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. 2012 Sep;64(9):2964-74.
doi: 10.1002/art.34503.

Protein kinase Cδ oxidation contributes to ERK inactivation in lupus T cells

Gabriela J Gorelik  1 , Sushma Yarlagadda, Dipak R Patel, Bruce C Richardson
Affiliations

Protein kinase Cδ oxidation contributes to ERK inactivation in lupus T cells

Gabriela J Gorelik et al. Arthritis Rheum. 2012 Sep.

Erratum in

  • Arthritis Rheum. 2014 Mar;66(3):769. Patel, Dipak R [added]

Abstract

Objective: CD4+ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T(505) in the protein kinase Cδ (PKCδ) activation loop. However, the mechanisms that prevent PKCδ T(505) phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKCδ, contributing to impaired ERK pathway signaling in lupus T cells.

Methods: CD4+ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation.

Results: Treating CD4+ T cells with peroxynitrite nitrated PKCδ, preventing PKCδ T(505) phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKCδ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKCδ, but not unmodified PKCδ, was refractory to PMA-stimulated T(505) phosphorylation, similar to PKCδ in peroxynitrite-treated cells.

Conclusion: Oxidative stress causes PKCδ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKCδ as a link between oxidative stress and the T cell epigenetic modifications in lupus.

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Conflict of interest statement

Financial conflict of interest: There is no financial conflict of interest to disclose.

Figures

Fig. 1
Fig. 1
PDK-1 is not affected in lupus T cells. CD4+ T cells from healthy donors (control) or from lupus patients were stimulated or not with PMA for 15 min. 20 μg of whole cell lysates were subjected to SDS-PAGE fractionation, transferred to nitrocellulose membranes and probed with a polyclonal antibody against PKC δ p-T505, as described in Materials and Methods. Membranes were stripped and reprobed with anti-phospho-PDK1. PKCδ and PDK-1 were used as controls for the corresponding total protein expression. Panel A shows a representative blot comparing phosphorylation of PDK1 in a lupus patient with a normal donor. Panel B represents the mean ± S.E of four similar experiments performed in different cell preparations from normal and SLE patients. * p≤0.02 lupus vs normal
Fig. 2
Fig. 2
Peroxynitrite decreases PKCδ T505 phosphorylation while increasing protein nitration in T cells. CD4+ T cells from healthy donors were untreated or treated with 20 – 80 μM peroxynitrite for 15 min and then PMA stimulated as indicated. Whole cell proteins were obtained and subjected to Western blot analysis. A. Representative immunoblot showing phosphorylation at the activation loops of PKCδ, PKCθ and PKCα using antibodies specific to the phosphorylated PKC isoforms as indicated. B. Quantitative analysis of PKCδ T505 from 4 healthy donors in four different experiments (mean ± S.E.M). Values were normalized to total PKCδ. PMA-stimulated CD4+ T cells but without ONOO− treatment (solid bar) were considered as 100%. C. Representative experiment using anti 3-nitrotyrosine antibody to probe PMA stimulated T cell lysates. D. Relative quantitation of 3-nitrotyrosine proteins from four similar experiments (mean ± S.E.M.). Untreated cells and PMA-unstimulated cells (□) were used as controls (*p < 0.01 ONOO− treated vs untreated).
Fig. 3
Fig. 3
Differential PKCδ phosphorylation induced by peroxynitrite. A. Representative experiment showing the effect of different ONOO− concentrations on PKCδ phosphorylation. CD4+ T cells were isolated from healthy controls and treated with ONOO− at the concentrations specified. Following treatment the cells were stimulated with 50 ng/ml PMA for 15 min and PMA-stimulated peroxynitrite-untreated cells were used as control. Protein lysates were then subjected to electrophoresis, transferred to nitrocellulose and the membranes probed with anti-p-T505-PKCδ. The blot was then stripped and reprobed with anti-p-Y311-PKC δ. NaOH was used as vehicle control and added to the culture for 15 min before PMA stimulation at the same final concentration as in the ONOO− solution. B. Differential dose-response curves of p-PKCδ following ONOO- treatment. The graph represents the mean ± SEM of p-PKCδ expression relative to PKCδ of four independent experiments (*p≤0.04; ONOO-treated vs ONOO-untreated cells).
Fig. 4
Fig. 4
Peroxynitrite decreases ERK phosphorylation. A. CD4+ T cells from normal donors were treated or not for 15 min with peroxynitrite at the indicated concentrations then stimulated with PMA. Cells left untreated were used as control (cont). Whole cell extracts were fractionated by SDS-PAGE and followed by immunoblot using anti p-T505 PKC δ. After stripping the blot was re-probed with anti p- T202/Y204-ERK. No variation in total PKCδ or ERK protein expression is observed. This blot is representative of 4 independent experiments. B. Quantitative immunoblot analysis of 4 different experiments. PMA-stimulated non-peroxynitrite treated cells were considered as 1. Values are the mean ± SEM. * p ≤ 0.001 peroxynitrite vs non-peroxynitrite.
Fig. 5
Fig. 5
PKCδ nitration in lupus T cells. CD4+ T cells from three healthy donors (control) were untreated or treated with peroxynitrite followed by PMA stimulation. In parallel CD4+ T cells from six active and four inactive lupus patients were PMA-stimulated. Total lysates were immunoprecipitated then the supernatants and precipitates were immunoblotted with anti-p-T505 PKC δ and membranes reprobed with anti-total PKCδ. A: The bar graph shows the quantitative densitometric analysis of total PKC δ (■) and p-T505 PKCδ ( formula image) in the supernatant (spnt) and the precipitate (pp) using CD4+ T cells from healthy donors and patients with active disease. * p≤0.01, ** p≤0.04, #≤0.02. Values are the mean ± SEM of six independent experiments. B: The table shows nitrated PKCδ (total PKCδ content in pp) and p-T505PKC δ (p-PKCδ in spnt + pp) as percent of total PKCδ (expressed in arbitrary units) in spnt and pp in each experimental condition. Values are the mean ± SEM of six experiments. * p values vs control. C: The graph shows the correlation between the nitrated PKCδ levels in lupus patients and the SLEDAI scores. p value was determined by analysis of variance (ANOVA).
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