Phosphorylation of Munc-18/n-Sec1/rbSec1 by Protein Kinase C

Fujita, Yasuyuki; Sasaki, Takuya; Fukui, Koji; Kotani, Hirokazu; Kimura, Toshihiro; Hata, Yoshinobu; Südhof, Thomas C.; Scheller, Richard H.; Takai, Yoshimi

doi:10.1074/jbc.271.13.7265

Cited by 242 publications

(131 citation statements)

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“…For verification of specificity of the assay, N-terminal His 6 -tagged Munc18-2 was produced in Sf9 cells using the Bac to Bac system (Life Technologies Inc.) and purified on Ni-NTA-agarose (Qiagen) according to the manufacturer's protocol. The purified protein, or bovine serum albumin as a control, were added in increasing amounts to the in vitro translated 35 S-labeled wild-type Munc18-2 before applying the specimens in the coated wells. After assessing the linearity of signal over the counts/min range studied for all the Munc18-2 variants, the actual measurements were carried out using 150,000 cpm of Munc18-2 radioactivity per well.…”

Section: Methodsmentioning

confidence: 99%

“…After 1 h incubation at 37°C, the filter units were moved to fresh complete medium. To assess the level of Munc18-2 overexpression, samples of the cells were directly lysed in SDS-PAGE loading buffer, and the quantity of Munc18-2 relative to that in cells infected with the control (amyloid precursor protein) SFV was determined by Western blotting, 35 S-protein A, and Fujifilm BAS-1500 quantitation. Assay of Munc18-2 Stability-For analysis of Munc18-2 stability, Caco-2 cells grown on plastic were infected with recombinant SFVs for 4 h. Thereafter the cells were pulse labeled with […”

Section: Methodsmentioning

confidence: 99%

“…The myc-Munc18-2 variants were expressed in the cells using a 4-h SFV infection. Protein complexes were immunoprecipitated with anti-myc mAb 9E10, and the amount of syntaxin 3 and Munc-18-2 in the precipitates was analyzed by Western blotting, 35 S-protein A and the Fuji BAS-1500 system. A, Western blots of the immunoprecipitates stained using polyclonal antibodies against Munc18-2 (bottom panel) or syntaxin 3 (top panel).…”

Section: Figmentioning

confidence: 99%

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Munc18-2, a Functional Partner of Syntaxin 3, Controls Apical Membrane Trafficking in Epithelial Cells

Riento¹,

Kauppi²,

Keränen³

et al. 2000

Journal of Biological Chemistry

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The Sec1-related proteins bind to syntaxin family tSNAREs with high affinity, thus controlling the interaction of syntaxins with their cognate SNARE partners. Munc18-2 is a Sec1 homologue enriched in epithelial cells and forms a complex with syntaxin 3, a t-SNARE localized to the apical plasma membrane. We generated here a set of Munc18-2 point mutants with substitutions in conserved amino acid residues. The mutants displayed a spectrum of different syntaxin binding efficiencies. The in vitro and in vivo binding patterns were highly similar, and the association of the Munc18-2 variants with syntaxin 3 correlated well with their ability to displace SNAP-23 from syntaxin 3 complexes when overexpressed in Caco-2 cells. Even the Munc18-2 mutants that do not detectably bind syntaxin 3 were membrane associated in Caco-2 cells, suggesting that the syntaxin interaction is not the sole determinant of Sec1 protein membrane attachment. Overexpression of the wild-type Munc18-2 was shown to inhibit the apical delivery of influenza virus hemagglutinin (HA). Interestingly, mutants unable to bind syntaxin 3 behaved differently in the HA transport assay. While one of the mutants tested had no effect, one inhibited and one enhanced the apical transport of HA. This implies that Munc18-2 function in apical membrane trafficking involves aspects independent of the syntaxin 3 interaction.It is generally accepted that intracellular membrane trafficking requires compartment-specific membrane-anchored proteins denoted collectively as soluble NSF attachment protein receptors (SNAREs), 1 as well as the general factors N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs), or their homologues (1, 2). The SNARE proteins present on the transport vesicles (v-SNAREs; related to the neuronal synaptobrevin/VAMP proteins) and the target membranes (t-SNAREs; homologues of the neuronal syntaxin and SNAP-25 proteins) assemble into stable core complexes through formation of coiled coil helix bundles, a process which is closely connected to the fusion of membrane bilayers (3-5).Proteins of the Sec1 family (reviewed in Ref. 6) are suggested to play a crucial role in the control of SNARE complex assembly. These proteins bind with high affinity to specific syntaxins, thus modulating the capability of these t-SNAREs to interact with their cognate SNARE partners. In vitro binding assays or in vivo overexpression of Sec1 homologues have provided evidence for an inhibitory role of Sec1 proteins in SNARE complex formation and membrane trafficking (7-12). On the other hand, a wealth of evidence shows that Sec1 action is required for normal physiological function of the intracellular trafficking pathways: loss-of-function mutations in the Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster Sec1-related proteins have been demonstrated to lead to specific blocks in vesicle transport, the phenotypic effects often implying disturbance in consumption of transport vesicles (13)(14)(15)(16)(17)(18)(19)(20)...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

See 1 more Smart Citation

Munc18-2, a Functional Partner of Syntaxin 3, Controls Apical Membrane Trafficking in Epithelial Cells

Riento¹,

Kauppi²,

Keränen³

et al. 2000

Journal of Biological Chemistry

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show abstract

“…In neurons, Munc18a (also called nSec1) binds to syntaxin-1 with very high affinity and blocks the ability of syntaxin-1 to bind to VAMP-2 and SNAP-25 in vitro (14). PKC phosphorylation of Munc18a inhibits its binding to syntaxin (16,17), and its dissociation from syntaxin allows binding VAMP-2 and SNAP-25 (14). One role of Munc18 may therefore be as an inhibitor of syntaxin interactions with SNAP-25 and VAMP, and this could be released by PKC phosphorylation (16,17).…”

mentioning

confidence: 99%

“…PKC phosphorylation of Munc18a inhibits its binding to syntaxin (16,17), and its dissociation from syntaxin allows binding VAMP-2 and SNAP-25 (14). One role of Munc18 may therefore be as an inhibitor of syntaxin interactions with SNAP-25 and VAMP, and this could be released by PKC phosphorylation (16,17). We have previously reported the SNARE isoforms of VAMP (VAMP-2 and -3) (18,19), SNAP-25 (SNAP-23) (20), and syntaxin (syntaxin-2 through -4) (21) expressed in the pancreatic acinar cell.…”

mentioning

confidence: 99%