Major phosphorylation site (Ser(55)) of neurofilament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture

Ser(55) of neurofilament L (NF-L) is reported to be partly phosphorylated in neurons and to be phosphorylated by cyclic AMP-dependent protein kinase (PKA). Bovine NF-L was phosphorylated by PKA in a low concentration of MgCl2 (0.3 mM) and digested by trypsin, Trypsin-digested fragments were assigned by MALDI/TOF (matrix-assisted laser desorption and ionization/time-of-flight) mass spectrometry. Phosphorylation sites were found at Ser(41), Ser(55), and Ser(62) in the head region, with Ser(55) considered the preferred site. A site-specific phosphorylation-dependent antibody against Ser(55) rendered NF-L phosphorylated at Ser(55) detectable in primary cultured rat neurons. One-hour treatment with 20 nM okadaic acid increased the phosphorylation level of Ser(55), and co-treatment with 10 mu M forskolin enhanced it. However, forskolin alone did not elevate the phosphorylation level. As a consequence, NF-L may be phosphorylated at Ser(55) by PKA or by a PKA-like kinase in vivo; however, the phosphorylation level of Ser(55) may be modulated by certain phosphatases sensitive to okadaic acid.