A neurofilament-associated kinase phosphorylates only a subset of sites in the tail of chicken midsize neurofilament protein.
Although neurofilaments are among the most highly phosphorylated proteins extant, relatively little is known about the kinases involved in their phosphorylation. The majority of the phosphates present on the two higher-molecular-mass neurofilament subunits are added to multiply repeated sequence motifs in the tail. We have examined the specificity of a neurofilament-associated kinase (NFAK) partially purified from chicken spinal cord that selectively phosphorylates the middle-molecular-mass neurofilament subunit, NF-M. Two-dimensional phosphopeptide mapping of 32P-labeled NF-M shows that, in vitro, NFAK phosphorylates a subset of peptides phosphorylated in vivo in cultured neurons. The absence of a complete complement of labeled phosphopeptides following in vitro phosphorylation, compared with phosphorylation in vivo, is not due to a lack of availability of phosphorylation sites because the same maps are obtained when enzymatically dephosphorylated NF-M is used as an in vitro substrate. Phosphopeptide maps from in vitro-phosphorylated NF-M and those from a recombinant fusion protein containing only a segment of the tail piece of chicken NF-M reveal identical labeled peptides. The fusion protein lacks a segment containing 17 KXX(S/T)P putative phosphorylation sites contained in the tail of chicken NF-M but contains a segment that includes four KSPs and a KSD site also present in the intact tail. These results suggest (a) that NFAK mediates the phosphorylation of some, but not all, potential phosphorylation sites within the tail of NF-M and (b) that multiple kinases are necessary for complete phosphorylation of the NF-M tail.
Hollander
B A
BA
Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville 32610.
Ayyub
C
C
Shaw
G
G
Bennett
G S
GS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002474:Cell-Free System; D002478:Cells, Cultured; D002645:Chickens; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005727:Ganglia, Spinal / Q000378:metabolism; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013116:Spinal Cord / Q000201:enzymology / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D016900:Neurofilament Proteins; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; C084357:neurofilament-associated kinase