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. Author manuscript; available in PMC: 2016 Aug 1.
Published in final edited form as: J Immunol Methods. 2015 Apr 7;423:3–11. doi: 10.1016/j.jim.2015.03.018

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

Orla Maguire a,*, Kieran O'Loughlin a, Hans Minderman a
PMCID: PMC4522232  NIHMSID: NIHMS693621  PMID: 25862606

Abstract

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 has been used to study the relationship between phosphorylation motifs and NF-κB 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 in the relationship between p65 phosphorylation motifs and its nuclear localization indicate that these parameters have different significance 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). TNFα- 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-p65s529) increased rapidly following stimulation and that nuclear localization of P-p65s529 followed the nuclear localization pattern of total p65. However, in the presence of tacrolimus, P-p65s529 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.

Keywords: NF-κB, p65, serine 529, phosphorylation, nuclear localization, imaging flow cytometry

Introduction

Phosphorylation of key proteins is an essential epigenetic event in cellular processes including those regulating proliferation, metabolism, and signal transduction (reviewed in (Banerjee and Chakravarti, 2011)). The study of phosphorylation in signal transduction events is especially important in pathways that are known to be dysregulated in disease states. Recent studies have defined a role for measuring the kinetics of phosphorylation events in not only the diagnosis/prognosis of diseases (Mackenzie et al., 2012; Sonnenblick et al., 2012; Woost et al., 2011), but also the efficacy of therapies, such as with the bcr/abl kinase inhibitor Imatinib, directed towards epigenetic alterations. (Druker et al., 2001; Hedley et al., 2008).

Nuclear Factor -kappa-light-chain-enhancer of B cells (NF-κB) is a transcription factor activated by a variety of stimuli and is involved in key processes including proliferation, stress response, and cytokine production (Huang et al., 2010; Zheng et al., 2011). Its dysregulation is often implicated in inflammation, auto-immune disease, infections, and cancer (Ahmed, 2010; Brown et al., 2008; Nair et al., 2009; Wu and Zhou, 2010).

Activation of the NF-κB transcription factor pathway requires a number of phosphorylation steps to allow transient cytoplasmic to nuclear localization (Oeckinghaus and Ghosh, 2009) and once nuclear, phosphorylated NF-κB binds to promoter sequences of target genes to regulate their expression. Phosphorylation is a rapid and dynamic process, occurring within minutes of activation. Its rapid and transient nature makes it difficult to accurately measure phosphorylation states relevant to biological effects using techniques such as western blotting that due to their labor intensiveness or sample size requirements use limited sampling approaches.

Flow cytometry enables the determination of phosphorylation events in heterogeneous cell populations by co-staining cell populations with phospho-specific antibodies and antibodies for immunophenotyping (Chow et al., 2005; Lin et al., 2010; Perez et al., 2004). A caveat of conventional flow cytometry is the inability to determine the intracellular origin of a measured signal which may be especially relevant when studying signaling events.

In the NF-κB signaling cascade, phosphorylation of p65 is required for nuclear translocation and transcriptional activation but different sites of phosphorylation have different significance in this regard. For example, phosphorylation at the serine 536 position is required for nuclear translocation while phosphorylation of serine 529 affects transcriptional activity and serine 276 is involved in co-activator recruitment at target gene promoters (Mattioli et al., 2004; Wang and Baldwin, 1998; Zhong et al., 1998). Thus, in the context of studying pharmacodynamic drug effects on NF-κB activity, the relevance of using nuclear translocation of p65 as a parameter of response depends on the p65 phosphorylation sites that are affected.

Tacrolimus (TAC) is one of the cornerstones of contemporary immunosuppressive drug regimen. By inhibiting the activity of calcineurin, TAC inhibits the T cell receptor-mediated activation of the nuclear factor of activated T cells (NFAT), one of the key signaling pathways controlling the immune response on a cellular level. In addition to TAC's effect on calcineurin and the NFAT signaling pathway, TAC has recently been described to also affect the NF-κB signaling pathway by inhibiting the phosphorylation of p65 at the serine 529 position (Vafadari et al., 2013). In order to demonstrate the applicability and relevance of IFC to simultaneously evaluate specific phosphorylation motifs and nuclear translocation, TAC exposure of healthy donor T cells was used as the experimental model system with the expectation that TAC induced inhibition of p65 at the serine 529 position should be detectable without affecting nuclear translocation of total p65.

