Abstract
Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.
Highlights
Modification of signal transduction pathways within the synapse following neuronal activity is thought to be a mechanism underlying learning and memory in higher organisms
Efforts to identify PP1 regulators using a yeast twohybrid screen of a rat brain cDNA library yielded spinophilin, a protein that is localized to dendritic spines [22]
These studies suggest that yotiao, which associates with the NR1A subunit of the NMDA receptor [11], assembles a PKAPP1 signaling module that regulates synaptic transmission mediated by the NMDA receptor
Summary
Materials—Thrombin and Staphyloccocus V8 protease were purchased from Sigma. Microcystin LR was purchased from Calbiochem. Bovine brain was homogenized in 5 volumes of 0.25 M STM buffer (0.25 M sucrose, 5 mM Tris-HCl, pH 7.2, 1 mM MgCl2, 0.1% (v/v) -mercaptoethanol, 0.1 mM EDTA, 1 mM benzamidine, and 1 mM PMSF). Bovine spinal cord was homogenized in 2 volumes of 1 mM sodium phosphate, pH 8.0, 5 mM EDTA, and 1 mM -mercaptoethanol and centrifuged at 100,000 ϫ g for 30 min. The sample was applied to a DEAE-cellulose column and the neurofilaments eluted with a gradient of 25–200 mM NaCl. Fractions containing neurofilaments, determined by their electrophoretic migration and immunoreactivity with anti-NF-L, NF-M, and NF-H antibodies, were pooled and dialyzed into 10 mM PIPES, pH 6.8, 150 mM NaCl, 1 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.1 mM PMSF, and 1 g/ml leupeptin. The assay was restricted to a maximum of 20% release of total phosphate to ensure linearity
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