The relationship of NM23 (NME) metastasis suppressor histidine phosphorylation to its nucleoside diphosphate kinase, histidine protein kinase and motility suppression activities

Abstract

The NM23/NME gene was identified as a metastasis suppressor. It's re-expression inhibited cancer cell motility and suppressed metastasis, without effecting primary tumor size in multiple model systems. The mechanisms of NME suppression of motility and metastasis are incompletely known. Of particular interest, has been NME histidine 118 phosphorylation, involved in nucleoside diphosphate kinase (NDPK) and histidine protein kinase (HPK) activities. Using recently developed monoclonal antibodies to phosphohistidine, we have addressed the correlation of NME phosphohistidine with motility suppression, and distinguished the NDPK and HPK contributions. While general levels of NME correlated with its 1-phosphohistidine form in two cell line model systems, two exceptions were noted: Tumor cells actively migrating in scratch assays, even if expressing high levels of NME1, were low in its 1-phosphohistidine form. Site-directed mutagenesis of NME1 histidine 118 and proline 96 was examined by transfection experiments and partial purification of recombinant proteins. NME1P96S overexpressing tumor cells exhibited high motility and migration phenotypes despite high 1-phosphohistidine content and NDPK activity; HPK activity using succinate thiokinase as a substrate was poor. The data suggest the importance of NME 1-phosphohistidine levels in potential mechanistic pathways of metastasis suppression and point toward the HPK activity of NME1 downstream of autophosphorylation.

A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

“…Based on earlier studies it has been suggested that pHis might be more abundant in proteins than expected 29 and the existence of His kinases and pHis phosphatases in human cells also implies the existence of non-conventional phosphorylation-based signaling systems [30][31][32][33] . Our work shows for the first time that under non-acidic conditions, an overall coverage of SONAte phosphopeptide enrichment using HAP can reveal more non-conventional than conventional phosphorylation sites ( Fig.…”

NME/NM23/NDPK and Histidine Phosphorylation

“…To distinguish between these two functions, the consequences of mutating Pro96 of NME1 has been investigated. Mutation of Pro96 to serine causes conformational changes in NME affecting its binding to protein substrates [114]. This mutation was first identified in Drosophila as the "killer of prune" mutation since it was lethal in combination with the prune gene mutation [115][116][117].…”

“…Therefore, mutation of H118 abrogates both nucleotide and protein kinase activity, and mutation of Pro96 compromises only its protein kinase activity [53]. Recent findings indicate that NME1 s ability to suppress motility depends on its protein kinase function, as neither mutant is able to inhibit migration of transfected MDA-MB-231 and MDA-MB-435 breast cancer cells [114]. Interestingly, migrating cells overexpressing NME1 had lower levels of its 1-pHis form compared to stationary cells.…”

The Function of NM23-H1/NME1 and Its Homologs in Major Processes Linked to Metastasis

“…This includes cell migration [29], growth and differentiation [20,30,31], signal transduction, transcriptional regulation [32,33], and apoptosis [34]. In addition, other molecular activities have been assigned to NM23/NDPKs, such as histidine-dependent protein kinase (histidine phosphotransferase) activity [35][36][37][38], unusual nuclease activity [39,40], and lipid bilayer-binding [41]. Taken together, multiple functions have been assigned to the NM23/NDPK protein family and many interaction partners were identified by different in vitro methods [29].Thus NM23 gene family display pleiotropic functions and their effect on metastatic progression might be contradictory in many cancers.…”