MATERIALS AND METHODS

All analysis files mentioned are available upon request.

Cell culture and NF-κB activation

Cell lines were purchased from ATCC. Jurkat human lymphoblastic leukemia cells were maintained in RPMI-1640 media (Mediatech Inc., Manassas, VA) supplemented with 10% fetal bovine serum (PAA Laboratories Pty Ltd, Queensland, Australia), 2 mM L-glutamine, 20 U/mL penicillin, and 20 μg/mL streptomycin (Mediatech Inc., Manassas, VA). HL-60 human promyelocytic leukemia cells were maintained in Iscoves media supplemented with 20% fetal bovine serum, L-glutamine, penicillin, and streptomycin, as above. Cell lines were maintained at exponential growth at 37°C in a fully humidified atmosphere of 5% CO2 in air.

For NF-κB activation, cell densities were adjusted to 1×106 cells/mL. Cells were exposed to 10 ng/mL Tumor Necrosis Factor alpha (TNFα) (Invitrogen, Carlsbad, CA) or 20 ng/mL PMA (Sigma, St. Louis, MO) / 1.5 μm Ionomycin (Sigma, St. Louis, MO) for various time-points (see results) at 37°C, 5% CO2 in air. Following activation, cells were fixed for 10 minutes in 4% methanol-free formaldehyde (FA) (Polysciences Inc, Warrington, PA) at room temperature and stained as outlined below.

Antibodies and staining

P-p65s529 was detected by direct labeling. Antibody was diluted in permeabilization wash buffer (PWB) consisting of 0.1% Triton-X-100 (EMD Biosciences, La Jolla, CA) in sterile 1× phosphate buffered saline (PBS). The Mouse monoclonal P-p65s529–AF488 antibody (BD Biosciences, San Diego, CA) was diluted 1:10 in PWB and incubated with cells at room temperature for 30 minutes. Antibodies were removed, cells washed with PWB, and resuspended in 100 μL sterile 1× PBS.

Total p65 and NFAT1 were detected by indirect labeling. Antibodies were diluted in PWB as above. The primary Rabbit polyclonal NF-κB/p65 antibody (SantaCruz Biotechnology Inc, Santa Cruz, CA) was diluted 1:20 in PWB. Rabbit polyclonal NFAT1 antibody (Cell Signaling, Danvers, MA) was diluted 1:50 in PWB. Samples were incubated for 20 minutes at room temperature. Primary antibodies were removed, cells washed with PWB, and 1:200 dilution of secondary AF647 conjugated F(ab’)2 fragment donkey anti rabbit IgG antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was added and incubated at room temperature in the dark for 20 minutes. Secondary antibody was removed, cells washed with PWB, and resuspended in 100 μL sterile 1× PBS.

Just prior to data acquisition on the ImageStream, DAPI (Invitrogen, Carlsbad, CA) was added to all samples (0.5 μg/mL final concentration), to stain the nucleus.

NF-κB activation in healthy donor PBL in vitro

Healthy donor peripheral blood (PBL) was collected in accordance with a protocol approved by the Institutional Review Board at Roswell Park Cancer Institute. PBL was drawn in sodium heparinized tubes and allowed to ‘rest’ at room temp for at least 1 hour. 500 μL PBL was used per sample and activation was initiated by adding either TNFα (100 ng/mL final concentration) or 50 ng/mL PMA (Sigma, St. Louis, MO) / 3.75 μM Ionomycin (Sigma, St. Louis, MO) for various time-points (see results) at 37°C, 5% CO2 in air. A control sample was included in the absence of activation stimulants. Following incubation, samples were immediately fixed using 20:1 ratio (9.5 mLs) of 4% FA diluted in 1× PBS for 10 minutes at room temperature to preserve phosphorylation status. Following fixation, red blood cells were lysed using 9.5mLs 1× BD Lyse Buffer according to the manufacturer's recommendations (BD Phosflow, BD Biosciences). Cells were then stained for NF-κB/p65 and P-p65s529 as outlined above.

