Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density.
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.
Terry-Lorenzo
R T
RT
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Inoue
M
M
Connor
J H
JH
Haystead
T A
TA
Armbruster
B N
BN
Gupta
R P
RP
Oliver
C J
CJ
Shenolikar
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D006801:Humans; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013116:Spinal Cord / Q000378:metabolism; D013569:Synapses / Q000378:metabolism*
D016900:Neurofilament Proteins; D011994:Recombinant Proteins; C044882:neurofilament protein L; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1