Background/Purpose A-Kinase anchoring Protein AKAP79 associates to and regulates the activity of PKA, PKC and calcineurin, key regulators of T cell activation. We have previously described that AKAP79 is overexpressed in T cells from systemic lupus erythematosus lupus patients and inhibits IL2 transcription in Jurkat T cells (Criado et al., A&R 63 (Suppl10):S916). In the present study, we aim to analyze the effect of AKAP79 levels on IL2 production in SLE and normal T cells and characterize the role of the associations to PKA, PKC and calcineurin in T cell activation and IL2 transcription.

Methods T cells were isolated by negative selection from SLE patients and healthy controls (HC). RNA was purified, retrotranscribed to cDNA and levels of AKAP79 and β-actin transcripts were quantified by quantitative real-time PCR using Sybr Green technology. AKAP79 protein expression was quantified in T cell lysates by ELISA. AKAP79 mutants deficient for association to PKA (ΔPKA-AKAP79), PKC (ΔPKC-AKAP79) and calcineurin (ΔCN-AKAP79) were generated with QuickChange Site Directed Mutagenesis kit and confirmed by sequencing. Jurkat T cells were transduced with dual promoter pRRL-AKAP79/GFP- or control pRRL-GFP-expressing lentiviruses. For functional assays, T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies or PMA/ionomycin. RNA was isolated, IL2 transcripts quantified by quantitative real-time PCR (qPCR) and results calculated as fold change over non-stimulated cell (Mean +/- SEM). Statistical differences between groups were analysed by ANOVA and non-parametric Mann-Whitney U test, using GraphPad Prism software.

Results  IL2 transcription induced by anti-CD3/CD28 stimulation was not significantly different between SLE (n=6) and HC (n=6) T cells. However, a negative correlation between levels of AKAP79 protein and induction of IL2 transcription was found in T cells, regardless of their origin (R²= 0.53, **p=0.0076). This was confirmed in Jurkat T cells by transduction with different amounts of AKAP79 (R²= 0.57, *p=0.03, n= 8). Complementation of anti-CD3/CD28 stimulation by PMA, but not ionomycin, restored IL2 transcription in Jurkat cells overexpressing AKAP79, suggesting that inhibition of PKC by AKAP79 was responsible for the observed reduction of IL2 transcription. Consistent with this interpretation, overexpression of ΔPKC-AKAP79 partially rescued IL2 transcription when compared to WT-AKAP79 but ΔPKA-AKAP79 and ΔCN-AKAP79 had no significant effect (GFP: 11.59 +/- 2.21, AKAP79: 1.67 +/- 0.61, ΔPKC-AKAP79:  4.86 +/- 1.24, ΔPKA-AKAP79: 0.73 +/- 0.29, ΔCN-AKAP79:  0.88 +/- 0.35). Likewise, analysis of the kinetics of Erk activation in response to antiCD3/CD28 showed that Erk activation was blocked by AKAP79 overexpression and was recued by ΔPKC-AKAP79.

Conclusion Overexpression of AKAP79 inhibits IL2 transcription and Erk activation in a PKC-dependent manner. Thus, targeting AKAP79-PKC interaction could provide a therapeutic approach to modulate T cell activation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/association-of-a-kinase-anchoring-protein-79-akap79-to-pkc-mediates-inhibition-of-il2-transcription-and-erk-activation-in-t-cells/