Abstract
Vesicular (v)- and target (t)-SNAREs play essential roles in intracellular membrane fusion through the formation of cytoplasmic α-helical bundles. Several v-SNAREs have a Longin N-terminal extension that, by promoting a closed conformation, plays an autoinhibitory function and decreases SNARE complex formation and membrane fusion efficiency. The molecular mechanism leading to Longin v-SNARE activation is largely unknown. Here we find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise. In search of a potential downstream mechanism, we found that TI-VAMP is phosphorylated in vitro by c-Src kinase on tyrosine 45 of the Longin domain. Accordingly, a mutation of tyrosine 45 into glutamate, but not phenylalanine, activates both t-SNARE binding and exocytosis. Activation of TI-VAMP-mediated exocytosis thus relies on tyrosine phosphorylation.
Highlights
The mechanism of activation of Longin vesicular sensitive factor attachment protein receptors (SNAREs), TI-VAMP/VAMP7, which is autoinhibited by its Longin domain, is largely unknown
We find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise
We found that TI-VAMP exocytosis is regulated by long lasting treatment with insulin or insulin-like growth factor-1 (IGF-1) and that c-Src kinase is involved in the Tyr-45 phosphorylation of the Longin domain (LD) of TI-VAMP
Summary
The mechanism of activation of Longin vesicular SNAREs, TI-VAMP/VAMP7, which is autoinhibited by its Longin domain, is largely unknown. Significance: Exocytosis mediated by TI-VAMP can be regulated by release of Longin domain autoinhibition. Several v-SNAREs have a Longin N-terminal extension that, by promoting a closed conformation, plays an autoinhibitory function and decreases SNARE complex formation and membrane fusion efficiency. We find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise. We found that TI-VAMP exocytosis is regulated by long lasting treatment with insulin or insulin-like growth factor-1 (IGF-1) and that c-Src kinase is involved in the Tyr-45 phosphorylation of the LD of TI-VAMP. We demonstrated that the Tyr-45 mutation in Glu of TI-VAMP is more active for plasma membrane t-SNARE binding This modification further promotes axonal growth and TI-VAMPmediated exocytosis in hippocampal neurons
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