Expression and phosphorylation of neurofilament proteins in rat pheoichromocytoma (PC 12) cells

The in vitro rephosphorylation of phosphatase-treated neurofilament (NF) subunits was assessed. Enzymatically dephosphorylated NF-L and NF-M proved as good or better substrates for phosphorylation by various exogenous protein kinases, while dephosphorylated NF-H was refractory to phosphorylation, suggesting that a conformational change accompanies the dephosphorylation of NF-H but not NF-L or NF-M.The biosynthesis and phosphorylation of NF subunits were investigated in PC 12 cells. While all three NF subunits were synthesized in PC 12 cells, NF-M and NF-H were synthesized as hypophosphorylated precursors. NF-M was shown to undergo a time-dependent decrease in electrophoretic mobility in both control and NGF treated cells, consistent with its undergoing post-translational phosphorylation. Nerve growth factor (NGF) administration resulted in increased biosynthesis of NF-L and NF-M, increased stability of both NF-L and the slowest migrating form of NF-M, as well as increased incorporation of phosphate into NF-H. A soluble pool of NF subunits was also found in PC 12 cells. Soluble NF-M and NF-H were synthesized as faster migrating electrophoretic forms. Soluble NF-M underwent a time-dependent increase in phosphorylation state, indicating that the extent of phosphorylation does not significantly affect the solubility of this NF subunit.The effect of NGF on NF gene expression PC 12 cells was further explored. NGF treatment resulted in time-dependent increases in the levels of all three NF subunits. However, increases in NF-H levels were delayed relative to those of NF-L and NF-M. NGF treatment resulted in 12- and 14-fold increases in NF-L and NF-M mRNA, respectively, as well as increased transcriptional activity of both genes. The stability of NF-M mRNA was also increased in NGF treated PC 12 cells. However, NGF treatment was found not to affect the levels of NF-H mRNA nor the transcription rate of the NF-H gene, indicating that the effect of NGF on the biosynthesis of NF-H in PC 12 cells may occur at the translational or post-translational level