The Metastasis Suppressor Protein Nme1 Is a Concentration-Dependent Modulator of Ca²⁺/Calmodulin-Dependent Protein Kinase II

Abstract

Nucleoside diphosphate kinases (Nmes/NDPKs) have been implicated in a multitude of cellular processes including an important role in metastasis suppression and several enzymatic activities have been assigned to the Nme family. Nevertheless, for many of these processes, it has not been possible to establish a strong connection between Nme enzymatic activity and the relevant biological function. We hypothesized that, in addition to its known enzymatic functions, members of the Nme family might also regulate signaling cascades by acting on key signal transducers. Accordingly, here we show that Nme1 directly interacts with the calcium/calmodulin-dependent kinase II (CaMKII). Using purified proteins, we monitored the phosphorylation of a number of CaMKII substrates and determined that at nanomolar levels Nme1 enhances the phosphorylation of T-type substrates; this modulation shifts to inhibition at low micromolar concentrations. Specifically, the autophosphorylation of CaMKII at Thr286 is completely inhibited by 2μM Nme1, a feature that distinguishes Nme1 from other known endogenous CaMKII inhibitors. Importantly, CaMKII inhibition does not require phosphotransfer activity by Nme1 since the kinase-dead Nme1 H118F mutant is as effective as the wild-type form of the enzyme. Our results provide a novel molecular mechanism whereby Nme1 could modulate diverse cellular processes in a manner that is independent of its known enzymatic activities.

NME/NM23/NDPK and Histidine Phosphorylation

“…A simultaneous study revealed that NME1 interacts directly with the calcium-calmodulin-dependent kinase II (CAMKII). However, the H118 catalytic site mutant was as effective as the WT form of the enzyme, implying that CAMKII regulation does not involve its phosphotransfer activity [118]. Moreover, human recombinant NME1 H118F and P96S mutants were unable to phosphorylate KSR, and in transfected HEK293T cells, the P96S mutant failed to suppress unlike WT NME1, suggesting that NME1 acts as a protein kinase transferring its histidine phosphate to a serine in the KSR pathway [54].…”