The polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin1A (Syx), was shown by us to act as a fusion clamp in PC12 cells, making release dependent on stimulation by Ca2+. By using a Syx-based FRET probe, we demonstrated that 5RK is absolutely required for a depolarization-induced Ca+2-dependent, close-to-open transition (CDO) of Syx that involves the vesicular SNARE synaptobrevin2 and occurs concomitantly with Ca2+-triggered release. Here, we investigated the mechanism underlying the 5RK requirement, and identified phosphorylation of Syx at Ser-14 (S14) by protein kinase CK2 as a crucial molecular determinant. Following biochemical verification that both endogenous Syx and CSYS are constitutively S14 phosphorylated in PC12 cells, dynamic FRET analysis of phospho-null and phospho-mimetic mutants of CSYS and the use of a CK2 inhibitor revealed that it is the S14 phosphorylation that confers the 5RK requirement. Concomitant amperometric analysis of catecholamine release revealed that the phospho-null mutants do not support release, spontaneous and evoked. Collectively, these results identify a functionally important CK2 phosphorylation site in Syx that is required for 5RK-regulation of CDO and for concomitant Ca2+-triggered release.
bioRxiv Subject Collection: Neuroscience