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http://dx.doi.org/10.5352/JLS.2006.16.7.1199

Inhibition of ERK1/2 Activation and Cytoskeleton Rearrangement by the Recombinant Protein of Plasminogen Kringle 5  

Ha, Jung-Min (Cancer Research Institute and Department of Biomedical Sciences College of Medicine, The Catholic University of Korea)
Kim, Hyun-Kyung (Cancer Research Institute and Department of Biomedical Sciences College of Medicine, The Catholic University of Korea)
Kim, Myoung-Rae (Cancer Research Institute and Department of Biomedical Sciences College of Medicine, The Catholic University of Korea)
Joe, Young-Ae (Cancer Research Institute and Department of Biomedical Sciences College of Medicine, The Catholic University of Korea)
Publication Information
Journal of Life Science / v.16, no.7, 2006 , pp. 1199-1206 More about this Journal
Abstract
Plasminogen kringle 5 is a potent inhibitor of endothelial tell proliferation like an endogenous angiogenesis inhibitor, angiostatin consisting of plasminogen kringles 1-4. In this study, we produced the recombinant protein of plasminogen kringle 5 (PK5) employing an Pichia expression system and examined its. effect on~endothelial cell migration and its possible inhibitory mechanism. PK5 was expressed in Pichia pastoris GS115 by fusion of the cDNA spanning from Thr456 to Phe546 to the secretion signal sequence of a-factor prepro-peptide. After methanol induction, the secreted PK5 was purified by using S-spin column. SDS-PACE analysis of the purified protein showed one major band of approximately 10kDa. In in vitro migration assays, the purified protein inhibited dose-dependently the migration of human umbilical endothelial cells (HUVECs) induced by basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) with an $IC_{50}$ of approximately 500nM. Accordingly, it inhibited bfGF-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in HUVECs at 500nM. In addition, it also potently inhibited bFGF-induced cytoskeletal rearrangement of HUVECs. Thus, these results suggest that Pichia-produced PK5 effectively inhibits endothelial cell migration, in part by suppression of ERK1/2 activation and blocking cytoskeleton rearrangement.
Keywords
Angiogenesis inhibitor; endothelial cell; migration; cytoskeletal rearrangement; plasminogen kringle;
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