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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 subcellular location in activated T cells. Jurkat cells were transfected with GFP inert protein control, PKCθ or Coro1A wild-type cDNA expression plasmids, respectively. (A) Co-localization of transfected PKCθ and Coro1A occurred at a rate of approximately 76% in intact cells as measured by immunofluorescence. Jurkat T cells were stimulated for 20 min with anti-CD3/anti-CD28 antibodies and analysed by subsequent staining with protein-specific antibodies. A representative image is shown. (B) Translocation of PKCθ to the plasma membrane is inhibited by overexpression of Coro1A. Jurkat cells were transfected with Coro1A or GFP, respectively. After 21 hrs cells were stimulated with anti-CD3/anti-CD28 antibodies for 20 min or left unstimulated, as indicated and subcellular distribution of endogenous PKCθ was determined by immunoblotting. The cell fractions are defined as the soluble (s) fraction, the particulate (pt) fraction and the Triton-X100 non-soluble (ns) fraction, which were prepared as described in the Additional file 1 (Supplementary Methods). The p59 fyn protein was detected to control for cell fractionation. (C) Lipid rafts were prepared by fractionation of sucrose gradients and immunostained for PKCθ and Coro1A. As a marker for the raft fraction, the marker ganglioside M1 of each fraction was quantified in a dot blot employing HRP-Choleratoxin B (not shown). A representative experiment of 3 independent experiments is shown.
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Fig3: Coro1A modulates PKCθ-mediated subcellular location in activated T cells. Jurkat cells were transfected with GFP inert protein control, PKCθ or Coro1A wild-type cDNA expression plasmids, respectively. (A) Co-localization of transfected PKCθ and Coro1A occurred at a rate of approximately 76% in intact cells as measured by immunofluorescence. Jurkat T cells were stimulated for 20 min with anti-CD3/anti-CD28 antibodies and analysed by subsequent staining with protein-specific antibodies. A representative image is shown. (B) Translocation of PKCθ to the plasma membrane is inhibited by overexpression of Coro1A. Jurkat cells were transfected with Coro1A or GFP, respectively. After 21 hrs cells were stimulated with anti-CD3/anti-CD28 antibodies for 20 min or left unstimulated, as indicated and subcellular distribution of endogenous PKCθ was determined by immunoblotting. The cell fractions are defined as the soluble (s) fraction, the particulate (pt) fraction and the Triton-X100 non-soluble (ns) fraction, which were prepared as described in the Additional file 1 (Supplementary Methods). The p59 fyn protein was detected to control for cell fractionation. (C) Lipid rafts were prepared by fractionation of sucrose gradients and immunostained for PKCθ and Coro1A. As a marker for the raft fraction, the marker ganglioside M1 of each fraction was quantified in a dot blot employing HRP-Choleratoxin B (not shown). A representative experiment of 3 independent experiments is shown.

Mentions: Mechanistically, in transient Jurkat transfection assays, PKCθ and Coro1A co-localized in intact Jurkat T cells (Figure 3A), and Coro1A overexpression inhibited both plasma membrane and lipid raft recruitment of PKCθ in CD3/CD28-activated cells (Figure 3B/C). While we cannot exclude additional Coro1A functions affecting NF-κB activation independent of PKCθ, based on the experiments described above, we conclude that Coro1A, which is in a complex with PKCθ, modulates PKCθ functionally.Figure 3


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 subcellular location in activated T cells. Jurkat cells were transfected with GFP inert protein control, PKCθ or Coro1A wild-type cDNA expression plasmids, respectively. (A) Co-localization of transfected PKCθ and Coro1A occurred at a rate of approximately 76% in intact cells as measured by immunofluorescence. Jurkat T cells were stimulated for 20 min with anti-CD3/anti-CD28 antibodies and analysed by subsequent staining with protein-specific antibodies. A representative image is shown. (B) Translocation of PKCθ to the plasma membrane is inhibited by overexpression of Coro1A. Jurkat cells were transfected with Coro1A or GFP, respectively. After 21 hrs cells were stimulated with anti-CD3/anti-CD28 antibodies for 20 min or left unstimulated, as indicated and subcellular distribution of endogenous PKCθ was determined by immunoblotting. The cell fractions are defined as the soluble (s) fraction, the particulate (pt) fraction and the Triton-X100 non-soluble (ns) fraction, which were prepared as described in the Additional file 1 (Supplementary Methods). The p59 fyn protein was detected to control for cell fractionation. (C) Lipid rafts were prepared by fractionation of sucrose gradients and immunostained for PKCθ and Coro1A. As a marker for the raft fraction, the marker ganglioside M1 of each fraction was quantified in a dot blot employing HRP-Choleratoxin B (not shown). A representative experiment of 3 independent experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4390099&req=5

Fig3: Coro1A modulates PKCθ-mediated subcellular location in activated T cells. Jurkat cells were transfected with GFP inert protein control, PKCθ or Coro1A wild-type cDNA expression plasmids, respectively. (A) Co-localization of transfected PKCθ and Coro1A occurred at a rate of approximately 76% in intact cells as measured by immunofluorescence. Jurkat T cells were stimulated for 20 min with anti-CD3/anti-CD28 antibodies and analysed by subsequent staining with protein-specific antibodies. A representative image is shown. (B) Translocation of PKCθ to the plasma membrane is inhibited by overexpression of Coro1A. Jurkat cells were transfected with Coro1A or GFP, respectively. After 21 hrs cells were stimulated with anti-CD3/anti-CD28 antibodies for 20 min or left unstimulated, as indicated and subcellular distribution of endogenous PKCθ was determined by immunoblotting. The cell fractions are defined as the soluble (s) fraction, the particulate (pt) fraction and the Triton-X100 non-soluble (ns) fraction, which were prepared as described in the Additional file 1 (Supplementary Methods). The p59 fyn protein was detected to control for cell fractionation. (C) Lipid rafts were prepared by fractionation of sucrose gradients and immunostained for PKCθ and Coro1A. As a marker for the raft fraction, the marker ganglioside M1 of each fraction was quantified in a dot blot employing HRP-Choleratoxin B (not shown). A representative experiment of 3 independent experiments is shown.
Mentions: Mechanistically, in transient Jurkat transfection assays, PKCθ and Coro1A co-localized in intact Jurkat T cells (Figure 3A), and Coro1A overexpression inhibited both plasma membrane and lipid raft recruitment of PKCθ in CD3/CD28-activated cells (Figure 3B/C). While we cannot exclude additional Coro1A functions affecting NF-κB activation independent of PKCθ, based on the experiments described above, we conclude that Coro1A, which is in a complex with PKCθ, modulates PKCθ functionally.Figure 3

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