Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release

Genç, Özgür; Kochubey, Olexiy; Toonen, Ruud F.; Verhage, Matthijs; Schneggenburger, Ralf

doi:10.7554/elife.01715

Cited by 68 publications

(102 citation statements)

References 65 publications

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“…Together, our proteomics analysis identifies several putative substrates of PKCB such as MUNC18 (Brochetta et al , 2014; Genc et al , 2014), SNAP23 (Polgár et al , 2003; Lorentz et al , 2012), STIM1 (Pozo‐Guisado et al , 2010), and MYH9 (Ludowyke et al , 2006) (Appendix Table S1) that may enhance mast cell secretion as a positive arm in the incoherent feed‐forward regulation (Fig 4F). Surprisingly, we also discovered that VAMP8 has an evolutionarily conserved phosphorylation motif in the center of the SNARE fusion complex that acts in parallel as a negative regulatory arm in the same circuit to prevent fusion (Fig 4G).…”

Section: Discussionmentioning

confidence: 91%

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

et al. 2016

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Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α‐helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

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Section: Discussionmentioning

confidence: 91%

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

et al. 2016

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“…This view is corroborated by early work on superpriming (32), which implicates the Munc13-Rim-Rab3 interaction as another requirement for this SV maturation process. On the other hand, recent evidence assigns decisive roles to specific isoforms of PKC (6,28) and PKC-mediated phosphorylation of Munc18 (29) to the induction of PTP. However, assuming that PTP and PdBu-induced potentiation act through the same mechanism (evidence provided here) and given the finding that all effects of PdBu are eliminated by disrupting its interaction with Munc13 (26), we may conclude that Munc13 executes both forms of potentiation by regulating the energy barrier for SV fusion (54).…”

Section: Discussionmentioning

confidence: 99%

“…Modulation of presynaptic voltage-gated Ca 2+ channels by slow transmitter systems is probably the most powerful mechanism of changing release probability (p) of synaptic vesicles (SVs) (19)(20)(21). Changes in intrinsic [Ca 2+ ] i sensitivity of the release apparatus also contribute and have been investigated in the context of the phospholipase-C-diacylglycerol (PLC-DAG) signaling pathway (22-26) and posttetanic potentiation (15,(27)(28)(29)(30), but the influence of this modulation on STP is less well understood.Here, we describe heterogeneity of STP among synapses in the medial nucleus of the trapezoid body (MNTB)-the calyces of Held. AP-evoked excitatory postsynaptic currents (EPSCs) vary in amplitude by more than a factor of 5 among calyx synapses and display divergent STP patterns (31).…”

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confidence: 99%

Superpriming of synaptic vesicles as a common basis for intersynapse variability and modulation of synaptic strength

Taschenberger

Woehler

Neher

2016

Proc. Natl. Acad. Sci. U.S.A.

