Annexin 2 Promotes the Formation of Lipid Microdomains Required for Calcium-regulated Exocytosis of Dense-Core Vesicles

Chasserot‐Golaz, Sylvette; Vitale, Nicolas; Umbrecht-Jenck, Emeline; Knight, D. E.; Gerke, Volker; Bader, Marie‐France

doi:10.1091/mbc.e04-07-0627

Cited by 127 publications

(163 citation statements)

References 58 publications

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“…Resting and stimulated PC12 cells were fixed and further processed for immunofluorescence as described (40,41). Stained cells were visualized with a Zeiss confocal microscope LSM 510.…”

Section: Methodsmentioning

confidence: 99%

The Coffin–Lowry syndrome-associated protein RSK2 is implicated in calcium-regulated exocytosis through the regulation of PLD1

Zeniou-Meyer

Liu

Béglé

et al. 2008

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

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Exocytosis of neurotransmitters and hormones occurs through the fusion of secretory vesicles with the plasma membrane. This highly regulated process involves key proteins, such as SNAREs, and specific lipids at the site of membrane fusion. Phospholipase D (PLD) has recently emerged as a promoter of membrane fusion in various exocytotic events potentially by providing fusogenic cone-shaped phosphatidic acid. We show here that PLD1 is regulated by ribosomal S6 kinase 2 (RSK2)-dependent phosphorylation. RSK2 is activated by a high K + -induced rise in cytosolic calcium. Expression of inactive RSK2 mutants or selective knockdown of endogenous RSK2 dramatically affects the different kinetic components of the exocytotic response in chromaffin cells. RSK2 physically interacts with and stimulates PLD activity through the phosphorylation of Thr-147 in the PLD1 amino-terminal phox homology domain. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis. We propose that PLD-related defects in neuronal and endocrine activities could contribute to the effect observed after the loss-of-function mutations in Rsk2 that lead to Coffin–Lowry syndrome, an X-linked form of growth and mental retardation.

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“…Resting and stimulated PC12 cells were fixed and further processed for immunofluorescence as described (40,41). Stained cells were visualized with a Zeiss confocal microscope LSM 510.…”

Section: Methodsmentioning

confidence: 99%

The Coffin–Lowry syndrome-associated protein RSK2 is implicated in calcium-regulated exocytosis through the regulation of PLD1

Zeniou-Meyer

Liu

Béglé

et al. 2008

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

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“…This plasmid allows also the identification of the subpopulation of cells that transiently express the siRNAs through the immunostaining of GH. In transfected PC12 cells, GH is stored in secretory granules and released by exocytosis in response to cell stimulation (14,24). Western blot analysis revealed that transient expression of PLD1 siRNA reduced the level of PLD1 but did not affect the level of actin or GH (Fig.…”

Section: Pa Is Produced At the Plasma Membrane When Exocytosis Ismentioning

confidence: 92%

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

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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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“…Secretagogue-evoked stimulation triggers its rapid translocation to the plasma membrane (Fig. 6A), where it stabilizes lipid micro-domains required for exocytosis (Chasserot-Golaz et al, 2005). In Tat-expressing PC12 cells, neither ATP (Fig.…”

Section: Tat Inhibits Chromaffin and Pc12 Cell Exocytosismentioning

confidence: 99%

HIV-1 Tat protein inhibits neurosecretion by binding to phosphatidylinositol 4,5-bisphosphate

Tryoen-Tóth

Chasserot‐Golaz

et al. 2013

Journal of Cell Science

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Summary HIV-1 transcriptional activator (Tat) enables viral transcription and is also actively released by infected cells. Extracellular Tat can enter uninfected cells and affect some cellular functions. Here, we examine the effects of Tat protein on the secretory activity of neuroendocrine cells. When added to the culture medium of chromaffin and PC12 cells, Tat was actively internalized and strongly impaired exocytosis as measured by carbon fiber amperometry and growth hormone release assay. Expression of Tat mutants that do not bind to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] did not affect secretion, and overexpression of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), the major PtdIns(4,5)P 2 synthesizing enzyme, significantly rescued the Tat-induced inhibition of neurosecretion. This suggests that the inhibition of exocytosis may be the consequence of PtdIns(4,5)P 2 sequestration. Accordingly, expression of Tat in PC12 cells interfered with the secretagogue-dependent recruitment of annexin A2 to the plasma membrane, a PtdIns(4,5)P 2 -binding protein that promotes the formation of lipid microdomains that are required for exocytosis. In addition Tat significantly prevented the reorganization of the actin cytoskeleton necessary for the movement of secretory vesicles towards plasma membrane fusion sites. Thus, the capacity of extracellular Tat to enter neuroendocrine cells and sequester plasma membrane PtdIns(4,5)P 2 perturbs several PtdIns(4,5)P 2 -dependent players of the exocytotic machinery, thereby affecting neurosecretion. We propose that Tat-induced inhibition of exocytosis is involved in the neuronal disorders associated with HIV-1 infection.

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Annexin 2 Promotes the Formation of Lipid Microdomains Required for Calcium-regulated Exocytosis of Dense-Core Vesicles

Cited by 127 publications

References 58 publications

The Coffin–Lowry syndrome-associated protein RSK2 is implicated in calcium-regulated exocytosis through the regulation of PLD1

The Coffin–Lowry syndrome-associated protein RSK2 is implicated in calcium-regulated exocytosis through the regulation of PLD1

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

HIV-1 Tat protein inhibits neurosecretion by binding to phosphatidylinositol 4,5-bisphosphate

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