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bioRxiv · 10.1101/2020.08.20.258863

CK2 Phosphorylation is required for Regulation of Syntaxin 1A activity in Ca2+-triggered release in neuroendocrine cells

Barak-Broner, N.·Singer-Lahat, D.·Chikvashvili, D.·Lotan, I.
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Abstract

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. Summary statementMany phospho-proteins participate in vesicle exocytosis. We show that a recently identified structural transition of syntaxin1A that accompanies Ca2+-regulated exocytosis in neuroendocrine cells is controlled by CK2 phosphorylation of syntaxin1A.

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Barak-Broner, N., Singer-Lahat, D., Chikvashvili, D., Lotan, I.. 2020-08-21. CK2 Phosphorylation is required for Regulation of Syntaxin 1A activity in Ca2+-triggered release in neuroendocrine cells. https://doi.org/10.1101/2020.08.20.258863

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