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Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Li J, Hardy K, Phetsouphanh C, Tu WJ, Sutcliffe EL, McCuaig R, Sutton CR, Zafar A, Munier CM, Zaunders JJ, Xu Y, Theodoratos A, Tan A, Lim PS, Knaute T, Masch A, Zerweck J, Brezar V, Milburn PJ, Dunn J, Casarotto MG, Turner SJ, Seddiki N, Kelleher AD, Rao S - J. Cell. Sci. (2016)

Bottom Line: Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation.This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood.Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Education, Science, Technology & Mathematics, University of Canberra, Canberra, Australian Capital Territory 2617, Australia Department of Microbiology & Immunology, The Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria 3010, Australia.

No MeSH data available.


Related in: MedlinePlus

p65 Ser536 phosphorylation by nuclear PKC-θ. (A) Immunoblotting of p65, p65 phosphorylated at Ser486 (p65s486p) and Ser536 (p65s536p) in the nuclear extract (NE), and IKK, phosphorylated IκB-α on Ser32 or Ser36 (pIκB-α) and total p65 levels in the cytoplasmic extract (CE) in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. Representative blot of three independent repeats (also see Fig. S4D,E). (B) Nuclear-to-cytoplasmic ratios (NE/CE) of p65 protein levels immunoblotted in nuclear versus cytoplasmic fractions was calculated for the cells described in A (mean±s.e.m., n=3). *P≤0.05, **P≤0.01 (two-way ANOVA). (C) Confocal microscopy of p65 nuclear localization during activation with PMA and Ca2+ ionophore for 0, 0.5, 1 and 2 h in Jurkat T cells transfected with vector only (VO) or the cytoplasm-restricted PKC-θ (NLS) mutant plasmids. Cells were stained with DAPI and anti-p65 antibodies. Representative images of these constructs. Scale bars: 20 μm. (D) Nuclear translocation of p65 (Fn/Fc) expressed as a ratio of nuclear (Fn) and cytoplasmic (Fc) fluorescence with background subtraction (mean±s.e.m., n>20 cells). **P≤0.01; ***P≤0.001; ****P<0.0001; ns, not significant (Mann–Whitney test). (E) Gene expression of p50 (NFKB1), p65 (RELA), and (IKBKE) in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with VO, WT and NLS plasmids (mean±s.e.m., n=2). (F) Immunoblotting of p65 and p65 phosphorylated serine 536 (p65 Ser536p) levels in nuclear extract from DMSO or 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (P/I) (n=6). (G) GAPDH-normalized gene expression of IL2, TNF, TNFSF9 and SATB1 in the vehicle-control or 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (P/I) (mean±s.e.m., n=3). *P≤0.05, **P≤0.01, ***P≤0.001 (one-tailed Student's t-test). (H) ChIP-PCR analysis of p65 Ser536p at the promoters of IL2, TNF, TNFSF9 and an intronic region of SATB1 in Jurkat T cells expressing VO, WT and NLS plasmids after primary (1°) and secondary (2°) stimulations. ChIP enrichment ratio relative to the no-antibody control is shown (mean±s.e.m., n=3). *P≤0.05, **P≤0.01, ***P≤0.001 (one-tailed Student's t-test).
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JCS181248F6: p65 Ser536 phosphorylation by nuclear PKC-θ. (A) Immunoblotting of p65, p65 phosphorylated at Ser486 (p65s486p) and Ser536 (p65s536p) in the nuclear extract (NE), and IKK, phosphorylated IκB-α on Ser32 or Ser36 (pIκB-α) and total p65 levels in the cytoplasmic extract (CE) in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. Representative blot of three independent repeats (also see Fig. S4D,E). (B) Nuclear-to-cytoplasmic ratios (NE/CE) of p65 protein levels immunoblotted in nuclear versus cytoplasmic fractions was calculated for the cells described in A (mean±s.e.m., n=3). *P≤0.05, **P≤0.01 (two-way ANOVA). (C) Confocal microscopy of p65 nuclear localization during activation with PMA and Ca2+ ionophore for 0, 0.5, 1 and 2 h in Jurkat T cells transfected with vector only (VO) or the cytoplasm-restricted PKC-θ (NLS) mutant plasmids. Cells were stained with DAPI and anti-p65 antibodies. Representative images of these constructs. Scale bars: 20 μm. (D) Nuclear translocation of p65 (Fn/Fc) expressed as a ratio of nuclear (Fn) and cytoplasmic (Fc) fluorescence with background subtraction (mean±s.e.m., n>20 cells). **P≤0.01; ***P≤0.001; ****P<0.0001; ns, not significant (Mann–Whitney test). (E) Gene expression of p50 (NFKB1), p65 (RELA), and (IKBKE) in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with VO, WT and NLS plasmids (mean±s.e.m., n=2). (F) Immunoblotting of p65 and p65 phosphorylated serine 536 (p65 Ser536p) levels in nuclear extract from DMSO or 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (P/I) (n=6). (G) GAPDH-normalized gene expression of IL2, TNF, TNFSF9 and SATB1 in the vehicle-control or 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (P/I) (mean±s.e.m., n=3). *P≤0.05, **P≤0.01, ***P≤0.001 (one-tailed Student's t-test). (H) ChIP-PCR analysis of p65 Ser536p at the promoters of IL2, TNF, TNFSF9 and an intronic region of SATB1 in Jurkat T cells expressing VO, WT and NLS plasmids after primary (1°) and secondary (2°) stimulations. ChIP enrichment ratio relative to the no-antibody control is shown (mean±s.e.m., n=3). *P≤0.05, **P≤0.01, ***P≤0.001 (one-tailed Student's t-test).

