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Major Phosphorylation Site (Ser 55 ) of Neurofilament L by Cyclic AMP‐Dependent Protein Kinase in Rat Primary Neuronal Culture
Author(s) -
Nakamura Yu,
Hashimoto Ryota,
Kashiwagi Yujiro,
Aimoto Saburo,
Fukusho Eriko,
Matsumoto Naohiko,
Kudo Takashi,
Takeda Masatoshi
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0740949.x
Subject(s) - okadaic acid , phosphorylation , forskolin , protein kinase a , phosphatase , chemistry , kinase , biochemistry , protein phosphorylation , trypsin , microbiology and biotechnology , neurofilament , biology , enzyme , in vitro , immunohistochemistry , immunology
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 MgCl 2 (0.3 m M ) 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 n M okadaic acid increased the phosphorylation level of Ser 55 , and co‐treatment with 10 μ 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.