scholarly journals Overexpression of RelA in transgenic mouse thymocytes: specific increase in levels of the inhibitor protein I kappa B alpha.

1995 ◽  
Vol 15 (7) ◽  
pp. 3523-3530 ◽  
Author(s):  
P Perez ◽  
S A Lira ◽  
R Bravo
Keyword(s):  
Transgenic Mice ◽  
Binding Activity ◽  
Mrna Levels ◽  
Inhibitor Protein ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Specific Increase ◽  
Stimulation Of

RelA (p65) is one of the strongest activators of the Rel/NF-kappa B family. As a first step to elucidate the mechanisms that regulate its activity in vivo, we have generated transgenic mice overexpressing RelA in the thymus. Although the levels of RelA were significantly increased in thymocytes of transgenic mice, the overall NF-kappa B-binding activity in unstimulated cells was not augmented compared with that in control thymocytes. This could be explained by the dramatic increase of endogenous I kappa B alpha levels observed in RelA-overexpressing cells in both cytoplasmic and nuclear compartments. The ikba mRNA levels were not augmented by overexpressed RelA, but I kappa B alpha inhibitor was found to be stabilized through association with RelA. Although a fraction of RelA was associated with cytoplasmic p105, no changes in the precursor levels were observed. Upon stimulation of RelA-overexpressing thymocytes with phorbol 12-myristate 13-acetate and lectin (phytohemaglutinin), different kappa B-binding complexes, including RelA homodimers, were partially released from I kappa B alpha. Association of RelA with I kappa B alpha prevented complete degradation of the inhibitor. No effect of phorbol 12-myristate 13-acetate-lectin treatment was detected on RelA associated with p105. Our data indicate that cytoplasmic retention of overexpressed RelA by I kappa B alpha is the major in vivo mechanism controlling the potential excess of NF-kappa B activity in long-term RelA-overexpressing thymocytes.

scholarly journals In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B.

1995 ◽  
Vol 15 (3) ◽  
pp. 1294-1301 ◽  
Author(s):  
I Alkalay ◽  
A Yaron ◽  
A Hatzubai ◽  
S Jung ◽  
A Avraham ◽  
...  
Keyword(s):  
T Cell Activation ◽  
Cell Activation ◽  
Extended Period ◽  
Activation Process ◽  
Nf Kappa B ◽  
Inflammatory Reactions ◽  
I Kappa B Alpha ◽  
Cytoplasmic Complex ◽  
Stimulation Of

NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

scholarly journals Domain organization of I kappa B alpha and sites of interaction with NF-kappa B p65.

1995 ◽  
Vol 15 (4) ◽  
pp. 2166-2172 ◽  
Author(s):  
E Jaffray ◽  
K M Wood ◽  
R T Hay
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Ankyrin Repeat ◽  
Inhibitor Protein ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Terminal Domain ◽  
Dna Binding Activity ◽  
Protease Cleavage ◽  
Flexible Region

The DNA-binding activity and cellular distribution of the transcription factor NF-kappa B are regulated by the inhibitor protein I kappa B alpha. I kappa B alpha belongs to a family of proteins that contain multiple repeats of a 30- to 35-amino-acid sequence that was initially recognized in the erythrocyte protein ankyrin. Partial proteolysis has been used to study the domain structure of I kappa B alpha and to determine the sites at which it interacts with NF-kappa B. The data reveal a tripartite structure for I kappa B alpha in which a central, protease-resistant domain composed of five ankyrin repeats is flanked by an unstructured N-terminal extension and a compact, highly acidic C-terminal domain that is connected to the core of the protein by a flexible linker. Functional analysis of V8 cleavage products indicates that I kappa B alpha molecules lacking the N-terminal region can interact with and inhibit the DNA-binding activity of the p65 subunit of NF-kappa B, whereas I kappa B alpha molecules which lack both the N- and C-terminal regions are incapable of doing so. Protease cleavage of the N terminus of I kappa B alpha was unaffected by the presence of the p65 subunit of NF-kappa B, whereas bound p65 blocked cleavage of the flexible linker connecting the C-terminal domain to the ankyrin repeat-containing core of the protein. This linker region is highly conserved within the human, rat, pig, and chicken homologs of I kappa B alpha, and while it has been suggested that it represents a sixth ankyrin repeat, it is also likely that this is a flexible region of the protein that interacts with NF-kappa B.

scholarly journals BCL3 encodes a nuclear protein which can alter the subcellular location of NF-kappa B proteins.

