Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin

Development ◽  
2001 ◽  
Vol 128 (8) ◽  
pp. 1403-1414 ◽  
Author(s):  
J.A. Lorenzen ◽  
S.E. Baker ◽  
F. Denhez ◽  
M.B. Melnick ◽  
D.L. Brower ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Receptor Tyrosine Kinase ◽  
Nuclear Import ◽  
S2 Cells ◽  
Wild Type ◽  
Tyrosine Kinase Signaling ◽  
Its Gene ◽  
Drosophila Homolog ◽  
Drosophila S2 Cells ◽  
Mutant Phenotypes

The initiation of gene expression in response to Drosophila receptor tyrosine kinase signaling requires the nuclear import of the MAP kinase, D-ERK. However, the molecular details of D-ERK translocation are largely unknown. In this regard, we have identified D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin. DIM-7 exhibits a dynamic nuclear localization pattern that overlaps the spatial and temporal profile of nuclear, activated D-ERK. Co-immunoprecipitation experiments show that DIM-7 associates with phosphorylated D-ERK in Drosophila S2 cells. Furthermore, moleskin mutations enhance hypomorphic and suppress hypermorphic D-ERK mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated D-ERK. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin (β) homolog Ketel, also reduce the nuclear localization of activated D-ERK. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated D-ERK.

scholarly journals Baculovirus Transregulator IE1 Requires a Dimeric Nuclear Localization Element for Nuclear Import and Promoter Activation

2002 ◽  
Vol 76 (18) ◽  
pp. 9505-9515 ◽  
Author(s):  
Victoria A. Olson ◽  
Justin A. Wetter ◽  
Paul D. Friesen
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Direct Interaction ◽  
Productive Infection ◽  
Homologous Region ◽  
Wild Type ◽  
Nuclear Entry ◽  
Early Protein ◽  
Biochemical Fractionation ◽  
Localization Element

ABSTRACT Immediate-early protein IE1 is a principal regulator of viral transcription and a contributor to origin-specific DNA replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Since these viral functions involve interaction of dimeric IE1 with palindromic homologous region (hr) enhancer-origin elements of the AcMNPV genome within the nucleus, it is presumed that proper nuclear transport of IE1 is essential for productive infection. To investigate the mechanisms of IE1 nuclear import, we analyzed the effect of site-directed mutations on IE1 subcellular distribution. As demonstrated by fluorescence microscopy and biochemical fractionation of plasmid-transfected cells, wild-type IE1 localized predominantly to the nucleus. Substitution or deletion of amino acid residues within a positively charged domain (residues 534 to 538) adjacent to IE1's oligomerization motif impaired nuclear import and caused loss of transactivation. Moreover, upon coexpression, these import-defective mutations prevented nuclear entry of wild-type IE1. In contrast, double-mutated IE1 defective for both nuclear import and dimerization failed to block nuclear entry or transactivation by wild-type IE1. Thus, import-defective IE1 dominantly interfered with wild-type IE1 by direct interaction and cytosolic trapping. Collectively, our data indicate that the small basic domain encompassing residues R537 and R538 constitutes a novel nuclear localization element that functions only upon IE1 dimerization. These findings support a model wherein IE1 oligomerizes within the cytosol as a prerequisite for nuclear entry and subsequent high-affinity interaction with the symmetrical binding sites comprising AcMNPV hr enhancer-origin elements.

scholarly journals Insertion of a Classical Nuclear Import Signal into the Matrix Domain of the Rous Sarcoma Virus Gag Protein Interferes with Virus Replication

2004 ◽  
Vol 78 (24) ◽  
pp. 13534-13542 ◽  
Author(s):  
Rachel A. Garbitt ◽  
Karen R. Bone ◽  
Leslie J. Parent
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Rous Sarcoma Virus ◽  
Virus Assembly ◽  
Wild Type ◽  
Nuclear Targeting ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Junction Molecules ◽  
Sarcoma Virus

ABSTRACT The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a “classical ” nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the gag gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.

scholarly journals Wild-Type Levels of Nuclear Localization and Human Immunodeficiency Virus Type 1 Replication in the Absence of the Central DNA Flap

2002 ◽  
Vol 76 (23) ◽  
pp. 12078-12086 ◽  
Author(s):  
Ana Limón ◽  
Noriko Nakajima ◽  
Richard Lu ◽  
Hina Z. Ghory ◽  
Alan Engelman
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Numerous factors have been implicated in the nuclear localization of retroviral preintegration complexes. Whereas sequences in human immunodeficiency virus type 1 (HIV-1) matrix, Vpr, and integrase proteins were initially reported to function specifically in nondividing cells, other recently identified sequences apparently function in dividing cells as well. One of these, the central DNA flap formed during reverse transcription, is specific to lentiviruses. It was previously reported that flap-negative (F−) HIV-1LAI was completely defective for viral spread in the MT-4 T-cell line, yet F− HIV-1 vectors were only 2- to 10-fold defective in various single-round transduction assays. To address these different findings, we analyzed the infectivity and nuclear localization phenotypes of two highly related T-cell-tropic strains, HIV-1NL4-3 and a derivative of HIV-1HXBc2 deficient for both Vpr and Nef. In stark contrast to the previous report, F− derivatives of both strains replicated efficiently in MT-4 cells. F− HIV-1NL4-3 also spread like wild-type HIV-1NL4-3 in infected Jurkat and primary T-cell cultures. In contrast, F− HIV-1HXBc2 was replication defective in primary T cells. Results of real-time quantitative PCR assays, however, indicated that F− HIV-1HXBc2 entered primary T-cell nuclei as efficiently as its wild-type counterpart. Thus, the F− HIV-1HXBc2 growth defect did not appear to correlate with defective nuclear import. Consistent with this observation, wild-type nef restored replication to F− HIV-1HXBc2 in primary T cells. Our results indicate that the central DNA flap does not play a major role in either preintegration complex nuclear import or HIV-1 replication in a variety of cell types.

scholarly journals Nuclear import of the human androgen receptor

1993 ◽  
Vol 293 (3) ◽  
pp. 761-768 ◽  
Author(s):  
G Jenster ◽  
J Trapman ◽  
A O Brinkmann
Keyword(s):  
Androgen Receptor ◽  
Cell Line ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Subcellular Distribution ◽  
Wild Type ◽  
Cytoplasmic Staining ◽  
Localization Signal ◽  
Human Androgen Receptor

Nuclear import of the human androgen receptor was investigated by immunocytochemical analysis of androgen receptor deletion and substitution mutants, which were transiently expressed in COS-1 cells. The signal responsible for nuclear import is encoded by amino-acid residues 608-625 and is functionally similar to the bipartite nucleoplasmin nuclear-localization signal. Although the subcellular distribution of androgen receptors mutated in the DNA-binding domain was unchanged compared with the wild-type androgen receptor, in the presence of ligand these mutations resulted in part of the receptor population forming clusters. Depending on the presence or absence of the bipartite nuclear localization signal, clusters were formed in the nucleus or in the cytoplasm, respectively. Expression of the wild-type androgen receptor in different cell lines revealed a cell-line-specific subcellular distribution of the unliganded receptor. The androgen receptor was predominantly nuclear when expressed in HeLa cells, whereas mainly cytoplasmic staining was observed when it was expressed in COS-1 cells. In the presence of hormone, the androgen receptor was located in the nucleus, independent of the cell line that was expressing the receptor. Anti-androgens and various steroid hormones induced the nuclear localization of the wild-type androgen receptor in a dose-dependent way, without activating transcription of an androgen-regulated reporter gene. This indicates that the inability of the tested compounds to activate transcription is not due to inhibited nuclear import.

The strawberry notch gene functions with Notch in common developmental pathways

Development ◽  
1993 ◽  
Vol 119 (2) ◽  
pp. 377-395 ◽  
Author(s):  
C.A. Coyle-Thompson ◽  
U. Banerjee
Keyword(s):  
Notch Pathway ◽  
Developmental Pathways ◽  
Wild Type ◽  
Phenotypic Analysis ◽  
Extra Copy ◽  
Its Gene ◽  
Gene Functions ◽  
Specific Manner ◽  
Notch Gene ◽  
Mutant Phenotypes

Genetic and phenotypic analysis of strawberry notch suggests that its gene product is required during embryogenesis and oogenesis, and for the development of the eye, wing and leg. Several lines of evidence suggest that strawberry notch participates together with Notch in many common pathways. A number of strawberry notch mutant phenotypes are similar to those of Notch mutants and can be rescued by an extra copy of wild-type Notch. In addition, mutations in strawberry notch interact strongly with Notch mutants in a tissue-specific manner. Mutations in the strawberry notch and Notch loci also show very similar interactions with genes like Hairless, Delta, groucho, Serrate, and deltex that have all been proposed to participate in Notch related pathways. The genetic evidence presented here suggests that strawberry notch participates with members of the Notch pathway in facilitating developmentally relevant cell-cell communications.

scholarly journals Yeast Chromatin Assembly Complex 1 Protein Excludes Nonacetylatable Forms of Histone H4 from Chromatin and the Nucleus

