[Show abstract][Hide abstract] ABSTRACT: Importin α is involved in the nuclear import of proteins. It also contributes to spindle assembly and nuclear membrane formation, however, the underlying mechanisms are poorly understood. Here, we studied the function of importin α7 by gene targeting in mice and show that it is essential for early embryonic development. Embryos lacking importin α7 display a reduced ability for the first cleavage and arrest completely at the two-cell stage. We show that the zygotic genome activation is severely disturbed in these embryos. Our findings indicate that importin α7 is a new member of the small group of maternal effect genes.
PLoS ONE 03/2011; 6(3):e18310. DOI:10.1371/journal.pone.0018310 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Notch signaling pathway is an important regulation system for the development and self-renewal of different tissues. A specific feature of this signaling cascade is the function of Notch as a surface receptor and regulator of gene expression. Hence, Notch activation and signal transduction requires the proteolytic release of the Notch intracellular domain (NICD), which activates the transcription of cell-specific genes after its transport into the nucleus. To date, little is known about the mechanisms that mediate NICD nuclear import. We here show that transport of NICD into the nucleus is mediated by the canonical importin α/β1 pathway. GST pull-down experiments revealed that NICD binds via one of its four potential nuclear localization signals to importins α3, α4, and α7, but not to α1 and α5. siRNA-mediated knockdown experiments showed that importins α3, α4 (and to a lesser extent, α7) mediate nuclear import of NICD and thus are directly involved in Notch signaling.
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The online version of this article (doi:10.1007/s00018-010-0378-7) contains supplementary material, which is available to authorized users.
Cellular and Molecular Life Sciences CMLS 05/2010; 67(18):3187-96. DOI:10.1007/s00018-010-0378-7 · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-inducible factors are crucial in the regulatory process of oxygen homeostasis of vertebrate cells. Inhibition of prolyl hydroxylation of HIF-alpha subunits by prolyl-hydroxylases (PHD1, PHD2 and PHD3) leads to transcription of a greater number of hypoxia responsive genes. We have investigated the subcellular distribution and the molecular mechanisms regulating the intracellular allocation of PHD1 and PHD2. As reported earlier we find PHD1 located exclusively in the nucleus. We demonstrate that nuclear import of PHD1 occurs importin alpha/beta dependently and relies on a nuclear localisation signal (NLS). By contrast PHD2 is cycling between nucleus and cytoplasm, and nuclear import seems to be independent of "classical" importin alpha/beta receptors. Furthermore, we reveal that the exit of PHD2 from the nucleus requires CRM1 and the N-terminal 100 amino acids of the protein. Our findings provide new insights into the mechanisms of the regulation of the oxygen sensor cascade of PHDs in different cellular compartments.
Biochemical and Biophysical Research Communications 08/2009; 387(4):705-11. DOI:10.1016/j.bbrc.2009.07.090 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelin-1 is a powerful mitogen for various tumor and non-tumor cells. Its signaling cascade induces the inflammatory NF-kappaB complex, leading to expression of a number of target genes. In this context, MAPK p38 has been regarded as a potential phosphate donor for the p65 subunit of NF-kappaB. In the present study in HeLa cells, we have found that ET-1 induced signalling activates the NF-kappaB transcription complex (TC) in the nucleus at 6 h specifically via ET-A - but not ET-B receptor. The TC contains p65, p38 (alpha and beta) - binding to the NLS of p65 in the cytoplasm - as well as p50, but no IkappaBalpha. Specific p38 inhibition by SB203580 or by siRNA interferes markedly with gene expression of several target genes. Complex formation occurs in the cytoplasm, and both transcription factors transmigrate as a complex in the nucleus. Overexpression of p38, treatment with Chrysin, MG132, or dimethylformamide shows dependence of TC on p38 as partner. In other tumor cells lines studied, ET-1 activates TC, with p38 as an important complex partner of p65. TC-induction by ET-1 contains about twice the amount of p38 than by TNFalpha. Thus, p38 may be an additional therapeutic target to control inflammatory gene expression in tumor cells.
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-alpha subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1alpha into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors importin alpha/beta. HIF-1alpha, -1beta, and HIF-2alpha are binding to importin alpha1, alpha3, alpha5, and alpha7. The direct interaction of HIF-1alpha to alpha importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.
