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Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition.

ABSTRACT: NF-κB is a key transcription factor that dictates the outcome of diverse immune responses. How NF-κB is regulated by multiple activating receptors that are engaged during natural killer (NK)-target cell contact remains undefined. Here we show that sole engagement of NKG2D, 2B4 or DNAM-1 is insufficient for NF-κB activation. Rather, cooperation between these receptors is required at the level of Vav1 for synergistic NF-κB activation. Vav1-dependent synergistic signalling requires a separate PI3K-Akt signal, primarily mediated by NKG2D or DNAM-1, for optimal p65 phosphorylation and NF-κB activation. Vav1 controls downstream p65 phosphorylation and NF-κB activation. Synergistic signalling is defective in X-linked lymphoproliferative disease (XLP1) NK cells entailing 2B4 dysfunction and required for p65 phosphorylation by PI3K-Akt signal, suggesting stepwise signalling checkpoint for NF-κB activation. Thus, our study provides a framework explaining how signals from different activating receptors are coordinated to determine specificity and magnitude of NF-κB activation and NK cell responses.

SUBMITTER: Kwon HJ

PROVIDER: S-EPMC4894962 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition.

Kwon Hyung-Joon HJ Choi Go-Eun GE Ryu Sangryeol S Kwon Soon Jae SJ Kim Sun Chang SC Booth Claire C Nichols Kim E KE Kim Hun Sik HS

Nature communications 20160525

NF-κB is a key transcription factor that dictates the outcome of diverse immune responses. How NF-κB is regulated by multiple activating receptors that are engaged during natural killer (NK)-target cell contact remains undefined. Here we show that sole engagement of NKG2D, 2B4 or DNAM-1 is insufficient for NF-κB activation. Rather, cooperation between these receptors is required at the level of Vav1 for synergistic NF-κB activation. Vav1-dependent synergistic signalling requires a separate PI3K- ...[more]

PMID: 27221592

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Project description:Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹⁻³⁸ fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹⁻³⁸ fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.

Project description:BackgroundIntrahepatic and extrahepatic metastases contribute to the high recurrence rate and mortality of hepatocellular carcinoma (HCC). Constitutive activation of nuclear factor-κB (NF-κB) is a crucial feature of HCC. NF-κB p65 (p50-p65) is the most common dimeric form. Ser536 acts as an essential phosphorylation site of RelA/p65. However, the effect of RelA/p65 Ser536 phosphorylation on progression and metastases during intermediate and advanced HCC has not been reported.MethodsPhosphorylation of RelA/p65 (p-p65 Ser536) and NF-κB p65 were detected by using immunohistochemical staining in HCC tissue samples. The biological effects of RelA/p65 Ser536 phosphorylation were evaluated by using xenograft and metastasis models. NF-κB p65 nuclear translocation was detected by using Western blotting. The binding of NF-κB p65 to the BCL2, SNAIL, and MMP9 promoters was detected by using chromatin immunoprecipitation. The biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition were assessed by using tetrazolium-based colorimetry, colony formation, EdU incorporation, flow cytometry, cell wound healing, and transwell assay.ResultsNF-κB p65 is highly expressed, while p-p65 Ser536 is not well expressed in intermediate and advanced HCC tissues. In vivo experiments demonstrated that a phosphorylation-mimetic mutant of RelA/p65 Ser536 (p65/S536D) prevents tumor progression and metastasis. In vitro experiments showed that p65/S536D inhibits proliferation, migration, and invasion. Mechanistically, RelA/p65 Ser536 phosphorylation inhibits NF-κB p65 nuclear translocation and reduces NF-κB p65 binding to the BCL2, SNAIL, and MMP9 promoters.ConclusionsRelA/p65 Ser536 phosphorylation was detrimental to NF-κB p65 entry into the nucleus and inhibited HCC progression and metastasis by reducing BCL2, SNAIL, and MMP9. The phosphorylation site of RelA/p65 Ser536 has excellent potential to be a promising target for NF-κB-targeted therapy in HCC.

Project description:Novel plant DING proteins (full-length 38 kDa p38SJ, and 27 kDa p27SJ) exhibit phosphatase activity and modulate HIV-1 gene transcription. Previously, we demonstrated that DING regulates HIV-1 gene transcription by dephosphorylation and inactivation of CTD RNA polymerase II, the major elongating factor of HIV-1 Long Terminal Repeats (LTR). Because the transcription of HIV-1 is controlled by several viral and cellular factors, including p65/p50 subunits of NF-κB, we hypothesized that DING phosphatase can also affect the phosphorylation and activity of p65 NF-κB, in addition to C-terminal Domain (CTD) of RNA Polymerase II (RNAPII), to suppress HIV-1 gene transcription and inhibit HIV-1 infection. Here, we describe the inhibition of HIV-1 infection and the p65/p50 NF-κB phosphorylation by DING protein, analyzed by ELISA and northern-blot assays, western-blot assays, cell fractionation, and promoter-reporter assays in DING-expressing cells, using a pTet-on inducible system. Results from HIV-1 infection assays demonstrate a strong inhibition of HIV-1 and HIV-LTR RNA expression by DING protein, determined by p24 ELISA and by northern blot assay. Results from the western blot assays and cell fractionation assays show that there is an increase in the level of hypo-phosphorylated form of p65 NF-κB in DING-expressing cells. Both fractions of p65/p50, nuclear or cytoplasmic, are affected by DING phosphatase, but more cytoplasmic accumulation of p65 NF-κB was found in the presence of DING, suggesting that subsequent activation and nuclear import of active NF-κB is affected by DING. The major portion of nuclear p65 was dephosphorylated in DING-expressing cells. The promoter-reporter assay demonstrated that DING-mediated dephosphorylation and dysregulation of NF-κB p65 lead to the suppression of its binding to HIV-1 LTR, and resulted in the inhibition of p65-mediated activation of LTR transcription. Mapping of the region within LTR that was affected by DING revealed that both, NF-κB and CTD RNA Polymerase II binding sites were important, and cooperativity of these cellular factors was diminished by DING. In addition, mapping of the region within DING-p38SJ that affected LTR transcription, revealed that phosphate-binding domain is essential for this inhibitory activity. We have demonstrated the effect of DING phosphatases on HIV-1 infection, phosphorylation of p65 NF-κB, and transcription of HIV-1 LTR. Our studies suggest that one possible mechanism by which DING can regulate the expression of HIV-1 LTR can be through dysregulation of the transcription factor NF-κB p65 by preventing its phosphorylation and translocation to the nucleus and binding to the HIV-1 LTR, an action that could contribute to the utility of DING p38SJ as an antiviral agent. Importantly, DING not only inhibits HIV-1 LTR gene transcription in the presence of increased p65 NF-κB, but also suppresses HIV-1 infection. DING protein improved inhibitory effects of the known anti-retroviral drugs, Tenofovir (TFV) and Emtricitabine (FTS) on HIV-1, since in the combination with these drugs; the suppression of HIV-1 by DNG was significantly higher when it was in combination with these drugs, compared to controls or cases without DING. Thus, our data support the use of neuroprotective DING proteins as novel therapeutic antiviral drugs that suppress HIV-1 LTR transcription by interfering with the function of NF-κB.

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Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition.

Publications

Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition.

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