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Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads.

ABSTRACT: Upon transforming growth factor beta (TGF-beta) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, we have studied the kinetics of Smad nucleocytoplasmic shuttling in a quantitative manner in vivo. We have obtained rate constants for import and export of Smad2 and show that the cytoplasmic localization of Smad2 in uninduced cells reflects its nuclear export being more rapid than import. We find that TGF-beta-induced nuclear accumulation of Smad2 is caused by a pronounced drop in the export rate of Smad2 from the nucleus, which is associated with a strong decrease in nuclear mobility of Smad2 and Smad4. TGF-beta-induced nuclear accumulation involves neither a release from cytoplasmic retention nor an increase in Smad2 import rate. Hence, TGF-beta-dependent nuclear accumulation of Smad2 is caused exclusively by selective nuclear trapping of phosphorylated, complexed Smad2. The proposed mechanism reconciles signal-dependent nuclear accumulation of Smad2 with its continuous nucleocytoplasmic cycling properties.

SUBMITTER: Schmierer B

PROVIDER: S-EPMC1280270 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads.

Schmierer Bernhard B Hill Caroline S CS

Molecular and cellular biology 20051101 22

Upon transforming growth factor beta (TGF-beta) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, we have studied the kinetics of Smad nucleocytoplasmic shuttling in a quantitative manner in vivo. We have obtained rate constants for import and export of Smad2 and show that the cytoplasmic localization of ...[more]

PMID: 16260601

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Project description:Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283 to 291 (designated NES2) and amino acids 602 to 608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256 to 274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1 and importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1- and importin α/β-mediated transport by specific inhibitors (LMB, importazole, and ivermectin) clearly blocked PPV replication. The mutant viruses with deletions of the NESs or NLS motif of NS1 by using reverse genetics could not be rescued, suggesting that the NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/β-mediated nuclear import pathway, and PPV proliferation was inhibited by blocking NS1 nuclear import or export. IMPORTANCE PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (>50 kDa), it cannot pass through the nuclear pore complex by diffusion alone and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and their dependence on the CRM1 pathway for nuclear export was demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/β nuclear import pathway.

Project description:Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19LSLRELAI26 of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein.IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein.

Dataset Information

Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads.

Publications

Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads.

Similar Datasets

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