Myosin phosphatase and RhoA-activated kinase modulate neurotransmitter release by regulating SNAP-25 of SNARE complex

<div><p>Reversible phosphorylation of neuronal proteins plays an important role in the regulation of neurotransmitter release. Myosin phosphatase holoenzyme (MP) consists of a protein phosphatase-1 (PP1) catalytic subunit (PP1c) and a regulatory subunit, termed myosin phosphatase targeting subunit (MYPT1). The primary function of MP is to regulate the phosphorylation level of contractile proteins; however, recent studies have shown that MP is localized to neurons, and is also involved in the mediation of neuronal processes. Our goal was to investigate the effect of RhoA-activated kinase (ROK) and MP on the phosphorylation of one potential neuronal substrate, the synaptosomal-associated protein of 25 kDa (SNAP-25). SNAP-25 is a member of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, along with synaptobrevin and syntaxin, and the primary role of SNAP25 is to mediate vesicle fusion. We showed that MYPT1 interacts with SNAP-25, as revealed by immunoprecipitation and surface plasmon resonance based binding studies. Mass spectrometry analysis and <i>in vitro</i> phosphorylation/dephosphorylation assays demonstrated that ROK phosphorylates, while MP dephosphorylates, SNAP-25 at Thr138. Silencing MYPT1 in B50 neuroblastoma cells increased phosphorylation of SNAP-25 at Thr138. Inhibition of PP1 with tautomycetin increased, whereas inhibition of ROK by H1152, decreased the phosphorylation of SNAP-25 at Thr138 in B50 cells, in cortical synaptosomes, and in brain slices. In response to the transduction of the MP inhibitor, kinase-enhanced PP1 inhibitor (KEPI), into synaptosomes, an increase in phosphorylation of SNAP-25 and a decrease in the extent of neurotransmitter release were detected. The interaction between SNAP-25 and syntaxin increased with decreasing phosphorylation of SNAP-25 at Thr138, upon inhibition of ROK. Our data suggest that ROK/MP play a crucial role in vesicle trafficking, fusion, and neurotransmitter release by oppositely regulating the phosphorylation of SNAP-25 at Thr138.</p></div

Treatment of B50 neuroblastoma cells with PP1 and ROK inhibitors.

<p>B50 cells were incubated with tautomycetin (TMC), a PP1 specific inhibitor, or H1152, a ROK inhibitor, for 1 hour and 30 minutes respectively. Untreated cells served as controls. (A) Viability of B50 cells were examined using MTT assay. Values are mean±SEM (n = 6), p = 0.4645. (B) Effect of inhibitors on protein phosphatase activity. Values are mean±SEM (n = 3), p<0.0001. (C) The effect of the inhibitory treatment on the phosphorylation level of Thr138 in SNAP-25 was assessed by Western blots using anti-SNAP25^pThr138. Values are mean±SEM (n = 3), p = 0.0027. Amounts of SNAP-25 and actin were also determined as loading controls. Relative changes in the phosphorylation levels were determined by densitometry of the bands. Means ±SEM (n = 5), ANOVA and Dunnett’s post hoc analysis, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.</p

Effect of PP1 and ROK inhibitors on the phosphorylation level of SNAP-25 at Thr-138 and on exocytosis in mouse cortical synaptosomes.

<p>(A) Flag-KEPI, a myosin phosphatase specific inhibitor protein, was introduced into the synaptosomes by the freeze-thaw method. To distinguish the effect of Flag-tag from Flag-KEPI, Flag-peptide was also introduced. Control synaptosomes underwent the freeze-thaw process, but no protein or peptide was added. Transduction of Flag-KEPI into synaptosomes was verified by western blot analysis using anti-Flag antibody. Relative phosphorylation levels were detected by anti-CPI17^pThr38 antibody. C-kinase activated phosphatase inhibitor protein of 17 kDa (CPI17) and KEPI are homologous MP inhibitor proteins; both need phosphorylation to become active. As the surroundings of the phosphorylation sites of CPI17 and KEPI are similar, anti-CPI17^pThr38 antibody can recognize the phosphorylated, active KEPI protein. ANOVA: p = 0.0389. (B) Changes in the extent of exocytosis in response to Flag-peptide or Flag-KEPI transduction was measured by incorporating FM 2–10 fluorescent dye into the synaptosomes, and determining the release of dye. Each point represents the mean±SEM of three parallel experiments (n = 3). Symbols: <i>a</i>: significant (p<0.05) compared to control; <i>b</i>: significant compared to Flag-peptide; t-test was used. By ANOVA, we detected significant differences among treated groups (p = 0.006). (C) Effects of Flag-peptide and Flag-KEPI transduction on the phosphorylation of SNAP-25 at Thr138. ANOVA: p = 0.0050. (D) Effect of KCl depolarisation, in the absence and presence of TMC or H1152 treatments, on the phosphorylation level of Thr138 in SNAP-25 analysed by western blots using anti-SNAP25^pThr138. The duration of treatment was 1 hour. Actin was assessed as loading control. ANOVA: p < 0.0001. (E) Effect of TMC and H1152 treatment of synaptosomes on the interaction of SNAP-25 and syntaxin. Synaptosomes were treated with the inhibitors as mentioned above then lysed. Immunoprecipitations from synaptosome lysates were performed using anti-syntaxin antibody. The amount of Protein-A Sepharose was the same in each tube. The amounts of precipitated syntaxin and SNAP-25 were detected by Western blots. ANOVA: p<0.0001. Statistical analyses were performed using the data of n = 3–5 parallel experiments and representative blots are shown here. Means ±SEM (n = 5), ANOVA and Dunnett’s post hoc analysis, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.</p

