scholarly journals I kappaB alpha physically interacts with a cytoskeleton-associated protein through its signal response domain.

1997 ◽  
Vol 17 (12) ◽  
pp. 7375-7385 ◽  
Author(s):  
P Crépieux ◽  
H Kwon ◽  
N Leclerc ◽  
W Spencer ◽  
S Richard ◽  
...  
Keyword(s):  
26S Proteasome ◽  
Cdna Clones ◽  
Human Homolog ◽  
Microtubule Organizing Center ◽  
Protein Motifs ◽  
Amino Terminal ◽  
Signal Response ◽  
Nf Kappab ◽  
Two Hybrid

The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I kappaB alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappaB alpha as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.

scholarly journals A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

1998 ◽  
Vol 9 (8) ◽  
pp. 2201-2216 ◽  
Author(s):  
Thu Nguyen ◽  
Dani B.N. Vinh ◽  
Douglas K. Crawford ◽  
Trisha N. Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Terminus ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Synthetic Lethal ◽  
Lethal Phenotype ◽  
Amino Terminal ◽  
N Terminus ◽  
Two Hybrid

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations inSPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, thespc97 alleles are synthetic lethal withspc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

scholarly journals Multiple Interactions in Sir Protein Recruitment by Rap1p at Silencers and Telomeres in Yeast

2001 ◽  
Vol 21 (23) ◽  
pp. 8082-8094 ◽  
Author(s):  
Paolo Moretti ◽  
David Shore
Keyword(s):  
Complex Formation ◽  
Mating Type ◽  
Small Region ◽  
Carboxyl Terminus ◽  
Silent Chromatin ◽  
Histone Binding ◽  
Amino Terminal ◽  
Sir Complex ◽  
Two Hybrid

ABSTRACT Initiation of transcriptional silencing at mating type loci and telomeres in Saccharomyces cerevisiaerequires the recruitment of a Sir2/3/4 (silent information regulator) protein complex to the chromosome, which occurs at least in part through its association with the silencer- and telomere-binding protein Rap1p. Sir3p and Sir4p are structural components of silent chromatin that can self-associate, interact with each other, and bind to the amino-terminal tails of histones H3 and H4. We have identified a small region of Sir3p between amino acids 455 and 481 that is necessary and sufficient for association with the carboxyl terminus of Rap1p but not required for Sir complex formation or histone binding.SIR3 mutations that delete this region cause a silencing defect at HMR and telomeres. However, this impairment of repression is considerably less than that displayed by Rap1p carboxy-terminal truncations that are defective in Sir3p binding. This difference may be explained by the ability of the Rap1p carboxyl terminus to interact independently with Sir4p, which we demonstrate by in vitro binding and two-hybrid assays. Significantly, the Rap1p-Sir4p two-hybrid interaction does not require Sir3p and is abolished by mutation of the carboxyl terminus of Rap1p. We propose that both Sir3p and Sir4p can directly and independently bind to Rap1p at mating type silencers and telomeres and suggest that Rap1p-mediated recruitment of Sir proteins operates through multiple cooperative interactions, at least some of which are redundant. The physical separation of the Rap1p interaction region of Sir3p from parts of the protein required for Sir complex formation and histone binding raises the possibility that Rap1p can participate directly in the maintenance of silent chromatin through the stabilization of Sir complex-nucleosome interactions.

Sequence, expression and localization of calmodulin-domain protein kinases inEimeria tenellaandEimeria maxima

Parasitology ◽  
1996 ◽  
Vol 113 (5) ◽  
pp. 439-448 ◽  
Author(s):  
P. P. J. Dunn ◽  
J. M. Bumstead ◽  
F. M. Tomley
Keyword(s):  
Host Cell ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Sequence Data ◽  
Host Cells ◽  
Cdna Clones ◽  
Host Cell Membrane ◽  
Amino Terminal ◽  
Domain Protein

SUMMARYWe have isolated and sequenced cDNA clones fromEimeria tenellaandEimeria maximawhich encode proteins that share homology with a recently described family of calmodulin-domain protein kinases. The primary sequence data show that each of the protein kinases can be divided into 2 main functional domains – an amino-terminal catalytic domain typical of serine/threonine protein kinases and a carboxy-terminal domain homologous to calmodulin, which is capable of binding calcium ions at 4 ‘EF-hand’ motifs. Expression of theE. tenellacalmodulin-domain protein kinase (EtCDPK) increased towards the end of oocyst sporulation, as judged by Northern and Western blotting, and indirect immunofluorescent antibody labelling showed that within a few minutes of adding sporozoites to target host cells inin vitroculture EtCDPK was found to be specifically associated with a filament-like structure that converges at the apical end of the parasite. Once the parasite entered the host cell EtCDPK appeared to be left on the host cell membrane at the point of entry, indicating a brief yet specific role for this molecule in the invasion of host cells byE. tenella.

