Phosphorylation of syntaxin 3B by CaMKII regulates the formation of t-SNARE complexes

Abstract

Ribbon synapses in the retina lack the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system, but instead contain the related isoform syntaxin 3B. Previous studies have demonstrated that syntaxin 3B is essential for synaptic vesicle exocytosis in ribbon synapses, but syntaxin 3B is less efficient than syntaxin 1A in binding the t-SNARE protein SNAP-25 and catalyzing vesicle fusion. We demonstrate here that syntaxin 3B is localized mainly on the presynaptic membrane of retinal ribbon synapses and that a subset of syntaxin 3B is localized in close proximity to the synaptic ribbon. We show further, that syntaxin 3B can be phosphorylated by the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We determine that the phosphorylation site is located close to the N-terminus at T14. Syntaxin 3B with a phosphomimetic mutation (T14E) had a stronger binding affinity for SNAP-25 compared with wild type syntaxin 3B. We propose that phosphorylation of syntaxin 3B by CaMKII can modulate the assembly of the SNARE complex in ribbon synapses of the retina, and might regulate the exocytosis of synaptic vesicles in ribbon synapses.

Keywords: Munc18; Retina; Ribbon synapse; SNAP-25; SNARE complex.

Figure 1. Analysis of the expression of syntaxin 3 protein in different tissues

Top panel: Tissue extracts from retina, brain, kidney and liver where separated by SDS-PAGE and analyzed by western blotting using the syntaxin 3 antibody. The predicted position of syntaxin 3B (33kDa) is labeled with an arrow. A weak band that migrates slightly lower and probably represents a breakdown product is labeled with asterisk. The analysis shows that syntaxin 3 is highly expressed in retina and weakly expressed in the kidney. No expression of syntaxin 3 protein was detectable in brain or liver under these conditions. Bottom panel: The expression of the ubiquitously expressed marker Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed to confirm equal protein concentrations in the tissue extracts.

Figure 2. Syntaxin 3B is found in synaptic layers of the mouse retina

Top panel: Retina sections were labeled with antibodies directed against syntaxin 3 and the ribbon synapses and nuclear markers Ribeye/CTPB2. The synaptic layers (inner plexiform layer (IPL) and outer plexiform layer (OPL)) show strong syntaxin 3B labeling. INL, Inner nuclear layer, ONL, outer nuclear layer; GCL, ganglion cell layer. Bottom panels: Higher magnifications of the OPl and IPL regions. Scale bar = 20 μm.

Figure 3. Syntaxin 3B in photoreceptor terminals partially colocalizes with ribeye

Retinal sections where double labeled with antibodies against syntaxin 3 and ribeye/CTBP2 or the synaptic vesicle marker SV2. Synaptic terminals are marked with asterisks. Some syntaxin 3B is colocalized with ribeye but a large fraction occupies ribeye free parts of the plasma membrane. Scale bar = 5 μm.

Figure 4. Syntaxin 3B is localized on the plasma membrane of bipolar cell terminals

Consecutive optical sections of isolated retinal bipolar cells terminals were double labeled for syntaxin 3B and ribeye/CTBP2. Arrowheads mark the position of individual synaptic ribbons. Scale bar = 5 μ m.

Figure 5. Syntaxin 3B is associated with the synaptic plasma membrane

Subcellular fractionation of mouse retina was performed to analyze the distribution of syntaxin 3B. Synaptosomes (P2) were isolated from mouse retina by differential centrifugation. The synaptosomes were then hypo-osmotically lysed and separated into synaptic plasma membrane fraction (LP1), synaptic vesicle fraction (LP2) and soluble presynaptic protein fractions (LS1 and LS2). Equal protein amounts were analyzed by SDS-PAGE and western blotting using specific antibodies directed against SV2B (ribbon synaptic vesicle marker), syntaxin 3, SNAP-25 and PSD-95 (postsynaptic density protein).

Figure 6. The N-terminal domain reduces binding of syntaxin 3B to SNAP25.

SNAP-25 and Munc18 binding to GST fusion proteins of syntaxins 1A and 3B without transmembrane domains and the isolated syntaxin 3B SNARE domain was analyzed using pulldown assays with retina extract followed by SDS-PAGE and western blotting using SNAP-25 antibodies and GST antibodies (loading control). The structure of the recombinant proteins is depicted below. Two different amounts of GST-syntaxin 3B protein were loaded to allow a better comparison. Recombinant GST fusion proteins of the predicted size are marked with arrows.