For Tacrolimus (TAC) inhibition, 500 μL PBL was pre-treated with 10 nM TAC for 1.5 hrs at 37°C and then activated with PMA/Ionomycin as above with continued incubation with tacrolimus for 30 minutes. Control samples were incubated without stimulants and without TAC. Following activation, cells were fixed, red blood cells lysed, and stained for NFAT1 (as above).

ImageStreamX Acquisition

Data acquisition was performed on an imaging flow cytometer (ImagestreamX; Amnis/EMD Millipore, Seattle, WA) between years 2012-2014. Images acquired include a brightfield image (Channel 1 and 9; 430-480nm), FITC (Channel 2; 480-560nm), DAPI (Channel 7; 430-505nm), and AF647 (Channel 11; 660-740nm). AF488 was excited by a 488nm laser with a 100mW output, DAPI was excited by a 405nm laser with a 10mW output, and AF647 was excited by a 658nm laser with a 50mW output. The selected laser outputs prevented saturation of pixels in the relevant detection channels as monitored by the corresponding Raw Max Pixel features during acquisition. For each sample, bright field, P-p65s529-AF488 with total p65-AF647 or NFAT1-AF647 and DAPI (nuclear stain) images were simultaneously collected for 20,000 events. Cell classifiers were set for the lower limit of size of the brightfield image to eliminate debris, the upper limit of size of the brightfield image to eliminate aggregates, a minimum intensity classifier on the DAPI channel to exclude non-cellular (DAPI negative) images, and, in healthy donor samples, an upper limit on scatter intensity to eliminate monocytes and granulocytes.

Compensation

In each experiment single color controls were stained for all fluorochromes and 500 events were collected with all relevant lasers on for each individual control. All channels were on, with brightfield LEDs and scatter laser off to accurately observe fluorescence overlap in all channels. Only those events exhibiting a positive signal in the channel of interest were collected (e.g., the P-p65s529-AF488 control was positive in channel 2). Each single color control file was then merged to generate a compensation matrix (an example of which is shown in Supplementary Figure 1), and all sample files were processed with this matrix applied.

Data Analysis

Following compensation for spectral overlap based on single color controls, analysis was performed with IDEAS®software version 5.0 and individual cell images were created using IDEAS® software version 6.1 (Amnis Corp, Seattle, WA). Cell populations were hierarchically gated and an example of the gating strategy is shown in Figure 1. Single cells (A) that were in focus (B) and were positive for both DAPI and p65 (C) were selected as described previously (George et al., 2006; Maguire et al., 2011). All measurements of P-p65s529 were made from this parent total p65 population. In healthy donor samples, lymphocytes were gated based on positivity for p65 and scatter. After gates were applied, 70-80% of the total number of acquired images were incorporated into the final analysis. Expression levels of each factor in the entire cell are represented as ‘Intensity’. Intensity in the IDEAS® software is calculated as the sum of the pixel values in the software-generated ‘combined mask’ minus the background pixel values (i.e., those not in the combined mask).

Figure 1. Hierarchical gating and analysis strategy used to determine transcription factor expression and nuclear translocation.

Figure 1

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Single cells are discriminated from debris and cell aggregates based on area and aspect ratio of the brightfield image (A). Of those cells, events which are in focus are selected on the basis of a high value of a contrast parameter (gradient RMS of the brightfield image) (B). Single, in focus cells that positive for both a nuclear signal and total p65 signal are then selected (C). At this point cell line samples would progress to step E (see below). In healthy donor PBMC samples gating for lymphocytes was performed based on p65 positivity and scatter properties (D). A software-generated ‘morphology mask’ based on the nuclear image (E) is then applied to the ‘Similarity’ feature comparing the transcription factor and nuclear images. A distribution of cells with varying ‘similarity scores’ is then graphed (F), and the mean and standard deviation recorded to measure the ‘Rd value’. The median similarity score (Median SS) of the example distribution is also shown.

