Neurofilament-L is a protein phosphatase-1-binding protein associated withneuronal plasma membrane and post-synaptic density

Far Westerns with digoxigenin conjugated protein phosphatase-1 (PP1) cataly tic subunit identified PP1-binding proteins in extracts from bovine, rat, a nd human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobi lized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing follo wing cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-bi nding protein as bovine neurofilament-1 (NF-L). NF-L was the major PP1-bind ing protein in purified preparations of bovine spinal cord neurofilaments a nd the cytoskeletal compartment known as post-synaptic density, purified fr om rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, in hibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP 1 catalytic subunit but not the activity of PP2A, another major serine/ thr eonine phosphatase. PP1 binding to bovine NF-L was mapped to the head regio n. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills m any of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoske leton of mammalian neurons.