Munc18-2 and Syntaxin 3 Control Distinct Essential Steps in Mast Cell Degranulation

Brochetta, Cristiana; Suzuki, Ryo; Vita, Francesca; Soranzo, Maria Rosa; Claver, Julien; Madjène, Lydia Celia; Attout, Tarik; Vitte, Joana; Varin‐Blank, Nadine; Zabucchi, Giuliano; Rivera, Juan; Blank, Ulrich

doi:10.4049/jimmunol.1301277

Cited by 49 publications

(78 citation statements)

References 72 publications

(128 reference statements)

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“…This is different compared with our previous attempt to see the role of syntaxin-11 in the mast cell exocytosis, where strong knockdown of endogenous syntaxin-11 did not result in any defect of degranulation . Our new findings are in accordance with the previous studies suggesting the importance of Munc18-2 and syntaxin-3 in mast cell degranulation (Brochetta et al, 2014;Tadokoro et al, 2007). Thus, we now state that, although Munc18 proteins regulate both syntaxin-3 and syntaxin-11 isoforms in mast cells, the former interactions provide more functional contributions to the mast cell exocytosis.…”

Section: Discussionsupporting

confidence: 93%

Chaperoning of closed syntaxin-3 through Lys46 and Glu59 in domain 1 of Munc18 proteins is indispensable for mast cell exocytosis

Bin

Jung

Kim

et al. 2015

Journal of Cell Science

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Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation -whose levels and intracellular localization are regulated by Munc18-2 -remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.

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Section: Discussionsupporting

confidence: 93%

Chaperoning of closed syntaxin-3 through Lys46 and Glu59 in domain 1 of Munc18 proteins is indispensable for mast cell exocytosis

Bin

Jung

Kim

et al. 2015

Journal of Cell Science

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“…To test the fusogenic activity of VAMP8, we used in vitro ensemble content‐ and lipid‐mixing assays with SNARE proteins reconstituted in proteoliposomes (Materials and Methods) (Diao et al , 2015). We mixed liposomes containing purified VAMP8 together with liposomes containing both purified SNAP23 and STX3 (Figs 3A and EV5A) (Sakiyama et al , 2009; Brochetta et al , 2014). As expected for SNARE‐mediated membrane fusion, wild‐type non‐phosphorylated VAMP8 mediated content‐mixing (Fig 3A and B) and lipid‐mixing (Fig EV5B).…”

Section: Resultsmentioning

confidence: 99%

“…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: 96%

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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“…1) Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in this process. [2][3][4][5][6][7][8][9] SNARE-binding proteins also regulate exocytotic release in mast cells. 9,10) Syntaxin-3, -4 and synaptosomal-associated protein 23 kDa (SNAP-23) are SNARE proteins on the plasma membrane (t-SNARE) in mast cells.…”

mentioning

confidence: 99%

“…[2][3][4][5][6][7][8][9] SNARE-binding proteins also regulate exocytotic release in mast cells. 9,10) Syntaxin-3, -4 and synaptosomal-associated protein 23 kDa (SNAP-23) are SNARE proteins on the plasma membrane (t-SNARE) in mast cells. [2][3][4] Vesicle associated membrane protein (VAMP)-2, -7, and -8 are SNARE proteins on the secretory vesicles membrane (v-SNARE).…”

mentioning

confidence: 99%

Effect of Complexin II on Membrane Fusion between Liposomes Containing Mast Cell SNARE Proteins

Tadokoro

Hirashima

Utsunomiya‐Tate

2016

Biological & Pharmaceutical Bulletin

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Mast cells are involved in allergic responses and undergo exocytotic release of inflammatory mediators in response to antigen stimulation. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in this membrane fusion process; some SNARE-binding proteins regulate SNARE-dependent liposome membrane fusion. SNARE-binding protein complexin II is expressed in mast cells, where it positively regulates exocytotic release after antigen stimulation. We found that complexin II suppressed SNARE-dependent membrane fusion between mast cell SNARE-containing liposomes. This inhibitory effect of complexin II was abolished when we used a structurally divergent mutant (R59H) complexin II, where Arg59 is substituted with histidine. These results suggest that complexin II negatively regulates SNARE-dependent exocytotic membrane fusion in mast cells, and this inhibitory effect is dependent upon Arg59.Key words mast cell; exocytosis; membrane fusion; soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE); degranulation Mast cells are involved in allergic reactions. Cross-linking of FcεRI, the high-affinity receptor for the Fc region of immunoglobulin E, by multivalent antigens increases intracellular Ca 2+ concentration, causing exocytotic inflammatory mediator release from mast cells. 1) Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in this process. 2-9) SNARE-binding proteins also regulate exocytotic release in mast cells. 9,10) Syntaxin-3, -4 and synaptosomal-associated protein 23 kDa (SNAP-23) are SNARE proteins on the plasma membrane (t-SNARE) in mast cells. 2-4) Vesicle associated membrane protein (VAMP)-2, -7, and -8 are SNARE proteins on the secretory vesicles membrane (v-SNARE). 3,4) These t-SNARE and v-SNARE interact with each other and form SNARE complex which induce exocytotic release. [4][5][6] Final stage of exocytosis is membrane fusion process between secretory granule membrane and plasma membrane. SNAP-23/syntaxin-3 or SANP23/syntaxin-4 incorporated liposomes fuse with VAMP-8 but not VAMP-7 incorporated liposomes. 11)Complexins possess an α-helix in their central region that binds to and regulates SNAREs. 12) Complexins I and II are expressed in neuronal cells and may be involved in neurotransmitter release. 13,14) Complexin affects neuronal SNARE-mediated membrane fusion between liposomes. 15,16) Complexin II, but not complexin I, is expressed in mast cells, and exocytotic release after antigen stimulation is suppressed in mast cells where complexin II has been knocked down. 10) To elucidate the molecular mechanism of SNARE-dependent exocytosis in mast cells, we examined effects of complexin II on mast cell SNARE-dependent liposomal membrane fusion that is a simple assay system without other factors derived from cells. 11,[17][18][19] MATERIALS AND METHODS Protein Expression and PurificationFull-length rat syntaxin-3, SNAP-23, VAMP-2 or -8, and complexin II were expressed in Escherichia coli (E....

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Munc18-2 and Syntaxin 3 Control Distinct Essential Steps in Mast Cell Degranulation

Cited by 49 publications

References 72 publications

Chaperoning of closed syntaxin-3 through Lys46 and Glu59 in domain 1 of Munc18 proteins is indispensable for mast cell exocytosis

Chaperoning of closed syntaxin-3 through Lys46 and Glu59 in domain 1 of Munc18 proteins is indispensable for mast cell exocytosis

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Effect of Complexin II on Membrane Fusion between Liposomes Containing Mast Cell SNARE Proteins

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