Phosphorylation of the head domain of neurofilament protein (NF-M): a factor regulating topographic phosphorylation of NF-M tail domain KSP sites in neurons.
In neurons the phosphorylation of neurofilament (NF) proteins NF-M and NF-H is topographically regulated. Although kinases and NF subunits are synthesized in cell bodies, extensive phosphorylation of the KSP repeats in tail domains of NF-M and NF-H occurs primarily in axons. The nature of this regulation, however, is not understood. As obligate heteropolymers, NF assembly requires interactions between the core NF-L with NF-M or NF-H subunits, a process inhibited by NF head domain phosphorylation. Phosphorylation of head domains at protein kinase A (PKA)-specific sites seems to occur transiently in cell bodies after NF subunit synthesis. We have proposed that transient phosphorylation of head domains prevents NF assembly in the soma and inhibits tail domain phosphorylation; i.e. assembly and KSP phosphorylation in axons depends on prior dephosphorylation of head domain sites. Deregulation of this process leads to pathological accumulations of phosphorylated NFs in the soma as seen in some neurodegenerative disorders. To test this hypothesis, we studied the effect of PKA phosphorylation of the NF-M head domain on phosphorylation of tail domain KSP sites. In rat cortical neurons we showed that head domain phosphorylation of endogenous NF-M by forskolin-activated PKA inhibits NF-M tail domain phosphorylation. To demonstrate the site specificity of PKA phosphorylation and its effect on tail domain phosphorylation, we transfected NIH3T3 cells with NF-M mutated at PKA-specific head domain serine residues. Epidermal growth factor stimulation of cells with mutant NF-M in the presence of forskolin exhibited no inhibition of NF-tail domain phosphorylation compared with the wild type NF-M-transfected cells. This is consistent with our hypothesis that transient phosphorylation of NF-M head domains inhibits tail domain phosphorylation and suggests this as one of several mechanisms underlying topographic regulation.
Zheng
Ya-Li
YL
Laboratory of Neurochemistry, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Li
Bing-Sheng
BS
Veeranna
Pant
Harish C
HC
D016475:3T3 Cells; D000818:Animals; D002525:Cerebellar Cortex / Q000166:cytology; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016900:Neurofilament Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000737:chemistry*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection
D016900:Neurofilament Proteins; C061195:neurofilament protein M; D005576:Colforsin; D004815:Epidermal Growth Factor; D017868:Cyclic AMP-Dependent Protein Kinases