Selective packaging of human growth hormone into synaptic vesicles in a rat neuronal (PC12) cell line.

Schweitzer, Erik S.; Kelly, RB

doi:10.1083/jcb.101.2.667

Cited by 93 publications

(78 citation statements)

References 39 publications

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“…hGH is stored in LDCV (Schweitzer and Kelly, 1985) and is released upon stimulation, serving as a marker for secretion in cotransfected cells (Holz et al, 2000). Plasmids encoding EEtagged wild-type PI3K-C2␣ (PI3K-C2␣-wt) and PI3K-C2␣ containing a point mutation in the catalytic domain (PI3K-C2␣-R1251P) were transfected in HEK cells where both proteins were expressed at comparable levels (Figure 4).…”

Section: Resultsmentioning

confidence: 99%

“…Because hGH is stored in secretory vesicles (Schweitzer and Kelly, 1985), this method has been successfully used to elucidate the role played in neurosecretion by many proteins of interest (Holz et al, 2000). The enhancement of evoked secretion following overexpression of wild-type PI3K-C2␣ suggests that PI3K-C2␣ activity controls the progression of LDCV toward a fusion-competent state.…”

Section: Involvement Of Pi3k-c2␣ Enzymatic Activity In Neurosecretionmentioning

confidence: 99%

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Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

Meunier

Osborne

Hammond

et al. 2005

MBoC

102

108

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Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2␣ (PI3K-C2␣) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2␣ is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2␣ function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2␣ enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2␣ mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2␣ is required for acquisition of fusion competence in neurosecretion. INTRODUCTIONThe analysis of the molecular mechanism controlling neuroexocytosis has been greatly helped by studies on neurosecretory cells (Dunn and Holz, 1983;Sarafian et al., 1987;Martin and Walent, 1989;Monck and Fernandez, 1994;Rettig and Neher, 2002). Early work has shed light on two major steps consisting of the reversible ATP-dependent priming of docked granules followed by their Ca 2ϩ -driven fusion with the plasma membrane and release of the granule content (Holz et al., 1989;Hay and Martin, 1992). The spatiotemporal control of intracellular Ca 2ϩ concentration together with capacitance measurements has allowed the identification of distinct pools of chromaffin granules involved in these distinct steps (Rettig and Neher, 2002). Further electrophysiological and biochemical approaches, such as the reconstitution of secretion in permeabilized neurosecretory cells (Martin and Walent, 1989), have highlighted the role of key factors involved in these two processes. Cytosolic and membrane proteins have been linked to priming and fusion, including the mammalian orthologues of the Caenorhabditis elegans UNC13 and UNC18 gene products, synaptotagmins, and members of the SNARE family and associated proteins (Jahn and Sudhof, 1999;Burgoyne et al., 2001;Brose and Rosenmund, 2002;Rettig and Neher, 2002).In contrast, less is known about the contribution of lipid dynamics during these processes with the exception of phosphatidic acid and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ]. The former is produced by phospholipase D, which is essential for secretion in neurons (Humeau et al., 2001) and in neuroendocrine cells . The inhibition of Ca 2ϩ -dependent catecholamine release after depletion of phosphatidylinositol highlighted the role of phosphoinositides during the secretory events (Eberhard et al., 1990). Moreover, phosphatidylinositol transfer protein and phosphatidylinositol-4-phosphate 5 kinase (PI4P5K) were shown to be required for the ATP-dependent priming of secretory granules in PC12 cells (Hay and Martin, 1993;Hay et al., 1995). In addition, phosphatidylinositol 4-kinase (PI4K), an integral membrane protein of chromaffin granules and synaptic vesicles, is r...

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Section: Resultsmentioning

confidence: 99%

Section: Involvement Of Pi3k-c2␣ Enzymatic Activity In Neurosecretionmentioning

confidence: 99%

Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

Meunier

Osborne

Hammond

et al. 2005

MBoC

102

108

View full text Add to dashboard Cite

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“…We used a transfection assay in which hGH was coexpressed in PC12 cells with p35 (to promote Cdk5 phosphorylation) together with wild-type or mutant (S327A) SEPT5. Because most transfected cells coexpress both proteins, the secretion of hGH can be used to study the effect of SEPT5 on secretion (Schweitzer and Kelly, 1985). We induced Ca 2ϩ -dependent regulated secretion of hGH from PC12 cells with high K ϩ that depolarizes the membrane and triggers exocytosis.…”

Section: Cdk5/p35 Phosphorylation Of Sept5 Modulates Regulated Exocytmentioning

confidence: 99%

Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

Amin¹,

Zheng²,

Kesavapany³

et al. 2008

J. Neurosci.

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Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system, where it is involved in neuronal migration, synaptic transmission, and survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known also as hCDCrel-1, as an interacting clone. Here we report that p35 associates with SEPT5 in GST (glutathione S-transferase)-pull-down and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A serine (S)-to-alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild-type and knock-out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding to syntaxin-1. Moreover, mutant nonphosphorylated SEPT5 potentiated regulated exocytosis more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 phosphorylation of human septin SEPT5 at S327 plays a role in modulating exocytotic secretion.

