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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 modulates PKCθ-mediated effector function. (A) IL-2 promoter luciferase reporter assay performed with Jurkat T cells transfected with the constitutively active mutant PKCθ A/E and wild-type or truncated Coro1A – as indicated. Transfected cells were stimulated with the calcium ionophore ionomycin overnight. The insertion in the upper left corner shows expression of recombinant Coro1A in Jurkat T cells analysed by an anti-tag immunoblot. GFP-expressing plasmid was used as an inert protein overexpression control. (B) NF-κB-dependent promoter luciferase reporter of transfected Jurkat T cells either stimulated with ionomycin or left untreated. (C) IL-2 promoter-dependent luciferase reporter of Jurkat cells stimulated with phorbol ester/ionomycin, transfected with constitutively active mutant PKCθ A/E and stimulated with ionomycin, or alternatively, transfected with constitutively active mutants of both PKCθ and Calcineurin (CaN) and/or treated by cytochalasin (Cyt) D and PKC LMWI AEB071/Sotratstaurin as indicated. (D) Cyclin D1 promoter-dependent luciferase reporter of Jurkat cells stimulated with ionomycin and transfected with constitutively active mutants of several PKC family members and with PKCθ A/E and wild-type or truncated Coro1A, respectively. The mean ± SE of three independent experiments is shown. Statistical significance was defined with p < 0.05 (Student’s t-test) and marked with one asterisk (*). A/E: constitutively active and K/R: dominant negative mutant of PKCθ; Rac1 N17: dominant-negative mutant of Rac1; Rlu - relative luciferase activity.
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Fig2: Coro1A modulates PKCθ-mediated effector function. (A) IL-2 promoter luciferase reporter assay performed with Jurkat T cells transfected with the constitutively active mutant PKCθ A/E and wild-type or truncated Coro1A – as indicated. Transfected cells were stimulated with the calcium ionophore ionomycin overnight. The insertion in the upper left corner shows expression of recombinant Coro1A in Jurkat T cells analysed by an anti-tag immunoblot. GFP-expressing plasmid was used as an inert protein overexpression control. (B) NF-κB-dependent promoter luciferase reporter of transfected Jurkat T cells either stimulated with ionomycin or left untreated. (C) IL-2 promoter-dependent luciferase reporter of Jurkat cells stimulated with phorbol ester/ionomycin, transfected with constitutively active mutant PKCθ A/E and stimulated with ionomycin, or alternatively, transfected with constitutively active mutants of both PKCθ and Calcineurin (CaN) and/or treated by cytochalasin (Cyt) D and PKC LMWI AEB071/Sotratstaurin as indicated. (D) Cyclin D1 promoter-dependent luciferase reporter of Jurkat cells stimulated with ionomycin and transfected with constitutively active mutants of several PKC family members and with PKCθ A/E and wild-type or truncated Coro1A, respectively. The mean ± SE of three independent experiments is shown. Statistical significance was defined with p < 0.05 (Student’s t-test) and marked with one asterisk (*). A/E: constitutively active and K/R: dominant negative mutant of PKCθ; Rac1 N17: dominant-negative mutant of Rac1; Rlu - relative luciferase activity.

Mentions: After having observed a complex formation between PKCθ and Coro1A, we next asked the question about the functional relevance of this interaction. Therefore, it was analysed whether Coro1A does influence the transcriptional activation of genes that are established downstream targets of PKCθ such as IL-2 and Cyclin D1. In functional analyses using IL-2 promoter luciferase reporter assays, overexpression of wild-type Coro1A but not the COOH-deletion mutant, lacking the actin-binding domain, negatively interferes with PKCθ-dependent IL-2 transactivation in Jurkat T cells (Figure 2A). Thus, even though the actin-binding function of Coro1A is not necessary for its interaction with PKCθ (Figure 1), it appears to be of relevance for Coro1A modulating PKCθ function. In these experiments, Jurkat T cells co-transfected with the constitutively active mutant PKCθ A149E and wild-type or truncated Coro1A, were stimulated with the calcium ionophore, ionomycin. Co-transfection with the dominant-negative PKCθ K409R mutant or the dominant-negative mutant of Rac1, Rac1 N17, which leads to inhibition of IL-2 reporter transcription via actin polymerization defects served as positive controls. Those findings suggest that actin is part of a functional PKCθ:Coro1A axis identified in the Jurkat T cell line. In addition, wild-type but not the deletion mutant of Coro1A repressed the induction of an NF-κB-dependent promoter luciferase reporter (Figure 2B). This effect could be phenocopied both by cell-permeable pharmacological inhibitors of actin polymerisation and PKC function, respectively (Figure 2C). Similarly, Cyclin D1 promoter reporter activation (that was PKC isotype-selectively dependent on PKCθ function) was attenuated by wild-type Coro1A co-expression (Figure 2D).Figure 2


