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Novel protein kinase C θ: coronin 1A complex in T lymphocytes.

Siegmund K, Thuille N, Posch N, Fresser F, Baier G - Cell Commun. Signal (2015)

Bottom Line: Functionally, wild-type but not Coro1A lacking its actin-binding domain negatively interferes with PKCθ-dependent NF-κB, Cyclin D1 and IL-2 transactivation when analysed with luciferase promoter activation assays in Jurkat T cells.This could be phenocopied by pharmacological inhibitors of actin polymerization and PKC, respectively.In addition, we show that CD3(+) T cells isolated from Coro1A-deficient mice show impaired IKK/NF-κB transactivation.

View Article: PubMed Central - PubMed

Affiliation: Department for Pharmacology and Genetics, Division of Translational Cell Genetics, Medical University Innsbruck, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria. kerstin.siegmund@i-med.ac.at.

ABSTRACT

Background: Protein kinase C-θ (PKCθ) plays an important role in signal transduction down-stream of the T cell receptor and T cells deficient of PKCθ show impaired NF-κB as well as NFAT/AP-1 activation resulting in strongly decreased IL-2 expression and proliferation. However, it is not yet entirely clear, how the function of PKCθ - upon T cell activation - is regulated on a molecular level.

Findings: Employing a yeast two-hybrid screen and co-immunoprecipitation analyses, we here identify coronin 1A (Coro1A) as a novel PKCθ-interacting protein. We show that the NH2-terminal WD40 domains of Coro1A and the C2-like domain of PKCθ are sufficient for the interaction. Furthermore, we confirm a physical interaction by GST-Coro1A mediated pull-down of endogenous PKCθ protein. Functionally, wild-type but not Coro1A lacking its actin-binding domain negatively interferes with PKCθ-dependent NF-κB, Cyclin D1 and IL-2 transactivation when analysed with luciferase promoter activation assays in Jurkat T cells. This could be phenocopied by pharmacological inhibitors of actin polymerization and PKC, respectively. Mechanistically, Coro1A overexpression attenuates both lipid raft and plasma membrane recruitment of PKCθ in CD3/CD28-activated T cells. Using primary CD3(+) T cells, we observed that (opposite to PKCθ) Coro1A does not localize preferentially to the immunological synapse. In addition, we show that CD3(+) T cells isolated from Coro1A-deficient mice show impaired IKK/NF-κB transactivation.

Conclusions: Together, these findings both in Jurkat T cells as well as in primary T cells indicate a regulatory role of Coro1A on PKCθ recruitment and function downstream of the TCR leading to NF-κB transactivation.

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Coro1A is not recruited into the IS and its gene ablation strongly reduces NF-κB responses. (A, B) Confocal microscopy of Coro1A and PKCθ in primary human T cells. A T cell clone (KS140) specific for the tetanus toxin peptide (TT830–843; QYIKANSKFIGITE) and a T cell clone (6396p5.1.2) specific for the measles virus fusion protein peptide (F254–268; GDLLGILESRGIKAR) were used with autologous Epstein–Barr virus (EBV)-transformed B cells as APC. Quantification of Coro1A subcellular localization on 59 synapses is shown as bar graph. (C) CD3+ T cells were isolated from either wild-type or Coro1a knockout mice. After 2 hour resting ex vivo the cells were stimulated with soluble anti-CD3/CD28 and cross-linking anti-hamster IgG antibodies or PDBu for 5 and 15 minutes. Whole cell lysates (supplemented with phosphatase inhibitor) were subjected to SDS-Page and immunoblotting against phosphorylated IκBα, actin and Coro1A. (D) CD3+ T cells were isolated and stimulated as described in (C), but the stimulation time was increased to 8 hours. Nuclear extracts were prepared and analysed by electromobility shift assays (EMSA) for NF-κB binding to DNA. Experiments were repeated at least two times, with similar results.
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Fig4: Coro1A is not recruited into the IS and its gene ablation strongly reduces NF-κB responses. (A, B) Confocal microscopy of Coro1A and PKCθ in primary human T cells. A T cell clone (KS140) specific for the tetanus toxin peptide (TT830–843; QYIKANSKFIGITE) and a T cell clone (6396p5.1.2) specific for the measles virus fusion protein peptide (F254–268; GDLLGILESRGIKAR) were used with autologous Epstein–Barr virus (EBV)-transformed B cells as APC. Quantification of Coro1A subcellular localization on 59 synapses is shown as bar graph. (C) CD3+ T cells were isolated from either wild-type or Coro1a knockout mice. After 2 hour resting ex vivo the cells were stimulated with soluble anti-CD3/CD28 and cross-linking anti-hamster IgG antibodies or PDBu for 5 and 15 minutes. Whole cell lysates (supplemented with phosphatase inhibitor) were subjected to SDS-Page and immunoblotting against phosphorylated IκBα, actin and Coro1A. (D) CD3+ T cells were isolated and stimulated as described in (C), but the stimulation time was increased to 8 hours. Nuclear extracts were prepared and analysed by electromobility shift assays (EMSA) for NF-κB binding to DNA. Experiments were repeated at least two times, with similar results.

