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J. Biol. Chem., Vol. 277, Issue 39, 36032-36039, September 27, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/39/36032    most recent M206674200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sasaki, T. Articles by Hisanaga, S.-i. Search for Related Content PubMed PubMed Citation Articles by Sasaki, T. Articles by Hisanaga, S.-i. Social Bookmarking                      What's this?

In Vivo and in Vitro Phosphorylation at Ser-493 in the Glutamate (E)-segment of Neurofilament-H Subunit by Glycogen Synthase Kinase 3^*

Takahiro Sasaki^§, Masato Taoka^¶, Koichi Ishiguro, Atsuko Uchida^**, Taro Saito, Toshiaki Isobe^¶, and Shin-ichi Hisanaga

From the  Department of Biological Sciences and ^¶ Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minami-ohsawa, Hachiohji, Tokyo 192-0397, Japan, the  Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo 194-8511, Japan, and the  Integrated Proteomics System Project, Pioneer Research on Genome the Frontier, MEXT, c/o Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minami-ohsawa, Hachiohji, Tokyo 192-0397, Japan

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) 3. GSK3 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. GSK3 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 GSK3 than other proline-directed protein kinases, Cdk5/p35 and ERK. GSK3 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 GSK3 in rat spinal cords.

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