LOCALIZATION OF SITES IN THE TAIL DOMAIN OF THE MIDDLE MOLECULAR-MASSNEUROFILAMENT 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 selectivel y the middle molecular mass NF subunit (NF-M). Here we show that the m ajor kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhi bition of NFAK phosphorylation by the selective CKI inhibitor CKI-7, ( 2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKI under simi lar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKI. We have also identified Ser(502), Ser(52 8), and Ser(536) as phosphorylation sites by NFAK/CKI in vitro, each o f which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser(528) a nd Ser(536) and flanking amino acids are highly conserved in higher ve rtebrate NF-M sequences. Neither Ser(502) nor Ser(536) has been identi fied previously as NF-M phosphorylation sites.