Mechanisms of Receptor‐Mediated Nuclear Import and Nuclear Export

Pemberton, Lucy F.; Paschal, Bryce M.

doi:10.1111/j.1600-0854.2005.00270.x

Cited by 632 publications

(620 citation statements)

References 128 publications

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“…Here, we demonstrate that the situation is different for proteins that are able to traverse the nuclear envelope by both carrier-dependent and carrier-independent modes, such as importin-a and importin-b (5,39,40). Although the importinb antibody interfered with the binding of importin-a, the antibody-decorated importin-b was nevertheless capable of entering the nucleus.…”

Section: Discussionmentioning

confidence: 86%

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

et al. 2008

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The observation that some antibodies can enter the nucleus after their microinjection into the cytoplasm established the principle of protein nucleocytoplasmic shuttling. Here, we introduce the concept of stationary antibodies for studying nuclear transport, particularly of native proteins. Contrary to the aforementioned translocating immunoglobulins, stationary antibodies do not cross the nuclear envelope. They are distinguished by their ability to trigger the nucleocytoplasmic redistribution of their antigen. What determines these apparently contradictory outcomes has not been explored. We studied a stationary STAT1 antibody and a translocating importin-b antibody. The stationary phenotype resulted from the inhibition of carrier-independent transport. This was not due to crosslinking or precipitation of antigen, because the antigen-antibody complex remained highly mobile. Rather, decoration with stationary antibody precluded actual nuclear pore passage of antigen. In addition, both antibodies inhibited the carrier-dependent translocation via importin-a, but by diverse mechanisms. The translocating antibody blocked the association with importin-a, whereas the stationary antibody prevented the phosphorylation of its antigen, and thus functioned upstream of the importin-a binding step. We identified a stationary antibody to green-fluorescent protein (GFP) and probed the translocation of GFP fusions of STAT1, thyroid hormone receptor and histones, demonstrating general application of this approach. Our results provide an experimental rationale for the use of antibodies as unique tools for dissecting protein nuclear translocation. As the microinjection of stationary antibodies extends to analyses of native proteins, this method can complement and validate results obtained with fluorescent-labeled derivatives. ' 2008 International Society for Advancement of Cytometry Key terms antibody; native protein; microinjection; nucleocytoplasmic shuttling; import; export; GFP TRANSPORT of proteins and macromolecules between the nucleus and the cytoplasm of eukaryotic cells is an important mechanism for cytoplasmic regulation of nuclear activities. Nucleocytoplasmic exchange occurs through specialized channels called ''nuclear pore complexes'' (NPCs), which perforate the double layer of the nuclear envelope (1). The NPCs function as selectivity filters allowing passive diffusion of molecules up to $40 kDa, whereas components of higher molecular weight are usually restricted from autonomous exchange (2,3). A well-characterized pathway for nuclear transport of proteins relies on the GTPase Ran and the transport factors (also called carriers or karyopherins) importin-a and importin-b or CRM1 for import or export, respectively (4,5). In addition, certain large proteins can enter the nucleus, independent of metabolic energy and transport factors via direct interactions with constituents of the nuclear pore (6), and an increasing number of proteins, e.g. STAT1 and importin-b are known to use both pathways consecutively or i...

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Section: Discussionmentioning

confidence: 86%

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

et al. 2008

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“…For dephosphorylation GST fusion proteins bound to glutathione-Sepharose were washed in PBS containing 1 % Triton-X-100 (v/v), 0.5 mM DTT, and 0.5 mM PMSF, and incubated in a dephosphorylation buffer (Tris 20 mM, NaCl 100 mM, MgCl 2 5 mM, DTT 1 mM, BSA 0.5 mg/ml, CaCl 2 1 mM) with recombinant calcineurin (Calbiochem) and 1 lM calmodulin. After electrophoresis, polyacrylamide gels were dried and incorporated 32 P measured with Fuji Phosphoimager, FLA7000, together with [c-32 P]ATP spotted on bench coat paper for quantification.…”

