Lithium Chloride Inhibits the Phosphorylation of Newly Synthesized Neurofilament Protein, NF‐M, in Cultured Chick Sensory Neurons

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Abstract
The middle and high molecular weight members of the neurofilament triplet, NF‐M and NF‐H, undergo extensive posttranslational polyphosphorylation, a process requiring 24 h or more for completion. We have investigated ways of perturbing this process in intact cells and have found that phosphorylation of newly synthesized NF‐M in cultured chick sensory neurons is inhibited by Li+. [35S]Methionine pulse‐chase experiments were carried out with pure neuronal cultures, and the phosphorylation of newly synthesized NF‐M was monitored by following the accompanying change, with chase time, in apparent size and charge of the polypeptide. Addition of LiCl to the medium inhibited this mobility shift in a dose‐dependent manner over concentrations between 2 and 25 mM. Incorporation of 32P into NF‐M, as well as NF‐H, was also inhibited, whereas incorporation into the low molecular weight neurofilament protein, β‐tubulin, and total protein was unaffected. Protein synthesis was not altered.Exposure to 25 mM LiCl for up to 72 h was not toxic, and the inhibition of NF‐M phosphorylation was completely reversible. When 25 mM Li+ was added after NF‐M had become partially phosphorylated, further progression was blocked, but there was no net dephosphorylation or degradation of NF‐M. Additional experiments suggest that this action of Li+ is probably not due to effects on second messenger levels or to effects on tubulin metabolism and assembly state presented in our accompanying article, but rather to interference by Li+ itself, with the phosphorylation of NF‐M and NF‐H by specific neurofilament kinase(s).