Phosphorylation of SNAP-25 at Ser187 mediates enhancement of exocytosis by a phorbol ester in INS-1 cells

Abstract

Activation of diacylglycerol (DAG) signaling pathways with phorbol esters dramatically enhances Ca2+-triggered exocytosis from both endocrine cells and neurons, however the relevant targets of DAG are controversial. A possible effector mechanism for this signaling pathway is phosphorylation of SNAP-25 (25 kDa synaptosome-associated protein) at Ser187 by PKC. Here, we investigated the role of Ser187 in the enhancement of exocytosis by the phorbol ester PMA (phorbol 12-myristate 13-acetate). We used patch-clamp measurements of membrane capacitance together with photorelease of caged-Ca2+ and membrane depolarization to study exocytosis. Expression of the nonphosphorylatable S187C SNAP-25 mutant did not attenuate the enhancement of exocytosis by PMA in either bovine chromaffin cells or the INS-1 insulin-secreting cell line. To test the effects of Ser187 mutations under conditions in which the endogenous SNAP-25 is disabled, we expressed botulinum toxin serotype E to cleave SNAP-25 in INS-1 cells. Coexpression of a toxin-resistant mutant (TR), but not wild-type SNAP-25, was able to rescue PMA-modulated exocytosis. Coexpression of the toxin with the TR-S187C SNAP-25 mutant was able to completely block the enhancement of exocytosis by PMA in response to photoelevation of [Ca2+]i to low microM levels or to a depolarizing train. The phospho-mimetic S187E mutation enhanced the small, fast burst of exocytosis evoked by photelevation of Ca2+, but, like PMA, had smaller effects on exocytosis evoked by a depolarizing train. This work supports the hypothesis that phosphorylation of Ser187 of SNAP-25 by PKC is a key step in the enhancement of exocytosis by DAG.

Figure 1.

Expression of Ser187 SNAP-25 mutants does not attenuate the enhancement of exocytosis by PMA in chromaffin cells or INS-1 cells. Whole-cell recordings were made from GFP-positive cells. Flash photolysis of caged Ca²⁺ at the time indicated by the arrow leads to a burst of exocytosis (ΔC_m) from the HCSP. This is followed 300 ms later by a train of depolarizing pulses to trigger Ca²⁺ influx (I_Ca) and release of granules that are less responsive to Ca²⁺ (the RRP). A, The top shows sample responses from chromaffin cells expressing GFP–SNAP-25 with the S187C mutation using the Semliki Forest virus expression system in either the absence or presence of 100 nm PMA. The bottom summarizes data obtained from chromaffin cells with the number of cells (N) indicated below each experimental condition. Q_Ca is the integral of the cumulative I_Ca for the 10 depolarizing pulses, whereas ΔC₁₀ is the total increase in capacitance in response to the depolarizing pulses. The data in the absence of PMA are from the study of Yang et al. (2007). B, The top shows sample responses from INS-1 cells expressing GFP–SNAP-25 with the TR and S187C mutations in either the absence or presence of 100 nm PMA. The bottom summarizes data obtained from INS-1 cells.

Figure 2.

A strategy for expressing TR mutations of SNAP-25 that are resistant to BoNT/E cleavage. A, Schematic representation of the SNAP-25 mutants used in this study. GFP is fused to the N terminus of SNAP-25 to serve as a reporter of protein expression. BoNT/E cleaves SNAP-25 between Arg180 and Ile181, but toxin resistance can be conferred with a TR set of mutations (R176P, D179K, M182T). Finally, mutation of Ser187 to Cys precludes phosphorylation by PKC, whereas the Glu mutation is phosphomimetic. B, Four micrographs of confocal fluorescence from live cells demonstrate that expressed GFP–SNAP-25 is primarily localized to the plasma membrane of INS-1 cells. The top left micrograph depicts expression pattern of WT GFP–SNAP-25, whereas the top right micrograph depicts INS-1 cells cotransfected with BoNT/E and GFP–SNAP-25. The bottom micrographs depict expression of GFP–SNAP-25 with the TR/S187C mutations with (bottom right) and without (bottom left) cotransfection with BoNT/E. Scale bar, 10 μm. C, Sample confocal immunofluorescence images demonstrate that TR SNAP-25 and BoNT/E are coexpressed in the same cells. The cells were treated with anti-BoNT/E antibody and excited with a 488 nm laser line to image GFP–SNAP-25 (green pseudocolor), whereas a 559 nm laser line was used to identify BoNT/E immunoreactivity (red pseudocolor). The images were merged to show the coexpression of BoNT/E and TR SNAP-25 in the same cells. Scale bars, 10 μm.

Figure 3.