The spatial relationship between the transcription factors and nuclear images was measured using the ‘Similarity’ feature in the IDEAS® software, as described previously (George et al., 2006; Maguire et al., 2011). Briefly, a ‘Morphology’ mask is created to conform to the shape of the nuclear DAPI image, and a ‘Similarity Score’ (SS) feature is defined. The SS is a log-transformed Pearson's correlation coefficient between the pixel values of two image pairs, and provides a measure of the degree of nuclear localization of a factor by measuring the pixel intensity correlation between the factor-of-interest images and the DAPI images within the masked region. Cells with a low SS exhibit poor correlation between the images (corresponding with a predominant cytoplasmic distribution of the factor), whereas cells with a high SS exhibit positive correlation between the images (corresponding with a predominant nuclear distribution of the factor). The relative shift in this distribution between two populations (e.g., control versus treated cells) was calculated using the Fisher's Discriminant ratio (Rd value). Specifically, the mean SS of the test population (time-points > 0) minus the mean SS of the control population (time =0) then divided by the sum of the standard deviations of both populations.

RESULTS

NF-κB activation by phosphorylation and nuclear localization can be measured by IFC

In order to determine whether activation of the P-p65s529 phosphorylation site could be measured by IFC simultaneously with total p65, the NF-κB/p65 signaling pathway was stimulated in the Jurkat cell line by exposure to PMA/Ionomycin for 30 minutes (a time-point previously determined to be optimal for total p65 nuclear translocation (Maguire et al., 2011)). As expected, total p65 expression did not change however the signal intensity relative to unstimulated cells, increased significantly for P-p65s529. An example of typical histogram overlays observed for the intensity of both p65 and P-p65s529 is shown in Figure 2A. Nuclear localization of total p65 and P-p65s529 was also measured following activation for 30 minutes with PMA/Ionomycin (Figure 2B) using the similarity score (SS), a measure of the degree of change in similarity between the protein and nuclear images, detailed in the materials and methods. The median similarity score (Med SS) of the distributions is also provided. Representative images of the untreated cells (Unstim) show cytoplasmic localization and PMA/Ionomycin-induced activation shows nuclear localization occurring for both total p65 and P-p65s529 (Figure 2C). The SS for each individual image is shown on its right in yellow.

Figure 2. P-p65s529 expression and nuclear translocation of p65 as parameters of response to PMA/Ionomycin-induced NF-κB activation.

Figure 2

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Activation of p65 was triggered in the Jurkat cell line using PMA/Ionomycin for 30 minutes. Expression level, measured by intensity, increases above unstimulated levels for P-p65s529 but not total p65 as shown by histogram overlays for both total p65 (left graph) and P-p65s529 (right graph) (A). Translocation into the nucleus, measured by similarity score (SS), significantly increases for both total p65 and P-p65s529 when cells are stimulated with PMA/Ionomycin (B). Median similarity score is also shown for each population. Representative images of nuclear translocation in both untreated (Unstim) and cells treated with PMA/Ionomycin are shown (C). From left to right in each panel, BF = brightfield images of each cell, followed by the nuclear image (blue) total p65 (red) and P-p65s529 (green) images, and the far right column is the merged image of the nucleus with total p65 and P-p65s529. The SS for total p65 and P-p65s529 is shown on the right of the individual image (yellow).

Time kinetics of total p65 and P-p65s529 expression and nuclear localization in Jurkat T-cell line

In order to determine whether the activation kinetics of P-p65s529 measured by IFC compared to those of total p65, Jurkat cells were treated with either TNFα (Figure 3A+C) or PMA/Ionomycin (Figure 3B+D) for up to 60 minutes. Expression levels for P-p65s529 following TNFα activation significantly increased rapidly peaking at 10 minutes. As expected, total p65 expression levels did not change (Figure 3A). P-p65s529 expression then declined returning to baseline at around 40 minutes. Example images from 0, 10, 30, and 60 minutes samples are shown in Supplementary Figure 2). The kinetics of PMA/Ionomycin-induced p65 activation differed from that of TNFα in Jurkat cells. Expression of P-p65s529 was more gradual, peaking at 30 minutes, but similar to the TNFα data there is no expression change for total p65 (Figure 3B). The PMA/Ionomycin-induced activation remains significantly above baseline 60 minutes post-treatment.

Figure 3. Time kinetics of activation-induced total p65 and P-p65s529 expression and nuclear localization in Jurkat cells.

