(1/1572) Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own.
Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners. (+info)
(2/1572) Nuclear and nucleolar targeting of human ribosomal protein S25: common features shared with HIV-1 regulatory proteins.
The nuclear and nucleolar targeting properties of human ribosomal protein S25 (RPS25) were analysed by the expression of epitope-tagged RPS25 cDNAs in Cos-1 cells. The tagged RPS25 was localized to the cell nucleus, with a strong predominance in the nucleolus. At the amino terminus of RPS25, two stretches of highly basic residues juxtapose. This configuration shares common features with the nucleolar targeting signals (NOS) of lentiviral RNA-binding transactivators, including human immunodeficiency viruses' (HIV) Rev proteins. Deletion and site-directed mutational analyses demonstrated that the first NOS-like stretch is dispensable for both nuclear and nucleolar localization of RPS25, and that the nuclear targeting signal is located within the second NOS-like stretch. It has also been suggested that a set of continuous basic residues and the total number of basic residues should be required for nucleolar targeting. Signal-mediated nuclear/nucleolar targeting was further characterized by the construction and expression of a variety of chimeric constructs, utilizing three different backbones with RPS25 cDNA fragments. Immunofluorescence analyses demonstrated a 17 residue peptide of RPS25 as a potential nuclear/nucleolar targeting signal. The identified peptide signal may belong to a putative subclass of NOS, characterized by compact structure, together with lentiviral RNA-binding transactivators. (+info)
(3/1572) Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation.
p21(Cip1/WAF1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21(Cip1/WAF1) is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21(Cip1/WAF1) in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21(Cip1/WAF1), and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21(Cip1/WAF1) demonstrated G1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21(Cip1/WAF1), concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21(Cip1/WAF1) forms a complex with the apoptosis signal-regulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21(Cip1/WAF1) lacking the nuclear localization signal (DeltaNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21(Cip1/WAF1) itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21(Cip1/WAF1), which are determined by its intracellular distribution and are dependent on the stage of differentiation. (+info)
(4/1572) Discrete domains mediate the light-responsive nuclear and cytoplasmic localization of Arabidopsis COP1.
The Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) protein plays a critical role in the repression of photomorphogenesis during Arabidopsis seedling development. We investigated the control of COP1 partitioning between nucleus and cytoplasm, which has been implicated in the regulation of COP1 activity, by using fusion proteins between COP1 and beta-glucuronidase or the green fluorescent protein. Transient expression assays using onion epidermal cells and data from hypocotyl cells of stably transformed Arabidopsis demonstrated that COP1 carries a single, bipartite nuclear localization signal that functions independently of light. Nuclear exclusion was mediated by a novel and distinct signal, bordering the zinc-finger and coiled-coil motifs, that was able to redirect a heterologous nuclear protein to the cytoplasm. The cytoplasmic localization signal functioned in a light-independent manner. Light regulation of nuclear localization was reconstituted by combining the individual domains containing the nuclear localization signal and the cytoplasmic localization signal; the WD-40 repeat domain of COP1 was not required. However, phenotypic analysis of transgenic seedlings suggested that the constitutively nuclear-localized WD-40 repeat domain was able to mimic aspects of COP1 function, as indicated by exaggerated hypocotyl elongation under light conditions. (+info)
(5/1572) Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1alpha.
Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents proteasomal degradation of HIF-1alpha, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. Here, we have used ARNT-mutant mouse hepatoma and embryonic stem cells to examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1alpha in hypoxia. As shown by immunofluorescence, HIF-1alpha accumulation in the nucleus of hypoxic cells was independent of the presence of ARNT, suggesting that nuclear translocation is intrinsic to HIF-1alpha. Co-immunoprecipitation of HIF-1alpha together with ARNT could be performed in nuclear extracts but not in cytosolic fractions, implying that formation of the HIF-1 complex occurs in the nucleus. A proteasome inhibitor and a thiol-reducing agent could mimic hypoxia by inducing HIF-1alpha in the nucleus, indicating that escape from proteolytic degradation is sufficient for accumulation and nuclear translocation of HIF-1alpha. During biochemical separation, both HIF-1alpha and ARNT tend to leak from the nuclei in the absence of either subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined. (+info)
(6/1572) SPA1, a WD-repeat protein specific to phytochrome A signal transduction.
The five members of the phytochrome photoreceptor family of Arabidopsis thaliana control morphogenesis differentially in response to light. Genetic analysis has identified a signaling pathway that is specifically activated by phytochrome A. A component in this pathway, SPA1 (for "suppressor of phyA-105"), functions in repression of photomorphogenesis and is required for normal photosensory specificity of phytochrome A. Molecular cloning of the SPA1 gene indicates that SPA1 is a WD (tryptophan-aspartic acid)-repeat protein that also shares sequence similarity with protein kinases. SPA1 can localize to the nucleus, suggesting a possible function in phytochrome A-specific regulation of gene expression. (+info)
(7/1572) The carboxyl terminus of RNA helicase A contains a bidirectional nuclear transport domain.
Human RNA helicase A was recently identified to be a shuttle protein which interacts with the constitutive transport element (CTE) of type D retroviruses. Here we show that a domain of 110 amino acids at the carboxyl terminus of helicase A is both necessary and sufficient for nuclear localization as well as rapid nuclear export of glutathione S-transferase fusion proteins. The import and export activities of this domain overlap but are separable by point mutations. This bidirectional nuclear transport domain (NTD) has no obvious sequence homology to previously identified nuclear import or export signals. However, the Ran-dependent nuclear import of NTD was efficiently competed by excess amounts of the nuclear localization signal (NLS) peptide from simian virus 40 large T antigen, suggesting that import is mediated by the classical NLS pathway. The nuclear export pathway accessed by NTD is insensitive to leptomycin B and thus is distinct from the leucine-rich nuclear export signal pathway mediated by CRM1. (+info)
(8/1572) Identification of the sequence responsible for the nuclear localization of human Cdc6.
The Cdc6 is the essential protein for the initiation of DNA replication. Cdc6 is localized in the G1 nucleus, and abnormal nuclear localization of this protein induces irregular initiation of DNA replication. We identified here that amino acids K57 and R58 in the human Cdc6 protein play an important role in the nuclear localization of the protein. The fundamental features of the mechanism regulating the localization of Cdc6 seem to be maintained in yeast, Xenopus, and human, since the amino acid sequence surrounding K57 and R58, (S/T)PXKR(L/I), is conserved in these species. Substitution of amino acid residue S54 with E and not Q blocked partially the nuclear localization of the protein, implying that the phosphorylation at S54 is involved in the regulating mechanism of the cell cycle-dependent localization of Cdc6. (+info)