Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex.

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.     

Nuclear Localization of the Saccharomyces cerevisiae HMG Protein NHP6A Occurs by a Ran-Independent Nonclassical Pathway

The Saccharomyces cerevisiae non-histone protein 6-A (NHP6A) is a member of the high-mobility group 1/2 protein family that bind and bend DNA of mixed sequence. NHP6A has only one high-mobility group 1/2 DNA binding domain and also requires a 16-amino-acid basic tail at its N-terminus for DNA binding. We show in this report that nuclear accumulation of NHP6A is strictly correlated with its DNA binding properties since only nonhistone protein 6 A–green fluorescent protein chimeras that were competent for DNA binding were localized to the nucleus. Despite the requirement for basic residues within the N-terminal segment for DNA binding and nuclear accumulation, this region does not appear to contain a nuclear localization signal. Moreover, NHP6A does not bind to the yeast nuclear localization signal receptor SRP1 and nuclear targeting of NHP6A does not require the function of the 14 different importins. Unlike histone H2B1 which contains a classical nuclear localization signal, entry of NHP6A into the nucleus was found to be independent of Ran as judged by coexpression of Ran GTPase mutants and was shown to occur at 0 °C after a 15-min induction. These unusual properties lead us to suggest that NHP6A entry into the nucleus proceeds by a nonclassical Ran-independent pathway.     

Nuclear translocation of intracellular domain of Protogenin by proteolytic cleavage

Protogenin (PRTG) is a transmembrane protein of immunoglobulin superfamily, which has multiple roles in embryogenesis as a receptor or an adhesion molecule. In this study, we present sequential proteolytic cleavage of PRTG. The cleavage first occurs at the extracellular domain, then at the interface of the transmembrane and the intracellular domain by γ-secretase, which results in the release of the intracellular domain of PRTG (PRTG-ICD). PRTG-ICD contains putative nuclear localization signal (NLS) at its N-terminal, and translocates to the nucleus in cultured cells and in the neuroepithelial cells of chick embryos. We propose that the PRTG-ICD is cleaved by γ-secretase and translocates to the nucleus, which is potentially implicated in signaling for neural differentiation and in cell adhesion mediated by PRTG.     

Prox1 is differentially localized during lens development

Prox1, the vertebrate cognate of Drosophila Prospero, is a homeodomain protein essential for the development of the lens, liver and lymphatic system. While it is well established that the subcellular distribution of Prospero changes during development, this had not been demonstrated for Prox1. Here, high-resolution confocal microscopy demonstrated that Prox1 protein is predominately cytoplasmic in the lens placode as well as the lens epithelium and germinative zone throughout development. However during fiber cell differentiation, Prox1 protein redistributes to cell nuclei. Finally, as lens fiber cells condense their chromatin in response to lens denucleation, Prox1 remains in the nucleus but does not appear to interact with DNA. Thus, it appears that the function of Prox1, like that of its Drosophila cognate Prospero, is at least partially controlled by changes in its subcellular distribution during development.     

Nuclear import and DNA-binding activity of RFX1. Evidence for an autoinhibitory mechanism.

RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent 'acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC.     

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.     

Mutations in the ATP-binding domain affect the subcellular distribution of mitotic centromere-associated kinesin (MCAK)

Mitotic centromere-associated kinesin (MCAK) is important for anaphase chromosome segregation. MCAK is diffusely localized to both the cytoplasm and the nucleus during interphase. At prophase MCAK is recruited to mitotic centromeres. It is associated with centromeres throughout mitosis and then returns to exhibiting a diffuse nuclear and cytoplasmic localization during interphase. MCAK has several predicted nuclear localization sequences. The subcellular distribution of expressed deletion constructs of GFP-MCAK suggest that the nucleocytoplasmic ratio of MCAK protein is dependent on a balance between several predicted nuclear localization sequences (NLS) and a putative nuclear exclusion sequence (NES) in the amino-terminal region of MCAK. Amino acid substitutions in the ATP-binding domain of the MCAK motor affect nuclear localization, which, in turn, influences the degree of centromere binding.     

Cyclin/CDK regulates the nucleocytoplasmic localization of the human papillomavirus E1 DNA helicase

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.     

Mechanisms of nuclear localization of the progesterone receptor

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638–642 and was shown to be constitutively active. When amino acids 638–642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or antihormone), it was almost fully active. A second putative nuclear localization signal is located in the DNA binding domain activated either through ligand binding or through production of constitutive receptor. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand the receptor was transferred into the nucleus as a monomer. After administration of hormone (or antihormone) a “cytoplasmic” monomer was transferred into the nucleus through interaction with a “nuclear” monomer. These interactions occurred through the steroid binding domains of both monomers.