Mentions: Having established that nuclear PKC-θ is important for inducible gene induction, we sought to establish how PKC-θ regulates p65. We investigated Ser486 and Ser536 phosphorylation because these events are central to canonical NF-κB signaling (Hochrainer et al., 2013). As anticipated, the overall nuclear p65 translocation increase in Jurkat T cells overexpressing the vector only and wild-type PKC-θ (WT) plasmids was greater during secondary activation than upon primary activation. In contrast, p65 translocation was inhibited in NLS-PKC mutant cells (Fig. 6A,B; Fig. S4D,E). Despite the increase of both nuclear p65 Ser486p and Ser536p in stimulated cells, the NLS-PKC mutation selectively reduced the levels of nuclear p65 and p65 Ser536p after primary and secondary activation compared to controls, without affecting Ser486p levels (Fig. S4E). This reduced p65 and Ser536p corresponded to gene-specific transcriptional inhibition shown in Fig. 5D. When nuclear-to-cytoplasmic ratios were considered, nuclear p65 increased during primary and secondary activation in vector only and WT cells but it was abolished in the NLS mutant (Fig. 6C,D), suggesting that a lack of chromatinized PKC-θ prevents nuclear p65 retention. Immunofluorescence showed that nuclear p65 was significantly reduced in PKC-NLS-transfected cells compared to the control during primary and secondary activation (Fig. S4F,G). Furthermore, reduced p65 nuclear translocation persisted over time (0.5–2 h), with no p65 shuttling detected above non-stimulated levels in the NLS-PKC mutant (Fig. 6C,D). This defective NF-κB signaling in the PKC-NLS mutant was not simply due to deregulated NF-κB production, because IKK (IKBKE), p50 (NFKB1) and p65 (RELA) transcription were the same in vector only, WT, and NLS-PKC cells (Fig. 6E). These data led us to hypothesize that one function of nuclear PKC-θ is to maintain p65 nuclear retention through Ser536 phosphorylation in a signal-dependent manner.Fig. 6.


Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Li J, Hardy K, Phetsouphanh C, Tu WJ, Sutcliffe EL, McCuaig R, Sutton CR, Zafar A, Munier CM, Zaunders JJ, Xu Y, Theodoratos A, Tan A, Lim PS, Knaute T, Masch A, Zerweck J, Brezar V, Milburn PJ, Dunn J, Casarotto MG, Turner SJ, Seddiki N, Kelleher AD, Rao S - J. Cell. Sci. (2016)