Expression of TR, but not WT, GFP–SNAP-25 can rescue exocytosis in INS-1 cells coexpressing BoNT/E. A, Sample traces from INS-1 cells cotransfected with BoNT/E and WT (GFP–SNAP-25; black trace) or TR SNAP-25 (red trace). B, Bar graph summary of data obtained from cells expressing WT or TR SNAP-25 with or without coexpression of BoNT/E.

Figure 4.

Incubation of INS-1 cells in PMA leads to rapid phosphorylation of SNAP-25 at Ser187. The S187E phosphomimetic mutant is detected by a Ser187 phospho-specific antibody. A, The time course of SNAP-25 phosphorylation during incubation of INS-1 cells in 100 nm PMA for the indicated period of time. Control lanes include sham incubations (Bath solution) and incubation in the vehicle DMSO for 30 min. Two groups of samples were prepared in parallel and separated using electrophoresis by SDS-PAGE gels. After transfer to membranes, they were probed with either the Ser187 phospho-specific antibody (Pi–SNAP-25; top) or an antibody that recognized the N terminus of SNAP-25 (SNAP-25; middle), respectively. One of the membranes was then stripped and probed with a β-actin antibody (bottom). B, Detection of expressed SNAP-25 mutants in INS-1 cells. INS-1 cells either incubated in PMA (100 nm) or standard bath solution for 30 min before lysis. CON refers to nontransfected cells, whereas WT refers to transfection with GFP–SNAP-25.

Figure 5.

Mutations of Ser187 blocks the enhancement of exocytosis by PMA if endogenous SNAP-25 is disabled by coexpression of BoNT/E. The kinetics of exocytosis from the HCSP are not affected by Ser187 mutations or PMA incubation. All experiments were performed in fluorescent-positive cells cotransfected with BoNT/E and the indicated GFP–SNAP-25 mutant. Cells were used within 1 h after addition of 100 nm PMA. A, Sample traces from cells expressing TR SNAP-25 and BoNT/E incubated in the absence (black trace) or presence (red trace) of PMA. B, Sample traces from cells expressing TR/S187C and BoNT/E incubated in the absence (black trace) or presence (red trace) of PMA. C, Bar graph summarizing data from the indicated number of cells. D, Plot of the rate constant of exocytosis from the HCSP plotted against the post-flash [Ca²⁺]_i for cells expressing either TR (circles), TR/S187C (squares), or TR/S187E (triangles). Filled symbols represent cells exposed to 100 nm PMA. The rate constant was obtained from an exponential fit to the C_m trace after flash photolysis of caged Ca²⁺.

Figure 6.

Immunoblots demonstrate that BoNT/E transfection cleaves transfected WT GFP–SNAP-25 and endogenous SNAP-25 but not TR SNAP-25. Antibodies used for immunoblots are the same as described for Figure 4. Expressed GFP–SNAP-25 is identified as a 52 kDa band, whereas endogenous SNAP-25 is ∼25 kDa. A, BoNT/E transfection abolishes the 52 kDa band recognized by the Ser187 phospho-specific antibody in GFP–SNAP-25 transfected cells. Transfection with BoNT/E leads to a second, lower-molecular-weight SNAP-25 band (highlighted by boxes) that presumably indicates truncated SNAP-25. CON refers to nontransfected cells, whereas WT refers to transfection with GFP–SNAP-25. B, BoNT/E cotransfection does not affect the relative intensity of the 52 kDa band recognized by the Ser187 phospho-specific antibody in TR SNAP-25 or TR/S187E transfected cells. C, Quantification of the relative intensity of the 52 kDa band representing phosphorylated GFP–SNAP-25. The intensity of the 52 kDa band recognized by the Ser187 phospho-specific antibody was divided by the intensity of the 52 kDa band recognized by the N-terminal SNAP-25 antibody. The relative intensity was then normalized to a value of 1.0 under the condition of exposure to PMA without cotransfection with BoNT/E (filled bars).

Figure 7.

Single cell immunofluorescence measurements demonstrate that expression of TR SNAP-25 does not block cleavage of endogenous SNAP-25 by BoNT/E. INS-1 cells were transfected with the TR/S187C SNAP-25 mutant plus or minus BoNT/E as indicated. PMA treatment for 30 min was used to induce phosphorylation of endogenous SNAP-25. Cells were fixed and probed with the pSer187 phospho-specific SNAP-25 antibody. The cells were excited with a 488 nm laser line to image exogenously expressed GFP–SNAP-25 mutant (TR/S187C; green pseudocolor), whereas a 559 nm laser line was used to identify endogenous Pi–SNAP-25 immunoreactivity (red pseudocolor). Coexpression of BoNT/E leads to a loss of Pi–SNAP-25 immunoreactivity presumably because the C terminus containing pSer187 is cleaved by the toxin. Scale bars, 10 μm. A, Sample cell images. B, Bar graph summarizing the fluorescent ratio (pSer187/GFP–SNAP-25) for the indicated number of cells.