Mix and match of the tumor metastasis suppressor Nm23 protein isoformsin vitroandin vivo

Nm23/NME was identified 30 years ago as the first metastatic gene suppressor family. Despite extensive studies, the mechanism of action behind the observed antimetastatic potential of Nm23 has remained largely unresolved. Human Nm23 is present in various isoforms, of which Nm23-H1 and Nm23-H2 are by far the most dominant. Both isoforms are multifunctional enzymes involved in important cellular processes, through their nucleic acid binding ability, their protein-protein interactions and/or their histidine kinase activity. Although Nm23-H1 and Nm23-H2 exhibit 88% sequence homology, they often are considered to have distinct biological functions. Here, we developed an efficient and robust purification protocol to pull-down Nm23 isoforms in their native state. We applied this protocol to purify both overexpressed isoform pure as well as endogenous Nm23 proteins from several human cell lines and mouse brain tissue. Subsequent native mass spectrometry (MS) analysis revealed that all purified Nm23 samples form hexamers, whereby the endogenous protein assembly is primarily present as heterohexamers formed by statistical association of the Nm23-H1 and Nm23-H2 isoforms. Therefore, we conclude that isoform-pure hexameric Nm23 complexes scarcely exist in vivo. We also used native and top-down MS to investigate the histidine autophosphorylation activity of purified Nm23 assemblies. Our data in fine challenge the biological relevance of studying the genes/proteins Nm23-H1 and Nm23-H2 individually.