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“…Given our current results, it is possible that PKA phosphorylation of syntaphilin at serine 43 has a significant effect on its binding to syntaxin-1A, thus turning off the inhibitory function of syntaphilin through cAMP-dependent signal cascades. To determine whether this phosphorylation event is implicated in the regulation of Ca 2ϩ -dependent exocytosis, the effect of expression of syntaphilin or its mutant on SNARE-mediated vesicle fusion was evaluated using a hGH release assay in transfected PC12 cells (38,39). In transfected PC12 cells hGH is concentrated in large dense core vesicles and undergoes Ca 2ϩ -dependent exocytosis in response to depolarization by high K ϩ .…”

Section: Physiological Effect Of Syntaphilin and Its S43d Mutant On Camentioning

confidence: 99%

Phosphorylation of Syntaphilin by cAMP-dependent Protein Kinase Modulates Its Interaction with Syntaxin-1 and Annuls Its Inhibitory Effect on Vesicle Exocytosis

Boczan

Leenders

Sheng

2004

Journal of Biological Chemistry

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cAMP-dependent protein kinase (PKA) can modulate synaptic transmission by acting directly on the neurotransmitter secretory machinery. Here, we identify one possible target: syntaphilin, which was identified as a molecular clamp that controls free syntaxin-1 and dynamin-1 availability and thereby regulates synaptic vesicle exocytosis and endocytosis. Deletion mutation and site-directed mutagenesis experiments pinpoint dominant PKA phosphorylation sites to serines 43 and 56. PKA phosphorylation of syntaphilin significantly decreases its binding to syntaxin-1A in vitro. A syntaphilin mutation of serine 43 to aspartic acid (S43D) shows similar effects on binding. To characterize in vivo phosphorylation events, we generated antisera against a peptide of syntaphilin containing a phosphorylated serine 43. Treatment of rat brain synaptosomes or syntaphilintransfected HEK 293 cells with the cAMP analogue BIMPS induces in vivo phosphorylation of syntaphilin and inhibits its interaction with syntaxin-1 in neurons. To determine whether PKA phosphorylation of syntaphilin is involved in the regulation of Ca 2؉ -dependent exocytosis, we investigated the effect of overexpression of syntaphilin and its S43D mutant on the regulated secretion of human growth hormone from PC12 cells. Although expression of wild type syntaphilin in PC12 cells exhibits significant reduction in high K ؉ -induced human growth hormone release, the S43D mutant fails to inhibit exocytosis. Our data predict that syntaphilin could be a highly regulated molecule and that PKA phosphorylation could act as an "off" switch for syntaphilin, thus blocking its inhibitory function via the cAMP-dependent signal transduction pathway.Neurotransmitter release involves a series of protein interactions between the membranes of synaptic vesicles and presynaptic terminals, culminating in the calcium-dependent fusion of the two membranes (for reviews see Refs. 1-4). The synaptic vesicle-associated protein synaptobrevin interacts with two membrane-associated proteins, SNAP 1 -25 and syntaxin, to form a stable SNARE complex (5-12). Formation of the SNARE complex has been proposed to bring the synaptic vesicle and plasma membranes into close apposition and provide the energy that drives the mixing of the two lipid bilayers (6, 7, 10). Calcium influx into the presynaptic terminal triggers the calcium sensor of the fusion machinery, upon which the SNARE core complex binding matures from a trans-state to a cis-state, resulting in the complete fusion of the two membranes and the release of neurotransmitter. The very stable cis-SNARE core complex is then dissociated by the action of ␣-SNAP and the ATPase N-ethylmaleimide-sensitive factor (10). The assembly and disassembly of the SNARE complex must be highly regulated to gain plasticity in neurotransmitter release. Recent studies have made significant progress in understanding this regulatory mechanism by the isolation of the SNARE complex interacting proteins, including Munc18/nSec1/rbSec1, complexins, Munc-13, tomosyn, cysteine st...

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Selective packaging of human growth hormone into synaptic vesicles in a rat neuronal (PC12) cell line.

Cited by 93 publications

References 39 publications

Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

Phosphorylation of Syntaphilin by cAMP-dependent Protein Kinase Modulates Its Interaction with Syntaxin-1 and Annuls Its Inhibitory Effect on Vesicle Exocytosis

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