Localization of sites in the tail domain of the middle molecular mass neurofilament subunit phosphorylated by a neurofilament-associated kinase and by casein kinase I.
We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKl) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKl inhibitor CKl-7, (2) the similarity in substrate specificity of NFAK and authentic CKl, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKl under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKl. We have also identified Ser502, SER528, and Ser536 as phosphorylation sites by NFAK/CKl in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKl phosphorylation consensus sequences, and Ser528 and Ser536 and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser502 nor Ser536 has been identified previously as NF-M phosphorylation sites.
Department of Neuroscience, University of Florida College of Medicine, Gainesville, USA.
B A
BA
Hollander
G S
GS
Bennett
G
G
Shaw
D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases; D002642:Chick Embryo; D002645:Chickens / Q000235:genetics; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D007546:Isoquinolines; D016900:Neurofilament Proteins; D047428:Protein Kinase Inhibitors; C058815:N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; D011494:Protein Kinases; C084357:neurofilament-associated kinase; D047388:Casein Kinases