Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa B kinases in NCTC 2544 keratinocytes.
In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory kappa B 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 NF kappa B-DNA binding and the activation of the upstream kinases IKK alpha and -beta. 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)-PKC alpha, 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-PKC alpha or PKC zeta had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC alpha but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta 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.
Kanke
T
T
Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, United Kingdom.
Macfarlane
S R
SR
Seatter
M J
MJ
Davenport
E
E
Paul
A
A
McKenzie
R C
RC
Plevin
R
R
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004789:Enzyme Activation; D006801:Humans; D051550:I-kappa B Kinase; D015603:Keratinocytes / Q000201:enzymology*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
D017931:DNA Primers; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human