Casein kinase II is responsible for phosphorylation of NF-L at Ser-473.
Ser-473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF-L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full-length dephosphorylated NF-L as the substrate, and employed MALDI/TOF (matrix-assisted laser desorption and ionization/time of flight) mass spectrometry and a site-specific phosphorylation-dependent antibody recognizing Ser-473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser-473 in vitro, while neither GSK-3beta nor calcium/calmodulin-dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF-L indicated that CK II was the kinase mediating Ser-473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser-473 in vitro. The antibody also demonstrated that NF-L phosphorylated at Ser-473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser-473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser-473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF-L in vivo.
Nakamura
Y
Y
Department of Clinical Neuroscience, Psychiatry, Osaka University Graduate School of Medicine, Suita-shi, Japan. nakamura@psy.med.osaka-u.ac.jp
Hashimoto
R
R
Kashiwagi
Y
Y
Wada
Y
Y
Sakoda
S
S
Miyamae
Y
Y
Kudo
T
T
Takeda
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D047390:Casein Kinase II; D002417:Cattle; D007150:Immunohistochemistry; D016900:Neurofilament Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016900:Neurofilament Proteins; C044882:neurofilament protein L; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases