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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
Author(s) -
Hollander Brian A.,
Bennett Gudrun S.,
Shaw Gerry
Publication year - 1996
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1996.66010412.x
Subject(s) - neurofilament , protein subunit , casein kinase 2 , casein kinase 2, alpha 1 , phosphorylation , kinase , chemistry , casein kinase 1 , microbiology and biotechnology , biochemistry , biology , protein kinase a , mitogen activated protein kinase kinase , immunohistochemistry , gene , immunology
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 (CKI) family based on the following criteria: (1) inhibition 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 similar conditions, and (4) immunological cross‐reactivity of NFAK with an antibody raised against CKI. We have also identified Ser 502 , Ser 528 , and Ser 536 as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser 528 and Ser 536 and flanking amino acids are highly conserved in higher vertebrate NF‐M sequences. Neither Ser 502 nor Ser 536 has been identified previously as NF‐M phosphorylation sites.