期刊
JOURNAL OF NEUROCHEMISTRY
卷 74, 期 3, 页码 949-959出版社
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1046/j.1471-4159.2000.0740949.x
关键词
neurofilament; neurofilament L; cyclic AMP-dependent protein kinase; neuronal culture; matrix-assisted laser desorption and ionization; phosphorylation; okadaic acid
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.
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