Section: Gst Protein Cloningmentioning

confidence: 99%

“…Cytonuclear trafficking of proteins larger than 40 kDa results from an active transport mediated by karyopherins, which facilitate translocation of cargos through the nuclear pore and release them in the nucleus, in the case of importins, or in the cytoplasm in the case of exportins [28,29]. The best characterized mechanisms for nuclear import and export are based on the association of cargos that contain nuclear localization signal (NLS) or a hydrophobic nuclear export sequence (NES) with importins and the exportin chromosome region maintenance 1 (CRM1), respectively [30][31][32]. Here we characterize the role of NLS, NES, and other sequences in Pyk2 cytonuclear trafficking and its control by S778 phosphorylation by PKA and dephosphorylation by calcineurin.…”

Section: Introductionmentioning

confidence: 99%

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

Faure

Ramos

Girault

2012

Cell. Mol. Life Sci.

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Cytonuclear signaling is essential for long-term alterations of cellular properties. Several pathways involving regulated nuclear accumulation of Ser/Thr kinases have been described but little is known about cytonuclear trafficking of tyrosine kinases. Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic non-receptor tyrosine kinase enriched in neurons and involved in functions ranging from synaptic plasticity to bone resorption, as well as in cancer. We previously showed the Ca 2? -induced, calcineurin-dependent, nuclear localization of Pyk2. Here, we characterize the molecular mechanisms of Pyk2 cytonuclear localization in transfected PC12 cells. The 700-841 linker region of Pyk2 recapitulates its depolarizationinduced nuclear accumulation. This region includes a nuclear export motif regulated by phosphorylation at residue S778, a substrate of cAMP-dependent protein kinase and calcineurin. Nuclear import is controlled by a previously identified sequence in the N-terminal domain and by a novel nuclear targeting signal in the linker region. Regulation of cytonuclear trafficking is independent of Pyk2 activity. The region regulating nuclear localization is absent from the non-neuronal shorter splice isoform of Pyk2. Our results elucidate the mechanisms of Ca 2? -induced nuclear accumulation of Pyk2. They also suggest that Pyk2 nuclear accumulation is a novel type of signaling response that may contribute to specific long-term adaptations in neurons.

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“…1 In this context, the observation that steroid activation of signaling pathways regulates nuclear/cytoplasmic shuttling of glucocorticoid, progesterone and androgen receptors 2 as well as orphan estrogen receptor alpha-related protein 3 has a strong impact.…”

Section: Introductionmentioning

confidence: 99%

Tyrosine phosphorylation of estradiol receptor by Src regulates its hormone-dependent nuclear export and cell cycle progression in breast cancer cells

et al. 2012

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We report that in breast cancer cells, tyrosine phosphorylation of the estradiol receptor alpha (ERalpha) by Src regulates cytoplasmic localization of the receptor and DNA synthesis. Inhibition of Src or use of a peptide mimicking the ERalpha p-Tyr537 sequence abolishes ERalpha tyrosine phosphorylation and traps the receptor in nuclei of estradiol-treated MCF-7 cells. An ERalpha mutant carrying a mutation of Tyr537 to phenylalanine (ER537F) persistently localizes in nuclei of various cell types. In contrast with ERalpha wt, ER537F does not associate with Ran and its interaction with Crm1 is insensitive to estradiol. Thus, independently of estradiol, ER537F is retained in nuclei, where it entangles FKHR-driving cell cycle arrest. Chromatin immunoprecipitation analysis reveals that overexpression of ER537F in breast cancer cells enhances FKHR interaction with cyclin D1 promoter. This mutant also counteracts cell transformation by the activated forms of Src or PI3-K. In conclusion, in addition to regulating receptor localization, ERalpha phosphorylation by Src is required for hormone responsiveness of DNA synthesis in breast cancer cells.

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Mechanisms of Receptor‐Mediated Nuclear Import and Nuclear Export

Cited by 632 publications

References 128 publications

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

Tyrosine phosphorylation of estradiol receptor by Src regulates its hormone-dependent nuclear export and cell cycle progression in breast cancer cells

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