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.