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

Abstract

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.

Phosphorylation of nm23-H1 by CKI induces its complex formation with h-prune and promotes cell motility

“…Similarly, in vivo in mammalian cells, nm23-H2 (the more suitable protein to be analysed by triptic digestion) was phosphorylated on S125 and on either S120 or S122; again, transient phosphorylation on S120 cannot be excluded, and this phosphorylation could be important to enhance the acid context that is necessary for CKII phosphorylation on S122 (Meggio et al, 1993). A recent study has also shown that autophosphorylation of the nm23-H1-S120G protein corrected its folding defect (Mocan et al, 2007). But what fraction of the nm23 proteins is phosphorylated in living cells, and how is this fraction is regulated in response to different stimuli are still open questions.…”

Aggregation of the neuroblastoma-associated mutant (S120G) of the human nucleoside diphosphate kinase-A/NM23-H1 into amyloid fibrils

“…Previous studies have shown that the S120G-NDPK-HA molten globule-folding intermediate is in equilibrium with the native monomer. This equilibrium may be easily controlled by protein phosphorylation (Mocan et al 2007), chemical chaperones (Georgescauld et al 2009), mutations, and quaternary structure assembly (Lascu et al 1997). Amyloid fibril formation by NDPK-HA is certainly a good model for studying the role of molten globule-folding intermediates in amyloidogenesis.…”

“…Proteins The expression and purification of the NDPK-HA and S120G-NDPK-HA (Lascu et al 1997;Mocan et al 2007) and of Dictyostelium NDP kinase (Cervoni et al 2003) were described earlier.…”

NDPKA is not just a metastasis suppressor – be aware of its metastasis-promoting role in neuroblastoma

“…33 This folding defect can be corrected when NDPK-A S120G is phosphorylated by ATP or by phosphoramidate. 34 When forming a complex with ADP, no significant structural changes are observed between NDPK-A S120G and the wild-type. 35 In addition to affecting the subunit assembly, the S120G mutation changes the interaction of NDPK-A with other cellular proteins.…”