scholarly journals Evidence for Separable Functions of Srp1p, the Yeast Homolog of Importin α (Karyopherin α): Role for Srp1p and Sts1p in Protein Degradation

2000 ◽  
Vol 20 (16) ◽  
pp. 6062-6073 ◽  
Author(s):  
Michelle M. Tabb ◽  
Prasad Tongaonkar ◽  
Loan Vu ◽  
Masayasu Nomura
Keyword(s):  
Protein Degradation ◽  
Nuclear Localization ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Localization Function ◽  
Importin Α ◽  
Yeast Homolog ◽  
Two Hybrid

ABSTRACT Srp1p (importin α) functions as the nuclear localization signal (NLS) receptor in Saccharomyces cerevisiae. Thesrp1-31 mutant is defective in this nuclear localization function, whereas an srp1-49 mutant exhibits defects that are unrelated to this localization function, as was confirmed by intragenic complementation between the two mutants. RPN11and STS1 (DBF8) were identified as high-dosage suppressors of the srp1-49 mutation but not of thesrp1-31 mutation. We found that Sts1p interacts directly with Srp1p in vitro and also in vivo, as judged by coimmunoprecipitation and two-hybrid analyses. Mutants of Sts1p that cannot interact with Srp1p are incapable of suppressingsrp1-49 defects, strongly suggesting that Sts1p functions in a complex with Srp1p. STS1 also interacted with the second suppressor, RPN11, a subunit of the 26S proteasome, in the two-hybrid system. Further, degradation of Ub-Pro-β-galactosidase, a test substrate for the ubiquitin-proteasome system, was defective in srp1-49 but not insrp1-31. This defect in protein degradation was alleviated by overexpression of either RPN11 or STS1 insrp1-49. These results suggest a role for Srp1p in regulation of protein degradation separate from its well-established role as the NLS receptor.

scholarly journals The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein degradation in vivo.

1995 ◽  
Vol 15 (11) ◽  
pp. 6311-6321 ◽  
Author(s):  
D J DeMarini ◽  
F R Papa ◽  
S Swaminathan ◽  
D Ursic ◽  
T P Rasmussen ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Genetic Selection ◽  
26S Proteasome ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
20S Proteasome ◽  
Amino Terminal ◽  
A Subunit

The yeast Sen1 protein was discovered by virtue of its role in tRNA splicing in vitro. To help determine the role of Sen1 in vivo, we attempted to overexpress the protein in yeast cells. However, cells with a high-copy SEN1-bearing plasmid, although expressing elevated amounts of SEN1 mRNA, show little increase in the level of the encoded protein, indicating that a posttranscriptional mechanism limits SEN1 expression. This control depends on an amino-terminal element of Sen1. Using a genetic selection for mutants with increased expression of Sen1-derived fusion proteins, we identified mutations in a novel gene, designated SEN3. SEN3 is essential and encodes a 945-residue protein with sequence similarity to a subunit of an activator of the 20S proteasome from bovine erythrocytes, called PA700. Earlier work indicated that the 20S proteasome associates with a multisubunit regulatory factor, resulting in a 26S proteasome complex that degrades substrates of the ubiquitin system. Mutant sen3-1 cells have severe defects in the degradation of such substrates and accumulate ubiquitin-protein conjugates. Most importantly, we show biochemically that Sen3 is a subunit of the 26S proteasome. These data provide evidence for the involvement of the 26S proteasome in the degradation of ubiquitinated proteins in vivo and for a close relationship between PA700 and the regulatory complexes within the 26S proteasome, and they directly demonstrate that Sen3 is a component of the yeast 26S proteasome.

Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

scholarly journals Inhibition of Androgen Receptor-Mediated Transcription by Amino-Terminal Enhancer of split

2001 ◽  
Vol 21 (14) ◽  
pp. 4614-4625 ◽  
Author(s):  
Xin Yu ◽  
Peng Li ◽  
Robert G. Roeder ◽  
Zhengxin Wang
Keyword(s):  
Androgen Receptor ◽  
Nuclear Extract ◽  
Basal Transcription ◽  
Hela Nuclear Extract ◽  
Protein Protein Interaction ◽  
Transcription System ◽  
Amino Terminal ◽  
Two Hybrid ◽  
Enhancer Of Split

ABSTRACT A yeast two-hybrid assay has identified an androgen-dependent interaction of androgen receptor (AR) with amino-terminal enhancer of split (AES), a member of the highly conserved Groucho/TLE family of corepressors. Full-length AR, as well as the N-terminal fragment of AR, showed direct interactions with AES in in vitro protein-protein interaction assays. AES specifically inhibited AR-mediated transcription in a well-defined cell-free transcription system and interacted specifically with the basal transcription factor (TFIIE) in HeLa nuclear extract. These observations implicate AES as a selective repressor of ligand-dependent AR-mediated transcription that acts by directly interacting with AR and by targeting the basal transcription machinery.

scholarly journals Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen.

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230 ◽  
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential

1991 ◽  
Vol 11 (4) ◽  
pp. 1912-1920
Author(s):  
S Katzav ◽  
J L Cleveland ◽  
H E Heslop ◽  
D Pulido
Keyword(s):  
Leucine Zipper ◽  
Ectopic Expression ◽  
Cdna Clones ◽  
3T3 Cells ◽  
Amino Terminal ◽  
Helix Loop Helix ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

vav, a novel human oncogene, was originally generated in vitro by replacement of its normal 5' coding sequences with sequences from pSV2neo DNA, cotransfected as a selectable marker (S. Katzav, D. Martin-Zanca, and M. Barbacid, EMBO J. 8:2283-2290, 1989). The vav proto-oncogene is normally expressed in cells of hematopoietic origin. To determine whether the 5' rearrangement of vav or its ectopic expression in NIH 3T3 cells contributes to its transforming potential, we isolated murine and human proto-vav cDNA clones as well as human genomic clones corresponding to the 5' end of the gene. Normal proto-vav was poorly transforming in NIH 3T3 cells, whereas truncation of its 5' end greatly enhanced its transforming activity. The relative failure of full-length proto-vav cDNA clones to transform NIH 3T3 cells indicates that the transforming activity of vav is not simply due to ectopic expression. Analysis of the predicted amino terminus of the vav proto-oncogene shows that it contains a helix-loop-helix domain and a leucine zipper motif similar to that of myc family proteins, though it lacks a basic region that is usually found adjacent to helix-loop-helix domains. Loss of the helix-loop-helix domain of proto-vav, either by truncation or by rearrangement with pSV2neo sequences, activates its oncogenic potential.

scholarly journals Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential.

1991 ◽  
Vol 11 (4) ◽  
pp. 1912-1920 ◽  
Author(s):  
S Katzav ◽  
J L Cleveland ◽  
H E Heslop ◽  
D Pulido
Keyword(s):  
Leucine Zipper ◽  
Ectopic Expression ◽  
Cdna Clones ◽  
3T3 Cells ◽  
Amino Terminal ◽  
Helix Loop Helix ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

vav, a novel human oncogene, was originally generated in vitro by replacement of its normal 5' coding sequences with sequences from pSV2neo DNA, cotransfected as a selectable marker (S. Katzav, D. Martin-Zanca, and M. Barbacid, EMBO J. 8:2283-2290, 1989). The vav proto-oncogene is normally expressed in cells of hematopoietic origin. To determine whether the 5' rearrangement of vav or its ectopic expression in NIH 3T3 cells contributes to its transforming potential, we isolated murine and human proto-vav cDNA clones as well as human genomic clones corresponding to the 5' end of the gene. Normal proto-vav was poorly transforming in NIH 3T3 cells, whereas truncation of its 5' end greatly enhanced its transforming activity. The relative failure of full-length proto-vav cDNA clones to transform NIH 3T3 cells indicates that the transforming activity of vav is not simply due to ectopic expression. Analysis of the predicted amino terminus of the vav proto-oncogene shows that it contains a helix-loop-helix domain and a leucine zipper motif similar to that of myc family proteins, though it lacks a basic region that is usually found adjacent to helix-loop-helix domains. Loss of the helix-loop-helix domain of proto-vav, either by truncation or by rearrangement with pSV2neo sequences, activates its oncogenic potential.

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