Protein Kinase C-Dependent Phosphorylation of Synaptosome-Associated Protein of 25 kDa at Ser¹⁸⁷Potentiates Vesicle Recruitment

Abstract

Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser(187), in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser(187) accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.

Synaptobrevin2 is the v-SNARE required for cytotoxic T-lymphocyte lytic granule fusion

“…4). Also, it should be noted that the affinity of SNARE proteins to its partners is regulated by phosphorylation and leads to a different pool size of releasable granules 31,32 . As phosphorylation is a well known regulator of IS formation and function, it is plausible that CTLs regulate SNARE protein affinities dynamically depending on the physiological state.…”

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

“…Whole-cell patch clamp, ratiometric intracellular calcium ([Ca 2ϩ ] i ) measurements, flash photolysis of caged Ca 2ϩ , and amperometry and membrane capacitance measurements were performed as described previously (Nagy et al, 2002). Data were analyzed using Igor Prosoftware (Wavemetrics, Lake Oswego, OR).…”

“…Bovine chromaffin cell preparation was performed essentially as described previously (Nagy et al, 2002). The primary antibodies used were rabbit anti-SNAP-25 (1:3000; catalog no.…”

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

“…One phosphorylation site in the SNARE domain of SNAP25, S187, has been widely studied (Shimazaki et al , 1996). Phosphorylation of S187 increases secretion (Shu et al , 2008) and the crystal structure of the SNARE complex reveals that S187 forms part of the outside surface area of the α‐helical bundle (Fasshauer et al , 1998; Sutton et al , 1998; Nagy et al , 2002). Additional SNARE domain phosphorylation sites have been reported: SNAP23 (S23, T24, and S160) (Polgár et al , 2003) and VAMP2 (T35 and S61) (Hirling & Scheller, 1996; Li et al , 2009), but their involvement in secretion has not been well defined.…”