1994 ◽  
Vol 14 (6) ◽  
pp. 3915-3926 ◽  
Author(s):  
Q Zhang ◽  
J A Didonato ◽  
M Karin ◽  
T W McKeithan
Keyword(s):  
Nuclear Localization Signal ◽  
Nuclear Protein ◽  
Subcellular Location ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Nf Kappa B ◽  
Subnuclear Localization ◽  
I Kappa B Alpha ◽  
Localization Signal

BCL3 is a candidate proto-oncogene involved in the recurring translocation t(14;19) found in some patients with chronic lymphocytic leukemia. BCL3 protein acts as an I kappa B in that it can specifically inhibit the DNA binding of NF-kappa B factors. Here, we demonstrate that BCL3 is predominantly a nuclear protein and provide evidence that its N terminus is necessary to direct the protein into the nucleus. In contrast to I kappa B alpha (MAD3), BCL3 does not cause NF-kappa B p50 to be retained in the cytoplasm; instead, in cotransfection assays, it alters the subnuclear localization of p50. The two proteins colocalize, suggesting that they interact in vivo. Further immunofluorescence experiments showed that a mutant p50, lacking a nuclear localization signal and restricted to the cytoplasm, is brought into the nucleus in the presence of BCL3. Correspondingly, a wild-type p50 directs into the nucleus a truncated BCL3, which, when transfected alone, is found in the cytoplasm. We tested whether BCL3 could overcome the cytoplasmic retention of p50 by I kappa B alpha. Results from triple cotransfection experiments with BCL3, I kappa B alpha, and p50 implied that BCL3 can successfully compete with I kappa B alpha and bring p50 into the nucleus; thus, localization of NF-kappa B factors may be affected by differential expression of I kappa B proteins. These novel properties of BCL3 protein further establish BCL3 as a distinctive member of the I kappa B family.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Interaction of the v-Rel oncoprotein with NF-kappaB and IkappaB proteins: heterodimers of a transformation-defective v-Rel mutant and NF-2 are functional in vitro and in vivo.

1996 ◽  
Vol 16 (3) ◽  
pp. 1169-1178 ◽  
Author(s):  
D W White ◽  
G A Pitoc ◽  
T D Gilmore
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Cellular Protein ◽  
Lymphoid Cells ◽  
Spleen Cells ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Chicken Spleen

The v-Rel oncoprotein of the avian Rev-T retrovirus is a member of the Rel/NF-kappa B family of transcription factors. The mechanism by which v-Rel malignantly transforms chicken spleen cells is not precisely known. To gain a better understanding of functions needed for transformation by v-Rel, we have now characterized the activities of mutant v-Rel proteins that are defective for specific protein-protein interactions. Mutant v-delta NLS, which has a deletion of the primary v-Rel nuclear localizing sequence, does not interact efficiently with I kappa B-alpha but still transforms chicken spleen cells approximately as well as wild-type v-Rel, indicating that interaction with I kappa B-alpha is not essential for the v-Rel transforming function. A second v-Rel mutant, v-SPW, has been shown to be defective for the formation of homodimers, DNA binding, and transformation. However, we now find that v-SPW can form functional DNA-binding heterodimers in vitro and in vivo with the cellular protein NF-kappa B p-52. Most strikingly, coexpression of v-SPW and p52 from a retroviral vector can induce the malignant transformation of chicken spleen cells, whereas expression of either protein alone cannot. Our results are most consistent with a model wherein Rel homodimers or heterodimers must bind DNA and alter gene expression in order to transform lymphoid cells.

scholarly journals Hierarchical phosphorylation of the translation inhibitor 4E-BP1

2001 ◽  
Vol 15 (21) ◽  
pp. 2852-2864 ◽  
Author(s):  
Anne-Claude Gingras ◽  
Brian Raught ◽  
Steven P. Gygi ◽  
Anna Niedzwiecka ◽  
Mathieu Miron ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Translation Initiation ◽  
Binding Activity ◽  
Two Dimensional ◽  
Initiation Phase ◽  
Sds Page ◽  
Translation Inhibitor ◽  
Hierarchical Phosphorylation ◽  
Stimulation Of

In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5′ end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.

scholarly journals I(kappa)B(gamma) inhibits DNA binding of NF-kappaB p50 homodimers by interacting with residues that contact DNA.

1996 ◽  
Vol 16 (11) ◽  
pp. 6477-6485 ◽  
Author(s):  
S Bell ◽  
J R Matthews ◽  
E Jaffray ◽  
R T Hay
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Protein Footprinting ◽  
I Kappa B Alpha ◽  
Dna Binding Activity ◽  
Cellular Genes ◽  
Phosphate Backbone ◽  
Nf Kappab ◽  
Contact Dna

