Major phosphorylation site (Ser55) of neurofilament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture.
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.
Nakamura
Y
Y
Department of Clinical Neuroscience, Psychiatry, Osaka University Graduate School of Medicine, Suita-shi, Japan. nakamura@psy.med.osaka-u.ac.jp
Hashimoto
R
R
Kashiwagi
Y
Y
Aimoto
S
S
Fukusho
E
E
Matsumoto
N
N
Kudo
T
T
Takeda
M
M
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000235:genetics* / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D019319:Okadaic Acid / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats
D004791:Enzyme Inhibitors; D016900:Neurofilament Proteins; D010446:Peptide Fragments; D010710:Phosphates; C044882:neurofilament protein L; D005576:Colforsin; D019319:Okadaic Acid; D017868:Cyclic AMP-Dependent Protein Kinases