CK2 Phosphorylation Is Required for Regulation of Syntaxin 1A Activity in Ca2+-Triggered Release in Neuroendocrine Cells |
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Authors: | Noa Barak-Broner Dafna Singer-Lahat Dodo Chikvashvili Ilana Lotan |
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Affiliation: | 1.Department of Neurobiology Biochemistry & Biophysics, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv-Yafo 69978, Israel;2.Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv-Yafo 69978, Israel; (D.S.-L.); (D.C.);3.Sagol School of Neuroscience, Tel Aviv University, Ramat Aviv, Tel Aviv-Yafo 69978, Israel |
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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. |
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Keywords: | syntaxin CK2 phosphorylation PC12 cells exocytosis amperometry FRET evoked release |
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