ササキ　タカヒロ   Sasaki Takahiro   佐々木　孝寛    所属   医学部 医学科（東京女子医科大学病院）    職種   助教
論文種別	原著
言語種別	英語
査読の有無	査読あり
表題	In vivo and in vitro phosphorylation at Ser-493 in the glutamate (E)-segment of neurofilament-H subunit by glycogen synthase kinase 3beta.
掲載誌名	正式名：The Journal of biological chemistry 略　 称：J Biol Chem ISSNコード：00219258/00219258
掲載区分	国外
巻・号・頁	277(39),pp.36032-9
著者・共著者	Sasaki Takahiro†*, Taoka Masato, Ishiguro Koichi, Uchida Atsuko, Saito Taro, Isobe Toshiaki, Hisanaga Shin-Ichi
担当区分	筆頭著者,責任著者
発行年月	2002/09
概要	Neurofilament (NF), a major neuronal intermediate filament, is composed of three subunits, NF-L, NF-M, and NF-H. All three subunits contain a well conserved glutamate (E)-rich region called "E-segment" in the N terminus of the tail region. Although the E-segments of NF-L and NF-M are phosphorylated by casein kinases, it has not been observed in NF-H. Using mass spectrometric analysis, we identified phosphorylation of the E-segment of NF-H, prepared from rat spinal cords, at Ser-493 and Ser-501 in the Ser-Pro sequences. The E-segment kinase was isolated from rat brain extract using column chromatography and identified as glycogen synthase kinase (GSK) 3beta. GSK3beta was shown to phosphorylate at Ser-493 in vitro by phosphopeptide mapping and site-directed mutagenesis, and in vivo in HEK293 cells using the phospho-Ser-493 antibody, but did not phosphorylate Ser-501. GSK3beta preferred Ser-493 to the KSP-repeated sequences for phosphorylation sites in the NF-H tail domain. Moreover, Ser-493 was a better phosphorylation site for GSK3beta than other proline-directed protein kinases, Cdk5/p35 and ERK. GSK3beta in the spinal cord extract was associated with NF cytoskeletons. Taken together, we concluded that Ser-493 in the E-segment of NF-H is phosphorylated by GSK3beta in rat spinal cords.
DOI	10.1074/jbc.M206674200
PMID	12130654