In vivo and in vitro phosphorylation at Ser-493 in the glutamate (E)-segment of neurofilament-H subunit by glycogen synthase kinase 3beta.
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.
Sasaki
Takahiro
T
Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minami-ohsawa, Hachiohji, Tokyo 192-0397, Japan. sasaki-takahiro@c.metro-u.ac.jp
Taoka
Masato
M
Ishiguro
Koichi
K
Uchida
Atsuko
A
Saito
Taro
T
Isobe
Toshiaki
T
Hisanaga
Shin-Ichi
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000737:chemistry*; D010766:Phosphorylation; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013116:Spinal Cord / Q000378:metabolism; D013997:Time Factors
D016900:Neurofilament Proteins; C051774:neurofilament protein H; D018698:Glutamic Acid; D012694:Serine; D011392:Proline; D051360:Cyclin-Dependent Kinase 5; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; C495905:CDK5 protein, human; C495907:Cdk5 protein, rat; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3; D010450:Endopeptidases