Human nucleoside diphosphate (NDP) kinase A is a ‘house-keeping’ enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5′-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro. A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5′-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro. We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli. Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro. The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.
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March 13 2007
Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of human nucleoside diphosphate kinase A/Nm23-H1 Available to Purchase
Iulia Mocan;
Iulia Mocan
*Institut de Biochimie et Génétique Cellulaires (UMR 5095), Université Victor Segalen Bordeaux2 and CNRS, 33077 Bordeaux Cedex, France
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Florian Georgescauld;
Florian Georgescauld
*Institut de Biochimie et Génétique Cellulaires (UMR 5095), Université Victor Segalen Bordeaux2 and CNRS, 33077 Bordeaux Cedex, France
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Philippe Gonin;
Philippe Gonin
*Institut de Biochimie et Génétique Cellulaires (UMR 5095), Université Victor Segalen Bordeaux2 and CNRS, 33077 Bordeaux Cedex, France
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Didier Thoraval;
Didier Thoraval
*Institut de Biochimie et Génétique Cellulaires (UMR 5095), Université Victor Segalen Bordeaux2 and CNRS, 33077 Bordeaux Cedex, France
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Laura Cervoni;
Laura Cervoni
†Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’ and the Center of Molecular Biology of Consiglio Nazionale delle Ricerche, Università degli Studi ‘La Sapienza’, 00185 Rome, Italy
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Anna Giartosio;
Anna Giartosio
†Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’ and the Center of Molecular Biology of Consiglio Nazionale delle Ricerche, Università degli Studi ‘La Sapienza’, 00185 Rome, Italy
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Sandrine Dabernat-Arnaud;
Sandrine Dabernat-Arnaud
‡EA 483, Université Victor Segalen Bordeaux2, 33076 Bordeaux Cedex, France
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Marc Crouzet;
Marc Crouzet
*Institut de Biochimie et Génétique Cellulaires (UMR 5095), Université Victor Segalen Bordeaux2 and CNRS, 33077 Bordeaux Cedex, France
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Marie-Lise Lacombe;
Marie-Lise Lacombe
§Unité 680 INSERM, Faculté de Médecine Pierre et Marie Curie, site Saint-Antoine, 75012 Paris, France
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Ioan Lascu
Ioan Lascu
1
*Institut de Biochimie et Génétique Cellulaires (UMR 5095), Université Victor Segalen Bordeaux2 and CNRS, 33077 Bordeaux Cedex, France
1To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
July 27 2006
Revision Received:
November 20 2006
Accepted:
December 08 2006
Accepted Manuscript online:
December 08 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2007
Biochem J (2007) 403 (1): 149–156.
Article history
Received:
July 27 2006
Revision Received:
November 20 2006
Accepted:
December 08 2006
Accepted Manuscript online:
December 08 2006
Citation
Iulia Mocan, Florian Georgescauld, Philippe Gonin, Didier Thoraval, Laura Cervoni, Anna Giartosio, Sandrine Dabernat-Arnaud, Marc Crouzet, Marie-Lise Lacombe, Ioan Lascu; Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of human nucleoside diphosphate kinase A/Nm23-H1. Biochem J 1 April 2007; 403 (1): 149–156. doi: https://doi.org/10.1042/BJ20061141
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