Kanke T, Macfarlane S R, Seatter M J, Davenport E, Paul A, McKenzie R C, Plevin R
Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, United Kingdom.
J Biol Chem. 2001 Aug 24;276(34):31657-66. doi: 10.1074/jbc.M100377200. Epub 2001 Jun 18.
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.
在本研究中，我们通过刺激人角质形成细胞系NCTC2544中新型G蛋白偶联受体蛋白酶激活受体-2，研究了应激激活蛋白（SAP）激酶和抑制性κB激酶（IKK）的调节作用。胰蛋白酶和肽SLIGKV刺激c-Jun氨基末端激酶和p38丝裂原活化蛋白激酶活性呈时间依赖性增加。胰蛋白酶还刺激NF-κB与DNA结合以及上游激酶IKKα和IKKβ的激活。佛波醇12-肉豆蔻酸酯13-乙酸酯也强烈激活SAP激酶和IKK亚型，提示可能存在蛋白激酶C介导的调节机制参与胰蛋白酶的作用。用选择性蛋白激酶C（PKC）抑制剂GF109203X和Gö6983预孵育，或转染显性负性（DN）-PKCα，可消除佛波醇12-肉豆蔻酸酯13-乙酸酯介导的c-Jun氨基末端激酶活性，尽管其仅部分抑制对胰蛋白酶的反应。相反，Gö6983比GF109203X更能降低胰蛋白酶刺激的p38丝裂原活化蛋白激酶活性，尽管DN-PKCα或PKCζ无显著作用。此外，PKC抑制剂部分降低胰蛋白酶刺激的IKKα活性，但消除IKKβ的活性，而DN-PKCα而非DN-PKCζ显著降低胰蛋白酶刺激的Flag-IKKβ活性。本研究首次表明蛋白酶激活受体-2介导对SAP激酶和IKK信号的刺激，以及PKC亚型在各信号通路调节中的不同作用。
