The Rel protein DIF mediates the antifungal but not the antibacterial host defense in Drosophila

We have isolated two Drosophila lines that carry point mutations in the gene coding for the NF-KB-like factor DIF. Like mutants of the Toll pathway, Dif mutant flies are susceptible to fungal but not to bacterial infections. Genetic epistasis experiments demonstrate that Dif mediates the Toll-dependent control of the inducibility of the antifungal peptide gene Drosomycin. Strikingly, DIF alone is required for the antifungal response in adults, but is redundant in larvae with Dorsal, another Rel family member. In Drosophila, Dif appears to be dedicated to the antifungal defense elicited by fungi and gram-positive bacteria. We discuss in this light the possibility that NF-KB1/p50 might be required more specifically in the innate immune response against gram-positive bacteria in mammals.     

Toll receptor-mediated Drosophila immune response requires Dif, an NF-kappaB factor

The induction of immunity genes in Drosophila has been proposed to be dependent on Dorsal, Dif, and Relish, the NF-kappaB-related factors. Here we provide genetic evidence that Dif is required for the induction of only a subset of antimicrobial peptide genes. The results show that the presence of Dif without Dorsal is sufficient to mediate the induction of drosomycin and defensin. We also demonstrate that Dif is a downstream component of the Toll signaling pathway in activating the drosomycin expression. These results reveal that individual members of the NF-kappaB family in Drosophila have distinct roles in immunity and development.     

Nuclear import of the Drosophila Rel protein Dorsal is regulated by phosphorylation

In Drosophila, dorsal-ventral polarity is determined by a maternally encoded signal transduction pathway that culminates in the graded nuclear localization of the Rel protein, Dorsal. Dorsal is retained in the cytoplasm by the IkappaB protein, Cactus. Signal-dependent phosphorylation of Cactus results in the degradation of Cactus and the nuclear targeting of Dorsal. We present an in-depth study of the functional importance of Dorsal phosphorylation. We find that Dorsal is phosphorylated by the ventral signal while associated with Cactus, and that Dorsal phosphorylation is essential for its nuclear import. In vivo phospholabeling of Dorsal is limited to serine residues in both ovaries and early embryos. A protein bearing mutations in six conserved serines abolishes Dorsal activity, is constitutively cytoplasmic, and appears to eliminate Dorsal phosphorylation, but still interacts with Cactus. Two individual serine-to-alanine mutations produce unexpected results. In a wild-type signaling background, a mutation in the highly conserved PKA site (S312) produces only a weak loss-of-function; however, it completely destabilizes the protein in a cactus mutant background. Significantly, the phosphorylation of another completely conserved serine (S317) regulates the high level of nuclear import found in ventral cells. We conclude that the formation of a wild-type Dorsal nuclear gradient requires the phosphorylation of both Cactus and Dorsal. The strong conservation of the serines suggests that phosphorylation of other Rel proteins is essential for their proper nuclear targeting.     

Characterization of karyopherins in androgen receptor intracellular trafficking in the yeast model

Background: Mechanisms regulating androgen receptor (AR) subcellular localization represent an essential component of AR signaling. Karyopherins are a family of nucleocytoplasmic trafficking factors. In this paper, we used the yeast model to study the effects of karyopherins on the subcellular localization of the AR. Methods: Yeast mutants deficient in different nuclear transport factors were transformed with various AR based, GFP tagged constructs and their localization was monitored using microscopy. Results: We showed that yeast can mediate androgen-induced AR nuclear localization and that in addition to the import factor, Importinα/β, this process required the import karyopherin Sxm1. We also showed that a previously identified nuclear export sequence (NES^AR) in the ligand binding domain of AR does not appear to rely on karyopherins for cytoplasmic localization. Conclusions: These results suggest that while AR nuclear import relies on karyopherin activity, AR nuclear export and/or cytoplasmic localization may require other undefined mechanisms.     

