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Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment.

ABSTRACT: The DNA binding activity of NF-?B is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-?B activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF. The phosphorylation and subsequent degradation of I-?B? as well as the serine 536 phosphorylation and nuclear translocation of p65 NF-?B were insufficient for VCAM-1 expression in response to TNF. The requirement for p50 NF-?B in TNF-induced VCAM-1 expression may be associated with its interaction with and phosphorylation by DNA-PK, which appears to be dominant over the requirement for p65 NF-?B activation. p50 NF-?B binding to its consensus sequence increased its susceptibility to phosphorylation by DNA-PK. Additionally, DNA-PK activity appeared to increase the association between p50/p50 and p50/p65 NF-?B dimers upon binding to DNA and after binding of p50 NF-?B to the VCAM-1 promoter. Analyses of the p50 NF-?B protein sequence revealed that both serine 20 and serine 227 at the amino terminus of the protein are putative sites for phosphorylation by DNA-PK. Mutation of serine 20 completely eliminated phosphorylation of p50 NF-?B by DNA-PK, suggesting that serine 20 is the only site in p50 NF-?B for phosphorylation by DNA-PK. Re-establishing wild-type p50 NF-?B, but not its serine 20/alanine mutant, in p50 NF-?B(-/-) fibroblasts reversed VCAM-1 expression after TNF treatment, demonstrating the importance of the serine 20 phosphorylation site in the induction of VCAM-1 expression. Together, these results elucidate a novel mechanism for the involvement of DNA-PK in the positive regulation of p50 NF-?B to drive VCAM-1 expression.

SUBMITTER: Ju J

PROVIDER: S-EPMC3003413 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment.

Ju Jihang J Naura Amarjit S AS Errami Youssef Y Zerfaoui Mourad M Kim Hogyoung H Kim Jong G JG Abd Elmageed Zakaria Y ZY Abdel-Mageed Asim B AB Giardina Charles C Beg Amer A AA Smulson Mark E ME Boulares A Hamid AH

The Journal of biological chemistry 20101021 52

The DNA binding activity of NF-κB is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-κB activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF. The phosphorylation and subsequent degradation of I-κBα as well as the serine 536 phosphorylation and nuclear translocation of p65 NF-κB were insufficient for VCAM-1 expression in response to TNF. The requirement for p50 NF-κB in TNF-induced VCAM- ...[more]

PMID: 20966071

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Project description:Picroliv, an iridoid glycoside derived from the plant Picrorhiza kurroa, is used traditionally to treat fever, asthma, hepatitis, and other inflammatory conditions. However, the exact mechanism of its therapeutic action is still unknown. Because nuclear factor-kappaB (NF-kappaB) activation plays a major role in inflammation and carcinogenesis, we postulated that picroliv must interfere with this pathway by inhibiting the activation of NF-kappaB-mediated signal cascade. Electrophoretic mobility shift assay showed that pretreatment with picroliv abrogated tumor necrosis factor (TNF)-induced activation of NF-kappaB. The glycoside also inhibited NF-kappaB activated by carcinogenic and inflammatory agents, such as cigarette smoke condensate, phorbol 12-myristate 13-acetate, okadaic acid, hydrogen peroxide, lipopolysaccharide, and epidermal growth factor. When examined for the mechanism of action, we found that picroliv inhibited activation of IkappaBalpha kinase, leading to inhibition of phosphorylation and degradation of IkappaBalpha. It also inhibited phosphorylation and nuclear translocation of p65. Further studies revealed that picroliv directly inhibits the binding of p65 to DNA, which was reversed by the treatment with reducing agents, suggesting a role for a cysteine residue in interaction with picroliv. Mutation of Cys(38) in p65 to serine abolished this effect of picroliv. NF-kappaB inhibition by picroliv leads to suppression of NF-kappaB-regulated proteins, including those linked with cell survival (inhibitor of apoptosis protein 1, Bcl-2, Bcl-xL, survivin, and TNF receptor-associated factor 2), proliferation (cyclin D1 and cyclooxygenase-2), angiogenesis (vascular endothelial growth factor), and invasion (intercellular adhesion molecule-1 and matrix metalloproteinase-9). Suppression of these proteins enhanced apoptosis induced by TNF. Overall, our results show that picroliv inhibits the NF-kappaB activation pathway, which may explain its anti-inflammatory and anticarcinogenic effects.

Dataset Information

Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment.

Publications

Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment.

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