N-Myristoyltransferase 2 Is Regulated by Polyphosphate-Driven Lysine Interactions.

Inorganic polyphosphate (polyP) is a ubiquitous polymer increasingly recognized as a regulator of protein function through lysine polyphosphate modification (KPM), a reversible interaction with lysine-rich protein regions. Although numerous KPM targets have been identified, the functional consequences of polyP binding remain poorly understood. N-myristoyltransferase 2 (NMT2), an essential enzyme that catalyzes protein N-myristoylation and regulates membrane-associated signaling pathways, was previously identified as a candidate KPM target. Here, we investigated the molecular and functional relationship between polyP and NMT2. Using a fluorescence-based coenzyme A release assay, we found that polyP directly down-regulated NMT2 enzymatic activity in a dose-dependent manner, with long-chain polyP (polyP700) producing significantly greater inhibition than medium-chain polyP (polyP100). In cells, both exogenous polyP treatment and induction of endogenous polyP synthesis reduced phosphorylation of Src, a downstream signaling protein whose activation depends on N-myristoylation, supporting inhibition of NMT2 function in vivo. PolyP also decreased the viability of NMT2-overexpressing HeLa cells in a dose-dependent manner, suggesting functional consequences for cellular fitness. Biochemical analysis further revealed that polyP induced a pronounced electrophoretic mobility shift in NMT2 that was completely reversed by a 25-residue lysine-rich competitor peptide, consistent with a specific and reversible interaction mediated through lysine-enriched regions of the enzyme. These findings identify NMT2 as a functional target of polyP and demonstrate that polyP negatively regulates NMT2 activity, downstream Src signaling, and cellular viability. Our results expand the emerging concept of lysine polyphosphate modification and establish polyP as a previously unrecognized regulator of protein lipidation-dependent signaling pathways.