Increased solubility of high-molecular-mass neurofilament subunit by suppression of dephosphorylation: its relation to axonal transport.
To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200-kDa subunit (NF-H) in the axon was soluble in the 1% Triton-containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68-kDa subunit (NF-L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF-H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP-dependent protein kinase, which resulted in NF disassembly. The carboxy-terminal phosphorylation state of NF-H probed with the phosphorylation-sensitive antibodies was also not directly related to solubility. The solubility of NF-H did not differ along the nerve. In contrast, the solubility of L-[35S]methionine-labeled, transported NF-H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF-H solubility is positively correlated with the rate of NF transport.
Tsuda
M
M
Department of Molecular and Cellular Neurobiology, Gunma University School of Medicine, Gunma-ken, Japan.
Tashiro
T
T
Komiya
Y
Y
D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D008297:Male; D016900:Neurofilament Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000737:chemistry / Q000201:enzymology; D017830:Octoxynol; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D013126:Spinal Nerve Roots / Q000166:cytology
D016900:Neurofilament Proteins; C051774:neurofilament protein H; D017830:Octoxynol; D017868:Cyclic AMP-Dependent Protein Kinases; D010744:Phosphoric Monoester Hydrolases