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 modulates PKCθ-mediated effector function. (A) IL-2 promoter luciferase reporter assay performed with Jurkat T cells transfected with the constitutively active mutant PKCθ A/E and wild-type or truncated Coro1A – as indicated. Transfected cells were stimulated with the calcium ionophore ionomycin overnight. The insertion in the upper left corner shows expression of recombinant Coro1A in Jurkat T cells analysed by an anti-tag immunoblot. GFP-expressing plasmid was used as an inert protein overexpression control. (B) NF-κB-dependent promoter luciferase reporter of transfected Jurkat T cells either stimulated with ionomycin or left untreated. (C) IL-2 promoter-dependent luciferase reporter of Jurkat cells stimulated with phorbol ester/ionomycin, transfected with constitutively active mutant PKCθ A/E and stimulated with ionomycin, or alternatively, transfected with constitutively active mutants of both PKCθ and Calcineurin (CaN) and/or treated by cytochalasin (Cyt) D and PKC LMWI AEB071/Sotratstaurin as indicated. (D) Cyclin D1 promoter-dependent luciferase reporter of Jurkat cells stimulated with ionomycin and transfected with constitutively active mutants of several PKC family members and with PKCθ A/E and wild-type or truncated Coro1A, respectively. The mean ± SE of three independent experiments is shown. Statistical significance was defined with p < 0.05 (Student’s t-test) and marked with one asterisk (*). A/E: constitutively active and K/R: dominant negative mutant of PKCθ; Rac1 N17: dominant-negative mutant of Rac1; Rlu - relative luciferase activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390099&req=5

Fig2: Coro1A modulates PKCθ-mediated effector function. (A) IL-2 promoter luciferase reporter assay performed with Jurkat T cells transfected with the constitutively active mutant PKCθ A/E and wild-type or truncated Coro1A – as indicated. Transfected cells were stimulated with the calcium ionophore ionomycin overnight. The insertion in the upper left corner shows expression of recombinant Coro1A in Jurkat T cells analysed by an anti-tag immunoblot. GFP-expressing plasmid was used as an inert protein overexpression control. (B) NF-κB-dependent promoter luciferase reporter of transfected Jurkat T cells either stimulated with ionomycin or left untreated. (C) IL-2 promoter-dependent luciferase reporter of Jurkat cells stimulated with phorbol ester/ionomycin, transfected with constitutively active mutant PKCθ A/E and stimulated with ionomycin, or alternatively, transfected with constitutively active mutants of both PKCθ and Calcineurin (CaN) and/or treated by cytochalasin (Cyt) D and PKC LMWI AEB071/Sotratstaurin as indicated. (D) Cyclin D1 promoter-dependent luciferase reporter of Jurkat cells stimulated with ionomycin and transfected with constitutively active mutants of several PKC family members and with PKCθ A/E and wild-type or truncated Coro1A, respectively. The mean ± SE of three independent experiments is shown. Statistical significance was defined with p < 0.05 (Student’s t-test) and marked with one asterisk (*). A/E: constitutively active and K/R: dominant negative mutant of PKCθ; Rac1 N17: dominant-negative mutant of Rac1; Rlu - relative luciferase activity.
Mentions: After having observed a complex formation between PKCθ and Coro1A, we next asked the question about the functional relevance of this interaction. Therefore, it was analysed whether Coro1A does influence the transcriptional activation of genes that are established downstream targets of PKCθ such as IL-2 and Cyclin D1. In functional analyses using IL-2 promoter luciferase reporter assays, overexpression of wild-type Coro1A but not the COOH-deletion mutant, lacking the actin-binding domain, negatively interferes with PKCθ-dependent IL-2 transactivation in Jurkat T cells (Figure 2A). Thus, even though the actin-binding function of Coro1A is not necessary for its interaction with PKCθ (Figure 1), it appears to be of relevance for Coro1A modulating PKCθ function. In these experiments, Jurkat T cells co-transfected with the constitutively active mutant PKCθ A149E and wild-type or truncated Coro1A, were stimulated with the calcium ionophore, ionomycin. Co-transfection with the dominant-negative PKCθ K409R mutant or the dominant-negative mutant of Rac1, Rac1 N17, which leads to inhibition of IL-2 reporter transcription via actin polymerization defects served as positive controls. Those findings suggest that actin is part of a functional PKCθ:Coro1A axis identified in the Jurkat T cell line. In addition, wild-type but not the deletion mutant of Coro1A repressed the induction of an NF-κB-dependent promoter luciferase reporter (Figure 2B). This effect could be phenocopied both by cell-permeable pharmacological inhibitors of actin polymerisation and PKC function, respectively (Figure 2C). Similarly, Cyclin D1 promoter reporter activation (that was PKC isotype-selectively dependent on PKCθ function) was attenuated by wild-type Coro1A co-expression (Figure 2D).Figure 2

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