Mentions: Next, we investigated the subcellular localization of Coro1A and PKCθ upon T cell activation. For this purpose human T cell blasts from immunized donors were incubated with APCs loaded or not with the corresponding peptide and analysed by confocal microscopy for the localization of PKCθ and Coro1A with regard to the IS (stained by antibodies against (p)tyrosine). Of note, while as already published, activation-induced PKCθ recruitment to the IS was consistently observed by confocal microscopy [19], Coro1A was not recruited to the IS. Coro1A was rather excluded from the IS in approximately 65% of antigen:APC-stimulated T cell blasts (Figure 4A/B), suggesting a role as negative regulator in TCR signaling.Figure 4


Novel protein kinase C θ: coronin 1A complex in T lymphocytes.

Siegmund K, Thuille N, Posch N, Fresser F, Baier G - Cell Commun. Signal (2015)

Coro1A is not recruited into the IS and its gene ablation strongly reduces NF-κB responses. (A, B) Confocal microscopy of Coro1A and PKCθ in primary human T cells. A T cell clone (KS140) specific for the tetanus toxin peptide (TT830–843; QYIKANSKFIGITE) and a T cell clone (6396p5.1.2) specific for the measles virus fusion protein peptide (F254–268; GDLLGILESRGIKAR) were used with autologous Epstein–Barr virus (EBV)-transformed B cells as APC. Quantification of Coro1A subcellular localization on 59 synapses is shown as bar graph. (C) CD3+ T cells were isolated from either wild-type or Coro1a knockout mice. After 2 hour resting ex vivo the cells were stimulated with soluble anti-CD3/CD28 and cross-linking anti-hamster IgG antibodies or PDBu for 5 and 15 minutes. Whole cell lysates (supplemented with phosphatase inhibitor) were subjected to SDS-Page and immunoblotting against phosphorylated IκBα, actin and Coro1A. (D) CD3+ T cells were isolated and stimulated as described in (C), but the stimulation time was increased to 8 hours. Nuclear extracts were prepared and analysed by electromobility shift assays (EMSA) for NF-κB binding to DNA. Experiments were repeated at least two times, with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Coro1A is not recruited into the IS and its gene ablation strongly reduces NF-κB responses. (A, B) Confocal microscopy of Coro1A and PKCθ in primary human T cells. A T cell clone (KS140) specific for the tetanus toxin peptide (TT830–843; QYIKANSKFIGITE) and a T cell clone (6396p5.1.2) specific for the measles virus fusion protein peptide (F254–268; GDLLGILESRGIKAR) were used with autologous Epstein–Barr virus (EBV)-transformed B cells as APC. Quantification of Coro1A subcellular localization on 59 synapses is shown as bar graph. (C) CD3+ T cells were isolated from either wild-type or Coro1a knockout mice. After 2 hour resting ex vivo the cells were stimulated with soluble anti-CD3/CD28 and cross-linking anti-hamster IgG antibodies or PDBu for 5 and 15 minutes. Whole cell lysates (supplemented with phosphatase inhibitor) were subjected to SDS-Page and immunoblotting against phosphorylated IκBα, actin and Coro1A. (D) CD3+ T cells were isolated and stimulated as described in (C), but the stimulation time was increased to 8 hours. Nuclear extracts were prepared and analysed by electromobility shift assays (EMSA) for NF-κB binding to DNA. Experiments were repeated at least two times, with similar results.
Mentions: Next, we investigated the subcellular localization of Coro1A and PKCθ upon T cell activation. For this purpose human T cell blasts from immunized donors were incubated with APCs loaded or not with the corresponding peptide and analysed by confocal microscopy for the localization of PKCθ and Coro1A with regard to the IS (stained by antibodies against (p)tyrosine). Of note, while as already published, activation-induced PKCθ recruitment to the IS was consistently observed by confocal microscopy [19], Coro1A was not recruited to the IS. Coro1A was rather excluded from the IS in approximately 65% of antigen:APC-stimulated T cell blasts (Figure 4A/B), suggesting a role as negative regulator in TCR signaling.Figure 4