2004 ◽  
Vol 24 (23) ◽  
pp. 10180-10192 ◽  
Author(s):  
Lynn Glowczewski ◽  
Jakob H. Waterborg ◽  
Judith G. Berman
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Fluorescent Protein ◽  
Chromatin Assembly ◽  
Histone H4 ◽  
Wild Type ◽  
Acetylation State ◽  
Assembly Factor ◽  
Heterochromatin Silencing ◽  
Green Fluorescent

ABSTRACT In yeast, the establishment and maintenance of a transcriptionally silent chromatin state are dependent upon the acetylation state of the N terminus of histone proteins. Histone H4 proteins that contain mutations in N-terminal lysines disrupt heterochromatin and result in yeast that cannot mate. Introduction of a wild-type copy of histone H4 restores mating, despite the presence of the mutant protein, suggesting that mutant H4 protein is either excluded from, or tolerated in, chromatin. To understand how the cell differentiates wild-type histone and mutant histone in which the four N-terminal lysines were replaced with alanine (H4-4A), we analyzed silencing, growth phenotypes, and the histone composition of chromatin in yeast strains coexpressing equal amounts of wild-type and mutant H4 proteins (histone H4 heterozygote). We found that histone H4 heterozygotes have defects in heterochromatin silencing and growth, implying that mutations in H4 are not completely recessive. Nuclear preparations from histone H4 heterozygotes contained less mutant H4 than wild-type H4, consistent with the idea that cells exclude some of the mutant histone. Surprisingly, the N-terminal nuclear localization signal of H4-4A fused to green fluorescent protein was defective in nuclear localization, while a mutant in which the four lysines were replaced with arginine (H4-4R) appeared to have normal nuclear import, implying a role for the charged state of the acetylatable lysines in the nuclear import of histones. The biased partial exclusion of H4-4A was dependent upon Cac1p, the largest subunit of yeast chromatin assembly factor 1 (CAF-1), as well as upon the karyopherin Kap123p, but was independent of Cac2p, another CAF-1 component, and other chromatin assembly proteins (Hir3p, Nap1p, and Asf1p). We conclude that N-terminal lysines of histone H4 are important for efficient histone nuclear import. In addition, our data support a model whereby Cac1p and Kap123 cooperate to ensure that only appropriately acetylated histone H4 proteins are imported into the nucleus.

scholarly journals Calmodulin-dependent nuclear import of HMG-box family nuclear factors: importance of the role of SRY in sex reversal

2010 ◽  
Vol 430 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Gurpreet Kaur ◽  
Aurelie Delluc-Clavieres ◽  
Ivan K. H. Poon ◽  
Jade K. Forwood ◽  
Dominic J. Glover ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Calcium Binding ◽  
Nuclear Translocation ◽  
Sex Reversal ◽  
Nuclear Accumulation ◽  
Common Mechanism ◽  
Wild Type ◽  
Hmg Box ◽  
First Time

The HMG (high-mobility group)-box-containing chromatin-remodelling factor SRY (sex-determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus is critically dependent on two NLSs (nuclear localization signals) that flank its HMG domain: the C-terminally located ‘β-NLS’ that mediates nuclear transport through Impβ1 (importin β1) and the N-terminally located ‘CaM-NLS’ which is known to recognize the calcium-binding protein CaM (calmodulin). In the present study, we examined a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localization of GFP (green fluorescent protein)–SRY fusion proteins in transfected cells compared with wild-type. The CaM antagonist CDZ (calmidazolium chloride) was found to significantly reduce wild-type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ's effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM-binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG-box-domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found for the first time to rely on CaM for optimal nuclear localization. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors.

The sal3+ Gene Encodes an Importin-β Implicated in the Nuclear Import of Cdc25 in Schizosaccharomyces pombe

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 689-703 ◽  
Author(s):  
Gordon Chua ◽  
Carol Lingner ◽  
Corey Frazer ◽  
Paul G Young
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
G1 Arrest ◽  
Mitotic Entry ◽  
Localization Sequence ◽  
Mutant Phenotypes

Abstract In Schizosaccharomyces pombe, the nuclear accumulation of Cdc25 peaks in G2 and is necessary for the proper timing of mitotic entry. Here, we identify the sal3+ gene product as an importin-β homolog that participates in the nuclear import of Cdc25. Loss of sal3+ results in a cell cycle delay, failure to undergo G1 arrest under nitrogen-starvation conditions, and mislocalization of Cdc25 to the cytosol. Fusion of an exogenous classical nuclear localization sequence (cNLS) to Cdc25 restores its nuclear accumulation in a sal3 disruptant and suppresses the sal3 mutant phenotypes. In addition, we show that enhanced nuclear localization of Cdc25 at endogenous levels of expression advances the onset of mitosis. These results demonstrate that the nuclear translocation of Cdc25 is important for the timing of mitotic entry and that Sal3 plays an important role in this process.

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