Abstract
The polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin1A (Syx), was previously shown by us to act as a fusion clamp in PC12 cells, as charge neutralization of 5RK promotes spontaneous and inhibits Ca2+-triggered release. Using a Syx-based FRET probe (CSYS), we demonstrated that 5RK is required for a depolarization-induced Ca+2-dependent opening (close-to-open transition; CDO) of Syx, which involves the vesicular SNARE synaptobrevin2 and occurs concomitantly with Ca2+-triggered release. Here, we investigated the mechanism underlying the CDO requirement for 5RK and identified phosphorylation of Syx at Ser-14 (S14) by casein kinase 2 (CK2) as a crucial molecular determinant. Thus, 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 the S14 phosphorylation confers the CDO requirement for 5RK. In accord, amperometric analysis of catecholamine release revealed that the phospho-null mutant does not support Ca2+-triggered release. These results identify a functionally important CK2 phosphorylation of Syx that is required for the 5RK-regulation of CDO and for concomitant Ca2+-triggered release. Further, also spontaneous release, conferred by charge neutralization of 5RK, was abolished in the phospho-null mutant.
Highlights
Ca2+ -triggered exocytosis is a fundamental cellular process mediated by the SNARE proteins, syntaxin 1A (Syx), synaptobrevin2 (Syb2), and SNAP-25 [1,2]
Acknowledging the potential relevance of casein kinase 2 (CK2)-induced S14 phosphorylation of Syx to exocytosis, we used dynamic fluorescence resonance energy transfer (FRET) and amperometry analyses to explore the possibility that S14 phosphorylation is involved in the molecular/cellular events that endow 5RK its central role
This study demonstrates the importance of CK2 phosphorylation of Syx at S14 in the regulation of a depolarization-induced Ca2+ -dependent opening of Syx, and in Ca2+ triggered release
Summary
Ca2+ -triggered exocytosis is a fundamental cellular process mediated by the SNARE proteins, syntaxin 1A (Syx), synaptobrevin (Syb2), and SNAP-25 [1,2]. In the ‘open’ conformation Syx can participate in the formation of SNARE complexes [4]. The N-peptide, harbors a specific region of interest at Ser-14 (S14), which is the sole target in Syx for phosphorylation by casein kinase. A number of observations over the past few years have suggested a potential role for CK2 phosphorylation of S14 in Syx in the direct modulation of exocytosis: (i) S14 phosphorylation increased through development in parallel with synapse development and maturation in rat brains [12];
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