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 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.
Ibañez
Cristina
C
Instituto Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain.
Blanes-Mira
Clara
C
Fernández-Ballester
Gregorio
G
Planells-Cases
Rosa
R
Ferrer-Montiel
Antonio
A
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009465:Neuromuscular Agents / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D013696:Temperature; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009465:Neuromuscular Agents; D011993:Recombinant Fusion Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D014443:Tyrosine; D019061:src-Family Kinases; D019274:Botulinum Toxins, Type A