The distribution of phosphorylation sites among identified proteolytic fragments of mammalian neurofilaments.
Neurofilaments were treated with chymotrypsin or with Staphylococcus aureus V8 protease (V8 protease) and the proteolytic fragments in soluble and particulate centrifugal fractions were identified by immune blotting, using antibodies raised against the Mr = 68,000 (P68), 145,000 (P145), and 200,000 (P200) subunits. The data provide further evidence that each of the three subunits has a different disposition within the filament. A Mr = 160,000 fragment of P200, which may correspond to the side arm projections on neurofilaments, was released into solution by chymotrypsin. In contrast, the proteolytic fragments of P68 and P145 were recovered mainly in the particulate centrifugal fraction, indicating that the two subunits are more closely associated with the filament backbone. Proteolytic cleavage studies on neurofilaments that were 32P-labeled in vivo indicated that the phosphorylated domains in P200 and P145 are localized in a restricted segment of each subunit, which occurs between the chymotryptic and V8 protease cleavage sites. No 32P was associated with the bulk of chymotryptic fragments, which are found in the particulate fraction, are about 40,000 daltons in size, and derive from all three neurofilament subunits. Most of the phosphorylation sites in neurofilaments are peripherally located in the projection domain of P200, suggesting that phosphorylation may modulate interactions between neurofilaments and other neuronal components.
D000818:Animals; D001369:Axons / Q000032:analysis*; D001933:Brain Stem / Q000032:analysis*; D002918:Chymotrypsin; D003599:Cytoskeleton / Q000032:analysis*; D010450:Endopeptidases; D046911:Macromolecular Substances; D008297:Male; D008666:Metalloendopeptidases; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord / Q000032:analysis*
D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D010450:Endopeptidases; D002918:Chymotrypsin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus