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 neurofilamen t L (NF-L), With peptides including the native phosphorylation site, it was not possible to locate responsible kinases, We therefore adopted full-leng th dephosphorylated NF-L as the substrate, and employed MALDI/TOF (matrix-a ssisted laser desorption and ionization/time of flight) mass spectrometry a nd a site-specific phosphorylation-dependent antibody recognizing Ser-473 p hosphorylation, 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 -3 beta nor calcium/calmodulin-dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NP-L indicate d that CK II was the kinase mediating Ser-473 phosphorylation in vitro as o pposed 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-4 73 was abundant in the neuronal perikarya of the rat cortex, indicating tha t phosphorylation of Ser-473 may take place there, This result may support the suggestion that CK II is the kinase responsible for Ser-473 phosphoryla tion, Despite many reports showing that CK I mediates phosphorylation of ne urofilaments, CK II may phosphorylate NF-L in vivo. (C) 1999 Federation of European Biochemical Societies.