Silencing of MYPT1 by siRNA in B50 neuroblastoma cell line.

<p>(A) Western blot images of cell lysates after treatments with 100 nM of siRNA targeting the endogenous MYPT1 subunit. The relative amount of MYPT1 was detected and determined with anti-MYPT1^1-296 antibody. (B) MTT cell viability assays were carried out 48 hours after transfecting cells with MYPT1 specific siRNA. (C) Assay of phosphatase activity in control and siRNA transfected cell lysates with ³²P-MLC20 substrate. (D) The phosphorylation of SNAP-25 on Thr138 was detected by anti-SNAP25^pThr138, and the levels of SNAP-25 as well as actin from control and transfected cells was also determined. The changes in the level of the phosphorylated SNAP-25 was determined comparing the relative density of the bands normalized for the loading controls. Means ±SEM (n = 5), student t test, *p<0.05; **p<0.01;; ****p<0.0001.</p

Phosphorylation and dephosphorylation of purified Flag-SNAP-25.

<p>(A) Mass spectrometry analysis of ROK-phosphorylated SNAP-25. Collision induced dissociation (CID) spectra representing SNAP-25 [136–161] peptides. Upper panel: m/z: 991.4868 (3+), ¹³⁶RVTNDARENEMDENLEQVSGIIGNLR¹⁶¹. Lower panel: m/z: 1018.1437 (3+), ¹³⁶RVT(Phospho)NDARENEMDENLEQVSGIIGNLR¹⁶¹. Site of phosphorylation is Thr-138 as proven by unmodified y₈ and phosphorylated b₅ fragment ions. Peptide fragments are labeled according to the nomenclature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177046#pone.0177046.ref034" target="_blank">34</a>]. -P stands for the 98-Da neutral loss of phosphoric acid characteristic to Ser/Thr phosphorylation, pr stands for the precursor ion and ⁰ denotes water loss of the respective fragment ions. (B) Phosphorylation of SNAP-25 by RhoA-activated kinase (ROK) in the presence of γ-³²P-ATP and Mg²⁺ as detailed in Materials and Methods. No ROK was introduced to control samples. Phosphorylation was detected by autoradiography. (C) Examination of SNAP-25 phosphorylation/dephosphorylation on Thr138 by <i>in vitro</i> kinase and phosphatase assays. Flag-SNAP-25 was incubated in the absence (control) or presence of ROK and then with or without MP. Phosphorylation level of Thr138 was investigated by Western blot using anti-SNAP25^pThr138 phosphorylation site-specific antibody. The loading of Flag-SNAP-25 was verified by anti-Flag antibody. (D) <i>In vitro</i> protein kinase assay of wild type (SNAP-25^wt) and Thr138Ala mutant (SNAP-25^T138A) of SNAP-25 by ROK. The level of SNAP-25^pThr138 was assessed by anti-SNAP-25^pThr138 antibody by Western blot.</p

Interaction between SNAP-25 and the MYPT1 subunit of myosin phosphatase.