Figure 7. Syntaxin 3B is phosphorylated by CaMKII at T14

A. GST-syntaxin fusion proteins were in vitro phosphorylated by CaMKII and Ɣ -³²P ATP, separated by SDS-PAGE and analyzed by autoradiography (³²P) and immunoblotting with GST antibodies. Two different amounts of syntaxin 3B were loaded to allow better comparison with the other proteins. B: Wildtype GST-syntaxin 3B as well as mutated proteins were phosphorylated and analyzed as in A. Wild type syntaxin 3B and mutant proteins GST-syntaxin 3B T81A, S145A, and S187A were phosphorylated similarly, while the mutant protein GST-syntaxin 3B T14A was not phosphorylated, demonstrating that T14 is the phosphorylation site in syntaxin 3B.

Figure 8. A phosphomimetic mutation of syntaxin 3B (T14E) increases the binding affinity to SNAP25

A. GST pulldown assays with retina extract were used to investigate the binding of SNAP-25 and Munc18 to wildtype GST-syntaxin 3B (WT), a phosphomimetic mutant (T14E) and a control mutant (T14A). The pulldown samples were analyzed by SDS-PAGE and immunoblotting with antibodies against Munc18, SNAP-25 and GST. The phosphomimetic T14E mutant protein had a much higher affinity to SNAP-25 than the wildtype or the T14A mutant protein but Munc18 was bound with similar efficiency by all three proteins. B. GST pulldown experiments were performed to directly test the binding of recombinant SNAP-25 to wildtype (WT) or mutant (T14E) GST-syntaxin 3B. The concentration of SNAP-25 in the assay is given above the lanes. The phosphomimetic T14E mutant protein bound recombinant SNAP-25 with a higher affinity than GST-syntaxin 3B. C. Multiple pulldown experiments were performed using recombinant SNAP-25 (0.08 μM) and the amount of bound SNAP-25 was quantified. Results were normalized to the amount bound by the wildtype protein. The T14E mutant protein showed a significantly higher affinity for recombinant SNAP-25 than the wild type protein. (WT: 100.0 ± 15.97%; T14E: 179.5 ± 18.63% (+/- SEM, n=3, p-value= 0.0317 (unpaired two tailed t test)).

Figure 9. Syntaxin 3B in the mouse retina is phosphorylated at T14

A. Left panel (1-3): Western blot analysis of mouse retina extract using a peptide antibody directed against a phosphopeptide derived from the N-terminus of syntaxin 3B detected a band of the predicted size of syntaxin 3B (1). Addition of the immunizing phospopeptide to the western blot blocked the signal (lane 3), whereas the corresponding unphosphorylated peptide had only a weak effect on the phospo-syntaxin 3B signal (lane 2). Right panel (4-6): Retina extract was incubated for 1 hr at 37⁰C in the presence (6) or absence (5) of alkaline phosphatase and analyzed by western blotting using the phospho-syntaxin 3 antibody. Unincubated extract was used as a control (4). Stability of syntaxin 3B protein during the incubation was confirmed by blotting with the syntaxin 3 antibody. B. Retina sections were labeled with the phospho-syntaxin 3 antibody with or without blocking antibodies as in A. Strong labeling of the ribbon synapse containing layers (IPL and OPL) with the phospho-specific antibodies indicates that syntaxin 3B in ribbon synapses is phosphorylated at T14. Scale bar = 20μm.

Figure 10. Model of the regulation of syntaxin 3B in ribbon synapses of the retina

In conventional synapses syntaxin 1A exists in two conformations: “open” and “closed”. The “closed” conformation has the Habc domain (green) folding back on the SNARE domain (blue) and cannot bind to SNAP-25 to form a t-SNARE complex. We propose that syntaxin 3B in ribbon synapses of the retina exists mainly in the closed conformation. Activation of CaMKII by elevated calcium leads to the phosporylation of syntaxin 3B in the proximity of the ribbon. The phosphorylation of syntaxin 3B stabilizes the open conformation that can then efficiently form a t-SNARE complex and increase synaptic vesicle exocytosis.