Major phosphorylation site (Ser55) of neurofilament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture |
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Authors: | Nakamura Y Hashimoto R Kashiwagi Y Aimoto S Fukusho E Matsumoto N Kudo T Takeda M |
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Affiliation: | Department of Clinical Neuroscience, Psychiatry, Osaka University Graduate School of Medicine, Suita-shi, Japan. nakamura@psy.med.osaka-u.ac.jp |
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Abstract: | Ser55 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 Ser41, Ser55, and Ser62 in the head region, with Ser55 considered the preferred site. A site-specific phosphorylation-dependent antibody against Ser55 rendered NF-L phosphorylated at Ser55 detectable in primary cultured rat neurons. One-hour treatment with 20 nM okadaic acid increased the phosphorylation level of Ser55, and co-treatment with 10 microM forskolin enhanced it. However, forskolin alone did not elevate the phosphorylation level. As a consequence, NF-L may be phosphorylated at Ser55 by PKA or by a PKA-like kinase in vivo; however, the phosphorylation level of Ser55 may be modulated by certain phosphatases sensitive to okadaic acid. |
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Keywords: | Neurofilament Neurofilament L Cyclic AMP-dependent protein kinase Neuronal culture Matrix-assisted laser desorption and ionization Phosphorylation Okadaic acid |
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