Figure 3

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Jurkat cells were stimulated with TNFα (A+C) or PMA/Ionomycin for various time durations up to 1 hour (B+D). Expression level is measured as fold change of intensity relative to untreated zero time-point set to 1. With TNFα, expression level of P-p65s529 peaks at 10 minutes, with no change in total p65 (A). Nuclear translocation, measured by Rd Value, of P-p65s529 peaks at 20 minutes and total p65 translocation peaks at 25 minutes (B). With PMA/Ionomycin, expression level of P-p65s529 peaks at 30 minutes, with no change in p65 (C). Nuclear translocation of both total p65 and P-p65s529 peaks at 40 minutes (D). Results are representative of 3 independent experiments, error bars represent SEM (*p<0.05, **p<0.01, ***p<0.001, Students t test).

In contrast, when measuring nuclear localization, TNFα induced both P-p65s529 and total p65 translocation significantly relative to time zero (untreated cells), with P-p65s529 peaking at 20 minutes, preceding a total p65 peak at 25 minutes (Figure 3C). At later time-points, the level of P-p65s529 in the nucleus returned close to untreated values, detailing the dynamic and short-lived nature of p65 phosphorylation events following activation by TNFα in this cell line, and mirroring the pattern of P-p65s529 expression. Total p65, however, is still significantly present in the nucleus after 60 minutes.

PMA/Ionomycin-activated kinetics also follow a different pattern than expression, where both total p65 and P-p65s529 localize in the nucleus more slowly, both peaking at 40 minutes (Figure 3D). Both factors then decline and have returned to cytoplasmic localization by 2 hours (data not shown). These results show that in this cell line, phosphorylation and nuclear localization measurements are comparable, thus confirming that nuclear translocation is an accurate alternative measure of activation at this step. The kinetics of activation by different stimulants however differs.

Similar results were observed when the same experiments were conducted on the promyelocytic leukemia HL-60. Expression of P-p65s529 increased following activation with TNFα and PMA/Ionomycin. Notably, IFC measured expression and nuclear translocation kinetics were more rapid in this cell line with activation causing a significant up-regulation at 2 minutes (Suppl Figure 2). Also of note in the HL-60 cell line, p65 nuclear localization could still be measured 4 hours post-treatment. The data thus demonstrate how the kinetics of NF-κB activation using the same activating trigger, be it determined by expression of P-p65s529 or nuclear presence of p65, can differ between cell lines.

Time kinetics of total p65 and P-p65s529 expression and nuclear localization in healthy donor lymphocytes

In order to determine whether IFC can accurately measure P-p65s529 expression in lymphocytes ex vivo, healthy donor PBL samples were treated with PMA/Ionomycin for up to 60 minutes (Figure 4). NF-κB activation was studied in the lymphocytes (gated based on p65 positivity and scatter properties). As expected, expression levels for total p65 following activation did not significantly rise above the untreated samples (Figure 4A, black line). Expression of P-p65s529 gradually increases, peaking at 20 minutes and then declining (Figure 4A, grey line). Nuclear translocation kinetics for total p65 showed a similar initial increase in nuclear localization peaking at 30 minutes (Figure 4B, black line). Nuclear translocation kinetics for P-p65s529 showed that P-p65s529 expression corresponds with its nuclear localization (Figure 4B, grey line). Interestingly, similar to the expression results, the P-p65s529 nuclear localization begins to decline after 20 minutes even when total p65 remains nuclear. Since this motif is involved in DNA binding to initiate the transcription of downstream target genes, its decline is likely necessary before total p65 leaves the nucleus.

Figure 4. Time kinetics of PMA/Ionomycin-induced p65 and P-p65s529 expression and nuclear localization in healthy donor lymphocytes.

Figure 4

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Whole blood samples from hematologically healthy donors were stimulated with PMA/Ionomycin for various time durations up to 1 hour. Expression level is measured as fold change of intensity relative to untreated zero time-point set to 1. No change in expression level of p65 is observed, however expression level of P-p65s529 peaks at 20 minutes (A). Nuclear translocation, measured by the Rd value, of total p65 peaks at 30 minutes whereas P-p65s529 peaks at 15 minutes. Both remain in the nucleus at 60 minutes. Results are representative of 3 independent experiments, error bars represent SEM (*p<0.05, **p<0.01, Students t test).

