Characterization of Additional Casein Kinase I Sites in the C-Terminal "Tail" Region of Chicken and Rat Neurofilament-M

Abstract: In previous studies we have identified Ser⁵⁰², Ser⁵²⁸, and Ser⁵³⁴ as target sites in chicken neurofilament middle molecular mass protein (NF-M) for casein kinase I (CKI) in vitro and have shown that these sites are also phosphorylated in vivo. We now make use of a combination of molecular biological and protein chemical techniques to show that two additional in vivo phosphorylation sites in chicken NF-M, Ser⁴⁶⁴ and Ser⁴⁷¹, can also be phosphorylated by CKI in vitro. These two sites are conserved in higher vertebrate NF-M molecules, and recombinant protein constructs containing the homologous rat NF-M peptides can be phosphorylated by CKI in vitro, suggesting that phosphorylation of these sites is conserved at least in higher vertebrates. The two new sites are adjacent to a conserved peptide sequence (VEE-IIEET-V) found once in higher vertebrate NF-M molecules and twice in lamprey NF-180. Variants of this sequence are also found in neurofilament low and high molecular mass proteins (NF-L and NF-H) and α-internexin, and in mammalian NF-L are known to be associated with in vivo phosphorylation sites. We speculate that CKI phosphorylation in general, and these sites in particular, may be important in neurofilament function.