Activation of mitogen-activated protein kinases (Erk1 and Erk2) cascade results in phosphorylation of NF-M tail domains in transfected NIH 3T3 cells

Neurofilaments (NFs) are neuron-specific intermediate filaments, and are th e major cytoskeletal component in large myelinated axons. Lysine-serine-pro line (KSP) repeats in the tail domains of high molecular weight NF proteins (NF-M and NF-H) are extensively phosphorylated in vivo in the axon. This p hosphorylation in the tail domain has been postulated to play an important role in mediating neuron-specific properties, including axonal caliber and conduction velocity. Recent studies have shown that the mitogen-activated p rotein kinases (extracellular signal-regulated kinases, Erk1 and Erk2) phos phorylate KSP motifs in peptide substrates derived from the NF-M and NF-H t ail domains in vitro. However, it is not clear whether activation of the mi togen activated protein (MAP) kinase pathway is able to phosphorylate these domains in vivo. To answer this question, a constitutively active form of mitogen-activated Erk activating kinase (MEK1) was cotransfected with an NF -M expression construct into NIH 3T3 cells. The activated mutant, but not t he dominant negative mutant, induced phosphorylation of NF-M. In addition, it was shown that epidermal growth factor, which induces the MAP kinase cas cade in NIH 3T3 cells, also activated endogenous Erk1 and Erk2 and NF-M tai l domain phosphorylation in the transfected cells. These results present di rect evidence that in-vivo activation of Erk1 and Erk2 is sufficient for NF -M tail domain phosphorylation in transfected cells.