IFC can measure the tacrolimus-induced inhibition of P-p65s529 phosphorylation where total p65 nuclear localization is unaffected

A recent study identified the immunosuppressive drug tacrolimus (TAC) as a novel inhibitor of p65 by direct inhibition of the P-p65s529 motif (Vafadari et al., 2013). This is unique since, to our knowledge, all other known inhibitors of P-p65s529 are in fact IκB kinase inhibitors. To determine whether IFC could accurately measure this inhibition, peripheral blood mononuclear cells (PBMCs) were treated with 10 nM TAC, a concentration we have previously determined to be sufficient to completely inhibit TAC's canonical target, nuclear factor of activated transcription (NFAT1) (Maguire et al., 2013). Signaling was then stimulated by PMA/Ionomycin for 10 or 30 minutes and activation was studied in the lymphocytes (gated based on p65 positivity and scatter properties) (Figure 5). PMA/Ionomycin activated expression of P-p65s529 peaks at 10 minutes and is declining at 30 minutes in these samples (Figure 5A, grey line) TAC significantly inhibits the expression of P-p65s529 at both 10 minutes (p=0.0112) and 30 minutes (p=0.004) (black line). The observed inhibition of expression correlated with an inhibition of nuclear localization of P-p65s529 by 30 minutes (Figure 5B). Although this nuclear localization is not statistically significant it does trend down. Total p65 activation by PMA/Ionomycin is unaffected by addition of TAC (Figure 5C). As expected, activation by PMA/Ionomycin results in a complete shift of NFAT1 into the nucleus at both time-points (Figure 5D, grey line). Presence of 10 nM TAC completely inhibits PMA/Ionomycin induced nuclear translocation (p<0.0001) (black line).

Figure 5. Tacrolimus (TAC)-inhibits PMA/Ionomycin-induced P-p65s529 expression without affecting nuclear translocation of p65 in healthy donor T-cells.

Figure 5

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Peripheral blood mononuclear cells from hematologically healthy donors were stimulated with PMA/Ionomycin for 10 minutes or 30 minutes. Expression level of P-p65s529, measured as fold change of intensity relative to untreated zero time-point set to 1, is inhibited at both 10 and 30 minutes post PMA/Ionomycin treatment (A). Nuclear translocation of P-p65s529 is inhibited at 30 minutes (B). Nuclear translocation of total p65 is unaffected by TAC at either time-point (C). As a control, the nuclear translocation of the transcription factor NFAT1 is completely inhibited by TAC (D). Results are representative of 3 independent experiments, error bars represent SEM (*p<0.05, **p<0.01, ****p<0.0001, Students t test)

Discussion

In this study the applicability of imaging flow cytometry (IFC) to simultaneously assess protein phosphorylation motifs and intracellular localization is demonstrated by determining the correlation between the phosphorylation of the NF-κB subunit p65 on the serine 529 residue (P- p65s529), the p65 nuclear localization, and the effects thereon of the immunosuppressant tacrolimus (TAC).

For the transcription factor NF-κB, there are a number of sites on the p65 sub-unit that are phosphorylated by different kinases depending upon the activating trigger of the signaling pathway. Phosphorylated p65 serines include serine 276 (Vermeulen et al., 2003), serine 311 (Duran et al., 2003), serine 468 (Schwabe and Sakurai, 2005), serine 529 (Wang et al., 2000), serine 536 (Sakurai et al., 1999). The functional significance of the different phosphorylation sites with regards to NF-kB transcriptional activity differ. The use of phospho-specific antibodies to detect expression levels of the various phosphorylated p65 motifs by flow cytometry has been used as a parameter of response to assess the activity of the NF-κB pathway in relation to disease states or drug intervention (Hernandez Mde et al., 2011; Lin et al., 2013; Simard et al., 2014; Vafadari et al., 2013). Alternatively, since activation of NF-κB is associated with relocation from the cytoplasm to nucleus, the nuclear localization of NF-κB has also been used as a parameter for activation. Although both the phosphorylation and nuclear localization of p65 are necessary for transcriptional activity, neither one by itself is sufficient to warrant transcription. Therefore in absence of an NF-κB transcription-dependent functional read out, it is important to assess both. The data demonstrate that in absence of TAC, the patterns of p65s529 phosphorylation are similar to those of its nuclear translocation but that the time kinetics of both can differ between cell lines and healthy donors and by different stimulants of activation. It is further demonstrated that TAC affects the serine 529 phosphorylation but without an effect on nuclear translocation of total p65. In the Jurkat cell line, the serine 529 phosphorylation and the nuclear translocation of total p65 are comparable. Serine 529 is located on the transactivation domain 1 (TA1) on p65 and is involved in the activation of transcription of NF-κB downstream target genes by promoter binding (Madrid et al., 2001; Wang and Baldwin, 1998; Wang et al., 2000). In a more tumorigenic cell line, the promyelocytic leukemic cell line, HL-60, the pattern of phosphorylation kinetics was quite different from those observed in Jurkat cells. Again, the phosphorylation event and nuclear translocation of total p65 occurred very rapidly within 2 minutes of TNFα activation. Although in this cell line both total p65 and P-p65s529 continued to be localized in the nucleus after 4 hours treatment, at 3 hours the P-p65ser529 expression levels had returned close to baseline levels possibly due to the more oncogenic nature of NF-kB in malignant cells, where activation is commonly sustained (Pickering et al., 2007).

