Interaction of the tail domain of high molecular weight subunits of neurofilaments with the COOH-terminal region of tubulin and its regulation by tau protein kinase II.
We previously showed that neurofilaments interact with microtubules (MTs) via their high molecular weight subunits (NF-H) after alkaline phosphatase treatment. Here we studied the effects of phosphorylation of NF-H on this interaction. tau protein kinase II, Ser/Thr protein kinase, phosphorylated NF-H in the tail domain, decreased its electrophoretic mobility to a native level, and also restored its property to be less interactive with MTs. Phosphorylation by cAMP-dependent protein kinase caused no shift of electrophoretic mobility or dissociation from MTs. We conclude that the tail domain of NF-H directly interacts with the MT surface, and the interaction is regulated via phosphorylation of the tail domain of NF-H by Ser/Thr protein kinase like tau protein kinase II. To characterize the binding domain of NF-H on MTs, subtilisin digestion of MTs and competition analysis with the MT binding fragment of tau protein were performed. The dissociation constant of NF-H to subtilisin MTs was higher than that to intact MTs. The maximum binding of NF-H was reduced when tau fragments existed. These results revealed that the COOH-terminal region of tubulin is involved in the binding to NF-H, and the NF-H and microtubule-associated protein binding domains are closely apposed on the surface of MTs.
Miyasaka
H
H
Department of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo, Japan.
Okabe
S
S
Ishiguro
K
K
Uchida
T
T
Hirokawa
N
N
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D015153:Blotting, Western; D002417:Cattle; D002918:Chymotrypsin; D051360:Cyclin-Dependent Kinase 5; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013116:Spinal Cord / Q000378:metabolism; D013381:Subtilisins; D014404:Tubulin / Q000378:metabolism*; D016875:tau Proteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D016900:Neurofilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014404:Tubulin; D016875:tau Proteins; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; C079134:tau protein kinase II; D013381:Subtilisins; D002918:Chymotrypsin