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 mu M okadaic aci d leads to a fragmentation of neurofilaments and a reduction in the el ectrophoretic mobilities of the three subunits on SDS-polyacrylamide g els (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 reducti on in electrophoretic mobility to inhibition of protein phos- phatase- 1. Okadaic acid treatment led to an increase in amino-terminal, relati ve to carboxyl terminal, domain phosphorylation in the low molecular w eight (NF-L) subunit in the Triton X-100-soluble and -insoluble fracti ons. The purified catalytic subunit of protein phosphatase-2A dephosph orylated P-32-labeled NF-L and the middle molecular weight subunit fro m okadaic acid-treated cultures, whereas the catalytic subunit of prot ein phosphatase-1 had no effect. In the case of NF-L, phosphate moieti es were preferentially removed from the amino terminal domain. These r esults show that the amino-terminal domain of NF-L can be phosphorylat ed in situ and implicate protein phosphatase-2A in the turnover of pho sphate moieties in this domain.