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Int J Mol Sci
2021 Dec 17;2224:. doi: 10.3390/ijms222413556.
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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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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.
Figure 1. Syx and CSYS are targets for CK2 phosphorylation. (A) Domain structure of Syx and the CSYS FRET probe. (B) CSYS is a target for CK2 phosphorylation similarly to native Syx. (a) Native PC12 cells were immunoprecipitated with anti-Syx antibody and immunoblotted either with anti-Syx antibody (IB-Syx; upper panel) or with Syx S14 phospho-specific antibody (IB- S14 phos; lower panel). Brain membranes (BM) were loaded as a control. (b) Following immunoprecipitation with anti-YFP antibody, proteins of PC12 cells transfected with CSYS were subjected to Western blot analysis with either anti-Syx antibody (IB-Syx; upper panel) or with Syx S14 phospho-specific antibody (IB-S14 phos; lower panel). Molecular markers are shown on the left. (c) Xenopus oocytes expressing CSYS and Syx proteins were immunoprecipitated with anti-Syx antibody (35S Syx; left panel) and immunoblotted with Syx S14 phospho-specific antibody (IB-S14 phos; right panel).
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