Dataset Information

Pre-folding IkappaBalpha alters control of NF-kappaB signaling.

ABSTRACT: Transcription complex components frequently show coupled folding and binding but the functional significance of this mode of molecular recognition is unclear. IkappaBalpha binds to and inhibits the transcriptional activity of NF-kappaB via its ankyrin repeat (AR) domain. The beta-hairpins in ARs 5-6 in IkappaBalpha are weakly-folded in the free protein, and their folding is coupled to NF-kappaB binding. Here, we show that introduction of two stabilizing mutations in IkappaBalpha AR 6 causes ARs 5-6 to fold cooperatively to a conformation similar to that in NF-kappaB-bound IkappaBalpha. Free IkappaBalpha is degraded by a proteasome-dependent but ubiquitin-independent mechanism, and this process is slower for the pre-folded mutants both in vitro and in cells. Interestingly, the pre-folded mutants bind NF-kappaB more weakly, as shown by both surface plasmon resonance and isothermal titration calorimetry in vitro and immunoprecipitation experiments from cells. One consequence of the weaker binding is that resting cells containing these mutants show incomplete inhibition of NF-kappaB activation; they have significant amounts of nuclear NF-kappaB. Additionally, the weaker binding combined with the slower rate of degradation of the free protein results in reduced levels of nuclear NF-kappaB upon stimulation. These data demonstrate clearly that the coupled folding and binding of IkappaBalpha is critical for its precise control of NF-kappaB transcriptional activity.

SUBMITTER: Truhlar SM

PROVIDER: S-EPMC2519148 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Pre-folding IkappaBalpha alters control of NF-kappaB signaling.

Truhlar Stephanie M E SM Mathes Erika E Cervantes Carla F CF Ghosh Gourisankar G Komives Elizabeth A EA

Journal of molecular biology 20080304 1

Transcription complex components frequently show coupled folding and binding but the functional significance of this mode of molecular recognition is unclear. IkappaBalpha binds to and inhibits the transcriptional activity of NF-kappaB via its ankyrin repeat (AR) domain. The beta-hairpins in ARs 5-6 in IkappaBalpha are weakly-folded in the free protein, and their folding is coupled to NF-kappaB binding. Here, we show that introduction of two stabilizing mutations in IkappaBalpha AR 6 causes ARs ...[more]

PMID: 18511071

Similar Datasets

Project description:AimTo study the effects of IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) on NF-kappaB, p53 and their downstream target genes. The relationship of NF-kappaB, p53, and IkappaBalpha was further discussed.MethodspECFP-IkappaBalpha, pECFP-IkappaBalphaM (amino acides 1-317, Ser32, 36A), pECFP-IkappaBalpha243N (amino acides 1-243), pECFP-IkappaBalpha244C (amino acides 244-317), pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-alpha-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection, location patterns of NF-kappaB, p53 and IkappaBalpha (IkappaBalphaM, IkappaBalpha243N, IkappaBalpha244C) were observed by a laser scanning microscope (LSM510/ConfoCor2, Zeiss). RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IkappaBalpha and its mutants on the transprition level of NF-kappaB, NF-kappaB downstream target gene TNF-alpha, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments beta-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader.ResultsCells that were transfected with p53-DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IkappaBalpha, CFP-IkappaBalphaM, and CFP-IkappaBalpha243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IkappaBalpha244C revealed a predominant nuclear localization. The mRNA levels of p65, TNF-alpha, p53 and Bax in CFP-IkappaBalpha transfected cells did not change significantly, while in YFP-p65/CFP-IkappaBalpha co-transfected cells, IkappaBalpha decreased the transcription of p65 downstream gene TNF-alpha (2.24+/-0.503) compared with the YFP-p65/CFP-C1 co-transfected cells (5.08+/-0.891) (P<0.05). Phosphorylation defective IkappaBalpha (IkappaBalphaM) decreased the transcription levels of all the four genes compared with the control (P<0.05). The N terminus of IkappaBalpha (IkappaBalpha243N) increased the transcription of NF-kappaB (1.84+/-0.176) and TNF-alpha (1.51+/-0.203) a little bit. However, the C terminus of IkappaBalpha (IkappaBalpha244C) increased the transcription of NF-kappaB, TNF-alpha, p53 and Bax significantly (8.29+/-1.662, 14.16+/-2.121, 10.2+/-0.621, 3.72+/-0.346) (P<0.05). The CCK-8 experiment also showed that IkappaBalpha244C and p53 synergistically mediate apoptosis.ConclusionsIkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) have different effects on NF-kappaB and p53 signaling pathways, according to their different structures. IkappaBalphaM bounds with NF-kappaB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and IkappaBalpha244C may be co-factor in inducing apoptosis. The C terminal of IkappaBalpha enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.

Dataset Information

Pre-folding IkappaBalpha alters control of NF-kappaB signaling.

Publications

Pre-folding IkappaBalpha alters control of NF-kappaB signaling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets