Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa b kinases in NCTC 2544 keratinocytes

T. Kanke, S.R. MacFarlane, M.J. Seatter, E.L. Davenport, A. Paul, R.C. McKenzie, R.J. Plevin

Research output: Contribution to journalArticle

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Abstract

In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory κB kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NFκB-DNA binding and the activation of the upstream kinases IKKα and -β. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKCα, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKCα or PKCζ had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKKα activity but abolished that of IKKβ, whereas DN-PKCα but not DN-PKCζ substantially reduced trypsin-stimulated Flag-IKKβ activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.
LanguageEnglish
Pages31657-31666
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number34
DOIs
Publication statusPublished - 2001

Fingerprint

PAR-2 Receptor
Heat-Shock Proteins
Keratinocytes
Trypsin
Protein Kinases
Phosphotransferases
Chemical activation
Protein Kinase C
JNK Mitogen-Activated Protein Kinases
seryl-leucyl-isoleucyl--glycyl-lysyl-valine
p38 Mitogen-Activated Protein Kinases
Protein Isoforms
Acetates
Protein C Inhibitor
Protein Kinase Inhibitors
G-Protein-Coupled Receptors
Transfection
Cells
Cell Line
Peptides

Keywords

  • stress-activated protein
  • protein kinases
  • kappa b kinases
  • keratinocytes

Cite this

@article{120f201ae5de4ed0b595ba018274b247,
title = "Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa b kinases in NCTC 2544 keratinocytes",
abstract = "In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory κB kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NFκB-DNA binding and the activation of the upstream kinases IKKα and -β. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and G{\"o}6983, or transfection of dominant negative (DN)-PKCα, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, G{\"o}6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKCα or PKCζ had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKKα activity but abolished that of IKKβ, whereas DN-PKCα but not DN-PKCζ substantially reduced trypsin-stimulated Flag-IKKβ activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.",
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year = "2001",
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pages = "31657--31666",
journal = "Journal of Biological Chemistry",
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Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa b kinases in NCTC 2544 keratinocytes. / Kanke, T.; MacFarlane, S.R.; Seatter, M.J.; Davenport, E.L.; Paul, A.; McKenzie, R.C.; Plevin, R.J.

In: Journal of Biological Chemistry, Vol. 276, No. 34, 2001, p. 31657-31666.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa b kinases in NCTC 2544 keratinocytes

AU - Kanke, T.

AU - MacFarlane, S.R.

AU - Seatter, M.J.

AU - Davenport, E.L.

AU - Paul, A.

AU - McKenzie, R.C.

AU - Plevin, R.J.

PY - 2001

Y1 - 2001

N2 - In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory κB kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NFκB-DNA binding and the activation of the upstream kinases IKKα and -β. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKCα, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKCα or PKCζ had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKKα activity but abolished that of IKKβ, whereas DN-PKCα but not DN-PKCζ substantially reduced trypsin-stimulated Flag-IKKβ activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.

AB - In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory κB kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NFκB-DNA binding and the activation of the upstream kinases IKKα and -β. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKCα, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKCα or PKCζ had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKKα activity but abolished that of IKKβ, whereas DN-PKCα but not DN-PKCζ substantially reduced trypsin-stimulated Flag-IKKβ activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.

KW - stress-activated protein

KW - protein kinases

KW - kappa b kinases

KW - keratinocytes

U2 - 10.1074/jbc.M100377200

DO - 10.1074/jbc.M100377200

M3 - Article

VL - 276

SP - 31657

EP - 31666

JO - Journal of Biological Chemistry

T2 - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 34

ER -