Bottom Line: Functionally, wild-type but not Coro1A lacking its actin-binding domain negatively interferes with PKCθ-dependent NF-κB, Cyclin D1 and IL-2 transactivation when analysed with luciferase promoter activation assays in Jurkat T cells.This could be phenocopied by pharmacological inhibitors of actin polymerization and PKC, respectively.In addition, we show that CD3(+) T cells isolated from Coro1A-deficient mice show impaired IKK/NF-κB transactivation.

View Article: PubMed Central - PubMed

Affiliation: Department for Pharmacology and Genetics, Division of Translational Cell Genetics, Medical University Innsbruck, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria. kerstin.siegmund@i-med.ac.at.

ABSTRACT

Background: Protein kinase C-θ (PKCθ) plays an important role in signal transduction down-stream of the T cell receptor and T cells deficient of PKCθ show impaired NF-κB as well as NFAT/AP-1 activation resulting in strongly decreased IL-2 expression and proliferation. However, it is not yet entirely clear, how the function of PKCθ - upon T cell activation - is regulated on a molecular level.

Findings: Employing a yeast two-hybrid screen and co-immunoprecipitation analyses, we here identify coronin 1A (Coro1A) as a novel PKCθ-interacting protein. We show that the NH2-terminal WD40 domains of Coro1A and the C2-like domain of PKCθ are sufficient for the interaction. Furthermore, we confirm a physical interaction by GST-Coro1A mediated pull-down of endogenous PKCθ protein. Functionally, wild-type but not Coro1A lacking its actin-binding domain negatively interferes with PKCθ-dependent NF-κB, Cyclin D1 and IL-2 transactivation when analysed with luciferase promoter activation assays in Jurkat T cells. This could be phenocopied by pharmacological inhibitors of actin polymerization and PKC, respectively. Mechanistically, Coro1A overexpression attenuates both lipid raft and plasma membrane recruitment of PKCθ in CD3/CD28-activated T cells. Using primary CD3(+) T cells, we observed that (opposite to PKCθ) Coro1A does not localize preferentially to the immunological synapse. In addition, we show that CD3(+) T cells isolated from Coro1A-deficient mice show impaired IKK/NF-κB transactivation.

Conclusions: Together, these findings both in Jurkat T cells as well as in primary T cells indicate a regulatory role of Coro1A on PKCθ recruitment and function downstream of the TCR leading to NF-κB transactivation.

Show MeSH
Related in: MedlinePlus