Golgi-associated protein kinase C-ϵ is delivered to phagocytic cups: Role of phosphatidylinositol 4-phosphate

Cheryl M. Hanes, Anna E. D'Amico, Takehiko Ueyama, Alexander C. Wong, Xuexin Zhang, W. Frederick Hynes, Margarida M. Barroso, Nathaniel C. Cady, Mohamed Trebak, Naoaki Saito, Michelle R. Lennartz

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Protein kinase C-ϵ (PKC-ϵ) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (ϵPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for ϵPS is unknown. Liposome assays revealed that the ϵPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P2. Wortmannin, but not LY294002, inhibits PKC-ϵ concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ϵ concentration at cups and the rate of phagocytosis. PKC-ϵ colocalizes with the trans-Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ϵ-expressing macrophages revealed a loss of Golgi-associated PKC-ϵ during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ϵ at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgiassociated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ϵ is tethered to the TGN via an ϵPS-PI4P interaction. The TGN-associated pool of PKC-ϵ concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that ϵPS binds PI4P and PI(3,5)P2 and that PI4P is necessary for PKC-ϵ localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis.

Original languageEnglish (US)
Pages (from-to)271-277
Number of pages7
JournalJournal of Immunology
Volume199
Issue number1
DOIs
StatePublished - Jul 1 2017

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Protein Kinase C
trans-Golgi Network
Phagocytosis
Membrane Fusion
Phosphotransferases
phosphatidylinositol 4-phosphate
Phagosomes
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Fc Receptors
Diglycerides
Liposomes
Immunoglobulin G
Macrophages
Phenotype

All Science Journal Classification (ASJC) codes

  • Immunology

Cite this

Hanes, Cheryl M. ; D'Amico, Anna E. ; Ueyama, Takehiko ; Wong, Alexander C. ; Zhang, Xuexin ; Hynes, W. Frederick ; Barroso, Margarida M. ; Cady, Nathaniel C. ; Trebak, Mohamed ; Saito, Naoaki ; Lennartz, Michelle R. / Golgi-associated protein kinase C-ϵ is delivered to phagocytic cups : Role of phosphatidylinositol 4-phosphate. In: Journal of Immunology. 2017 ; Vol. 199, No. 1. pp. 271-277.
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abstract = "Protein kinase C-ϵ (PKC-ϵ) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (ϵPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for ϵPS is unknown. Liposome assays revealed that the ϵPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P2. Wortmannin, but not LY294002, inhibits PKC-ϵ concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ϵ concentration at cups and the rate of phagocytosis. PKC-ϵ colocalizes with the trans-Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ϵ-expressing macrophages revealed a loss of Golgi-associated PKC-ϵ during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ϵ at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgiassociated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ϵ is tethered to the TGN via an ϵPS-PI4P interaction. The TGN-associated pool of PKC-ϵ concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that ϵPS binds PI4P and PI(3,5)P2 and that PI4P is necessary for PKC-ϵ localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis.",
author = "Hanes, {Cheryl M.} and D'Amico, {Anna E.} and Takehiko Ueyama and Wong, {Alexander C.} and Xuexin Zhang and Hynes, {W. Frederick} and Barroso, {Margarida M.} and Cady, {Nathaniel C.} and Mohamed Trebak and Naoaki Saito and Lennartz, {Michelle R.}",
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Hanes, CM, D'Amico, AE, Ueyama, T, Wong, AC, Zhang, X, Hynes, WF, Barroso, MM, Cady, NC, Trebak, M, Saito, N & Lennartz, MR 2017, 'Golgi-associated protein kinase C-ϵ is delivered to phagocytic cups: Role of phosphatidylinositol 4-phosphate', Journal of Immunology, vol. 199, no. 1, pp. 271-277. https://doi.org/10.4049/jimmunol.1700243

Golgi-associated protein kinase C-ϵ is delivered to phagocytic cups : Role of phosphatidylinositol 4-phosphate. / Hanes, Cheryl M.; D'Amico, Anna E.; Ueyama, Takehiko; Wong, Alexander C.; Zhang, Xuexin; Hynes, W. Frederick; Barroso, Margarida M.; Cady, Nathaniel C.; Trebak, Mohamed; Saito, Naoaki; Lennartz, Michelle R.

In: Journal of Immunology, Vol. 199, No. 1, 01.07.2017, p. 271-277.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Golgi-associated protein kinase C-ϵ is delivered to phagocytic cups

T2 - Role of phosphatidylinositol 4-phosphate

AU - Hanes, Cheryl M.

AU - D'Amico, Anna E.

AU - Ueyama, Takehiko

AU - Wong, Alexander C.

AU - Zhang, Xuexin

AU - Hynes, W. Frederick

AU - Barroso, Margarida M.

AU - Cady, Nathaniel C.

AU - Trebak, Mohamed

AU - Saito, Naoaki

AU - Lennartz, Michelle R.

PY - 2017/7/1

Y1 - 2017/7/1

N2 - Protein kinase C-ϵ (PKC-ϵ) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (ϵPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for ϵPS is unknown. Liposome assays revealed that the ϵPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P2. Wortmannin, but not LY294002, inhibits PKC-ϵ concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ϵ concentration at cups and the rate of phagocytosis. PKC-ϵ colocalizes with the trans-Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ϵ-expressing macrophages revealed a loss of Golgi-associated PKC-ϵ during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ϵ at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgiassociated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ϵ is tethered to the TGN via an ϵPS-PI4P interaction. The TGN-associated pool of PKC-ϵ concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that ϵPS binds PI4P and PI(3,5)P2 and that PI4P is necessary for PKC-ϵ localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis.

AB - Protein kinase C-ϵ (PKC-ϵ) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (ϵPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for ϵPS is unknown. Liposome assays revealed that the ϵPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P2. Wortmannin, but not LY294002, inhibits PKC-ϵ concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ϵ concentration at cups and the rate of phagocytosis. PKC-ϵ colocalizes with the trans-Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ϵ-expressing macrophages revealed a loss of Golgi-associated PKC-ϵ during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ϵ at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgiassociated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ϵ is tethered to the TGN via an ϵPS-PI4P interaction. The TGN-associated pool of PKC-ϵ concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that ϵPS binds PI4P and PI(3,5)P2 and that PI4P is necessary for PKC-ϵ localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis.

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