Phosphomimetic mutation of Ser-187 of SNAP-25 increases both syntaxin binding and highly Ca2+-sensitive exocytosis

Abstract

The phosphorylation targets that mediate the enhancement of exocytosis by PKC are unknown. PKC phosporylates the SNARE protein SNAP-25 at Ser-187. We expressed mutants of SNAP-25 using the Semliki Forest Virus system in bovine adrenal chromaffin cells and then directly measured the Ca2+ dependence of exocytosis using photorelease of caged Ca2+ together with patch-clamp capacitance measurements. A flash of UV light used to elevate [Ca2+](i) to several microM and release the highly Ca2+-sensitive pool (HCSP) of vesicles was followed by a train of depolarizing pulses to elicit exocytosis from the less Ca2+-sensitive readily releasable pool (RRP) of vesicles. Carbon fiber amperometry confirmed that the amount and kinetics of catecholamine release from individual granules were similar for the two phases of exocytosis. Mimicking PKC phosphorylation with expression of the S187E SNAP-25 mutant resulted in an approximately threefold increase in the HCSP, whereas the response to depolarization increased only 1.5-fold. The phosphomimetic S187D mutation resulted in an approximately 1.5-fold increase in the HCSP but a 30% smaller response to depolarization. In vitro binding assays with recombinant SNARE proteins were performed to examine shifts in protein-protein binding that may promote the highly Ca2+-sensitive state. The S187E mutant exhibited increased binding to syntaxin but decreased Ca2+-independent binding to synaptotagmin I. Mimicking phosphorylation of the putative PKA phosphorylation site of SNAP-25 with the T138E mutation decreased binding to both syntaxin and synaptotagmin I in vitro. Expressing the T138E/ S187E double mutant in chromaffin cells demonstrated that enhancing the size of the HCSP correlates with an increase in SNAP-25 binding to syntaxin in vitro, but not with Ca2+-independent binding of SNAP-25 to synaptotagmin I. Our results support the hypothesis that exocytosis triggered by lower Ca2+ concentrations (from the HCSP) occurs by different molecular mechanisms than exocytosis triggered by higher Ca2+ levels.

Figure 1.

Phosphorylation of purified recombinant SNAP-25 on Ser-187. Recombinant SNAP-25 was phosphorylated using either PKC or PKA with 100 μM ATP. Kinase reactions were stopped with an equal volume of Laemmli buffer, boiled, and resolved on SDS-PAGE. Phosphorylation on Ser-187 was assessed by immunoblotting with a phosphospecific antibody for pS187 of SNAP-25 (Pi-SNAP-25). (A) Comparison of PKA and PKC as Ser-187 kinases. (B) Comparison of monomeric SNAP-25 and heterodimeric syntaxin/SNAP-25 complexes as PKC substrates. (C) Effect of mutations at Ser-187 on Pi-SNAP-25 immunoreactivity.

Figure 2.

Measurement of the HCSP and depolarization-evoked exocytosis in chromaffin cells expressing GFP-SNAP-25 using the Semliki Forrest virus expression system. Whole-cell recordings were made from cells exhibiting GFP fluorescence. Flash photolysis of caged Ca²⁺ at the time indicated by the arrow leads to a burst of exocytosis (increase in membrane capacitance, ΔC_m) from the HCSP. This is followed 300 ms later by a train of 10 depolarizing pulses to trigger Ca²⁺ influx (I_Ca) in order to accelerate release of chromaffin granules that are less responsive to Ca²⁺ (the RRP). (A) Sample traces from an individual cell. (B) Averaged responses from 18 cells. Note that, whereas [Ca²⁺]_i is elevated uniformly throughout the cell upon photolysis of caged Ca²⁺, the near-membrane [Ca²⁺]_i that triggers release from the RRP is likely to be significantly higher than the spatially averaged [Ca²⁺]_i reported by the fluorescent indicators (middle trace) following depolarization-triggered Ca²⁺ influx (I_Ca).

Figure 3.

Expression of the phosphomimetic SNAP-25 mutation S187E leads to a larger HCSP size than the S187C mutation that precludes phosphorylation by PKC. (A) Sample responses of individual cells expressing either the S187C mutation (black traces) or the S187E mutation (red traces). (B) Averaged responses from cells expressing the S187C mutation (black traces, 35 cells) or S187E mutation (red traces, 40 cells).

Figure 4.

The HCSP is composed of granules with similar catecholamine content and release kinetics as the RRP. Carbon fiber amperometry to measure catecholamine release was combined with whole-cell capacitance measurements. Flash photolysis of NP-EGTA to elevate [Ca²⁺]_i to several μM to release the HCSP was followed 1 s later by 10 depolarizing pulses, 30 ms in duration, to release the RRP. Amperometric spikes (I_F, top trace) following flash photolysis where assigned to the HCSP whereas spikes following membrane depolarization were designated as RRP. (A) Sample experiment. (B) Average of 44 traces from 16 cells. Q (red line) represents the integral of the amperometric current. (C) Cumulative histogram of the charge (Q) of individual amperometric spikes from the HCSP (dashed line, 135 spikes, median = 0.80 pC) and the RRP (solid line, 274 spikes, median = 1.1 pC). (D) Cumulative histogram of the spike duration at half-maximal current (t_1/2) for the HCSP (dashed line, median = 6 ms) and the RRP (solid line, median = 8 ms)

Figure 5.

Binding of wild-type and mutant SNAP-25 to GST-syntaxin 1A. 200 ng of GST-syntaxin was bound to wells in glutathione-coated 96-well plates and incubated with the indicated concentrations of wild-type or mutant SNAP-25. Bound SNAP-25 was detected by ELISA using a monolonal RGS-His₆ antibody. Binding is expressed as a percentage of the maximum binding obtained. Data points are the mean of four experiments, ± SEM.

Figure 6.

Binding of wild-type and mutant SNAP-25 to GST-synaptotagmin 1. 200 ng of GST-synaptotagmin (cytoplasmic domain) was bound to wells in glutathione-coated 96-well plates and incubated with the indicated concentrations of wild-type or mutant SNAP-25. Bound SNAP-25 was detected by ELISA using a monolonal SNAP-25 antibody. Binding is expressed as a percentage of the maximum binding obtained. Data points are the mean of four experiments, ± SEM.

Figure 7.

Expression of the PKC phosphomimetic SNAP-25 mutant S187E in a background of the PKA phosphomimetic mutation T138E increases the size of the HCSP. (A) Sample responses of individual cells expressing the T138E mutation (black traces) or the double T138E/S187E mutation (EE, red traces). (B) Averaged responses from cells expressing either T138E (black traces, 26 cells) or EE (red traces, 30 cells).

Figure 8.

Summary data of granule pool sizes for cells expressing mutations of SNAP-25. Comparisons were made between cells expressing GFP-SNAP-25 mutants and those expressing “wild-type” GFP-SNAP-25. The dashed lines indicate values of ΔC₁₀ and the HCSP size for wt GFP-SNAP-25. A single asterisk indicates P < 0.01, whereas a double asterisk indicates P < 0.001. The numbers of cells (N) for each type of experiment are indicated.