Multiple functions of Nm23-H1 are regulated by oxido-reduction system.
Nucleoside diphosphate kinase (NDPK, Nm23), a housekeeping enzyme, is known to be a multifunctional protein, acting as a metastasis suppressor, transactivation activity on c-myc, and regulating endocytosis. The cellular mechanisms regulating Nm23 functions are poorly understood. In this study, we identified the modifications and interacting proteins of Nm23-H1 in response to oxidative stress. We found that Cys109 in Nm23-H1 is oxidized to various oxidation states including intra- and inter-disulfide crosslinks, glutathionylation, and sulfonic acid formation in response to H(2)O(2) treatment both in vivo and in vitro. The cross-linking sites and modifications of oxidized Nm23-H1 were identified by peptide sequencing using UPLC-ESI-q-TOF tandem MS. Glutathionylation and oxidation of Cys109 inhibited the NDPK enzymatic activity of Nm23-H1. We also found that thioredoxin reductase 1 (TrxR1) is an interacting protein of Nm23-H1, and it binds specifically to oxidized Nm23-H1. Oxidized Nm23 is a substrate of NADPH-TrxR1-thioredoxin shuttle system, and the disulfide crosslinking is reversibly reduced and the enzymatic activity is recovered by this system. Oxidation of Cys109 in Nm23-H1 inhibited its metastatic suppressor activity as well as the enzymatic activities. The mutant, Nm23-H1 C109A, retained both the enzymatic and metastasis suppressor activities under oxidative stress. This suggests that key enzymatic and metastasis suppressor functions of Nm23-H1 are regulated by oxido-reduction of its Cys109.
Lee
Eunsun
E
Department of Bioinspired Science, Division of Life and Pharmaceutical Sciences, College of Pharmacy, Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, Seoul, Korea.
Jeong
Jaeho
J
Kim
Sung Eun
SE
Song
Eun Joo
EJ
Kang
Sang Won
SW
Lee
Kong-Joo
KJ
D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D054778:NM23 Nucleoside Diphosphate Kinases / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry; D034622:RNA Interference; D053719:Tandem Mass Spectrometry / Q000379:methods
D003432:Cross-Linking Reagents; D004220:Disulfides; D054778:NM23 Nucleoside Diphosphate Kinases; D006861:Hydrogen Peroxide; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen