Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells

Nili, U.; Wit, Heidi de; Gulyas-Kovacs, Attila; Toonen, Ruud F.; Sørensen, Jakob Balslev; Verhage, Matthijs; Ashery, Uri

doi:10.1016/j.neuroscience.2006.08.014

Cited by 59 publications

(68 citation statements)

References 44 publications

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“…Thus, PKC activation exerts a dual role. This is in agreement with earlier studies on chromaffin cells [36,41]. Munc-18 is one of the central proteins involved at several levels in the exocytotic process [30] and has been shown to be phosphorylated by PKC [32].…”

Section: Discussionsupporting

confidence: 93%

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCα and PKCδ

et al. 2009

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Aims/hypothesis Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCα and PKCδ, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. Methods Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique.

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Section: Discussionsupporting

confidence: 93%

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCα and PKCδ

et al. 2009

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“…Unexpectedly, a nonphosphorylatable Syt1 variant had no effect on vesicle fusion in mouse embryonic chromaffin cells (21). However, we previously observed only limited contribution of PKC in mouse embryonic chromaffin cells compared with adult bovine chromaffin cells and neurons (8,22), suggesting that the role of PKC may differ between model systems.…”

mentioning

confidence: 77%

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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“…Capacitance measurements were performed 72 h after electroporation at 30 -32°C. Conventional whole cell recordings and capacitance measurements were performed as described (21,22) on control cells (expressing GFP) and cells expressing the PLD1 siRNA and GFP and analyzed with Igor Pro (Wavemetrics, Lake Oswego, OR). Flashes of UV light were generated by a flash lamp (TILL Photonics, Planegg, Germany), and fluorescence excitation light was generated by a monochromator (TILL Photonics).…”

Section: Methodsmentioning

confidence: 99%

“…7A). The exocytotic burst results from the fusion of a pool of release-competent granules, whereas the sustained phase represents granules that are mobilized to undergo priming during the calcium pulse and subsequent fusion (22,25). Exocytosis from chromaffin cells expressing PLD1 siRNAs was significantly attenuated compared with control cells: both the exocytotic burst (Fig.…”

Section: Reduction Of Endogenous Pld1 Inhibits Exocytosis By Reducingmentioning

confidence: 99%

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

Zeniou-Meyer

Zabari

Ashery

et al. 2007

Journal of Biological Chemistry

190

228

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Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNAmediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion. Phosphatidic acid (PA)2 is a pleiotropic bioactive lipid that has been proposed to activate selected enzymes (1), recruit proteins to membrane surfaces (2), and serve as a substrate for the formation of other signaling lipids (3). Most intriguingly, PA has also been shown to promote negative curvature in bi-layer membranes due to its small polar head-group in combination with two fatty-acyl side chains (4). The bulk of cellular PA is synthesized via two different acylation pathways, the glycerol 3-phosphate pathway and the dihydroxy acetone phosphate pathway, which are named according to their respective precursors. However, PA is also produced via hydrolysis of phosphatidylcholine by phospholipase D (PLD) (5) on a much faster time scale, and this latter source is thought to underlie the dynamic regulation of PA that allows it to function as a signaling lipid in agonist-stimulated cell biological responses such as secretion and changes in cellular morphology.In mammals, the classic PLD family is composed of a pair of membrane-associated proteins, PLD1 and PLD2. Both PLD isoforms require phosphatidylinositol 4,5-bisphosphate for their enzymatic activity. However, whereas PLD2 exhibits relatively high basal activity in isolation, full activation of PLD1 requires its stimulation by small GTPases of the ADP-ribosylation factor (ARF), Rho and Ral families, and protein kinase C (3, 6). PLD enzymes have been proposed to be involved in a number of cellular processes, including cell growth and survival, cell differentiation, and vesicular trafficking (3)....

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Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells

Cited by 59 publications

References 44 publications

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCα and PKCδ

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCα and PKCδ

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

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

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