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Simultaneous assessment of NF-?B/p65 phosphorylation and nuclear localization using imaging flow cytometry.

ABSTRACT: Aberrant activity of Nuclear Factor-kappaB (NF-?B) is associated with many diseases and is therapeutically targeted. Post-translational modifications, particularly phosphorylation of the RELA/p65 sub-unit, are essential for cytoplasmic to nuclear localization of NF-?B/p65 and initiation of transcription of downstream target genes. Immunoblot and phospho-flow cytometry have been used to study the relationship between phosphorylation motifs and NF-?B activation and microscopic analysis of nuclear localization of p65 is also used as a parameter for activation. The labor intensive nature of these approaches commonly limits the number of sampling points or replicates. Recent insights into the relationship between p65 phosphorylation motifs and their nuclear localization indicate that these parameters have different significances and should not be used interchangeably. In this study, we demonstrate feasibility and reproducibility of studying the relationship between p65 phosphorylation and nuclear translocation using imaging flow cytometry (IFC). TNF?- or PMA/Ionomycin-induced phosphorylation of p65 at serine 529 in cell line models and healthy donor lymphocytes served as the experimental model. IFC analysis demonstrated that expression of phosphorylated serine 529 (P-p65(s529)) increased rapidly following stimulation and that nuclear localization of P-p65(s529) followed the nuclear localization pattern of total p65. However, in the presence of tacrolimus, P-p65(s529) expression was inhibited without affecting nuclear localization of total p65. The data demonstrate the application of IFC to simultaneously assess phosphorylation of p65 and its cellular localization and the results obtained by this analysis corroborate current insights regarding the specific effect of tacrolimus on serine 529 phosphorylation.

SUBMITTER: Maguire O

PROVIDER: S-EPMC4522232 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry.

Maguire Orla O O'Loughlin Kieran K Minderman Hans H

Journal of immunological methods 20150407

Aberrant activity of Nuclear Factor-kappaB (NF-κB) is associated with many diseases and is therapeutically targeted. Post-translational modifications, particularly phosphorylation of the RELA/p65 sub-unit, are essential for cytoplasmic to nuclear localization of NF-κB/p65 and initiation of transcription of downstream target genes. Immunoblot and phospho-flow cytometry have been used to study the relationship between phosphorylation motifs and NF-κB activation and microscopic analysis of nuclear ...[more]

PMID: 25862606

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Project description:IntroductionNovel 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.MethodsHere, 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.ResultsResults 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.ConclusionWe 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.

Dataset Information

Simultaneous assessment of NF-?B/p65 phosphorylation and nuclear localization using imaging flow cytometry.

Publications

Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry.

Similar Datasets

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