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KMID : 0613820060160071199
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Journal of Life Science
2006 Volume.16 No. 7 p.1199 ~ p.1206
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Inhibition of ERK1/2 Activation and Cytoskeleton Rearrangement by the Recombinant Protein of Plasminogen Kringle 5
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Ha Jung-Min
Kim Hyun-Kyung
Kim Myoung-Rae
Joe Young-Ae
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Abstract
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Plasminogen kringle 5 is a potent inhibitor of endothelial cell 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 α-factor prepro-peptide. After methanol induction, the secreted PK5 was purified by using S-spin column. SDS-PAGE analysis of the purified protein showed one major band of approximately 10 kDa. 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 IC50 of approximately 500 nM. Accordingly, it inhibited bFGF-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in HUVECs at 500 nM. 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.
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KEYWORD
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Angiogenesis inhibitor, endothelial cell, migration, cytoskeletal rearrangement, plasminogen kringle
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