Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules.
We sought the mammalian neurofilament tail domain-specific kinase. Several well known kinases including cAMP-dependent protein kinase, protein kinase C, Ca(2+)-calmodulin-dependent protein kinase II, casein kinase I, and casein kinase II phosphorylated the high (NF-H) and middle molecular mass subunit (NF-M) of bovine neurofilaments, but they did not reduced the electrophoretic mobility of the dephosphorylated form of NF-M and NF-H by phosphorylation nor was the amount of phosphorylation increased by dephosphorylation of NF proteins, indicating that the phosphorylation sites by these kinases are not major in vivo phosphorylation sites at the tail domain. In contrast, cdc2 kinase phosphorylated specifically the dephosphorylated form of NF-H. 4 mol of phosphates were incorporated per mol of NF-H and this phosphorylation returned the electrophoretic mobility of the dephosphorylated form of NF-H to the position of the isolated, fully phosphorylated form of NF-H. Furthermore, the phosphorylation by cdc2 kinase dissociated the binding of dephosphorylated NF-H to microtubules. Phosphorylation sites were located at the carboxyl-terminal tail domain. The KSPXK motif, but not KSPXX, in the repetitive sequence was suggested to be the phosphorylation site by using synthetic peptides.
Laboratory of Cell and Developmental Biology, Faculty of Bioscience, Tokyo Institute of Technology, Yokohama, Japan.
S
S
Hisanaga
M
M
Kusubata
E
E
Okumura
T
T
Kishimoto
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D016900:Neurofilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013116:Spinal Cord / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D046911:Macromolecular Substances; D016900:Neurofilament Proteins; D010455:Peptides; D010750:Phosphoproteins; D011494:Protein Kinases; D016203:CDC2 Protein Kinase