Nuclear translocation of 2-amino-3-ketobutyrate coenzyme A ligase by cold and osmotic stress

Cells are continuously exposed to environmental stresses and respond to them to maintain cellular homeostasis. Failure to respond to these stresses may cause pathological states such as renal failure, complications of diabetes, and autoimmune diseases. Signal transduction induced by osmotic and cold stresses is not fully understood. In addition, mechanisms of these stress responses are yet to be elucidated. Activation of many signaling pathways induces translocation of proteins into the nucleus to transduce signals and regulate nuclear functions. By using inducible translocation trap (ITT), a reporter gene-based screening technique, nuclear translocation of 2-amino-3-ketobutyrate coenzyme A ligase (KBL) was detected in response to cold and osmotic stresses. Rapid nuclear translocation of KBL was confirmed by biochemical analysis and fluorescent microscopy. A large region of KBL was required for stress-induced nuclear translocation. The KBL reporter system will be a useful tool for the investigation of cold and osmotic stress responses.     

How regulated protein translocation can produce switch-like responses

It is widely appreciated that the regulated translocation of signaling proteins can increase the specificity and speed of signal transduction. It is less obvious that regulated translocation can also, in principle, turn a graded response into a more switch-like one. For example, if two or more signaling proteins are induced to translocate, the result can be a switch-like, ultrasensitive response. A switch-like response will also occur if translocation raises the local concentration of a signaling protein sufficiently to partially saturate the enzyme that inactivates it. These mechanisms are likely to make the mitotic activation of CDC2 (which is accompanied by the nuclear translocation of both CDC2–cyclin-B1 and its activator, CDC25C) and the growth-factor-induced activation of MAP kinase (which, upon sustained activation, concentrates in the nucleus and might thereby partially saturate the relevant MAP-kinase phosphatases) more switch-like.     

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120ctn, also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.     

Exploring the influence of cytosolic and membrane FAK activation on YAP/TAZ nuclear translocation

Membrane binding and unbinding dynamics play a crucial role in the biological activity of several nonintegral membrane proteins, which have to be recruited to the membrane to perform their functions. By localizing to the membrane, these proteins are able to induce downstream signal amplification in their respective signaling pathways. Here, we present a 3D computational approach using reaction-diffusion equations to investigate the relation between membrane localization of focal adhesion kinase (FAK), Ras homolog family member A (RhoA), and signal amplification of the YAP/TAZ signaling pathway. Our results show that the theoretical scenarios in which FAK is membrane bound yield robust and amplified YAP/TAZ nuclear translocation signals. Moreover, we predict that the amount of YAP/TAZ nuclear translocation increases with cell spreading, confirming the experimental findings in the literature. In summary, our in silico predictions show that when the cell membrane interaction area with the underlying substrate increases, for example, through cell spreading, this leads to more encounters between membrane-bound signaling partners and downstream signal amplification. Because membrane activation is a motif common to many signaling pathways, this study has important implications for understanding the design principles of signaling networks.     

A high-content glucocorticoid receptor translocation assay for compound mechanism-of-action evaluation

Ligand-induced cytoplasm to nucleus translocation is a critical event in the nuclear receptor (NR) signal transduction cascade. The development of green fluorescent proteins and their color variants fused with NRs, along with the recent developments in automated cellular imaging technologies, has provided unique tools to monitor and quantify the NR translocation events. These technology developments have important implications in the mechanistic evaluation of NR signaling and provide a powerful tool for drug discovery. The unique challenges for developing a robust NR translocation assay include cytotoxicity accompanied with chronic overexpression of NRs, basal translocation induced by serum present in culture medium, and interference from endogenous NRs, as well as subcellular dynamics. The authors have developed a robust assay system for the glucocorticoid receptor (GR) that was applied to a panel of nuclear receptor ligands. Using a high-content imaging system, ligand-induced, dose-dependent GR nuclear translocation was quantified and a correlation with other conventional assays established.     

Mechanisms of nuclear translocation of insulin

Insulin (Ins) and various other hormones and growth factors have been shown to be rapidly internalized and translocated to the cell nucleus. This review summarizes the mechanisms that are involved in the translocation of Ins to the nucleus, and discusses its possible role in Ins action, based on observations by the authors and others. Ins is internalized to endosomes by both receptor-mediated and fluid-phase endocytosis, the latter occurring only at high Ins concentrations. The authors recently demonstrated the caveolae are the primary cell membrane locations responsible for initiating the signal transduction cascade induced by Ins. Once Ins is internalized, Ins dissociates from the Ins receptor in the endosome, and is translocated to the cytoplasm, where most Ins is degraded by Ins-degrading enzyme (IDE), although how the polypeptides cross the lipid bilayer is unknown. Some Ins escapes the degradation and binds to cytosolic Ins-binding proteins (CIBPs), in addition to IDE. IDE and some CIBPs are known to be binding proteins for other hormones or their receptors, and are involved in gene regulation, suggesting physiological relevance of CIBPs in the signaling of Ins and other hormones. Ins is eventually translocated through the nuclear pore to the nucleus, where Ins tightly associates with nuclear matrix. The role of Ins internalization and translocation to the nucleus is still controversial, although there is substantial evidence to support its role in cellular responses caused by Ins. Many studies indicate that nuclear translocation of various growth factors and hormones plays an important role in cell proliferation or DNA synthesis. It would be reasonable to suggest that tial for the regulation of nuclear events by Ins.     

Importin beta mediates nuclear translocation of Smad 3

Smad proteins are intracellular mediators of transforming growth factor-beta (TGF-beta) and related cytokines. Although ligand-induced nuclear translocation of Smad proteins is clearly established, the pathway mediating this import is yet to be determined. We previously identified a nuclear localization signal (NLS) in the N-terminal region of Smad 3, the major Smad protein involved in TGF-beta signal transduction. This basic motif (Lys(40-)Lys-Leu-Lys-Lys(44)), conserved among all the pathway-specific Smad proteins, is required for Smad 3 nuclear import in response to ligand. Here we studied the nuclear import pathway of Smad 3 mediated by this NLS. We demonstrate that the isolated Smad 3 MH1 domain displays significant specific binding to importin beta, which is diminished or eliminated by mutations in the NLS. Full-size Smad 3 exhibits weak but specific binding to importin beta, which is enhanced after phosphorylation by the type I TGF-beta receptor. In contrast, no interaction was observed between importin alpha and Smad 3 or its MH1 domain, indicating that nuclear translocation of Smad proteins may occur through direct binding to importin beta. We propose that activation of all of the pathway-specific Smad proteins (Smads 1, 2, 3, 5, 8, and 9) exposes the conserved NLS motif, which then binds directly to importin beta and triggers nuclear translocation.     

细胞因子和生长因子信号转导途径中信号蛋白分子的核转运机制

信号蛋白分子的入核及出核转运是细胞因子和生长因子信号转导途径中的重要环节.核定位序列（NLS）是信号蛋白分子上与入核转运相关的氨基酸序列.核孔复合物（NPC）、核转运蛋白importin和能量供应体Ran/TC4在入核转运过程中也发挥了重要作用.另外,很多细胞因子和生长因子或其受体上所含有的NLS序列也具有核定位功能,并可能通过“伴侣机制”参与其他信号蛋白分子的入核转运.     

Cactus-independent nuclear translocation of Drosophila RELISH

Insects can effectively and rapidly clear microbial infections by a variety of innate immune responses including the production of antimicrobial peptides. Induction of these antimicrobial peptides in Drosophila has been well established to involve NF-kappaB elements. We present evidence here for a molecular mechanism of Lipopolysaccharide (LPS)-induced signaling involving Drosophila NF-kappaB, RELISH, in Drosophila S2 cells. We demonstrate that LPS induces a rapid processing event within the RELISH protein releasing the C-terminal ankyrin-repeats from the N-terminal Rel homology domain (RHD). Examination of the cellular localization of RELISH reveals that the timing of this processing coincides with the nuclear translocation of the RHD and the retention of the ankyrin-repeats within the cytoplasm. Both the processing and the nuclear translocation immediately precede the expression of antibacterial peptide genes cecropin A1, attacin, and diptericin. Over-expression of the RHD but not full-length RELISH results in an increase in the promoter activity of the cecropin A1 gene in the absence of LPS. Furthermore, the LPS-induced expression of these antibacterial peptides is greatly reduced when RELISH expression is depleted via RNA-mediated interference. In addition, loss of cactus expression via RNAi revealed that RELISH activation and nuclear translocation is not dependent on the presence of cactus. Taken together, these results suggest that this signaling mechanism involving the processing of RELISH followed by nuclear translocation of the RHD is central to the induction of at least part of the antimicrobial response in Drosophila, and is largely independent of cactus regulation.     

