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Tyrosine phosphorylation modulates the activity of clostridial neurotoxins
A V Ferrer-Montiel1, J M Canaves, B R DasGupta
1Department of Biology, University of California San Diego, La Jolla, California 92093-0366, USA.
Abstract:
Clostridial neurotoxins' metalloprotease domain selectively cleaves proteins implicated in the process of synaptic vesicle fusion with the plasma membrane and, accordingly, blocks neurotransmitter release into the synaptic cleft. Here we investigate the potential modulation of these neurotoxins by intracellular cascades triggered by environmental signals, which in turn may alter its activity on target substrates. We report that the nonreceptor tyrosine kinase Src phosphorylates botulinum neurotoxins A, B, and E and tetanus neurotoxin. Protein tyrosine phosphorylation of serotypes A and E dramatically increases both their catalytic activity and thermal stability, while dephosphorylation reverses the effect. This suggests that the biologically significant form of the neurotoxins inside neurons is phosphorylated. Indeed, in PC12 cells in which tyrosine kinases such as Src and PYK2 are highly abundant, stimulation by membrane depolarization in presence of extracellular calcium induces rapid and selective tyrosine phosphorylation of internalized light chain, the metalloprotease domain, of botulinum toxin A. These findings provide a conceptual framework to connect intracellular signaling pathways involving tyrosine kinases, G-proteins, phosphoinositides, and calcium with the action of botulinum neurotoxins in abrogating vesicle fusion and neurosecretion.
Insights
Environmental signals modulate clostridial neurotoxins. The nonreceptor tyrosine kinase Src phosphorylates botulinum neurotoxins and tetanus neurotoxin, enhancing their activity and stability within neurons.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Clostridial neurotoxins inhibit neurotransmitter release by cleaving proteins essential for synaptic vesicle fusion.
- The activity of these neurotoxins may be influenced by intracellular signaling pathways.
Purpose of the Study:
- To investigate the modulation of clostridial neurotoxins by intracellular cascades.
- To determine if environmental signals can alter neurotoxin activity.
Main Methods:
- Investigated the effect of nonreceptor tyrosine kinase Src on botulinum neurotoxins (A, B, E) and tetanus neurotoxin.
- Analyzed the impact of protein tyrosine phosphorylation on neurotoxin catalytic activity and thermal stability.
- Examined neurotoxin phosphorylation in PC12 cells stimulated by membrane depolarization and calcium.
Main Results:
- Src phosphorylates botulinum neurotoxins A, B, E, and tetanus neurotoxin.
- Tyrosine phosphorylation significantly increases the catalytic activity and thermal stability of botulinum neurotoxin serotypes A and E.
- Dephosphorylation reverses these effects, indicating phosphorylation is the biologically relevant state.
- Internalized botulinum toxin A light chain undergoes rapid, selective tyrosine phosphorylation in stimulated PC12 cells.
Conclusions:
- Intracellular tyrosine phosphorylation by kinases like Src represents a key regulatory mechanism for clostridial neurotoxins.
- This provides a link between intracellular signaling pathways (tyrosine kinases, calcium) and neurotoxin action on vesicle fusion and neurosecretion.