Modulation of botulinum neurotoxin A catalytic domain stability by tyrosine phosphorylation

Botulinum neurotoxin A (BoNT A) is a substrate of the Src family of tyrosine kinases. Here, we report that the BoNT A light chain (LC) is phosphorylated in the tyrosine-71 located at N-terminus. Covalent modification of this residue notably increases the thermal stability of the endopeptidase activi...

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Bibliographic Details
Published in:FEBS letters 2004-12, Vol.578 (1), p.121-127
Main Authors: Ibañez, Cristina, Blanes-Mira, Clara, Fernández-Ballester, Gregorio, Planells-Cases, Rosa, Ferrer-Montiel, Antonio
Format: Article
Language:English
Subjects:
Animals
Botulinum Toxins, Type A - chemistry
Botulinum Toxins, Type A - genetics
Botulinum Toxins, Type A - metabolism
Catalytic Domain
Clostridial
Excitability
Exocytosis
Membrane Proteins - metabolism
Models, Molecular
Nerve Tissue Proteins - metabolism
Neuromuscular Agents - chemistry
Neuromuscular Agents - metabolism
Neurotransmission
PC12 Cells
Phosphorylation
Protease
Rats
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
SNAP25
src-Family Kinases - metabolism
Synaptosomal-Associated Protein 25
Temperature
Tyrosine - metabolism
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Summary:Botulinum neurotoxin A (BoNT A) is a substrate of the Src family of tyrosine kinases. Here, we report that the BoNT A light chain (LC) is phosphorylated in the tyrosine-71 located at N-terminus. Covalent modification of this residue notably increases the thermal stability of the endopeptidase activity, without affecting its catalytic efficacy. Similarly, mutation of this residue specifically affected the protein stability but not its endopeptidase function. Fusion of the Tat-translocating domain to the N-terminus of the enzyme produced a cell permeable, functional enzyme, as evidenced by immunocytochemistry and by the cleavage of cytosolic SNAP25 in intact PC12 cells. Noteworthy, truncation of cellular SNAP25 was reduced in cells when the Src kinase activity was inhibited with a specific antagonist, implying that tyrosine phosphorylation of BoNT A LC modulates the in vivo proteolytic activity of the neurotoxin. Taken together, these findings substantiate the tenet that tyrosine phosphorylation of BoNT A LC could be an important modulatory strategy of the neurotoxin stability and suggest that the phosphorylated neurotoxin may be a relevant molecule in vivo.
ISSN:0014-5793
1873-3468
DOI:10.1016/j.febslet.2004.10.084