[Show abstract][Hide abstract] ABSTRACT: Resistance to anticancer drugs that target DNA topoisomerase II (topo II) isoforms alpha and/or beta is associated with decreased nuclear and increased cytoplasmic topo IIalpha. Earlier studies have confirmed that functional nuclear localization and export signal sequences (NLS and NES) are present in both isoforms. In this study, we show that topo II alpha and beta bind and are imported into the nucleus by importin alpha1, alpha3, and alpha5 in conjunction with importin beta. Topo IIalpha also binds exportin/CRM1 in vitro. However, wild-type topo IIalpha has only been observed in the cytoplasm of cells that are entering plateau phase growth. This suggests that topo IIalpha may shuttle between the nucleus and the cytoplasm with the equilibrium towards the nucleus in proliferating cells but towards the cytoplasm in plateau phase cells. The CRM1 inhibitor Leptomycin B increases the nuclear localization of GFP-tagged topo IIalpha with a mutant NLS, suggesting that its export is being inhibited. However, homokaryon shuttling experiments indicate that fluorescence-tagged wild-type topo II alpha and beta proteins do not shuttle in proliferating Cos-1 or HeLa cells. We conclude that topo II alpha and beta nuclear export is inhibited in proliferating cells so that these proteins do not shuttle.
Experimental Cell Research 03/2007; 313(3):627-37. DOI:10.1016/j.yexcr.2006.11.004 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The “classical” nuclear protein import pathway depends on importin α and importin β. Importin α binds nuclear localization
signal (NLS)-bearing proteins and functions as an adapter to access the importin β-dependent import pathway. In humans, only
one importin β is known to interact with importin α, while six α importins have been described. Various experimental approaches
provided evidence that several substrates are transported specifically by particular α importins. Whether the NLS is sufficient
to mediate importin α specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import
substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin α3, and the less
specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and
protein context contribute to the specificity of importin α binding and transport.
[Show abstract][Hide abstract] ABSTRACT: NF-kappaB transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin alpha3 and importin alpha4 as the main importin alpha isoforms mediating TNF-alpha-stimulated NF-kappaB p50/p65 heterodimer translocation into the nucleus. Importin alpha3 and alpha4 are close relatives in the human importin alpha family. We show that importin alpha3 isoform also mediates nuclear import of NF-kappaB p50 homodimer in nonstimulated cells. Importin alpha3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-kappaB p50 and p65 proteins. Importin alpha molecules are known to have armadillo repeats that constitute the N-terminal and C-terminal NLS binding sites. We demonstrate by site-directed mutagenesis that NF-kappaB p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin alpha3. In vitro competition experiments and analysis of cellular NF-kappaB suggest that NF-kappaB binds to importin alpha only when it is free of IkappaBalpha. The present study demonstrates that the nuclear import of NF-kappaB is a highly regulated process mediated by a subset of importin alpha molecules.
[Show abstract][Hide abstract] ABSTRACT: Nuclear import of HuR, a shuttling RNA-binding protein, is associated with reduced stability of its target mRNAs. Increased function of the AMP-activated protein kinase (AMPK), an enzyme involved in responding to metabolic stress, was recently shown to reduce the cytoplasmic levels of HuR. Here, we provide evidence that importin alpha1, an adaptor protein involved in nuclear import, contributes to the nuclear import of HuR through two AMPK-modulated mechanisms. First, AMPK triggered the acetylation of importin alpha1 on Lys(22), a process dependent on the acetylase activity of p300. Second, AMPK phosphorylated importin alpha1 on Ser(105). Accordingly, expression of importin alpha1 proteins bearing K22R or S105A mutations failed to mediate the nuclear import of HuR in intact cells. Our results point to importin alpha1 as a critical downstream target of AMPK and key mediator of AMPK-triggered HuR nuclear import.
[Show abstract][Hide abstract] ABSTRACT: Nuclear import of HuR, a shuttling RNA-binding protein, is associated with reduced stability of its target mRNAs. Increased
function of the AMP-activated protein kinase (AMPK), an enzyme involved in responding to metabolic stress, was recently shown
to reduce the cytoplasmic levels of HuR. Here, we provide evidence that importin α1, an adaptor protein involved in nuclear
import, contributes to the nuclear import of HuR through two AMPK-modulated mechanisms. First, AMPK triggered the acetylation
of importin α1 on Lys22, a process dependent on the acetylase activity of p300. Second, AMPK phosphorylated importin α1 on Ser105. Accordingly, expression of importin α1 proteins bearing K22R or S105A mutations failed to mediate the nuclear import of
HuR in intact cells. Our results point to importin α1as a critical downstream target of AMPK and key mediator of AMPK-triggered
HuR nuclear import.
Journal of Biological Chemistry 11/2004; 279(46):48376-48388. · 4.57 Impact Factor