p65 Ser536 phosphorylation by nuclear PKC-θ. (A) Immunoblotting of p65, p65 phosphorylated at Ser486 (p65s486p) and Ser536 (p65s536p) in the nuclear extract (NE), and IKK, phosphorylated IκB-α on Ser32 or Ser36 (pIκB-α) and total p65 levels in the cytoplasmic extract (CE) in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. Representative blot of three independent repeats (also see Fig. S4D,E). (B) Nuclear-to-cytoplasmic ratios (NE/CE) of p65 protein levels immunoblotted in nuclear versus cytoplasmic fractions was calculated for the cells described in A (mean±s.e.m., n=3). *P≤0.05, **P≤0.01 (two-way ANOVA). (C) Confocal microscopy of p65 nuclear localization during activation with PMA and Ca2+ ionophore for 0, 0.5, 1 and 2 h in Jurkat T cells transfected with vector only (VO) or the cytoplasm-restricted PKC-θ (NLS) mutant plasmids. Cells were stained with DAPI and anti-p65 antibodies. Representative images of these constructs. Scale bars: 20 μm. (D) Nuclear translocation of p65 (Fn/Fc) expressed as a ratio of nuclear (Fn) and cytoplasmic (Fc) fluorescence with background subtraction (mean±s.e.m., n>20 cells). **P≤0.01; ***P≤0.001; ****P<0.0001; ns, not significant (Mann–Whitney test). (E) Gene expression of p50 (NFKB1), p65 (RELA), and (IKBKE) in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with VO, WT and NLS plasmids (mean±s.e.m., n=2). (F) Immunoblotting of p65 and p65 phosphorylated serine 536 (p65 Ser536p) levels in nuclear extract from DMSO or 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (P/I) (n=6). (G) GAPDH-normalized gene expression of IL2, TNF, TNFSF9 and SATB1 in the vehicle-control or 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (P/I) (mean±s.e.m., n=3). *P≤0.05, **P≤0.01, ***P≤0.001 (one-tailed Student's t-test). (H) ChIP-PCR analysis of p65 Ser536p at the promoters of IL2, TNF, TNFSF9 and an intronic region of SATB1 in Jurkat T cells expressing VO, WT and NLS plasmids after primary (1°) and secondary (2°) stimulations. ChIP enrichment ratio relative to the no-antibody control is shown (mean±s.e.m., n=3). *P≤0.05, **P≤0.01, ***P≤0.001 (one-tailed Student's t-test).
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JCS181248F6: p65 Ser536 phosphorylation by nuclear PKC-θ. (A) Immunoblotting of p65, p65 phosphorylated at Ser486 (p65s486p) and Ser536 (p65s536p) in the nuclear extract (NE), and IKK, phosphorylated IκB-α on Ser32 or Ser36 (pIκB-α) and total p65 levels in the cytoplasmic extract (CE) in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. Representative blot of three independent repeats (also see Fig. S4D,E). (B) Nuclear-to-cytoplasmic ratios (NE/CE) of p65 protein levels immunoblotted in nuclear versus cytoplasmic fractions was calculated for the cells described in A (mean±s.e.m., n=3). *P≤0.05, **P≤0.01 (two-way ANOVA). (C) Confocal microscopy of p65 nuclear localization during activation with PMA and Ca2+ ionophore for 0, 0.5, 1 and 2 h in Jurkat T cells transfected with vector only (VO) or the cytoplasm-restricted PKC-θ (NLS) mutant plasmids. Cells were stained with DAPI and anti-p65 antibodies. Representative images of these constructs. Scale bars: 20 μm. (D) Nuclear translocation of p65 (Fn/Fc) expressed as a ratio of nuclear (Fn) and cytoplasmic (Fc) fluorescence with background subtraction (mean±s.e.m., n>20 cells). **P≤0.01; ***P≤0.001; ****P<0.0001; ns, not significant (Mann–Whitney test). (E) Gene expression of p50 (NFKB1), p65 (RELA), and (IKBKE) in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with VO, WT and NLS plasmids (mean±s.e.m., n=2). (F) Immunoblotting of p65 and p65 phosphorylated serine 536 (p65 Ser536p) levels in nuclear extract from DMSO or 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (P/I) (n=6). (G) GAPDH-normalized gene expression of IL2, TNF, TNFSF9 and SATB1 in the vehicle-control or 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (P/I) (mean±s.e.m., n=3). *P≤0.05, **P≤0.01, ***P≤0.001 (one-tailed Student's t-test). (H) ChIP-PCR analysis of p65 Ser536p at the promoters of IL2, TNF, TNFSF9 and an intronic region of SATB1 in Jurkat T cells expressing VO, WT and NLS plasmids after primary (1°) and secondary (2°) stimulations. ChIP enrichment ratio relative to the no-antibody control is shown (mean±s.e.m., n=3). *P≤0.05, **P≤0.01, ***P≤0.001 (one-tailed Student's t-test).
Mentions: Having established that nuclear PKC-θ is important for inducible gene induction, we sought to establish how PKC-θ regulates p65. We investigated Ser486 and Ser536 phosphorylation because these events are central to canonical NF-κB signaling (Hochrainer et al., 2013). As anticipated, the overall nuclear p65 translocation increase in Jurkat T cells overexpressing the vector only and wild-type PKC-θ (WT) plasmids was greater during secondary activation than upon primary activation. In contrast, p65 translocation was inhibited in NLS-PKC mutant cells (Fig. 6A,B; Fig. S4D,E). Despite the increase of both nuclear p65 Ser486p and Ser536p in stimulated cells, the NLS-PKC mutation selectively reduced the levels of nuclear p65 and p65 Ser536p after primary and secondary activation compared to controls, without affecting Ser486p levels (Fig. S4E). This reduced p65 and Ser536p corresponded to gene-specific transcriptional inhibition shown in Fig. 5D. When nuclear-to-cytoplasmic ratios were considered, nuclear p65 increased during primary and secondary activation in vector only and WT cells but it was abolished in the NLS mutant (Fig. 6C,D), suggesting that a lack of chromatinized PKC-θ prevents nuclear p65 retention. Immunofluorescence showed that nuclear p65 was significantly reduced in PKC-NLS-transfected cells compared to the control during primary and secondary activation (Fig. S4F,G). Furthermore, reduced p65 nuclear translocation persisted over time (0.5–2 h), with no p65 shuttling detected above non-stimulated levels in the NLS-PKC mutant (Fig. 6C,D). This defective NF-κB signaling in the PKC-NLS mutant was not simply due to deregulated NF-κB production, because IKK (IKBKE), p50 (NFKB1) and p65 (RELA) transcription were the same in vector only, WT, and NLS-PKC cells (Fig. 6E). These data led us to hypothesize that one function of nuclear PKC-θ is to maintain p65 nuclear retention through Ser536 phosphorylation in a signal-dependent manner.Fig. 6.

Bottom Line: Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation.This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood.Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Education, Science, Technology & Mathematics, University of Canberra, Canberra, Australian Capital Territory 2617, Australia Department of Microbiology & Immunology, The Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria 3010, Australia.

No MeSH data available.


Related in: MedlinePlus