Mutations in squirrel monkey glucocorticoid receptor impair nuclear translocation

To identify the determinants of impaired glucocorticoid receptor (GR) signaling in a model of glucocorticoid resistance, cloned GR from Guyanese squirrel monkeys (gsmGR) was tagged with enhanced green fluorescent protein, and nuclear translocation was examined in transfected COS1 cells. In keeping with evidence that gsmGR transactivational competence is impaired, we found that nuclear translocation is likewise diminished in gsmGR relative to human GR (hGR). Experiments with GR chimeras revealed that replacement of the gsmGR ligand binding domain (LBD) with that from hGR increased translocation. Truncated gsmGR constructs lacking the LDB after amino acid 552 also showed increased translocation even in the absence of cortisol. Three back-mutations of gsmGR to hGR (Thr551Ser, Ala616Ser, and Ser618Ala) in the LBD confirmed that these amino acids play a role in diminished translocation.     

Real-time imaging nuclear translocation of Akt1 in HCC cells

Akt is one of the critical mediators in cellular signaling, and overactivation of Akt related pathway frequently occurs in hepatocellular carcinoma (HCC). In this study, we presented that Akt was upregulated in HCC cell lines, and its active phosphorylated form was mainly located in the nucleus. Employing the laser confocal techniques for imaging intracellular protein dynamics, we monitored the transnuclear movement of GFP-tagged wild-type Akt1 (Akt1-WT-GFP) and its inactive mutant (Akt1-T308A/S473A-GFP) in live SMMC-7721 HCC cells, and both of fusion proteins were found to distribute over the cytoplasm and nucleus. Moreover, it was found that platelet derived growth factor (PDGF) was able to accelerate the nuclear translocation of wild-type Akt1 in HCC cells but failed to speed up the motion of the mutant. It was demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt1 facilitated the nuclear translocation of Akt1, but the phosphorylation at threonine 308 and serine 473 was not prerequisite.     

Nuclear translocation of active AKT is required for erythroid differentiation in erythropoietin treated K562 erythroleukemia cells

Erythroid differentiation of human erythroleukemia cell line K562 induced by erythropoietin is a complex process that involves modifications at nuclear level, including nuclear translocation of phosphatidyl-inositol 3-kinase. In this work we show that erythropoietin stimulation of K562 cells can induce nuclear translocation of active Akt, a downstream molecule of the phosphatidyl-inositol 3-kinase signaling pathway. Akt shows a peak of activity in whole cell homogenates at earlier stage when compared to the nucleus, which shows a peak delayed of 10 min. Akt increases its intranuclear amount and activity rapidly and transiently in response to EPO. Almost all Akt kinase that translocates to the nucleus shows a marked phosphorylation on serine 473. Nuclear enzyme translocation is blocked by the phosphatidyl-inositol 3-kinase inhibitor Ly294002 or Wortmannin. The specific Akt pharmacological inhibitor VI, VII and VIII that act as blocking enzyme activation inhibited translocation as well, whereas Akt inhibitor IX, that inhibits Akt activity, did not block Akt nuclear translocation. When cells were treated by means of siRNA sequences or with the Akt inhibitors the differentiation process was arrested, thus showing the requirement of the nuclear translocation of the active enzyme to differentiate. These findings strongly suggest that the intranuclear translocation of active Akt kinase represents an important step in the signaling pathway that mediates erythropoietin-induced erythroid differentiation.     

A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.     

TNFalpha induces rapid activation and nuclear translocation of telomerase in human lymphocytes

Maintenance of telomeres regulates chromosomal stability and cellular mitosis through a checkpoint mechanism. Continuous cell proliferation requires telomerase to maintain chromosomal stability and to counteract the cellular mitotic clock. Importantly, nuclear expression of telomerase activity is required for elongation of telomere sequences. In this study, we show that tumor necrosis factor alpha (TNFalpha) induces telomerase activity in the cytoplasm of peripheral blood lymphocytes (PBL) at 60 min, followed by translocation of activated telomerase to the nucleus at 120 min. Conversely, the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin blocks TNFalpha-induced activation of telomerase, whereas the specific NF-kappaB translocation inhibitor SN-50 blocks TNFalpha-induced nuclear translocation of activated telomerase. These studies suggest that activation and nuclear translocation of telomerase are regulated by PI3K/Akt/NF-kappaB signaling pathways in PBL.     

线粒体蛋白质运输机制

：线粒体的大多数蛋白质是由核基因编码、细胞质合成,而最终运输到线粒体。在此过程中,需要线粒体外膜和内膜的蛋白质运输机器(至少三种主要的移位酶复合物)来保证前体蛋白质的正确运输。