Increased phosphorylation of the amino-terminal domain of the low molecular weight neurofilament subunit in okadaic acid-treated neurons.
Treatment of rat dorsal root ganglion cultures with 1 microM okadaic acid leads to a fragmentation of neurofilaments and a reduction in the electrophoretic mobilities of the three subunits on SDS-polyacrylamide gels (Sacher, M. G., Athlan, E. S., and Mushynski, W. E. (1992) Biochem. Biophys. Res. Commun. 186, 524-530). Based on the observed response to varying concentrations of okadaic acid, fragmentation was inferred to be due to inhibition of protein phosphatase-2A activity and reduction in electrophoretic mobility to inhibition of protein phosphatase-1. Okadaic acid treatment led to an increase in amino-terminal, relative to carboxyl-terminal, domain phosphorylation in the low molecular weight (NF-L) subunit in the Triton X-100-soluble and -insoluble fractions. The purified catalytic subunit of protein phosphatase-2A dephosphorylated 32P-labeled NF-L and the middle molecular weight subunit from okadaic acid-treated cultures, whereas the catalytic subunit of protein phosphatase-1 had no effect. In the case of NF-L, phosphate moieties were preferentially removed from the amino-terminal domain. These results show that the amino-terminal domain of NF-L can be phosphorylated in situ and implicate protein phosphatase-2A in the turnover of phosphate moieties in this domain.
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
M G
MG
Sacher
E S
ES
Athlan
W E
WE
Mushynski
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004988:Ethers, Cyclic / Q000633:toxicity*; D005727:Ganglia, Spinal / Q000378:metabolism; D046911:Macromolecular Substances; D008387:Marine Toxins; D016900:Neurofilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D051381:Rats
D004988:Ethers, Cyclic; D046911:Macromolecular Substances; D008387:Marine Toxins; D016900:Neurofilament Proteins; D010080:Oxazoles; D010446:Peptide Fragments; D010750:Phosphoproteins; D019319:Okadaic Acid; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2