110

156

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Glutamatergic synapses show large variations in strength and shortterm plasticity (STP). We show here that synapses displaying an increased strength either after posttetanic potentiation (PTP) or through activation of the phospholipase-C-diacylglycerol pathway share characteristic properties with intrinsically strong synapses, such as (i) pronounced short-term depression (STD) during high-frequency stimulation; (ii) a conversion of that STD into a sequence of facilitation followed by STD after a few conditioning stimuli at low frequency; (iii) an equalizing effect of such conditioning stimulation, which reduces differences among synapses and abolishes potentiation; and (iv) a requirement of long periods of rest for reconstitution of the original STP pattern. These phenomena are quantitatively described by assuming that a small fraction of "superprimed" synaptic vesicles are in a state of elevated release probability (p ∼ 0.5). This fraction is variable in size among synapses (typically about 30%), but increases after application of phorbol ester or during PTP. The majority of vesicles, released during repetitive stimulation, have low release probability (p ∼ 0.1), are relatively uniform in number across synapses, and are rapidly recruited. In contrast, superprimed vesicles need several seconds to be regenerated. They mediate enhanced synaptic strength at the onset of burst-like activity, the impact of which is subject to modulation by slow modulatory transmitter systems.posttetanic potentiation | short-term plasticity | calyx of Held | Munc13 | phorbol ester G lutamatergic synapses display a variety of dynamic changes in response to stimulation with action potential (AP) trains, ranging from immediate short-term depression to facilitation followed by depression (1). Both pharmacological (2-6) and molecular (7-9) perturbations have been described, which change such patterns from one to the other in a given synapse. Short-term plasticity (STP) has been shown to underlie many basic signal processing tasks of circuits in the central nervous system (10-13) and rapid changes of STP have been considered "... to be an almost necessary condition for the existence of (short-lived) activity states in the central nervous system" (ref. 14, p. 247). The balance between facilitation and depression is shifted during posttetanic potentiation (PTP) (15) and behavioral states are dynamically regulated by STP (16). Regulation occurs through slow, modulatory transmitter systems (17, 18). However, many open questions regarding the mechanisms underlying such changes remain. Modulation of presynaptic voltage-gated Ca 2+ channels by slow transmitter systems is probably the most powerful mechanism of changing release probability (p) of synaptic vesicles (SVs) (19)(20)(21). Changes in intrinsic [Ca 2+ ] i sensitivity of the release apparatus also contribute and have been investigated in the context of the phospholipase-C-diacylglycerol (PLC-DAG) signaling pathway (22-26) and posttetanic potentiation (15,(27)(28)(29)(30), but the infl...

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“…4A). Paired-pulse plasticity critically depends on activation of Munc13-1 and PKC and PKC phosphorylation of Munc18-1, but Syt1 phosphorylation is dispensable (see above); potentiation by direct application of DAG (-analogs) depends on all these factors (8,11,12) and HFSinduced potentiation requires at least PKC activation (6,7,35), Munc18-1 phosphorylation (12), and Syt1 phosphorylation (Fig. 2), whereas the role of Munc13-1 is unknown.…”

Section: Potentiation Of Spontaneous Release Is Mediated By Multiplementioning

confidence: 99%

“…1A) (8,11). We previously identified Munc18-1 as an essential PKC substrate, because a nonphosphorylatable Munc18-1 mutant completely inhibits PKC-dependent STP (8,12). Importantly, a phosphomimetic mutation of Munc18-1 cannot fully bypass the requirement for PKC activation, indicating that other PKC substrates must contribute to this form of plasticity (8).…”

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confidence: 99%

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

Jong

Meijer

Saarloos

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAGinduced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1 T112A ), which prevents its PKCdependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca 2+ sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect pairedpulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1 Δ109-116 ), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.P resynaptic strength changes rapidly during repetitive stimulation [short-term plasticity (STP)] and activation of intracellular signal transduction pathways (1, 2). The diacylglycerol (DAG)/protein kinase C (PKC) cascade is one of the most potent pathways at the presynaptic terminal. Its activation leads to 50-100% potentiation of spontaneous and action potential (AP)-evoked release (3-5), and is critical for multiple forms of presynaptic plasticity (6-8). DAG directly activates the vesicle priming factor Munc13-1 (9, 10) and indirectly activates downstream effectors via PKC. Activation of both Munc13-1 and PKC is essential for this pathway to operate (Fig. 1A) (8, 11). We previously identified Munc18-1 as an essential PKC substrate, because a nonphosphorylatable Munc18-1 mutant completely inhibits PKC-dependent STP (8, 12). Importantly, a phosphomimetic mutation of Munc18-1 cannot fully bypass the requirement for PKC activation, indicating that other PKC substrates must contribute to this form of plasticity (8). These substrates have not been identified to date.Among other presynaptic PKC substrates is synaptotagmin-1 (Syt1, ref. 13). Syt1 is the vesicular Ca 2+ sensor that mediates fast AP-evoked release in the hippocampus (14) and drives a large fraction of spontaneous release (15). In the latter case, Syt1 competes with alternative sensors, in particular Doc2s, for SNARE binding ...

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Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release

Cited by 68 publications

References 65 publications

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Superpriming of synaptic vesicles as a common basis for intersynapse variability and modulation of synaptic strength

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

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