retain its nuclear export activity when it is tagged with 2ϫ GFP (21,36,37). Consistent with these previous reports, the 2ϫGFP⅐MAPKKNES positive control was exclusively localized in the cytoplasm in the absence of LMB, compared with nonspecific subcellular localization of GFP alone and GFP plus a scrambled peptide (Fig.…”

Section: Human Cdc7 Contains Two Nuclear Export Signals That Are Recosupporting

confidence: 88%

“…The pEGFP⅐MAPKKNES was constructed using 5Ј-TCGACT-TCAAAAAAAACTTGAAGAACTTGAACTT-3Ј and 5Ј-GAT-CAAGTTCAAGTTCTTCAAGTTTTTTTTGAAG-3Ј (21). To generate tandem GFP-tagged (2ϫGFP) constructs, pEGFP⅐ Cdc7NES1, pEGFP⅐Cdc7NES2, pEGFP⅐Cdc7NES1⅐NES2, and pEGFP⅐MEKKNES were digested with AgeI and then ligated with a 790-bp fragment generated by double-digesting pEGFP-C1 with AgeI and XmaI.…”

Section: Methodsmentioning

confidence: 99%

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Identification and Characterization of Human Cdc7 Nuclear Retention and Export Sequences in the Context of Chromatin Binding

Kim

Lee

2007

Journal of Biological Chemistry

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The Cdc7 serine/threonine kinase activates the initiation of DNA replication by phosphorylating MCM proteins that are bound to the origins of DNA replication. We reported previously that human Cdc7 nuclear import is mediated directly by importin-␤ through its binding to the Cdc7 nuclear localization sequence (NLS). Here, we report that human Cdc7 nuclear localization is regulated by two additional elements: nuclear retention (NRS) and export sequences (NES). Cdc7 proteins imported into the nucleus are retained in the nucleus by associating with chromatin, for which NRS-(306 -326) is essential. Importantly, this binding appears to be specific to the origin of DNA replication, because the binding of wild-type Cdc7 to origin is 2.4-fold higher than to non-origin DNA. Furthermore, an NRS-defective Cdc7 mutant could not be retained in the nucleus, although it was imported into the nucleus normally. Together, our data suggest that NRS plays an important role in the activation of DNA replication by Cdc7. The Cdc7 proteins unassociated with chromatin are bound by CRM1 via two NES elements: NES1 at 458 -467 within kinase insert III, and NES2 at 545-554 within the kinase IX domain. The primary function of the Cdc7-CRM1 association may be to translocate nuclear Cdc7 to the cytoplasm. However, the binding of CRM1 with Cdc7 at NES2 raises an interesting possibility that CRM1 may also down-regulate Cdc7 by masking its kinase domain.

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Section: Human Cdc7 Contains Two Nuclear Export Signals That Are Recosupporting

confidence: 88%

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Human Cdc7 Nuclear Retention and Export Sequences in the Context of Chromatin Binding

Kim

Lee

2007

Journal of Biological Chemistry

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“…Hodel et al show that various NLSs linked to GFP can be used as a model system to test nuclear import (5), while we have shown the same for NESs to test nuclear export (6).…”

Section: Import Export and Other Signals: Application To Drug Deliverysupporting

confidence: 61%

Controlling Protein Compartmentalization to Overcome Disease

2006

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Over the past decade, considerable progress has been made to improve our understanding of the intracellular transport of proteins. Mechanisms of nuclear import and export involving classical receptors have been studied. Signal sequences required for directing a protein molecule to a specific cellular compartment have been defined. Knowledge of subcellular trafficking of proteins has also increased our understanding of diseases caused due to mislocalization of proteins. A specific protein on deviating from its native cellular compartment may result in disease due to loss of its normal functioning and aberrant activity in the "wrong" compartment. Mislocalization of proteins results in diseases that range from metabolic disorders to cancer. In this review we discuss some of the diseases caused due to mislocalization. We further focus on application of nucleocytoplasmic transport to drug delivery. Various rationales to treat diseases by exploiting intracellular transport machinery have been proposed. Although the pathways for intracellular movement of proteins have been defined, these have not been adequately utilized for management of diseases involving mislocalized proteins. This review stresses the need for designing drug delivery systems utilizing these mechanisms as this area is least exploited but offers great potential.

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“…Signals exist on proteins for import into the nucleolus [27–29], import into the nucleus (nuclear localization signals) [30], and export from the nucleus to the cytoplasm (nuclear export signals) [31–34]. In addition, amino acid signaling sequences govern protein trafficking to the endoplasmic reticulum [35,36], Golgi apparatus [37], lysosomes [38], mitochondria [39], and peroxisomes [40].…”

Section: Nuclear Export Mechanismsmentioning

confidence: 99%

Nuclear export of proteins and drug resistance in cancer

Turner

Dawson

Sullivan

2012

Biochemical Pharmacology

309

363

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The intracellular location of a protein is crucial to its normal functioning in a cell. Cancer cells utilize the normal processes of nuclear-cytoplasmic transport through the nuclear pore complex of a cell to effectively evade anti-neoplastic mechanisms. CRM1-mediated export is increased in various cancers. Proteins that are exported in cancer include tumor-suppressive proteins such as retinoblastoma, APC, p53, BRAC1, FOXO proteins, INI1/hSNF5, galectin-3, Bok, nucleophosmin, RASSF2, Merlin, p21CIP, p27KIP1, N-WASP/FAK, estradiol receptor and Tob, drug targets topoisomerase I and IIα and BCR-ABL, and the molecular chaperone protein Hsp90. Here, we review in detail the current processes and known structures involved in the export of a protein through the nuclear pore complex. We also discuss the export receptor molecule CRM1 and its binding to the leucine-rich nuclear export signal of the cargo protein and the formation of a nuclear export trimer with RanGTP. The therapeutic potential of various CRM1 inhibitors will be addressed, including leptomycin B, ratjadone, KOS-2464, and specific small molecule inhibitors of CRM1, N-azolylacrylate analogs, FOXO export inhibitors, valtrate, acetoxychavicol acetate, CBS9106, and SINE inhibitors. We will also discuss examples of how drug resistance may be reversed by targeting the exported proteins topoisomerase IIα, BCR-ABL, and galectin-3. As effective and less toxic CRM1 export inhibitors become available, they may be used as both single agents and in combination with current chemotherapeutic drugs. We believe that the future development of low-toxicity, small-molecule CRM1 inhibitors may provide a new approach to treating cancer.

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Model system to study classical nuclear export signals

Cited by 21 publications

References 25 publications

Identification and Characterization of Human Cdc7 Nuclear Retention and Export Sequences in the Context of Chromatin Binding

Identification and Characterization of Human Cdc7 Nuclear Retention and Export Sequences in the Context of Chromatin Binding

Controlling Protein Compartmentalization to Overcome Disease

Nuclear export of proteins and drug resistance in cancer

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