Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167 |
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| Institution: | 1. Pulmonary Disease Program, Vascular Biology Center, Georgia Regents University, Augusta, GA 30912, USA;2. Department of Pediatrics, University of California at San Francisco, San Francisco, CA 94143, USA;3. Cardiovascular Research Institute, University of California at San Francisco, San Francisco, CA 94143, USA;1. Department of Applied Chemistry and Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 812-8581, Japan;2. Department of Chemistry and Biochemistry, Texas Christian University, Fort Worth, TX 76129, USA;3. Underwood International College, Integrated Science and Engineering Division, Yonsei University, Seoul, 03722, Republic of Korea;1. Health Physics and Dosimetry Laboratory, Department of Energy Engineering and Physics, Amir Kabir University of Technology, Tehran, Iran;2. Department of Nuclear Engineering, Science and Research Branch, Islamic Azad University, P.O. Box 14515-775, Tehran, Iran;3. Nuclear Science Research School, Nuclear Science and Technology Research Institute, Tehran 11365-3486, Iran;1. Northwest Agriculture and Forestry University, College of Life Science, Yangling, Shaanxi, China;2. Zhejiang Sci-Tech University, College of Life Science, Hangzhou, Zhejiang, China;1. Department of Plant Physiology, University of Agriculture in Krakow, Pod?u?na 3, Kraków 30-239, Poland;2. The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, Kraków 30-239, Poland |
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| Abstract: | Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H2O2) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H2O2 compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity. |
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| Keywords: | Posttranslational regulation Phosphorylation Catalase Free radicals |
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