Site-specific phosphorylation of the middle molecular weight human neurofilament protein in transfected non-neuronal cells

We expressed the human midsized neurofilament subunit (NF-M) using genomic DNA in mouse L cells and showed that it is transcribed and translated into a protein capable of assembly into the cytoskeleton and of forming a filamentous network that colocalizes with the endogenous vimentin filaments. Moreover, human NF-M expressed in L cells is phosphorylated at sites within the multiphosphorylation repeat (MPR), i.e., the major sites of phosphorylation of NF-M in vivo. We also expressed a genomic construct lacking the MPR domain in the native molecule and showed that this MPR(-) protein also was expressed and formed a filamentous network despite diminished incorporation of radiolabeled phosphate. Two major conclusions emerged from the work described in this paper: human NF-M is translated, assembled, and phosphorylated at physiological sites without the need of any other specific neuronal proteins; phosphorylation sites other than the MPR are present within NF-M which may play a role in synthesis, assembly, and degradation of NF protein in humans.