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Interaction of the IkappaBalpha C-terminal PEST sequence with NF-kappaB: insights into the inhibition of NF-kappaB DNA binding by IkappaBalpha.

ABSTRACT: The transcription factor NF-kappaB (p50/p65) binds either a kappaB DNA element or its inhibitor protein, IkappaBalpha, but these two binding events are mutually exclusive. The reason for this exclusivity is not obvious from the available crystal structure data. The C-terminal PEST-like sequence of IkappaBalpha appears to be involved in the process, but it is located in both of the published X-ray structures of the IkappaBalpha/NF-kappaB complex at a significant distance away from the DNA contact loop in the NF-kappaB DNA-binding domain. We have used nuclear magnetic resonance spectroscopy and differential isotopic labeling to probe the interactions between the p50/p65 NF-kappaB heterodimer and IkappaBalpha in solution. Our measurements are able to resolve a local structural discrepancy between the two crystal structures, and we confirm that the primary interaction of the IkappaBalpha PEST domain is with the DNA-binding domain of the p65 subunit. Mutagenesis of key arginine residues in the DNA contact sequence results in the loss of specific interaction of the PEST sequence with the p65 subdomain. We conclude that the local structure of the IkappaBalpha/NF-kappaB complex in the region of the PEST sequence is consistent with a direct interaction of this acidic sequence with the basic DNA contact sequence in p65, thus reducing the affinity of NF-kappaB for DNA by a competitive mechanism that is still to be elucidated fully.

SUBMITTER: Sue SC

PROVIDER: S-EPMC2688455 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Interaction of the IkappaBalpha C-terminal PEST sequence with NF-kappaB: insights into the inhibition of NF-kappaB DNA binding by IkappaBalpha.

Sue Shih-Che SC Dyson H Jane HJ

Journal of molecular biology 20090324 4

The transcription factor NF-kappaB (p50/p65) binds either a kappaB DNA element or its inhibitor protein, IkappaBalpha, but these two binding events are mutually exclusive. The reason for this exclusivity is not obvious from the available crystal structure data. The C-terminal PEST-like sequence of IkappaBalpha appears to be involved in the process, but it is located in both of the published X-ray structures of the IkappaBalpha/NF-kappaB complex at a significant distance away from the DNA contact ...[more]

PMID: 19327364

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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

Interaction of the IkappaBalpha C-terminal PEST sequence with NF-kappaB: insights into the inhibition of NF-kappaB DNA binding by IkappaBalpha.

Publications

Interaction of the IkappaBalpha C-terminal PEST sequence with NF-kappaB: insights into the inhibition of NF-kappaB DNA binding by IkappaBalpha.

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