NF-(kappa)B is an inducible transcription factor that activates many cellular genes involved in stress and immune response and whose DNA binding activity and cellular distribution are regulated by I(kappa)B inhibitor proteins. The interaction between NF-(kappa)B p50 and DNA was investigated by protein footprinting using chemical modification and partial proteolysis. Both methods confirmed lysine-DNA contacts already found in the crystal structure (K-147, K-149, K-244, K-275, and K-278) but also revealed an additional contact in the lysine cluster K-77-K-78-K-80 which was made on an extended DNA. Molecular modelling of such a DNA-protein complex revealed that lysine 80 is ideally placed to make phosphate backbone contacts in the extended DNA. Thus, it seems likely that the entire AB loop, containing lysines 77, 78, and 80, forms a C-shaped clamp that closes around the DNA recognition site. The same protein footprinting approaches were used to probe the interaction of p50 with the ankyrin repeat containing proteins I(kappa)B(gamma) and I(kappa)B(alpha). Lysine residues in p50 that were protected from modification by DNA were also protected from modification by I(kappa)B(gamma) but not I(kappa)B(alpha). Similarly, proteolytic cleavage at p50 residues which contact DNA was inhibited by bound I(kappa)B(gamma) but was enhanced by the presence of I(kappa)B(alpha). Thus, I(kappa)B(gamma) inhibits the DNA binding activity of p50 by direct interactions with residues contacting DNA, whereas the same residues remain exposed in the presence of I(kappa)B(alpha), which binds to p50 but does not block DNA binding.

scholarly journals Impaired noradrenaline-induced lipolysis in white fat of aP2-Ucp1 transgenic mice is associated with changes in G-protein levels

2002 ◽  
Vol 364 (2) ◽  
pp. 369-376 ◽  
Author(s):  
Pavel FLACHS ◽  
JiŘí NOVOTNÝ ◽  
Filip BAUMRUK ◽  
Kristina BARDOVÁ ◽  
Lenka BOUŘOVÁ ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Transgenic Mice ◽  
G Protein ◽  
Energy Status ◽  
Α Subunit ◽  
Fat Depots ◽  
Ucp1 Expression ◽  
White Fat ◽  
Stimulation Of

In vitro experiments suggest that stimulation of lipolysis by catecholamines in adipocytes depends on the energy status of these cells. We tested whether mitochondrial uncoupling proteins (UCPs) that control the efficiency of ATP production could affect lipolysis and noradrenaline signalling in white fat in vivo. The lipolytic effect of noradrenaline was lowered by ectopic UCP1 in white adipocytes of aP2-Ucp1 transgenic mice, overexpressing the UCP1 gene from the aP2 gene promoter, reflecting the magnitude of UCP1 expression, the impaired stimulation of cAMP levels by noradrenaline and the reduction of the ATP/ADP ratio in different fat depots. Thus only subcutaneous but not epididymal fat was affected. UCP1 also down-regulated the expression of hormone-sensitive lipase and lowered its activity, and altered the expression of trimeric G-proteins in adipocytes. The adipose tissue content of the stimulatory G-protein α subunit was increased while that of the inhibitory G-protein α subunits decreased in response to UCP1 expression. Our results support the idea that the energy status of cells, and the ATP/ADP ratio in particular, modulates the lipolytic effects of noradrenaline in adipose tissue in vivo. They also demonstrate changes at the G-protein level that tend to overcome the reduction of lipolysis when ATP level in adipocytes is low. Therefore, respiratory uncoupling may exert a broad effect on hormonal signalling in adipocytes.

scholarly journals Sex Differences in Estrogen-Dependent Transcription of Gonadotropin-Releasing Hormone (GnRH) Gene Revealed in GnRH Transgenic Mice

Endocrinology ◽  
2003 ◽  
Vol 144 (8) ◽  
pp. 3351-3358 ◽  
Author(s):  
Niren R. Thanky ◽  
Ruth Slater ◽  
Allan E. Herbison
Keyword(s):  
Transgenic Mice ◽  
Mrna Expression ◽  
Gene Transcription ◽  
Transgene Expression ◽  
Gonadal Steroids ◽  
Critical Element ◽  
Sexually Dimorphic ◽  
Mrna Levels ◽  
Estrogen Replacement

Abstract The mechanisms through which gonadal steroids exert feedback actions on the activity of the GnRH neurons are not understood. Using a series of GnRH-LacZ transgenic mice we have examined the manner in which gonadal steroids suppress GnRH mRNA expression in male and female mice. The long-term gonadectomy of 5.5-GNZ-3.5 transgenic mice resulted in significant increases in cellular GnRH mRNA expression (P < 0.05) and plasma LH concentrations (P < 0.01) in both sexes. However, cellular levels of LacZ mRNA and β-galactosidase, which provide an index of GnRH gene transcription, were only elevated in males after gonadectomy. This sexually differentiated response was also observed in mice gonadectomized for 2 wk. Estrogen replacement in gonadectomized males returned transgene expression to intact levels. Experiments in transgenic mice with 3′ and 5′ deleted GnRH-LacZ constructs revealed that the suppressive influence of estrogen on LacZ transcription in the male required a critical element located between −5.2 and −1.7 kb of the GnRH promoter. These studies show that the suppression of GnRH mRNA expression by estrogen in the male involves a decrease in GnRH gene transcription that is dependent on a distal GnRH promoter element. The same mechanism does not exist in females, indicating that gonadal steroids suppress GnRH mRNA levels in a sexually dimorphic manner.

Sign in / Sign up

Export Citation Format

Share Document