<p>(A) Immunoprecipitation was performed from B50 neuroblastoma cell lysates with antibodies specific for MYPT1, PP1cδ and SNAP-25 coupled to Protein-A Sepharose, and the precipitates were analyzed by Western blots using the indicated antibodies. In control, rabbit non-immune serum was coupled to Protein-A Sepharose. (B) Confocal microscopy of MYPT1 (green) and SNAP-25 (red). Images show the co-localization of SNAP-25 and MYPT1 in a merged image. (C-G) SPR-based binding assays of the interaction between MYPT1 and SNAP-25. Full-length GST-MYPT1^1-1004 (C) and a C-terminal GST-MYPT1^667-1004 fragment (D) were immobilized on sensor chips coupled with anti-GST. N-terminal fragments of His-MYPT1^1-296 (E) and His-MYPT1^1-633 (F) were immobilized on CM5 sensor chips by direct amine-coupling. Flag-SNAP-25 was injected over the surfaces at the concentrations indicated on the sensorgrams. Dissociation constants (K_D) derived by the BIAevaluation 3.1 software from the sensorgrams assuming a 1:1 binding model are indicated in the figures. The unit of dissociation constant values is M (n = 3). Schematic structure of the full-length, as well as the N-terminal and C-terminal fragments of MYPT1 used as ligands in the SPR measurements are shown (G).</p

Effect of PP1 and ROK inhibitors on the phosphorylation of SNAP-25 at Thr138 in brain slices.

<p>(A) Mouse cortical brain slices were prepared and treated with TMC or H1152 for 1 hour. To trigger depolarization, 8 mM KCl was added. Phosphorylation of SNAP-25 was investigated by Western blot using an anti-SNAP-25^pThr138 antibody. Amounts of SNAP-25 protein were also determined by anti-SNAP-25 antibody. Density of bands wase determined and normalized relative phosphorylation is illustrated. Means ±SEM (n = 5), ANOVA and Dunnett’s post hoc analysis, *p<0.05; **p<0.01; ***p<0.001. (B) Changes in SNAP-Thr138 phosphorylation in response to treatments as detailed in (A) were also imaged by confocal microscopy. The four sets of brain slices (control, KCl-treated, KCl + TMC treated, KCl + H1152 treated) were analysed with anti-SNAP-25^pThr138 antibody to assess differences in phosphorylation level, and also with anti-syntaxin antibody to identify syntaxin, a typical pre-synaptic protein. Co-localizations of syntaxin and phospho-SNAP-25 are also shown on merged images. Scale bar: 10 μm.</p

Altered Functional Protein Networks in the Prefrontal Cortex and Amygdala of Victims of Suicide

<div><p>Probing molecular brain mechanisms related to increased suicide risk is an important issue in biological psychiatry research. Gene expression studies on <em>post mortem</em> brains indicate extensive changes prior to a successful suicide attempt; however, proteomic studies are scarce. Thus, we performed a DIGE proteomic analysis of <em>post mortem</em> tissue samples from the prefrontal cortex and amygdala of suicide victims to identify protein changes and biomarker candidates of suicide. Among our matched spots we found 46 and 16 significant differences in the prefrontal cortex and amygdala, respectively; by using the industry standard <em>t</em> test and 1.3 fold change as cut off for significance. Because of the risk of false discoveries (FDR) in these data, we also made FDR adjustment by calculating the <em>q</em>-values for all the <em>t</em> tests performed and by using 0.06 and 0.4 as alpha thresholds we reduced the number of significant spots to 27 and 9 respectively. From these we identified 59 proteins in the cortex and 11 proteins in the amygdala. These proteins are related to biological functions and structures such as metabolism, the redox system, the cytoskeleton, synaptic function, and proteolysis. Thirteen of these proteins (CBR1, DPYSL2, EFHD2, FKBP4, GFAP, GLUL, HSPA8, NEFL, NEFM, PGAM1, PRDX6, SELENBP1 and VIM,) have already been suggested to be biomarkers of psychiatric disorders at protein or genome level. We also pointed out 9 proteins that changed in both the amygdala and the cortex, and from these, GFAP, INA, NEFL, NEFM and TUBA1 are interacting cytoskeletal proteins that have a functional connection to glutamate, GABA, and serotonin receptors. Moreover, ACTB, CTSD and GFAP displayed opposite changes in the two examined brain structures that might be a suitable characteristic for brain imaging studies. The opposite changes of ACTB, CTSD and GFAP in the two brain structures were validated by western blot analysis.</p> </div

Description of participants in the present study.

<p>NA: not available; S: suicide; C: control.</p

Representative gel image.

<p>The first dimension was carried out in pH 3–10 NL IPG strip and the second dimension was 24×20 cm 10% SDS PAGE. Part A shows the overlaid image, part B shows the standardized log abundance of a representative spot (2406, prefrontal cortex) on the different gels, part C shows 3D views of the individual spots (C1–C6: control brains; S1–S6: suicide brains).</p