Similar to the cell line models, activation of NF-κB with PMA/Ionomycin, in healthy donor lymphocytes, resulted in comparable P-p65s529 phosphorylation event and total p65 translocation. As expected, expression of total p65 did not change but expression of phosphorylated Ser529 increased. At 30 minutes the total p65 nuclear localization peaked, whereas P-p65s529 expression and nuclear localization was declining. Since serine 529 is involved in initiation of target gene transcription this decline may be necessary before p65 can return to the cytoplasm. Both remained present (above baseline) after 60 minutes. Increasingly, NF-κB is considered a therapeutic target particularly in diseases of immune function, and hematological malignancies (Breccia and Alimena, 2010; Haddad and Abdel-Karim, 2011). Since the time kinetics of phosphorylation events and nuclear translocation of total p65 can differ, the timing of sampling is critical in interpretation of detection of phospho-specific p65 species as a parameter of NF-κB signaling activity. It is also important to note that the choice of phosphorylation motif to be studied should be carefully considered depending on the model system and the cellular events studied. This study, and other recent literature, indicates that specific motifs are activated differently to differentially regulate their downstream targets. Serine 529, but not serine 536, is essential in stem cell differentiation (Yang et al., 2010), and serine 276, specifically, is involved in monocyte-derived macrophage survival (Wang et al., 2011). Serine 311 phosphorylation is abrogated in anergic T cells, and inhibis p65-induced IFNγ production without affecting serine 536 phosphorylation (Clavijo and Frauwirth, 2012). This study demonstrates that TAC can inhibit P-p65s529 phosphorylation but not the nuclear localization of p65. Interestingly, although expression decreases, it remains nuclear. Our previous studies have already shown that TAC inhibits IFNγ production (Maguire et al., 2013). Further studies into the pattern of this serine 529 inhibition and its specific effects on IFNγ and other downstream targets are ongoing in our lab.

In summary, the data demonstrate the application of IFC to simultaneously assess a phosphorylation motif of p65 and its relevance to the cellular localization of p65. The results obtained by IFC analysis corroborate the current insights derived from analysis with other platforms regarding the relation between the effect of TAC on serine 529 phosphorylation and the nuclear localization of p65. The IFC approach is much less labor intensive and time consuming than alternative analysis approaches like immunoblot or microscopy. Additionally, IFC provides the intracellular localization parameter which is highly relevant in case of the activity of transcription factors such as NF-κB which is unattainable by conventional flow cytometry analysis. The observed variable relationships between specific phosphorylation motifs and nuclear translocation events as a parameter of activation is likely not a phenomenon specific to NF-κB, but could be applicable also to other transcription factor pathways, including the MAPK (Desterke et al., 2011; Fischer et al., 2010) and Jak/STAT pathways (Diaz et al., 2011; Lesterhuis et al., 2011).

Supplementary Material

01

Highlights.

  • IFC can simultaneously measure phosphorylation and nuclear translocation of p65

  • Tacrolimus inhibits P-p65s529 phosphorylation but not nuclear translocation of p65

Acknowledgements

The authors wish to acknowledge Dr. Sherree Friend (Amnis Corp) for continued application support with regards to ImageStream data acquisition and analysis.

Research sponsored by NIH R33CA126667 (H.M.) and by the NCI Cancer Center Support Grant to the Roswell Park Cancer Institute (CA016056)

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