Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of the human nucleoside diphosphate kinase A/Nm23-H1

Iulia Mocan , Florian Georgescauld , Philippe Gonin , Didier Thoraval , Laura Cervoni , Anna Giartosio , Sanbrine Dabernat-Arnaud , Marc Crouzet , Marie-Lise Lacombe , Ioan Lascu (1)

1 Institut de Biochimie et Génétique Cellulaires

Human nucleoside diphosphate kinase A is a "housekeeping" 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 tumor dissemination. Its mutant S120G has been identified in high-grade neuroblastomas. We showed previously that the 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. Here, we report that protein autophosphorylation 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 nonhydrolysable ATP analog 5'-adenylylimido-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 E. 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 118 with an asparagine 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.

Mots clés: Life Sciences

Iulia Mocan, Florian Georgescauld, Philippe Gonin, Didier Thoraval, Laura Cervoni, et al.. Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of the human nucleoside diphosphate kinase A/Nm23-H1. Biochemical Journal, 2006, 403 (1), pp.149-156. ⟨10.1042/BJ20061141⟩. ⟨hal-00478638⟩