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Mol Biol Cell. Feb 1994; 5(2): 161–172.
PMCID: PMC301022
Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M, and NF-H by the catalytic subunit of cAMP-dependent protein kinase.
S Hisanaga, Y Matsuoka, K Nishizawa, T Saito, M Inagaki, and N Hirokawa
Department of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo, Japan.
Abstract
Phosphorylation of neurofilament-L protein (NF-L) by the catalytic subunit of cAMP-dependent protein kinase (A-kinase) inhibits the reassembly of NF-L and disassembles filamentous NF-L. The effects of phosphorylation by A-kinase on native neurofilaments (NF) composed of three distinct subunits: NF-L, NF-M, and NF-H, however, have not yet been described. In this paper, we examined the effects of phosphorylation of NF proteins by A-kinase on both native and reassembled filaments containing all three NF subunits. In the native NF, A-kinase phosphorylated each NF subunit with stoichiometries of 4 mol/mol for NF-L, 6 mol/mol for NF-M, and 4 mol/mol for NF-H. The extent of NF-L phosphorylation in the native NF was nearly the same as that of purified NF-L. However, phosphorylation did not cause the native NFs to disassemble into oligomers, as was the case for purified NF-L. Instead, partial fragmentation was detected in sedimentation experiments and by electron microscopic observations. This is probably not due to the presence of the three NF subunits in NF or to differences in phosphorylation sites because reassembled NF containing all three NF subunits were disassembled into oligomeric forms by phosphorylation with A-kinase and the phosphorylation by A-kinase occurred at the head domain of NF-L whether NF were native or reassembled. Disassembling intermediates of reassembled NF containing all three NF subunits were somewhat different from disassembling intermediates of NF-L. Thinning and loosening of filaments was frequently observed preceding complete disassembly. From the fact that the thinning was also observed in the native filaments phosphorylated by A-kinase, it is reasonable to propose the native NF is fragmented through a process of thinning that is stimulated by phosphorylation in the head domain of the NF subunits.
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