Short deletions in nuclear targeting sequences of African cassava mosaic virus coat protein prevent geminivirus twinned particle formation

Coat proteins (CPs) of geminiviruses are multifunctional proteins. Using transient expression experiments, we have recently identified putative sequence motifs of African cassava mosaic virus (ACMV) CP involved in nuclear import (NLS) and export (NES) (Virology 286 (2001) 373). Here, we report on the effect of corresponding deletion mutants in the context of infecting viruses. Since NLS and NES may overlap with DNA binding and multimerisation domains, we have investigated their effect on viral infection, particularly, on particle formation. All deletion mutants were infectious in Nicotiana benthamiana when co-inoculated with DNA B, but poorly sap-transmissible. Some of the mutants showed reduced levels of viral single-stranded DNA (ssDNA), whereas the amount of double-stranded DNA (dsDNA) was not greatly affected. None of these CP mutants was able to produce stable virus particles. In contrast, viruses with CP fused to Flag epitopes at the N- or C-terminus (CP:Flag or Flag:CP) were readily sap-transmissible and formed amorphous nucleoprotein particles but only few geminate structures. The relevance of the identified sequences in replicating viruses with reference to nuclear import and export as well as to particle stability and DNA binding is discussed.     

Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2

Analysis of mutants defective in meiotic chromosome pairing has uncovered a role for Caenorhabditis elegans chk-2 in initial establishment of pairing between homologous chromosomes during early meiotic prophase. chk-2 is also required for the major spatial reorganization of nuclei that normally accompanies the onset of pairing, suggesting a mechanistic coupling of these two events. Despite failures in pairing, nuclear reorganization, and crossover recombination, chk-2 mutants undergo many other aspects of meiotic chromosome morphogenesis and complete gametogenesis. Although chk-2 encodes a C. elegans ortholog of the Cds1/Chk2 checkpoint protein kinases, germ-line nuclei in chk-2 mutants are competent to arrest proliferation in response to replication inhibition and to trigger DNA damage checkpoint responses to ionizing radiation. However, chk-2 mutants are defective in triggering the pachytene DNA damage checkpoint in response to an intermediate block in the meiotic recombination pathway, suggesting that chk-2 is required either for initiation of meiotic recombination or for monitoring a specific subset of DNA damage lesions. We propose that chk-2 functions during premeiotic S phase to enable chromosomes to become competent for subsequent meiotic prophase events and/or to coordinate replication with entry into prophase.     

Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins

Adeno-associated virus (AAV) replicates in the nucleus of infected cells, and therefore multiple nuclear import events are required for productive infection. We analyzed nuclear import of the viral Rep proteins and characterized a nuclear localization signal (NLS) in the C-terminus. We demonstrate that basic residues in this region constitute an NLS that is transferable and mediates interaction with the nuclear import receptor importin alpha in vitro. Mutant Rep proteins are predominantly cytoplasmic and are severely compromised for interactions with importin alpha, but retain their enzymatic functions in vitro. Interestingly, mutations of the NLS had significantly less effect on importin alpha interaction and replication in the context of Rep78 than when incorporated into the Rep68 protein. Together, our results demonstrate that a bipartite NLS exists in the shared part of Rep68 and Rep78, and suggest that an alternate entry mechanism may also contribute to nuclear localization of the Rep78 protein.     

Vertebrate Rel proteins exhibit Dorsal-like activities in early Drosophila embryogenesis.

In Drosophila, the Toll/Dorsal pathway triggers the nuclear entry of the Rel protein Dorsal, which controls dorsoventral patterning in early embryogenesis and plays an important role in innate immunity of the adult fly. In vertebrates, the homologous Toll/IL-1 receptor signaling pathway directs the nuclear localization of Rel/NF-kappaB complexes, which activate genes involved in proliferation, apoptosis, and immune response. Recently, first evidence has been reported for the activity of vertebrate Rel proteins and a Toll-like signaling pathway in the dorsoventral patterning process of Xenopus laevis embryos. Given the evolutionary divergence of the fly and frog model organisms, these findings raise the question, to what extent the effector functions of this pathway have been conserved? Here, we report the ability of two Xenopus Rel proteins to partially substitute for several, but not all, functions of the Dorsal protein in Drosophila embryos. Our results suggest the interaction between Rel proteins and their cytoplasmic inhibitors as an important interface of evolutionary adaptation.     

The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors

We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7_39-98 localized mostly to the nucleus. The GST-11E7 and GST-11cE7_39-98 were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.