The effect of protein phosphatase inhibitors on neurofilament assembly

Treatment of dissociated cultures of rat dorsal root ganglia with 1 $ mu$M okadaic acid (OA), a serine/threonine protein phosphatase inhibitor, caused a decrease in the electrophoretic mobilities of all three neurofilament (NF) subunits, indicating an increase in their phosphorylation levels. In addition OA treatment led to the asynchronous solubilization of the subunits in Triton X-100 (Triton). This fragmentation corresponded with striking changes in the immunofluorescent staining pattern of NFs. Short-term OA treatment was fully reversible within 10 hrs. upon removal of the inhibitor from the culture medium.Based on the response to varying concentrations of both OA and calyculin A, another serine/threonine protein phosphatase inhibitor, fragmentation of NFs was inferred to be due to the inhibition of protein phosphatase-2A activity while the reduction in electrophoretic mobility of subunits was due to the additional inhibition of protein phosphatase-1. The OA-induced phosphate moieties on the low molecular weight NF subunit (NF-L) were localized, by chemical cleavage analysis, to the amino-terminal head domain and were removed in vitro by the catalytic subunit of protein phosphatase-2A but not by the catalytic subunit of protein phosphatase-1.Two-dimensional phosphopeptide mapping of the mid-sized NF subunit (NF-M) revealed that the OA-induced phosphate moieties on this subunit were also in the amino-terminal head domain. The Triton-soluble NF fragments were found, by immunoprecipitation under non-denaturing conditions, to be heteromers composed of NF-L/NF-M and NF-L/high molecular weight NF subunit. Similar heteromers were found in the small, naturally occurring Triton-soluble NF fraction from dorsal root ganglion cultures indicating that OA treatment amplifies a natural, dynamic process involving NF assembly/disassembly